Nicolas Dejeans

Kim Barroso

Martin E Fernandez-Zapico
email: fernandezzapico.martin@mayo.edu

Afshin Samali

Eric Chevet
email: eric.chevet@inserm.fr

Luciana L Almada

Rachel L O Olson

Holger W Auner

Rémy Pedeux

ACKNOWLEDGEMENT TABLE OF CONTENTS TABLE OF ILLUSTRATIONS TABLE OF ABREVATIONS ABSTRACT INTRODUCTION

Keywords: AAA+ ATPase, p97/VCP, Endoplasmic Reticulum Stress, Proteasis, Gene expression control Endoplasmic reticulum Cancer Stress Tumor EMT UPR, Proteasis, p97/VCP, Hedgehog, GLI1, HDACs, mSin3A

I Endoplasmic Reticulum 1 Endoplasmic Reticulum structure and functions. a) Structure b) Calcium storage and regulation c) Lipid synthesis d) Protein folding and maturation e) Protein export from the ER 2 Endoplasmic Reticulum signaling Epigenetic Regulation of Endoplasmic Reticulum Stress 3 Endoplasmic Reticulum Stress and cancer Novel roles of the unfolded protein response in the control of tumor development and aggressiveness II p97/VCP/CDC-48 1 Structure of p97/VCP. 2 Functions of p97/VCP/CDC-48. a) Regulation of Protein homeostasis b) Membrane fusion and trafficking c) Chromatin-associated functions 3 p97/VCP/CDC-48 role in transcription regulation

ACKNOWLEDGEMENT

The first part of my thesis work was realized under the direction of Dr. Eric Chevet in the Stress, Organites and Cancer Team at the INSERM unit U1053 directed by Jean Rosenbaum. I would like to thanks all the member of the INSERM unit 1053 with who I had the pleasure of working with during the first 2 years of my thesis and particularly the people from my team: Stéphanie, Nicolas, Saïd, Nestor and Eric who all have been really supportive and friendly.

Later I joined Dr Martin Fernandez-Zapico's laboratory in Rochester, USA and I would like to thank him for giving me the chance to have this very rewarding experience. I would also like to thank all the members of my team that have been extremely welcoming and friendly throughout all my stay, particularly: Martin, Lucianna, Ezequiel and Elisa.

When I came back to France I joined Eric Chevet's new team in Rennes, at the INSERM Oncogenesis Stress and Signaling directed by Patrick Legembre. I would like to thank all the members of my laboratory for this last year for their support and the good moments we have shared and particularly the members of my team: Asia, Tony, Eric, Gwen, Marie, Dimitrios, Alex.

I would like to thank Eric Chevet for giving me the opportunity to do this Ph.D, for motivating me when I needed, for always being available and enthusiastic about our work. I would also like to thank my friends and family who have supported me outside of work and particularly my father and the person who shares my life Marie. I also want to thank the member of the jury for taking the time to evaluate my work. The secretion pathway was studied in yeast and mammalian since the early 60's, which led to the identification of its different compartments and their hierarchy (1). The Endoplasmic Reticulum (ER) is the first compartment of the secretory pathway, it is composed of a network of membranes called cisternae organized in membrane-enclosed sacs or tube-like structures. The ER membranes are continuous with the outer nuclear membrane and two types of ER can be differentiated: the Rough and the Smooth Endoplasmic Reticulum. The Rough ER (RER) is covered with ribosomes on its cytosolic face which are the site of active protein translation.

OST

Future secreted or transmembrane proteins synthesized at the ribosomes bound to the ER can enter the ER lumen proteins to be folded and maturated, if these proteins reach their correct conformation they can escape the quality control machinery of the ER to be exported to their final destination.

The smooth ER (SRE) is identifiable by the lack of ribosomes on its surface and functions in metabolic processes such as the synthesis of lipids, phospholipids and steroids. Beyond being the first compartment of the secretory pathway and the site of lipid synthesis, the role of the ER extends to the regulation of calcium, detoxification and maintenance of protein homeostasis (2).

To ensure specific functions the ER is sub-compartmentalized and this is not only determined by

Figure 1: Electron microscopy image of the physical interaction between the ER and Mitochondria: Mitochondria Associated ER Membrane (MAM).

the type of ER (e.g. smooth or rough) but also by the localization of the ER membrane that can interact with the plasma membrane, the nucleus or other organites such as: Mitochondria at Mitochondria-Associated ER Membrane (MAM) domains (Figure 1), lysosomes and Golgi apparatus.

b) Calcium storage and regulation

The calcium ion (Ca 2+ ) is essential to human physiology as it is involved in various biological processes such as bone formation, hormone regulation, heart contraction or embryonic development as well as playing a major second messenger role in signal transduction pathways (3). Therefore cellular calcium concentration must be tightly controlled in a space-and timedependent manner. This is achieved by pumps and exchangers such as IP3R receptors (Inositol tri-phosphate Receptor 1-3) or SERCA proteins (Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase). In the cell, the ER is the main calcium storage compartment. Indeed the average ER calcium concentration is of 300 µM and it can go up to 1000 µM whereas the average cytosolic calcium concentration is of about 0.1 µM (3000-fold lower) (4). ER calcium homeostasis is essential, as too much Ca 2+ in the cytosol can impair the normal functions of the cell, and too little Ca 2+ in the ER lumen can impair ER functions and lead to ER stress. In addition this important difference in Ca 2+ concentration between the ER and the rest of the cell allows for its fast and controlled release in response to stimulus and as such Ca 2+ can act as a signal in many cellular processes such as secretion or motility. The release of the Ca 2+ from the ER to the cytosol is controlled by 2 receptor families: the RyR (Ryanodine receptor) and the IP3R receptors (Inositol tri-phosphate Receptor 1-3) (Figure 2) (5). Moreover, Ca 2+ can also be exchanged from the ER to the mitochondria at Mitochondria-Associated ER Membrane (MAM) domains thanks to VDAC (Voltage-dependent Anion Chanel) pump and IP3R receptor which is regulated by the protein receptor σ1R (Sigma-1 receptor) The entrance of the Ca 2+ into the ER relies on the SERCA proteins (Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase): ionic pumps that use the energy released by ATP hydrolysis to import the Ca 2+ (6) In human cells there are 3 SERCA genes (SERCA1-3) which encode up to 10 isoforms by alternative splicing, these isoforms are differentially expressed between cell types to answer specific needs (e.g.: skeletal muscle, cardiac muscle). In the ER lumen the Ca 2+ retention is mediated by the high capacity of many ER resident proteins to bind it such as: calreticulin (CALR), calnexin (CANX) or GRPs (Glucose Related Protein) (7).

Figure 2: Schematic representation of calcium storage and regulation by the ER in the cell -

The ER can release calcium in the cytosol through the RyR or IP3R receptors. To store calcium the ER relies on the SERCA pump. In the ER lumen the calcium is bound to ER resident proteins. The ER can also exchange calcium with the close mitochondria at MAM domains. These MAM domains are stabilized by Mitofusin which are mitochondria membrane proteins that can tether the Mitochondria to the ER.

c) Lipid synthesis

In the Eukaryotic cell the phospholipid synthesis mainly takes place in the Golgi apparatus and the ER, more precisely in the Smooth Reticulum Endoplasmic (SER) characterized by the low density of ribosomes surrounding the ER membrane. The phospholipids are the main components of all membranes and are used for the cell membrane and organites membranes such as endosomes, lysosomes, Golgi apparatus (8). Soluble cytosolic precursors are assembled on the cytosolic face of the SER, then flippase enzymes help the newly synthesized phospholipids to form a lipid bilayer (9). These lipids are then integrated into exocytosis vesicles for transportation before their release and merging with the destination membrane. However, thanks to the action of Lipid Transfer Proteins (LTP) if the destination of a lipid is a membrane close enough from the ER, like mitochondria at MAM domains there is no need for vesicle transportation (10). The SER is usually abundant in cells specialized in lipid metabolism: cells that synthesize steroid hormones from cholesterol. This is mainly achieved by the activation of transcription factors including SREBPs (Sterol Regulatory Element Binding Proteins). These transcription factors are synthesized as inactive precursors and are anchored in the ER membrane into protein complexes that are able to also bind to sterol lipids (SCAP, INSIG1, INSIG2) (11)(12). Upon cholesterol depletion, the SCAP/SREBP complex is recruited by COPII (Coat Protein Complex II) vesicles and exported to the Golgi apparatus where SREBP is cleaved by the S1P and S2P proteases thus releasing an active transcription factor in the cytosol (13). Alternatively, SREBP release can be induced by caspases 3 and 7. As a result, SREBP with the help of co-activators such as CBP (CREB Binding Protein), CBF (CCAAT-Binding Factor), NFY (Nuclear Factor Y) and Sp1 (Stimulating protein 1) activates the transcription of its target genes whose products are involved in lipid synthesis including cholesterol. The cholesterol synthesized in the ER can be exported in transport vesicles to any cellular membrane of which it is an essential component. If the cholesterol level at the membrane is diminishing, feedback loop exist to activate SREBP and resynthesize cholesterol.

d) Protein folding and maturation

Proteins that are destined for the secretory pathway possess a signal peptide at the beginning of their sequence (Figure 3). During their synthesis at the ribosomes bound to the ER when this peptide is synthesized the translation is halted by the binding of the Signal Recognition Particle (SRP) to the signal sequence (14a). The complex composed of the mRNA, SRP, the ribosome and newly synthesized peptide bind to the cytosolic face of the ER by interacting with membrane proteins such as the SRP receptor and the Translocation Associated Proteins (TRAP).

At this stage SRP dissociates from the nascent chain and translation resumes. Then the newly synthesized proteins can enter the ER lumen through a protein channel called the translocon. This channel is composed of SEC61 proteins and act as an aqueous channel through the hydrophobic membrane of the ER (14b). Inside the ER the signal peptide is cleaved by a signal peptidase that belongs to the serine protease family and the newly synthesized protein is then taken in charge by post translational modification (PTM) enzymes including PDIs (Protein disulphide isomerase) and Oligosaccharyl-transferase (OST). PDIs catalyze the formation of dislulfide bonds between cysteine residues within the protein and OST is a multimeric complex containing eight different proteins located at the ER membrane that can transfer a preassembled oligosaccharide to selected asparagine residues, a process also called N-glycosylation. These PTMs are crucial for the protein to find its correct conformation (15), but some also require the help of foldases or chaperones.

Foldases such as Grp58, PPI (Peptidyl prolyl cis-trans-isomerase) use the energy of ATP hydrolysis to catalyze structural transitions in the polypetide chain. In contrast, chaperones such as BiP, GRP78 or GRP170 create a suitable environment for the peptides by masking the hydrophobic sequences to prevent potential aggregation which could lead to a non-functional protein or by bringing closer different sequences of the peptide. If the protein is correctly folded and modified it is not held in the ER by the Quality Control (QC) machinery and can be exported to other compartments (15,16). However if the folding and maturation process fails, the protein is held in the ER by the QC machinery to get further folding chances, if the protein is terminally misfolded it is directed to the ERAD (Endoplasmic Reticulum Associated Degradation) pathway for degradation.

Figure 3: Co-translational translocation of a newly synthesized protein in the ER -1)

The mRNA is translated by the ribosome until the signal sequence is synthesized. The signal sequence is recognized by SRP. 2) The complex form by the mRNA/ribosome/nascent peptide and SRP is recruited at the ER membrane. 3) The nascent peptide is introduced inside the opened translocon. The signal peptidase cleaves the signal sequence. 4) Once the translation is finished the ribosome is released and the newly synthesized protein is taken in charge by PTM enzymes in the ER lumen..

e) Protein export from the ER

Proteins that enter the ER to be secreted can account for about 30-50% of all the protein synthesized by the cell (Figure 4) (17). Proteins that have passed the QC machinery are released from the ER at ER exit sites (ERES). These ERES are small membrane clusters that are contiguous with the ER membrane and that can bud to move to the Golgi (18). When these ERES start budding they are coated with COPII to form a vesicle, after their detachment from the ER these vesicles will flow to the Golgi where they will be recruited by surface receptor proteins.

The vesicles will then be uncoated and fuse with Golgi membrane thus releasing their cargo proteins which will progress into the secretory pathway.

Figure 4: Schematic representation of the secretory pathway -

The first compartment of the secretory pathway is the ER, proteins that are destined to be secreted are first synthesized at the ER membrane before they are folded in the ER lumen. They then exit the ER at ER exit sites and are transferred to the Golgi apparatus where they will complete their maturation. Finally proteins are exported to their final destination either to the cell membrane through transport vesicles or lysosomes for degradation (adapted from 19).

On the contrary terminally misfolded proteins are directed to the ERAD pathway for degradation (Figure 5) (20). The first step of the ERAD is substrate recognition: terminally misfolded proteins contains substructures such as exposed hydrophobic regions, unpaired cysteine residues or immature glycans which are recognized by ER sensor proteins of the ER quality control machinery. In mammalian cells two distinct ERAD pathways exist for glycoproteins and nonglycoproteins: Glycoproteins are recognized by lectin chaperones calnexin and calreticulin which can interact with ERp57 a glycoprotein specific thiol-disulfide oxidoreductase whereas nonglycoproteins are recognized and recruited to ERdj5 by BiP (21).

Evidences suggest that these misfolded proteins account for 30% of the proteins that enter the ER (22). Next the misfolded protein escorted by the chaperones is transported to a protein channel called retrotranslocon so it can be exported into the cytosol. The driving force required to extract the protein is provided by the AAA+ ATP p97/VCP/CDC-48 and its cofactors at the exit of the retrotranslocon (23). As the protein exits the dysloncon it is ubiquitinated (ubiquitin is a small 76 amino acid peptide) mainly by SYVN1 and MARCH6 two ER membrane anchored ubiquitin ligases. This polyubiquitin tag is then recognized by the 19S subunit of the proteasome and the polypeptide chain is fed into the central chamber of the 20S core region for proteolytic degradation.

Figure 5: Schematic representation of Endoplasmic Reticulum Associated Degradation. A) Misfolded proteins contain substructures that are recognized by ER chaperones of the ER quality control machinery. B) The misfolded protein is addressed to the retrotranslocon by ER chaperones. C) P97/VCP/CDC-48 thanks to the energy of ATP hydrolysis pulls the protein out of the ER through the retrotranslocon. D) As the protein exits it is polyubiquitinylated by E3 ubiquitin ligases. E) Once the ubiquitin modified protein is in the cytosol it is addressed to the proteasome for proteosomal degradation (adapted from 23).

2) Endoplasmic Reticulum signalling

As previously described, the ER homeostasis relies on several fine-tuned processes: protein folding, quality control, protein export or degradation and certain conditions such as change in ATP, calcium, redox state of the cell, or glucose can affect the folding of proteins in the ER. If the protein folding demand exceeds ER folding capacity thus disturbing the ER homeostasis it leads to a situation called ER stress. To restore its homeostasis the ER triggers an adaptive signalling response called the Unfolded Protein Response (UPR) (24). The following book chapter describes the signalling that emanates from the ER in response to ER stress: the UPR at the transcriptional, post-transcriptional, post-translation and epigenetic levels. This book chapter also highlights that the UPR signalling regulates pathways whose roles go beyond restoring the ER homeostasis. Finally, it provides an overview of the importance of ER stress induced transcription in diseases.

BOOK CHAPTER

3) Endoplasmic Reticulum Stress and cancer

Cancer cells proliferate quickly and have to cope with intrinsic stresses (oncogene expression, high DNA replication, high synthesis rates, oxidative stress) and extrinsic stresses (hypoxia, nutrient deprivation, change of their micro-environment, chemotherapeutic agents) which they need to overcome to survive. In most cancer cells due to the high protein folding demand and challenging conditions, the UPR is activated (25), although in normal cells if ER homeostasis cannot be restored the cell signals toward apoptosis, cancer cells have reduced their sensitivity to stress-mediated cell death through forced adaptation mechanisms (26).

In the previous book chapter we have described how ER stress-induced transcription affects proliferation and survival, two key features of cancers cells. The following review further discusses the impact of the UPR activation in different tumors and through examples we illustrate how cancer cells use the secretory pathway to acquire malignant traits such as inflammation, immunogenicity or angiogenesis.

Introduction

Tumor phenotypes including development and aggressiveness features can dramatically vary depending on the origin of tumor cells and context. The hallmarks of cancer defined by Hanahan and Weinberg [1] have helped to define these characteristics, which were also connected to the status of the secretory pathway (SP) [2,3]. As a consequence this essential cellular component has taken significant importance in the acquisition of cancer cell features. The SP ensures the biogenesis of proteins that are membrane-bound or secreted into the extracellular milieu. It is well accepted that approximately one-third of the polypeptides synthesized by a cell, enter the endoplasmic reticulum (ER), the first compartment of the SP [2,3]. However, the quantity of proteins entering the secretory pathway fluctuates, depending on the cell physiology, function and specific microenvironment. For instance, the synthesis of antibodies, extracellular matrix proteins, membrane receptors or secretory cyto/chemokines is cell-type specific and can impact the workload of the secretory machinery. Moreover, cell migration, differentiation or proliferation features can also create the demand for a higher need for protein secretion. Protein secretion fluctuations affect cell homeostasis, particularly cell amino acid, lipid and sugar metabolism and energy consumption. As such, a strong and reliable adaptive system is central for the cell to cope with the increased demand for protein folding in the ER. This adaptive system is named the unfolded protein response (UPR). In this review, we provide specific examples illustrating how the diversification of UPR signals in many human cell types, particularly in secretory cells, could impact typical cancer initiation, tumor development and cancer cell aggressiveness.

The UPR transmits stress signals from the ER lumen to the rest of the cell by three different proteins called PERK, ATF6 and IRE1. PERK (PKR-like endoplasmic reticulum kinase) is a transmembrane protein with a specific kinase activity in its cytosolic domain. Its main substrate is the translation initiation factor eIF2␣. Phosphorylation of eIF2␣ results in a decrease in translation as well as a preferential translation of key proteins such as CHOP and GADD34, two factors directly involved in the cellular decisions of life or death. The transmembrane protein ATF6 (Activating Transcription Factor 6) is an ER transcription factor. Under stress conditions, ATF6 is exported to the Golgi apparatus, cleaved and released from its membrane attachment by the proteases S1P and S2P, to play its role as nuclear Fig. 1. Cancer relevant UPR signaling components. Major UPR signaling modules are drawn. Relevance to cancer is indicated as follows: orange -proteins directly implicated in the modulation of cancer cell features, including initiation, angiogenesis, inflammation, immunogenicity or resistance. Green -proteins whose modulation or activation is observed in cancer tissues or involved with cancer development or aggressiveness, but whose role in the control of cancer features is not clearly defined. transcriptional activator. Finally, IRE1 (Inositol Requiring Enzyme 1), an ER resident type 1 transmembrane protein, has two enzymatic activities in its cytosolic domain: a serine/threonine kinase and an endoribonuclease activity. The endoribonuclease activity itself has two distinct molecular functions: (i) it participates in the unconventional splicing of the XBP1 transcription factor mRNA [4]; (ii) it degrades the mRNA of several secreted proteins, a process called RIDD (Regulated IRE1-Dependent Decay of RNA) [5]. The integration of signals from these three molecular pathways leads to a general transcription and translation reorientation, in favor of cell survival. Among the cellular processes regulated, the antioxidant capacity is increased, protein synthesis is decreased and the expression of ER chaperones/ER quality control proteins involved in protein folding (BiP, GRP94, CRT, PDIs) and in misfolded protein degradation is enhanced [6][7][8]. Finally, if ER homeostasis is not restored, ER stress can trigger apoptosis [9,10] (Fig. 1).

It is well established that differentiated cells such as neurons, blood cells, pancreatic ␤-cells, hepatocytes, all require a dedicated secretory pathway with appropriate specialized regulations [11]. In accordance with this, an increasing number of studies have shown a dependency of specific UPR components for the differentiation of particular cell types. For instance, the IRE1-XBP1 branch is required for the differentiation of pancreatic ␤ cells, plasma cells, or adipocytes [12][13][14][15][16] and disturbance of the PERK-ATF4 pathway triggers defects in oligodendrocytes, pancreatic and skeletal functions [17][18][19][20][21].

The "secretory switch" in transformed cells

Most cancers have to cope with increasing fluxes of proteins through their secretory pathway. This high secretory protein demand is caused by different hallmarks of cancer [2] and comprises all the processes that increase gene expression, in an unspecific manner, such as aneuploidy or the universal amplifier of transcription, MYC [22,23]. Hence, it is not surprising that aneuploidy was found to be associated to hypersensitivity to conditions interfering with protein synthesis and protein folding in yeast [24] and in human cancer cells [25], and that MYC transformation requires a reliable secretory pathway to mediate its oncogenic potential [26]. Moreover, cell transformation can result in an increase in proliferation and metabolic demand, thereby leading to nutrient (i.e. glucose, amino acids) depletion and subsequent ER stress [START_REF] Huber | p58(IPK)-Mediated attenuation of the proapoptotic PERK-CHOP pathway allows malignant progression upon low glucose[END_REF]. This means that during the cell transformation process, a "secretory switch" occurs and provides the transformed cells with novel secretory properties, which will in turn impact on cell homeostasis and interaction with the stroma.

What are the consequences of the "secretory switch" and associated ER stress on tumor-stroma interactions? First, it can lead to microenvironment architecture destabilization by remodeling of the extracellular matrix (ECM) through changes in ECM components abundance or matrix metalloproteinases (MMPs) expression, and consequently, to an increase in cancer dissemination and invasion [28,29]. Second, by modulating messenger (i.e. chemokines, cytokines) or contact/adhesion protein abundance (i.e. membrane receptors, integrins), the "secretory switch" and associated ER stress can trigger cancer cell proliferation, migration or tumor angiogenesis. Third, it can lead to remodeling of the immune response and ER stress transmission in the tumor microenvironment [30,31]. Fourth, it can modulate tumor immunogenicity by stimulating surface exposure of ER chaperones such as CRT [2,32,33] (Fig. 2).

UPR involvement in gastrointestinal cancer initiation

The UPR is a central pathway for intestinal functions and differentiation, and the human gastrointestinal tract represents an interesting example of UPR specialization. This is well illustrated by the immunostaining of UPR components in the normal intestine, which showed that UPR activation occurs in a heterogeneous manner in intestinal cell populations. Indeed, GRP78 abundance appeared high in transit amplifying cells (TA), low in intestinal stem cells (ISC) and heterogenous in Paneth cells [34]. This suggests that the UPR could be induced with intestinal cell differentiation or could represent a pathway driving differentiation. One element supporting the second hypothesis is that activation of the PERK/eIF2␣ axis in itself is sufficient to trigger the loss of ISC stemness [34]. Considering that ISCs are thought to represent the cells of origin for most colorectal cancers (CRCs) [35], this suggests that ER stress could have central implication for cancer initiation in the gastrointestinal tract.

Apart from the PERK/eIF2␣ arm, specialization of the UPR in the intestine is partly due to the IRE1 branch. Indeed, the gastrointestinal tract is the main tissue where the two IRE1 paralogs, IRE1␣ and IRE1␤ are expressed (in the epithelial cells) [36]. Although the functions of IRE1␤ are not entirely understood, IRE1␤-/-mice showed a role in the control of mucin production in goblet cells [37] and in resistance to chemically induced colitis [36]. Moreover, XBP1 deficiency or expression of XBP1 variants was associated with Crohn's disease and ulcerative colitis [START_REF] Kaser | XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[END_REF]. Interestingly, IRE1␤ and XBP1 deficiency were both associated with increased ER stress and inflammation of the intestine. A more recent report described the association between inflammatory bowel disease and tumorigenesis upon targeted deletion of XBP1 in the intestine [39]. In this model, XBP1 loss in epithelial cells results in an increase in colorectal cancer and colitis-associated cancer. This effect was attributed to an increase in ISC and TA cells, and a dysfunction of Paneth cells. ISC hyperproliferation was linked to increased WNT11 expression in Paneth cells and TA cells hyperproliferation to the activation of an ER stress-dependent interleukin/STAT3 pathway. Interestingly, in this study, a transgenic mouse model presenting a double deletion of XBP1 and IRE1␣ was found to not present ISC hyperproliferation observed in XBP1 deficient mice. Although one can ask the question of the roles of others components of the UPR, and especially IRE1␤ in these processes, the authors proposed that IRE1␣ is an important mediator of ER stress induced ISC expansion, in an XBP1independent manner, which might leave room for an instrumental role of RIDD. In addition, XBP1 deletion in Paneth cells revealed that the IRE1␣-TNF␣/NF-B pathway was central for ER stress-induced inflammation [40]. Parallel to these mechanisms, autophagy induced by the PERK/eIF2␣/ATF4 signaling axis partly restrained IRE1 activation and ER stress-induced intestinal inflammation. This was proposed as an explanation for the identification of mutations in autophagy components as risk factors in Crohn's disease [40]. Finally, adding to the role of the UPR in intestinal epithelium, XBP1 was also identified as a susceptibility locus associated with esophagus squamous cell carcinoma [41] and ER stress was shown to induce epithelial differentiation in precursor cells in the esophagus [42] and also may be linked to Barett's syndrome [43].

These studies provide good examples of (i) how the UPR can finetune the entire functions and differentiation of the gastrointestinal epithelia by integrating information from the microenvironment and (ii) how deregulation of this molecular pathway (XBP1 deletion) can impact inflammatory bowel disease and gastrointestinal cancer initiation.

UPR in blood cancers: transformation, progression and drug resistance

Blood associated cancers represent interesting models in which UPR signals might determine tumor phenotypes. First of all, the expression and activity of UPR components including BiP, IRE1␣, BLIMP1, and XBP1 are required for terminal differentiation of B cells into plasma cells and are found overexpressed in plasma cell-derived multiple myeloma (MM). MM evolves from a highly prevalent premalignant condition termed monoclonal gammopathy of undetermined significance (MGUS). A MGUS/MM phenotype was recently reported in transgenic mice with Em-directed expression of the XBP1 spliced isoform (XBP1s). This was corroborated with the aberrant expression of known human MM dysregulated genes and thus implicates XBP1s dysregulation in MM pathogenesis [44]. Interestingly, Cre-mediated and inducible deletion of BiP, BLIMP1 or XBP1 consistently induces cellular stresses and cell death in normal pre-B cells and in pre-B-cell acute lymphoblastic leukemia (ALL) driven by BCR-ABL and NRAS [45]. Moreover, two ALL clinical trials revealed that high XBP1s levels at the time of diagnosis predicted poor outcome. As such, pharmacological inhibition of IRE1-induced selective cell death in patient-derived pre-B ALL cells and significantly prolonged survival of transplanted mice. As a consequence, pre-B ALL cells appeared uniquely vulnerable to ER stress [45]. In a recent study, the Hypoxia Inducible Factor (HIF)-2␣ was implicated in the engraftment ability of human acute myeloid leukemia (AML) cells and in the maintenance of hematopoietic stem and progenitor cells (HSPCs). The mechanism controlling maintenance of HSPCs also involved ER stress signaling as HIF-2␣-deficient HSPCs displayed increased production of reactive oxygen species (ROS), which subsequently triggered apoptosis by activation of the UPR [46]. Hence these results might suggest an instrumental role of the UPR in HSPC differentiation program and in maintenance of the AML phenotype. In another model of blood-derived cancer, chronic myeloid leukemia (CML), apoptotic death triggered by the BCR-ABL inhibitor, imatinib, is activated downstream of ER stress [47]. Moreover, imatinib resistance in CML K562 cells was bypassed when preventing the activation of the ATF6 arm of the UPR, thus demonstrating the strong interconnection of these pathways in acquisition of tumor cell phenotypes [48]. Normal vs. cancer cell secretory pathway control and biological outcomes. In normal cells, the secretory pathway adapts to fluctuation of environmental stresses and intracellular needs, through a complex molecular signaling pathway: the unfolded protein response (UPR). This adaptation program is triggered by three ER transmembrane sensors, namely IRE1, PERK and ATF6. The cell secretory proteins needs vary depending on cell type, differentiation state or on the physiological context. In cancer cells, the secretory pathway is subjected to a strong environmental pressure due to environmental stress factors, such as hypoxia, oxidative stress or chemotherapies, and to oncogenic pressure (e.g. Myc/aneuploidy stimulation of transcription). Furthermore, in these cells, the increase in secretion demand is also dependent on a substantial requirement of energy and amino acid supply. The integration of both intrinsic and extrinsic challenges results in disturbance of the ER homeostasis causing the UPR to be constitutively active in these cells. Disturbance of the secretory pathway will, as a consequence, lead to the modulation of the secretion of proteins important for cancer features such as growth factors and their associated receptors, extracellular matrix proteins, matrix metalloproteinases, inflammatory factors, integrins, immunogenic factors or pro-angiogenic factors.

UPR and EMT: an intricate relationship

Epithelial to mesenchymal transition (EMT) enables carcinoma cells to acquire key malignant traits such as migratory and invasion properties, induces stem cell properties and drug resistance [49][50][51]. Hallmarks of this transition are repression of epithelial markers, up-regulation of mesenchymal markers and changes in morphology. During EMT, the phenotype of carcinoma cells is largely modified, for example the loss of epithelial polarity and zonula adherens mediated by the down-regulation of E-cadherin. E-cadherin is a cell adhesion protein and its cytosolic domain is associated at the membrane of the cell to ␤-catenin, which is a major player of the canonical WNT pathway [52]. Through EMT, diminution of E-cadherin leads to the release of ␤-catenin, resulting in its nuclear translocation. In the nucleus, ␤-catenin associates with transcription factors of the TCF/LEF family to regulate transcription of genes involved in EMT, migration and invasion.

Activation of the UPR has been involved in a growing number of cancers [53], but the link between UPR and EMT has been studied recently in breast cancers. Indeed, recent reports show an interrelationship between UPR signals and EMT, in a context specific manner [54][55][56].

Activation of the UPR is instrumental for EMT induction

This phenomenon was first reported in thyroid cells, in which tunicamycin or thapsigargin triggered signaling by the protooncogene tyrosine kinase SRC, caused dedifferentiation through the down-regulation of thyroid specific genes and induced an EMTlike phenotype. This included the change in the organization of the polarized epithelial monolayer, the formation of actin stress fibers, the loss of trans-epithelial resistance, the down-regulation of Ecadherin and the up-regulation of mesenchymal markers such as vimentin, ␣-smooth actin, ␣1I collagen and SNAI1/SIP1. Moreover the use of PP2, a SRC kinase inhibitor, prevented dedifferentiation and EMT, thus confirming the involvement of the SRC pathway [54]. Moreover, the UPR (induced by tunicamycin or overexpression of a variant protein) in alveolar epithelial cells was shown to trigger the SRC and ␤-catenin pathways [57]. Again, the use of PP2 also blocked the EMT and maintained the epithelial phenotype. Notably no increase in TGF-␤1, an important mediator of EMT, was observed in this report. Interestingly, in renal proximal tubular epithelial cells, thapsigargin induced an EMT whereas tunicamycin did not [58]. ER stressors that alter calcium fluxes between the ER lumen and the cytosol such as thapsigargin lead to an increased expression of TDAG51 and TGF-␤1. TDAG51 interacts with the cytoskeleton and induces shape changes as well as the activation of WNT signaling thereby leading to EMT. As such, overexpression of TDAG51 alone was able to induce an EMT phenotype in HK-2 cells. The serine/cysteine protease inhibitor SCCA1 is deregulated in many cancers associated with poor differentiation and aggressiveness. In mammary epithelial cells (MCF10A), overexpression of SCCA1 induces chronic UPR. This non-lethal chronic UPR activates NF-B that leads to IL-6 production, resulting in EMT-like phenotypes [59]. It has been recently reported that IL-6 signaling plays a critical role in driving EMT through cell autonomous inflammation [60,61]. In light of these reports, activation of UPR can lead to EMT trough several mechanisms including IL-6, SRC and/or WNT signaling, but how the UPR is triggered, either through alteration of calcium concentrations or increase of improperly folded proteins, might also determine the biological outcome. Thus, one might hypothesize that ER stressed cells enter dedifferentiation/EMT to change their phenotypes and consequently lower ER stress [57].

EMT induces activation of the UPR in colorectal and breast carcinoma

Cells subjected to EMT are also known to display an important secretory phenotype notably by changes in ECM protein secretion [62,63]. This could represent a cause for ER stress and UPR activation. In colorectal carcinoma cells (SW480, HCT116), stabilization of HIF1␣ through CoCl 2 -mediated inhibition of proline hydroxylase, or serum starvation, induces EMT and the subsequent activation of the UPR [64]. This mechanism is in part dependent upon ZEB-1, which is the main factor for EMT in colorectal carcinoma cells and a transcriptional repressor for E-cadherin [52,65,66]. In mammary epithelial cells, EMT induction by TWIST overexpression correlates with PERK constitutive activation [55]. Other branches of the UPR (i.e. IRE1␣ and ATF6) were not involved in this process. Interestingly, inhibition of PERK activity attenuated cells' ability to migrate and to form tumor spheres, thereby indicating that PERK might be involved in EMT-dependent cell malignancy. In addition, PERK signaling in EMT dedifferentiated cells leads to constitutive activation of NRF2, a master regulator of cellular response to oxidative damage, causing these cells to become chemoresistant through expression of antioxidant enzymes and drug efflux pumps [67][68][69]. These results might therefore explain the correlation observed between PERK activation status and highly aggressive and poorly differentiated breast cancer tumors. It is noteworthy that in tubular epithelial cells both EMT and UPR are activated simultaneously through reactive oxygen species (ROS) and SRC kinase-dependent pathways [70].

Targeting UPR as a novel approach to treat EMT chemoresistant cells

As there is a hierarchical relationship between UPR and EMT it could allow the development of new treatment strategies. Indeed, in diseases where EMT is induced by UPR, e.g. lung fibrosis, chronic kidney disease or breast cancer [57][58][59], targeting the UPR or the downstream activated pathways (e.g. SRC, WNT) with inhibitors might be an efficient way to prevent cells from undergoing EMT. In vitro, results were already observed with PP2 targeting the SRC kinase and preventing both PC C13 cells (thyroid cells) and alveolar epithelial cells from undergoing an EMT [54,57]. Currently, ER stress drugs are only used to treat multiple myeloma patients [71], however because EMT is associated with chemoresistance and invasiveness [49][50][51], there is a critical need to develop new approaches, therefore it might be very attractive to exploit the ER stress-sensitivity exhibited by cells subjected to EMT [64]. Indeed it was shown that in breast cancer cells several ER stressors such as tunicamycin, thapsigargin, DTT and A23187 render EMT undergoing cells more sensitive to cell death (up to 25 fold for thapsigargin). These treatments could even selectively eliminated EMT undergoing cells when co-cultivated with normal cells [55]. Also targeting the PERK pathway that is constitutively active in breast cancer [56,72] could also be a promising option. Indeed PERK is required for the cells to secrete new extracellular matrix and to enable EMT undergoing cells to invade, metastasize and form tumor spheres. PERK is also responsible for the constitutive activation of NRF2 in EMT undergoing human breast epithelial cells, causing cells to become MDR. Inhibition of PERK caused the decreased expression of 58 of the 142 NRF2 target genes and sensitized cells to chemotherapy. Together these observations suggest that targeting the UPR and its downstream effectors could be a key therapeutic strategy in the treatment of drug-resistant cancer cells.

UPR control of glioblastoma phenotypes

Glioblastoma (GBM) is the most common primary brain tumor in humans and remains incurable [73]. Despite the therapeutic efforts made in recent years, mortality is still close to 100% at 5 years. Different factors are involved in GBM aggressiveness, among which angiogenesis and tumor cell invasion/infiltration are critical [74,75]. Moreover, the mesenchymal phenotype is another hallmark of tumor aggressiveness in GBM [76,[START_REF] Carro | The transcriptional network for mesenchymal transformation of brain tumours[END_REF]. Remarkably, a single UPR component, IRE1␣, appeared to regulate these three features of GBM aggressiveness. Indeed, it was shown that expression of a dominant negative form of IRE1␣ triggered a mesenchymal drift in glioblastoma, characterized by modulation of the expression of extracellular matrix, angiogenesis, and inflammation proteins. This is in agreement with other studies reporting IRE1␣ coding gene somatic mutations in GBM [78,79] and ranking IRE1␣ as the fifth highest mutated kinase, carrying at least one driver mutation [78]. The mesenchymal drift accompanied by IRE1␣ inactivation was due to a set of pathways acting in a synergistically manner. In this model, IRE1␣-driven modulation of angiogenesis was attributed to the positive regulation of pro-angiogenic factors such as VEGF-A, IL-1␤ and IL-6 secretion [80,81] and the cleavage of the mRNA coding for the circadian gene PERIOD1 [82]. Adding to its role in the regulation of angiogenesis, PERIOD1 also regulated IRE1␣-dependent GBM infiltration [82]. Another IRE1␣ endoribonuclease substrate, the mRNA coding for the extracellular matrix protein SPARC was also found to be involved in modulation of GBM invasion ability in an autocrine fashion [START_REF] Dejeans | Autocrine control of glioma cells adhesion and migration through IRE1alphamediated cleavage of SPARC mRNA[END_REF]. Taken together, these data underline that IRE1␣ in itself can send both intracrine and autocrine signals to control the phenotype, the physiology and the aggressiveness of GBM.

UPR in triple negative breast cancer

Triple negative breast cancers (TNBC) represent the most aggressive breast cancer subtype, with high rates of tumor recurrence and poor overall survival [START_REF] Foulkes | Triple-negative breast cancer[END_REF]. Although the lack of expression of the estrogen, progesterone and HER2 receptor clusters, these tumors within the same group still comprise heterogeneous and poorly characterized breast cancers with no selective therapy [START_REF] Lehmann | Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies[END_REF]. Recently, a study by Chen and colleagues [START_REF] Chen | XBP1 promotes triple-negative breast cancer by controlling the HIF1alpha pathway[END_REF] revealed the contribution of the UPR to TNBC, in particular through the crosstalk with HIF-1␣, a transcription factor previously shown to be of particular importance in the hypoxic response in TNBC. Chen and colleagues characterize a new molecular mechanism, XBP1sdependent HIF-1␣ activation in TNBC, thereby indicating potential novel therapeutic strategies mediated through the inhibition of XBP1 in TNBC [START_REF] Lu | A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB[END_REF]. These observations could also be linked to the acquisition of a mesenchymal phenotype by breast tumor cells. Indeed, increased expression of XBP1 is associated with the progression of breast cancer and XBP1s is significantly over-expressed in matched metastatic tumors, which can act as a major regulator of EMT through SNAIL signaling [START_REF] Li | XBP1 induces snail expression to promote epithelial-to-mesenchymal transition and invasion of breast cancer cells[END_REF]. Moreover, as autophagy and UPR signaling also appear to be interconnected, combined chloroquine (CQ), a pharmacological inhibitor of autophagy, with other drugs known to act as ER stress enhancers (nelfinavir (an HIV protease inhibitor) and celecoxib (a cyclooxygenase-2 inhibitor) or its noncoxib analog 2,5-dimethyl-celecoxib (DMC)) were tested in TNBC. Addition of CQ resulted in synergistic enhancement of tumor cell killing by ER stress aggravators in vitro and in vivo, thus opening novel therapeutic avenues for TNBC [START_REF] Thomas | Preferential killing of triple-negative breast cancer cells in vitro and in vivo when pharmacological aggravators of endoplasmic reticulum stress are combined with autophagy inhibitors[END_REF].

Conclusions and future perspectives

Collectively, the aforementioned information sheds light on the intricate cancer signaling networks into which the UPR is involved. From this analysis it becomes evident that characterizing the UPR status in tumors might not only represent a good predictor of the disease outcome but also constitute an essential toolkit for better defining personalized treatments and following up treatment efficacy. As such, a thorough analysis of the UPR in tumors could be envisioned to firstly select the best and most relevant markers/predictors of tumor characteristics and then secondly to apply the most efficient targeted therapies to those tumors. Needless to say that in this context, therapies targeting the UPR itself could also be of interest either alone or as adjuvant therapies. In conclusion, the specificity of UPR signals and its impact on tumor phenotype represents an interesting avenue to better characterize carcinogenesis but also when documented in patients' tumors will constitute a novel basis for tumor typing and specialized treatments.

We have observed that many reports have implicated the UPR in different tumors as playing a pro-survival, pro-adaptation role therefore great efforts to target this pathway in cancer cells are being undertaken (27a). However the contribution of the UPR to malignancy is complex and differs from a given tumor type to another due to the cell characteristics and the conditions to which they are exposed. More mechanistic and physiological studies are therefore needed for each tumor to ensure the efficacy of pharmacological small molecules. In cancer cells, the stress induced by the accumulation of unfolded proteins in the ER triggers the UPR to restore protein homeostasis. The UPR signalling and the transcriptional reprogramming that accompany it, not only help the cancer cells to cope with the unfolded proteins but has also been shown to participate in the acquisition of malignant traits such as growth, angiogenesis, invasion and metastasis or inflammation. Although the molecular events triggered by the UPR are well described, the transcriptional mechanism underlying the transcriptional reprogramming still remains poorly defined.

The genomic instability and the high proliferation rate of cancer cells cause them to be overloaded with proteins including mutated proteins that cannot find their correct conformation.

As previously mentioned, such conditions trigger ER stress and the UPR, but another key player of maintenance of protein homeostasis is p97/VCP. Indeed, p97/VCP is not only part of the ERAD pathway but it is also involved in other degradation processes such as autophagy and works in concert with the Ubiquitin Proteasome System to degrade ubiquitinylated proteins. Interestingly p97/VCP was found overexpressed in many cancers, suggesting that its role in the regulation of protein homeostasis might be key for cancer cell survival.

II) p97/VCP/CDC-48

The cdc-48 gene was discovered thirty years ago in a genetic screen conducted in S. cerevisiae and named Cell Division Cycle 48 among other genes that caused cell cycle arrest (27b). CDC-48 mammalian homolog was later reported as a 97 kDa protein precursor for the small peptide valosin, and therefore name as valosin-containing protein (VCP) or p97. Although the valosin was a purification artefact unrelated to p97, the VCP nomenclature is still being used.

Studies on p97 are also conducted in other species like D. melanogaster where it is the name Transitional endoplasmic reticulum ATPase TER94 (TER94) that is often used, or C. elegans which also use the name cdc-48 but interestingly have two copies of the gene (cdc-48.1 and cdc-48.2).

1) Structure of p97/VCP

The p97 protein belongs to the ATPase Associated with various cellular Activity (AAA + ) family. AAA + proteins are characterized by the presence of AAA domains that contain Walker A and B motifs. Proteins containing such Walker motifs are found throughout evolution (from archae bacteria to human) which suggest that they are fundamental in all life forms (28).

Moreover, p97 is strongly and ubiquitously expressed in all multicellular organisms, for example in human p97/VCP proteins account for about 1% of the total cell proteins (29). The human p97/VCP is an 806 amino acid (AA) long protein and has four domains (30) (Figure 6A): the N domain starts at AA 1 to 187 and its role is to facilitate cofactors binding and substrate recognition -after a short linker the D1 domain (AA 208 to 459) contains Walker A and B motifs and although it possesses ATP activity it is believed that its main role is to help p97/VCP to find its quaternary structure -after a short linker the D2 domain (AA 481 to 761) also contains Walker A and B motifs and is the main site for ATP hydrolysis -finally the C terminal tail (AA 762 to 806) serves as a PTM site and binding site for p97/VCP cofactors In the cell p97/VCP is inactive as a monomer, but like other AAA+ proteins is active as a hexamer (Figure 6B) (28). Electron microscopy analyses revealed that p97/VCP/CDC-48 forms a hexameric ring which allows access for its cofactors to bind its N domain or C terminal tail.

Figure 6: Schematic representation p97/VCP domains and quaternary structure -A) p97/VCP 4 domains: N domain for cofactors binding, D1 domain for ATPase and quaternary structure, D2 domain main ATPase domain and C terminal tail for PTM and cofactors binding. B) p97/VCP function as a hexamer in the cells, the domain colours are conserved between A) and B), its structure quaternary structure allows ATPase activity and cofactors binding at its N domains and C terminal tails (adapted from 30).

In the rest of the text p97/VCP will refer to the human protein/gene, CDC-48 to the yeast protein/gene and CDC-48.1 or CDC-48.2 to the worm protein/gene and p97 will refer to the gene/protein of multiple or other species.

2) Functions of p97/VCP/CDC-48

p97's main function is to segregate (extract) a given protein from protein complex, organelle membrane or chromatin thereby facilitating its recycling or degradation by the proteasome (Figure 7)(30). p97 associates with various cofactors/adaptors and these interactions are key for its activity and functional diversity (Figure 8) (31). Most of p97 partners possess ubiquitin-X (UBX) domain or UBX-like domain and can bind to ubiquitin (32). Among them are many E3 ubiquitin ligases which are important for specific substrate recognition, but also E4B/UFD2 an ubiquitin ligase E4 which can extend shorter ubiquitin chain to promote proteasome degradation (33). Interestingly, p97 can also interact with deubiquitinating enzymes (DUB) that can remove ubiquitin from protein thus promoting substrate recycling over proteasome degradation (30). p97 adaptors are also important to promote its recruitment to a specific subcellular compartment as they can interact with protein from this subcellular compartment. For example Ufd1/Npl4 can interact with the ER transmembrane protein VIMP (VCP interacting membrane protein) to recruit p97 at the ER membrane so it can participate in ERAD (34). To date p97 functions can be grouped in three major categories all mediated by its segregase activity. These categories comprise regulation of protein homeostasis, membrane fusion and protein trafficking and chromatin-associated functions.

Figure 7: Schematic representation p97 activity -In this example p97 substrate: the "S" protein in brown is in complex with the protein B, once "S" is ubiquitinylated by the UPS it is recognized by p97 and its ubiquitin binding cofactors. p97 can then separate the protein "S" from its partner with the force provide by ATP hydrolysis. From there if the ubiquitin chain is maintained or extend the protein is sent to the proteasome for degradation, however if the ubiquitin chain is removed by deubiquitinase which can associate with p97, the protein is recycled (adapted from 30).

Figure 8: Schematic representation of p97 functions in cellular processes -A wide range of cellular processes required p97 mediated degradation such as: ERAD, mitochondria-associated degradation, autophagy, aggregates handling, chromatin-associated degradation and Endosomal trafficking (adapted from 30). a) Regulation of Protein homeostasis

Short-lived, misfolded or damaged proteins are degraded by the UPS. Ubiquitination occurs on lysine residues through the sequential activity of ubiquitin activating (E1), ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes. p97 thanks to its various cofactors/adaptors including several E3 enzymes is involved in the regulation of several protein homeostasis processes (Figure 8). the protein through the retrotranslocon to the cytosol, as the protein is pulled out it is also processed by the UPS (Figure 5). It is also believed that p97/VCP might play a role in the shuttling of the misfolded protein to the proteasome but this process is poorly defined.

Additionally, p97/VCP is capable of releasing membrane-bound transcription factors such as Nrf1 which are not degraded but instead translocate to the nucleus to activate transcription (35) Similarly, CDC-48 was shown to be required for the cleavage of SREBP from Golgi membrane in yeast (36).

-Mitochondria-associated degradation -It has also been showed in yeast that CDC-48 with its cofactor Vms1 (VCP/Cdc48-associated mitochondrial stress-responsive 1), Npl4, Ufd1 and the cooperation of the UPS are responsible for the quality control of proteins of the outer mitochondrial membrane (OOM) such as Fzo1 and Mcl1 (37). This observation is reinforced by the fact that CDC-48 cofactors Ufd1 and Npl4 are found highly enriched at the surface of the mitochondria.

-Co-translational degradation -p97/VCP/CDC-48 is also involved in a process called co-translational degradation, in fact during protein translation errors in the mRNAs or its degradation can cause the ribosome to stall and thus cannot complete the translation, the nascent peptide is then polyubiquitinylated to be recognized by p97/VCP/CDC-48-Ufd1-Npl4 and directed to the proteasome for degradation (31).

-Autophagy and aggregate handling -p97/VCP/CDC-48 was also reported to be part of other degradation mechanisms: autophagy and aggregate handling. Indeed several reports have shown that CDC-48 is required for autophagosomes maturation into autolysosomes, as in Cdc-48 knock down cells immature autophagosomes accumulate (38a, 38b)). Aggregation of misfolded proteins in the cytosol can be caused by denatured protein, heat shock or overexpression of insoluble/mutant protein, to cope with these misfolded proteins the cell stores them into aggresome before their degradation by autophagy. HDAC6 is key player in this process as it was

shown to be capable of shuttling ubiquitinylated misfolded proteins to aggresome along the microtubules (39). Interestingly, in 2006 it was shown that the p97/VCP-HDAC6 protein ratio could control the fate and destination of ubiquitinylated proteins: aggresome or proteasome (Figure 8). Indeed, HDAC6 can by interacting with its substrates hide their ubiquitin tags and promote aggregate formation which will be handled by autophagy. If the p97/VCP-HDAC6 ration is imbalanced p97/VCP can segregate HDAC6 from its ubiquitin substrates thus the ubiquitin tag is unmasked and the revealed protein is taken in charge by the UPS pathway. Later, another layer of complexity was added to the HDAC6-p97/VCP interaction as these proteins also control HSF1 a major transcription factor of the heat shock pathway. In fact, HSF1 was discovered to be part of an inactive complex in basal condition with p97/VCP, HDAC6 and HSPs (Heat Shock Proteins) (40) (Figure 10). However, upon accumulation of ubiquitinylated proteins in the cytosol (caused by heat shock or impairment of the proteasome) the complex dissociates thus HDAC6 and p97/VCP can cope with ubiquitinylated substrates and HSF1 activates transcription of its target genes which include chaperones, overall all the dissociated members work at restoring protein homeostasis.

Figure 10: Schematic representation of the role of HDAC6, p97/VCP and HSF1 in response to

accumulation of ubiquitinylated proteins -In basal conditions p97/VCP, HDAC6, HSF1 and HSP90 form an inactive complex. However upon accumulation of ubiquitinylated proteins the complex is dissociated. p97/VCP and its cofactors target ubiquitinylated substrates for proteosomal degradation. HSF1 translocates to the nucleus to activate its target genes which include chaperones. HDAC6 shuttles ubiquitinylated proteins along microtubules and promotes aggresomes formation. All these mechanisms help the cell to restore its protein homeostasis

b) Membrane fusion and trafficking

During the cell cycle both the Golgi apparatus and the ER experience dramatically change in shape and structure to the point where during mitosis the ER and Golgi are fragmented in vesicles and tubules that therefore need to be reassembled in the daughter cells. In this context, p97/VCP and its cofactors p47 and VCIP 135 are required for membrane fusion and reassembly of both the ER and Golgi apparatus (41). Moreover, p97/VCP was also shown to regulate the assembly of transitional ER, a process coordinated by p97/VCP phosphorylation: when not phosphorylated p97/VCP associates with p47 and syntaxin-5 an ER membrane protein involved in vesicle docking and fusion and promotes the assembly of transional ER whereas phosphorylation of p97/VCP inhibits these interactions thus the formation of transitional ER (42).

However, the precise role of p97/VCP in this process is not clear although it seems to involve binding and regulation of syntaxin-5 an ER membrane protein involved in vesicle docking and fusion. Recent studies have revealed a new role for p97/VCP in endocytic trafficking, indeed p97/VCP was found to interact with EEA1 (endosomal antigen-1) a protein responsible for the fusion of endocytic vesicles to form early endosomes (43). Upon p97/VCP silencing (pharmalogical and siRNA mediated) the EEA1 oligomeric state was disturbed causing a defect in early endosome maturation and size. Later another study linked p97/VCP to endocytic trafficking as p97/VCP and its cofactor UBXD1 were shown to interact with ubiquitinylatedcaveolin-1 (CAV1) a protein that is part of the endocytic machinery. In this study p97/VCP inhibition (pharmalogical or siRNA mediated) caused an accumulation of enlarged endosomes and a defect of CAV1 sorting into intraluminal vesicles. Although, their results slightly differ those two reports provide strong evidence of the involvement of p97/VCP in the endosomal trafficking but its precise role remains to be defined.

c) Chromatin-associated functions

p97 protein can be found in the nucleus of all species, in yeast CDC-48 translocation relies on the phosphorylation of its last tyrosine, indeed after phosphorylation CDC-48 tridimensional structure is changed and reveals a nuclear import sequence (44)(Figure 11).

However there is no tyrosine kinase in S. cerevisiae, only dual specificity protein kinases that phosphorylate serine/threonine or tyrosine residues and the enzyme responsible for this phosphorylation is still unknown.

In human, this tyrosine is conserved and can be phosphorylated by the protein kinase v-Src but this event regulates its activity during ERAD by preventing binding of p97/VCP partners: Ufd3 /or PNGase rather than inducing p97/VCP translocation. However, a recent report suggests that rather the N domain of the protein is required for p97/VCP translocation to the nucleus whereas the C terminal tail seems to control its export (45). In the nucleus, a wide range of cellular a nuclear protein by p97 (46). Indeed, extraction of the ubiquitin modified Aurora B kinase mediated by p97 and Ufd1-Npl4 is required for the formation of the nuclear envelope. As additional evidence that p97 and cofactors are required in this process, siRNA mediated silencing of Ufd1 or Npl4 caused defect in chromosome segregation and multi-lobed nuclei. DNA replication is a process tightly linked to the cell cycle progression as it ensures that both daughter cells will receive the correct amount of genetic material. p97 and its C. elegans orthologues CDC-48.1 and CDC-48.2 (duplicate gene) have been shown to participate to this process in an evolutionary conserved manner. Indeed in both human and worm CDT-1, a protein involved in the formation of the prereplication complex needs to be regulated by p97/CDC-48 to ensure that a single round of DNA amplification happens (47). This degradation of CDT-1 implicated p97/CDC-48 and its cofactor UFD-1/NPL-4, in fact worms lacking any of these proteins displayed increased level of CDT-1 are defective in S phase progression.

-DNA damage response -p97/VCP's role in DNA damage response is not simple as its substrates differ between different reparation mechanisms and different species. Historically, first evidence of p97/VCP implication in the DNA damage response came in 2000 when p97/VCP was found to interact with BRCA1 a well described tumour suppressor protein involved in double strand breaks (DBS) repair (48). In 2005 and 2007, the human p97 was identified as a novel substrate of PIKK (phosphatidylinositol 3-kinase-related kinase) kinases: ATM, ATR and DNA-PK, kinases that are known to orchestrate double strand break reparation (49)(50). The authors also reported an accumulation of serine 784 phosphorylated p97/VCP at the site of DNA doublestrand breaks. In 2007, a proteomic analysis of ATM substrates another well described kinase involved in DSB repair revealed p97/VCP and other proteins of the UPS as substrates Although these reports seem to point toward a role for p97/VCP in the DNA damage response it is only more recently that precise molecular event have been described.

- should be removed and replaced by a more faithful and processive DNA polymerase. Although, the precise mechanism is unknown, p97/VCP seems to be required in extraction of the translesion polymerase. Indeed, DVC1 (DNA damage-targeting VCP adaptor C1orf124) a newly discovered p97/VCP adaptor is believed to recruit p97/VCP/UFD1/NPL4 thanks to its interaction with PCNA at the replication fork and facilitate polymerase switching (51). In fact, depletion of DVC1 results in persistent association of PCNA with the translesion polymerase causing an increased amount of mutations because the polymerase cannot be switched. This suggests a role for p97/VCP/DVC1 in the rapid extraction of the translesion polymerase.

-DNA double strand breaks repair -The two ubiquitin-ligases RNF8 and RNF168 (E3) are known to orchestrate the DNA damage response at either double strand breaks or lesions in human cells. First, RNF8 is recruited at the damaged site and induces ubiquitinylation of chromatin-associated proteins which leads to the recruitment of RNF168 as it possesses ubiquitin binding domains (53). Then RNF168 induces ubiquitinylation of H2A and H2AX histones to which 53BP1 can bind, in fact 53BP1 needs to recognize both ub-H2A and di-methyl H4K20 to bind (54). Two reports have implicated p97/VCP and its ubiquitin binding cofactors UFD1-NPL4

as key players in this process (51) (Figure 13). However it is unclear how p97/VCP/UFD1/NPL4 is recruited as the first report suggests that this complex is recruited by K-48 ubiquitin modified proteins induced by RN8 whereas the second suggests that both RNF8 and RNF168 which induce K63 chains are required. In this context, it was shown that p97/VCP is required to extract L3MBTL1 ubiquitinylated by RNF8 from dimethyl H2K20 to allow binding of 53BP1 without which the DNA response would be interrupted. Additionally, JMJD2A/B a histone demethylase that can bind to the same mark is also ubiquitinylated by RNF8 and then extracted during the DNA damage response, however the implication of p97/VCP has not yet been addressed. In conclusion, p97/VCP can promote the recruitment of the repair factors to the damaged site by extracting proteins that are occupying the same histone mark.

3) p97/VCP/CDC-48 role in transcription regulation

In 2012, a study based on a global gene microarrays on patients with Paget's disease of bone a disease caused by mutations in the p97/VCP gene revealed that more than 60 genes are significantly deregulated in those patients (55). Analysis of the deregulated genes showed that and Spt23p (35). At the golgi membrane p97/VCP is required for the proteolytic activation of SREBP (36). As mentioned earlier, in the cytosol p97/VCP associates with HDAC6 and HSP90

to sequester HSF1 (40). Additionally, Chromatin Immunoprecipitation experiments, have revealed that p97/CDC-48 can associate directly at the chromatin with mono-ubiquitin transcription factors such as LexA-VP16, Met4 or Smad2/3 to prevent binding to their target promoter without degradation (57). Finally, other transcription factors often relevant for the study of cancer such as p53, H1F1NFKB were shown to interact with p97/VCP although the localisation of the interaction was not assessed (58,59,60). Other reports have revealed that p97 can also regulate gene expression by affecting certain histone marks. Although the role of ubiquitinylation of histone H2B is not well understood, this mark undergoes major changes during cell differentiation (61). Recently CDC-48 and its cofactor Ubx3 were shown to facilitate the transcription coupled recruitment of Lge1 a cofactor of the H2B ubiquitin ligase Bre1 (61). In another study, it was shown that accumulation of polyglutamine aggregates induces p97/VCP nuclear translocation. This translocation was associated with a global decrease of Histone H3 and H4 acetylation and as a result a diminution in transcription (62). Consistent with these observations, expression of mutant p97/VCP that cannot translocate to the nucleus were able to restore the histone H3 and H4 acetylation levels. These reports suggest an important role of p97/VCP/CDC-48 in the coordination of chromatin remodeling processes.

4) p97/VCP in diseases a)

IBMPFD and ALS -As p97/VCP is involved in a wide range of cellular processes and is a crucial keeper of the cell protein homeostasis it is not surprising that mutation in the gene p97/VCP that encode for the p97/VCP protein have been genetically linked to degenerative disorders (31,55). Indeed, autosomal dominant mutations in p97/VCP are Parkinsonism, ataxia, cataracts, dilated cardiomyopathy, hepatic fibrosis or hearing loss. More than 30 missense mutation in p97/VCP have been identified (31) and most of these mutations are clustered between the N and D1 domains of the protein (Figure 6), this region is thought to control the orientation of the N domain to which cofactors bind. As most mutations are in this region it suggests that it is important for the disease pathogenesis. Other mutations have been reported to affect p97/VCP basal ATP hydrolysis activity but not all ( 63), suggesting that it may not be the cause of the disease, moreover no mutation appear to impact the oligomerization of the protein (31). Additionally, two studies have reported that these disease-associated mutations affect p97/VCP interaction with cofactors suggesting that these mutation will not affect all p97/VCP function but only the function related to those cofactors (64). As p97/VCP knock-out mice are not viable, this could explain how patients or mice with these mutations develop normally, suggesting that p97/VCP functions in cycle control or cellular division are not affected by these mutations. Overall, these observations suggest that it is the imbalanced cofactor association of p97/VCP that is most likely to be responsible for the MSP. Common cellular features of all IBMPFD or ALS, are impaired ERAD and accumulation of ubiquitinylated protein aggregates indicate that the protein quality control system and protein aggregate clearance system are defective in these disorders. This is consistent with the fact that p97/VCP is required for autophagy and autophagosome biogenesis. Indeed depletion of p97/VCP or expression of dominant-negative p97/VCP mutant resulted in the accumulation of LC3II and immature autophagosomes [START_REF] Kaser | XBP1 links ER stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease[END_REF]. Additionally, accumulation of TDP-43 inclusions is also causal of FTD and ALS. TDP-43 is a RNA binding protein that can regulate processes such as transcription, pre-mRNA splicing, RNA transport and translation. Recently Ritso et al. showed a genetic interaction between p97 and TDP-43 and that p97 mutation caused accumulation of TDP-43 in the cytosol which had similar toxic effects (65). Moreover, TDP-43 mutations enhance the genetic interaction with p97 and also lead to cytotoxic accumulation of TDP-43. These results suggest that the degeneration caused by p97/VCP mutations is partly mediated by the toxic function of TDP-43 in the cytosol.

b)

Cancer -p97/VCP was found overexpressed in different cancers e.g.:

hepatocellular carcinoma, non-small cell lung carcinoma, osteosarcoma, oesophageal squamous cell carcinoma, gingival squamous cell carcinoma, prostate cancer, colorectal carcinomas, gastric carcinoma, pancreatic endocrine neoplasms, pancreatic cancer, and follicular thyroid cancer (66,67). Several studies have reported a correlation between the level of p97/VCP and adverse prognosis (67)(68). However, due to the wide range of cellular functions that p97/VCP regulates it is difficult to anticipate the exact contribution(s) of this protein in oncogenesis. Most of the functions in which p97/VCP is involved are relevant to cancer development for instance the control of transcription, cell-cycle progression, autophagy, proteostasis, endocytic processes or DNA-damage mechanisms. Observation of cancer cells with elevated p97/VCP revealed a decreased ability for the cell to undergo apoptosis which suggest an anti-apoptotic role for p97/VCP (69). Such a role is coherent with the fact that in cancer cells the genomic abnormalities and the resulting synthesis disturb the protein homeostasis and lead to proteotoxic stress. As p97/VCP is essential to restore protein homeostasis in the cell, elevated level of p97/VCP could prevent the proteotoxic stress from inducing apoptosis. Consistent with a pro-survival role of p97/VCP in cancer, another study have shown that p97/VCP is involved in the regulation of IKB, an inhibitor of NFKb (Nuclear Factor kappa B) a transcription factor which control genes involved in cell proliferation (70). In fact, Valla and colleagues have suggested that p97/CDC-48 and the UPS regulate NSCLC tumor genesis and metastasis via NFKB and p53 (58). This may apply to other cancers as increased levels of p97/VCP may cause the degradation of NFKB repressor and consequently promote cell proliferation. Numerous studies have also linked p97/VCP to the regulation of key cancer proteins such as p53, BRCA1, HIF1a, but it is still unclear how these regulations contribute to oncogenesis (48,58,59,60). However, p97/VCP inhibitors have shown encouraging results. In hepatocellular cancer, one of the only treatment available Sorafenib a multikinase inhibitor which was shown to target p97/VCP (71). Indeed Sorafenib prevents the phosphorylation of p97/VCP which results in stabilizing its membrane association, causing ER stress and ultimately apoptosis. Upon treatment with a reversible inhibitor of p97/VCP: Dbeq the cells were further sensitized to Sorafenib strengthening the idea that that Sorafenib apoptosis is indeed mediated by p97/VCP impairment. NMS-873 is an allosteric inhibitor that impairs p97/VCP enzymatic activity and was shown to activate the UPR, interfere with autophagy and induce cancer cell death in U2OS and HCT166 (72). More recently, CB-5083 which binds to the p97/VCP D2 domain had an important apoptotic effect in HCT116

xenografted mice (73). In fact, CB-5083 treatment caused accumulation of ubiquitinylated proteins, retention of ERAD substrates and irresolvable proteotoxic stress and as a consequence apoptosis mediated by the UPR. These studies suggest that targeting p97/VCP might be a viable strategy in certain cancers, however further tests are needed to ensure the safety of these drugs.

Indeed, recent studies have shown that ER stress which can be caused by p97/VCP inhibition or p97/VCP inhibition itself can promote aggressiveness of cancer cells such as induction of EMT.

HYPOTHESIS AND OBJECTIVES

ER stress is a condition often met by tumor cells as they are exposed to challenging conditions that alter protein homeostasis (i.e. intrinsic and extrinsic stress). Recently it has become clear that ER stress signalling and the UPR contribute to cancer biology and the acquisition of malignant characteristics such as inflammation, invasion and metastasis, angiogenesis or resistance to chemotherapy (74,75). Although the key initiation mechanisms of the UPR are now well established and comprise activation of the three sensors (IRE1, PERK, ATF6) which leads to the production of transcription factors (XBP1s, ATF4, ATF6f), the resulting transcriptional outputs are far more complex than expected. Indeed, the exact nature of the genes activated under ER stress-mediated cellular reprogramming and how these genes switch from a repressed to an activated state remains unclear. As a consequence, identifying the components that regulate ER stress genes might help to characterize the gene sets regulated during the UPR and by extent to better understand the contribution of this pathway to cancer development.

The initial observation that CDC-48.2 knockout worms are impaired for the induction of ER stress genes upon exposure to tunicamycin led us to propose that beyond its role in ERAD, CDC-48 might have a role during ER stress in the regulation of ER stress genes (76). Moreover, although p97/VCP overexpression is observed in many cancers and sometimes associated with poor prognosis in patients, the mechanisms by which this protein contributes to malignancy is unknown. As a consequence we hypothesized that since ER stress is involved in cancer development and p97/VCP is a regulator of ER stress genes expression, overexpression of p97/VCP in cancers might play an important role in cellular reprogramming occurring in this context and therefore may provide some elements of explanation to its contribution to malignancy.

To address these points the objectives of my thesis are 1) to demonstrate that p97/VCP/CDC-48 is a genuine regulator of ER stress genes expression, and 2) to understand the molecular mechanism underlying this regulation, including the identification of the genes regulated, the cofactors of p97/VCP/CDC-48 in this process, the client proteins and the cellular localisation of these interactions.

EXPERIMENTAL APPROACH

Co-immunoprecipitation:

Huh7 or HeLa were cultured in 10 cm plates for co-immunoprecipitation. Two plates were used for each experimental conditions as one was used as a control to check the binding of our target proteins on the magnetic beads. The plates were placed on ice and the media was removed. The cells were washed twice with 5mL of cold PBS (PBS, 14190-094, Life Technology). 500 µL of Lysis buffer (30 mM TrisHCl pH 7.5, 150 mM NacL, 1.5% CHAPS) containing protease and phosphatase inhibitors (Complete EDTA, 04693159001, Roche and PhosphoSTOP, 04906837001, Roche) was then added to each plates and the cells were scrapped every 10 mins for 30 mins. The lysates were transferred to 1.5 mL tubes and briefly vortex before being centrifuge at 17 000g for 15 mins at 4°C. The supernatant were collected and the pellets discarded. The protein concentration of the supernatants were measured and adjusted to 2µg/µL (if not possible to 1 µg/µL). For each immunoprecipitation 20µg was saved to be used as input and 1µg of specific antibody was added to 1000µg of the supernantants and incubated overnight on a rotating wheel at 4°C. The next day, 30µL magnetic beads (coupled with protein A (Dynabeads PG, 10003D, Thermo Fisher Scientific) for Rabbit antibody or with protein G (Dynabeads PA, 10001D, Thermo Fisher Scientific) for Mouse antibody) were washed 3 times in the lysis buffer and then incubated with the lysates for 15 mins at RT. Note that the magnetic bead manufacturer advise longer incubation time (45 to 60 mins) but it causes p97/VCP to bound to the beads. After 15 mins using the magnetic stand the magnetic beads were recuperated and the lysates discarded. The magnetic beads were then washed 3 times in the lysis buffer to remove aspecific bindings. Then 30µL of Laemmli buffer 1X was added to the beads, mixed by vortex and the mixtures were heated at 95°C for 5 mins. The supernatants were briefly centrifuge and separate from the magnetic beads using the magnetic stand and then loaded along with the input on a polyacrylamide gel of the appropriate concentration for immunoblotting.

Immunohistochemistry:

Huh7 cells were cultured on cover slips in 12 wells plates for immunohitochemistry. For each well/cover slip the media was removed and the cells were washed twice with cold PBS (PBS, 14190-094, Life Technology). After, the cells were incubated with a 4% Paraformaldehyde (PFA) in PBS solution for 10 mins and then were washed three times with cold PBS for 5 mins. The cell were permeabelise by incubation with a 0.1% Triton in PBS solution for 15 mins. After a quick wash the cells were incubated with H2O2 to inhibit the endogenous peroxidases. After a quick wash the cells were incubated with a 3% BSA in PBS for 1 hour to prevent aspecific binding of the primary antibody. After a quick wash the p97/VCP antibody (60316-1, Proteintech) was diluted at the 1:200 in the blocking solution (3%BSA in PBS) and incubated with the cells for 2 hours. After removing the antibody solution the cells were washed 3 times with PBS for 5 mins.

The cells were then incubated with a secondary antibody: Flex-HRP (EnVision System, K406987-2, Dako) for 30 mins and then washed 3 times with PBS for 5 mins. Then in the dark (to protect the signal from light) the cells were incubated with 3,3'-diaminobenzidine (DAB, EnVision System, K406987-2, Dako) for 10 mins. The cells were then wash with distilled water before being incubated with Hemalum for 3 mins to color the nucleus. After 2 washings with distilled water, the cells are washed with Ammonium Peroxide and then dehydrated by a succession of baths in 95° Alcohol for 1 min, 100° Alcohol for 1 min twice and Toluene for 5 min twice. Finally the cover slips were fixed using mounting medium on a slide before microscopic analysis.

Silencing using siRNA:

Huh7, HeLa, U87, U251, Panc1 cells were cultured in 6 wells plates before protein or RNA extraction. The day before the transfection 200 000 cells were plate in 1mL of medium (DMEM GlutaMAX, 10566-016, Thermo Fisher Scientific) complement with 10% SVF. The day of the transfection the old medium was replaced by 1 mL of fresh medium and for each reaction two tubes were prepared: in the first tube 150µL of OptiMem (31985062, Thermo Fisher Scientific)

was mixed with 4. All the other methods used during my thesis are described in the following method article or in the materials and methods section of the science articles (Article 3 and 4).

RESULTS

In 2008, in an attempt to identify new regulators of the UPR pathway, transgenic worms expressing a transcriptional reporter where the GFP is under the control of an ER stress promoter where subjected to a small RNA interference (RNAi) screening (76). The screening worked as follows: upon Tunicamycin treatment a pharmacological ER stressor (Tun) the worms become fluorescent, therefore if the fluorescence is modified by a RNAi it suggest that the silenced gene is an effector of the ER stress response: the UPR (Figure 14). Using this method we have identified CRP-1 a GTPase that belong to the Rho family as a regulator of ER stress genes in C.

elegans (76). In order to characterize the molecular mechanism behind this regulation CRP-1 partners were identified by mass spectrometry, among them several proteins involved in DNA remodelling and CDC-48 were found. Interestingly, CDC-48 and CRP-1 knock out worms displayed the same phenotype: impaired induction of ER stress genes induced by tunicamycin, suggesting a requirement of both CRP-1 and CDC-48 to induce these genes. These results suggested for the first time that CDC-48 role in ER may go beyond ERAD and ER formation, but that CDC-48 might also be involved in the regulation of ER stress genes.

To better understand how p97/CDC-48 regulates ER stress genes, we came back to the observation that CDC-48.2 knock out worms are impaired in their capacity to induced ckb-2p::gfp (our ER stress reporter) in the presence of Tun and performed a new screening to identify proteins that could restore this phenotype. The following paper present our findings.

Figure 14: Schematic representation of the ER stress regulators screening in C. elegans -C. elegans worms expressed a transgenic GPF under the control of an ER stress induced promoter. In ER stress condition induced by Tun the transgene is activated resulting in GFP production and observable fluorescence. B) Using these transgenic C. elegans we were able to identify potential regulators of the UPR by measuring fluorescence of the different RNAi treated worms.

In this study, I helped with the characterization of ER stress gene expression under RuvBL2/Reptin silencing conditions in both C. elegans and human cells. I also contribute to characterize the interaction between p97/VCP and RuvBL2/Reptin. Finally, I have tested the effect of RuvBL2 silencing during ER stress on the activation of the IRE1 pathway.

Introduction

The endoplasmic reticulum (ER) protein quality control system ensures the correct folding of transmembrane and secretory proteins before their export from this organelle [1].

Accumulation of improperly folded proteins in the ER triggers the unfolded protein response (UPR ER ) to restore ER homeostasis. This is achieved by enhancing ER-Associated Degradation (ERAD), increasing ER protein folding capacity, decreasing protein translation and inducing a defined gene expression profile (UPR ER genes) [2]. Although most of these molecular events are clearly established, the mechanism leading to the transcriptional regulation of specific genes under ER stress remains poorly understood.

Here, using as a model the nematode C. elegans, we identify a novel functional partner for p97/CDC- [4]. Using a C. elegans strain mutant for the p97/CDC-48 homolog cdc-48.2 (-/-) , we performed a genome-wide RNAi screen to identify proteins involved in the activation of UPR ER genes during ER stress. We found that the AAA+ ATPase RUVB-2 is a regulator of the ER stress response by repressing the transcription of select UPR ER genes in non-stressed conditions in both C. elegans and human cells. In response to ER stress, RUVB-2 is degraded in a CDC-48-dependent manner, thereby relieving repression of UPR ER genes. Altogether, our results identify a novel mechanism controlling gene expression downstream of p97/CDC-48 and unveil a novel function for RUVB-2 and its human homolog Reptin as a key regulator of the transcriptional response to ER stress.

Results and Discussion

A genome-wide screen identifies cdc-48 genetic interactors regulating ER stress-induced gene expression.

RNAi-mediated knockdown of cdc-48.1 or cdc-48.2 in C. elegans abolishes the ER stressinduced expression of a set of UPR ER genes including ckb-2 [4]. Using a transcriptional reporter expressing GFP under the control of the ckb-2 promoter, we confirmed the requirement for cdc-48.1 and cdc-48.2 in ER stress-induced gene transcription (Fig. 1A). Mutant cdc-48.1 (-/-) and cdc-48.2 (-/-) worms failed to respond to the ER stress inducer tunicamycin while ckb-2p::gfp fluorescence was increased more than 3-fold in wild-type (WT) worms (Fig. 1B). RNAi inactivation of ire-1, the main sensor of ER stress and mediator of UPR ER signalling, resulted in a significant decrease in fluorescence intensity in both ckb-2p::gfp and cdc-48.2 (-/-) ; ckb-2p::gfp worms (Fig. 1A, Table S1). These results confirm that ckb-2p::gfp transcription is IRE1dependent, as expected of a bona fide UPR ER reporter.

Because p97/CDC-48 is involved in protein degradation [5], we reasoned that it might modulate ER stress-induced ckb-2p transcription by eliminating a transcriptional repressor. To address this hypothesis, we designed an RNAi suppressor screen to identify genes whose knockdown could restore tunicamycin ckb-2p::gfp activation following ER stress in a cdc-48.2 (-/-) mutant background ( [4]; Fig. 1C). We performed the screen in liquid culture by feeding cdc-48.2 (-/-) ; ckb-2p::gfp synchronized L1 larvae with double stranded RNA (dsRNA)-expressing E. coli derived from the C. elegans ORFeome library that targets 11,698 open reading frames covering 62% of C. elegans genes [6]). We then exposed the worms to a concentration of tunicamycin (0.5 g/ml for 16 hours) leading to maximal ckb-2p::gfp induction in WT worms grown in liquid culture, and analyzed them by flow cytometry [7] to measure their length, number and fluorescence intensity. Each RNAi clone was tested in duplicate and the mean Z-score was calculated. Twohundred and forty one RNAi clones synergized with cdc48.2 (-/-) to decrease ckb-2p::gfp expression in our primary screen (mean Z-score value less than -1.5, or one of the two independent Z scores less than -3) (Fig. 1C). Of these, 59 clones significantly decreased GFP fluorescence below 0.75-fold (P<0.05) (Fig. 1D, Table S2). One-hundred and seventy-seven These were classified as potential suppressors of the cdc48.2 (-/-) phenotype.

To discriminate between ER stress-dependent and independent activation of ckb-2p::gfp transcription, we measured fluorescence intensity in cdc-48.2 (-/-) ; ckb-2p::gfp worms fed with candidate RNAi clones and treated either with tunicamycin or vehicle (DMSO; Fig. 1E).

Seventy-seven RNAi clones showing a similar increase in the fluorescence ratios under both conditions were considered ER stress-independent and not further analysed (Fig. 1F, Table S3).

By contrast, 100 RNAi clones which restored ckb-2p::gfp activation in the cdc48.2 (-/-) mutant background specifically under tunicamycin treatment were identified as ER-stress dependent suppressors of cdc48.2 (-/-) (Fig. 1E, Table S4). We next investigated if the genes targeted by these 2A

). Among these candidates, the AAA+ ATPase Ruvb2 was of particular interest.

To confirm the RNAi screen findings, we conducted a quantitative proteomic analysis to identify proteins whose levels are modified in cdc-48.2 (-/-) ; ckb-2p::gfp worms exposed to tunicamycin. We selected proteins represented by at least two peptides and that had a peptide ratio above 2 or below 0.5 between WT and mutant worms exposed to tunicamycin. Ninety three proteins increased and 15 proteins decreased in abundance in cdc-48.2 (-/-) mutants compared to the WT (Fig. 2B, Table S5). RUVB-2 was the only suppressor identified in our RNAi screen for which an increase in protein abundance could be detected in cdc-48.2 (-/-) ; ckb-2p::gfp compared to ckb-2p::gfp worms ((2.5± 0.5 fold increase, Fig. 2C). Because the quantity of ruvb-2 mRNA was not increased (Fig. S4A) under these conditions, the increased abundance of RUVB-2 in cdc-48.2 (-/-) mutants is likely due to attenuation of protein degradation rather than to increased transcription.

Conserved RUVB-2 and CDC-48 dependent regulation of UPR ER gene expression.

RNAi knockdown of ruvb-2 restored ckb-2p::gfp activation both in cdc-48.2 (-/-) and cdc-48.1 (-/-) mutant worms exposed to tunicamycin (Fig. 3A-B). This suggests that, under ER stress, the repressor RUVB-2 is degraded through a CDC-48.1-dependent mechanism to allow full ckb-2p::gfp induction. Moreover, knockdown of xbp-1 reduced ckb-2p::gfp expression in cdc-48.2 (-/-) ; ckb-2p::gfp worms treated with tunicamycin compared to the DMSO-treated ones (Fig. 3C).

Combined RNAi-mediated knock-down of xbp-1 and ruvb-2 decreased ckb-2p::gfp fluorescence to the same level observed using xbp-1 RNAi alone. This suggests that RUVB-2 is degraded through a CDC-48-dependent mechanism in response to tunicamycin, thus allowing XBP-1s to activate ckb-2 expression. Ruvb-2 inactivation also restored the expression of ER homeostasis regulators (CKB-2, F22E5.6, Y71F9AL.17/COPA-1) observed upon ER stress in WT animals [8] in cdc-48.2 (-/-) tunicamycin-treated worms (Fig. 3D andS4B). These results suggest that RUVB-2 represses the expression of select UPR ER target genes. We next tested whether this function was conserved in human cells. To this end, Huh7 cells transfected with the ER stress response element reporter gene (ERSE::tomato [9]) were knocked-down for Reptin using stable integration of a doxycycline-inducible short hairpin RNA (shRNA [10]) (Fig. 3E, left). Induction of Reptin shRNA synergized with tunicamycin treatment to activate ERSE::tomato transcription, demonstrating that Reptin can also a repress ER stress-mediated transcription in human cells. Of note, the silencing of the Reptin homolog Pontin did not affect the transcription of the ERSE::tomato reporter under basal conditions or upon tunicamycin-induced ER stress (Fig. 3E, right). We next quantified the mRNA amounts of 4 genes whose products are involved in the control of ER homeostasis (BiP, CHOP, EDEM1, ORP150). This revealed that Reptin silencing significantly increased the expression of BiP, CHOP and EDEM1 while did not affect that of ORP150 (Fig. 3F). Moreover, Reptin overexpression led to the significant repression of select genes (CHOP, EDEM1, ORP150) under basal conditions when compared to control transfected cells (Fig. S5). Altogether, these results suggest the existence of a conserved role for Reptin in repressing expression of ER stress response genes.

Post-translational control of Reptin expression by p97/CDC-48 impacts on ER stress response in human cells.

Further, we examined whether p97/CDC-48 could also acted by triggering the degradation of Reptin in response to ER stress as observed in C. elegans (Fig. 2). Reptin protein levels were significantly decreased upon tunicamycin treatment whereas p97/CDC-48, Pontin and Calnexin protein expression remained unaffected (Fig. 4A). Conversely, addition of the p97/CDC-48

inhibitor DBeQ, stabilized Reptin levels under ER stress (Fig. 4B). We then tested whether p97

and Reptin interacted physically using co-immunoprecipitation. These results were confirmed by determining Reptin's half-life upon stress (Table S6) and the values obtained under basal conditions were in the range of those determined in S. cerevisiae or S. pombe [11]. Reptin immunoprecipitates contained p97/CDC-48 and the interaction was modulated by tunicamycininduced ER stress, DBeQ or both (Fig. 4C). Interestingly, when the reverse experiment was carried out, Reptin was found in the p97/CDC-48 immunoprecipitate as well as a slower migrating Reptin immunoreactive species (Fig. 4D, arrow). Sequential immunoprecipitation with p97/CDC-48 and Reptin antibodies suggested that the latter corresponds to an ubiquitylated form of Reptin (Fig. 4E). Hence, p97/CDC-48 might control Reptin levels through an ubiquitindependent mechanism.

XBP1 mRNA splicing and ATF6 activation are partly regulated by a p97/reptin signalling axis

To follow up on the role of Reptin degradation upon ER stress in the expression of ER stress genes, we sought to test whether artificial modulation of Reptin expression also impacted the activation of the three UPR signalling arms. Reptin silencing slightly increased the expression of the ER stress upregulated chaperones GRP78 and GRP94, which are canonical targets of ATF6 and XBP1s signaling, under basal conditions (Fig. 5A), but did not affect tunicamycin-induced phosphorylation of eIF2 (Fig. 5B). ATF6 cleavage activation was increased after Reptin silencing in HuH7 cells (Fig. 5C). In accordance with this observation, reptin silencing also enhanced the expression of XBP1u mRNA under basal conditions, as could be expected since XBP1u is a target gene of ATF6 (Fig. 5D). Moreover, this occurred without affecting the expression levels of the newly discovered XBP1 mRNA ligase RtcB [12] (Fig. 5D). XBP1 mRNA splicing was also increased when Reptin was silenced both in basal conditions and ER stress (Fig. 5E). Conversely, DBeQ-mediated p97/CDC-48 inhibition (Fig. 5F) or siRNAmediated p97/CDC-48 silencing (Fig. S6) and the subsequent stabilization of Reptin led to reduced XBP1 mRNA splicing. Hence, partial stabilization of Reptin has a major impact on XBP1 mRNA splicing, which in turn impacts dramatically on the expression of various UPR ER genes. However we could not detect an interaction between Reptin and XBP1s protein (Fig. 5G).

Altogether, these results might indicate that Reptin is degraded through ubiquitin and p97/CDC-48-dependent mechanisms under ER stress and further support the role of Reptin in the control of select UPR ER genes through repression of XBP1 mRNA splicing and of ATF6 activation.

In the present work, we have uncovered a novel regulatory mechanism of UPR ER genes expression in response to ER stress conserved throughout metazoan evolution involving two AAA + ATPases, RUVB-2 (or Reptin) and CDC-48 (or p97). In this model, RUVB-2, which mostly localizes to the cytoplasm and the nucleus, plays an important role in the regulation of XBP1 mRNA splicing by a yet unknown mechanism. Upon ER stress, RUVB-2 is degraded through an ubiquitin and p97/CDC-48-dependent mechanism, thereby allowing the ER stress specific transcription factors ATF6 and XBP-1 to activate the transcription of UPR ER genes.

Beyond unravelling a novel UPR ER regulatory network, our data point towards the putative role of Reptin in non-conventional mRNA splicing. Our findings suggest that p97/CDC-48-induced degradation of target proteins plays an important role in the ER homeostasis control both, in the cytoplasm, to influence ERAD and to modulate UPR ER gene transcription [13].

Materials and Methods

RNAi screen -The RNAi feeding screen was performed in liquid culture using EM2 animals and carried out as previously described with some modifications [4]. RNAi clones from the Worm ORFeome version 1.1 library [6] were grown overnight at 37°C in 96-well plates. Each RNAi plate included a positive control (Y37D8A.10 encoding for a signal peptidase identified in a preliminary screen or BC14636 worms fed with the L4440 empty vector) and a negative control (gfp RNAi). RNAi expression was induced with 1mM IPTG for 1h before bacteria were added to the L1 larvae. Adult worms were bleached and the obtained L1 larvae (200) were added to each well of 96-well plates along with the induced bacteria and S-Medium, 50 μg/ml ampicillin, 1mM

IPTG buffer with a final well volume of 150 μl. The 96-well plates were incubated at 20°C with shaking. Forty-eight hours later ER stress was induced by tunicamycin (0.5 μg/ml) for 16 hours and measurements taken using the COPAS Biosort flow cytometer (Union Biometrica, Holliston, MA, USA). Experiments for each 96-well plate from the RNAi library were performed in COPAS measurements -The COPAS biosort analyzer was purchased from Union Biometrica (Holliston, MA, USA). Photomultiplicator tube control (PMT1) was set up at 600 so that the green fluorescence emission was not saturated in BC14636 worms exposed to tunicamycin (maximum signal) and still detectable in EM2 worms exposed to gfp RNAi (minimum signal).

Plates were read through a ReFLx module. Raw data extracted from COPAS included worm axial length (time of flight), worm number (extinction), and fluorescence (green fluorescence emission). Raw data were processed as previously described [14] and used for quantitative analyses. In the preceding paper, we have identified RUVBL2 as an evolutionary conserved repressor of certain ER stress genes including BiP, CHOP and EDEM in ER stress conditions. This repression relies at least in part on the inhibition of ATF6 activation and XBP1 mRNA splicing. Upon ER stress induction, the ubiquitin modified RUVBL2 is specifically degraded in a p97/VCP dependant manner causing an activation of the genes it normally represses. Although these results

have showed that the two AAA+ ATPase (RUVBL2 and p97/VCP) are regulating ER stress genes in basal and stress conditions they raised several questions:

-What is the molecular mechanism by which p97/VCP-RuvBL2 regulates ER stress genes: do they affect histones, transcription factors, other repressors?

-How does p97/VCP-RUVBL2 affect ATF6 activation and XBP1 mRNA splicing?

-Where does this interaction take place: in the nucleus or in the cytosol?

-Could p97/VCP-RUVBL2 regulate the transcription of other genes, even beyond ER stress conditions?

To address these questions we have tried to identify other actors that are part of the regulation of ER stress genes by p97/VCP-RUVBL2. Reasoning that these actors might also be regulated by p97/VCP and ER stress like RuvBL2 is. Therefore we looked back at our screening data: the 93 proteins that accumulate under ER stress in CDC-48.2 knock out worms and check for proteins that are described to play a role in gene expression regulation. Among the proteins involve in gene expression regulation one candidate in HAD-1 the orthologue of Human HDAC1 and HDAC2 particularly caught our attention. Indeed, HDAC1 was interesting for two reasons: i) it was described to be a negative regulator of BiP in basal conditions which is removed from BiP gene upon ER stress (77) ii) HDAC1 was described to interact physically with RUVBL2 in chromatin remodelling complex (78). This suggest that HDAC1 might cooperate with RuvBL2 to regulate expression of BiP and possibly other ER stress genes. As a result, we hypothesize that a p97/VCP-RUVBL2-HDAC1 complex might exist in the cell and be responsible for the regulation of certain ER stress genes including BiP. To test this, we will first check if HDAC1 or HDAC2 are regulated by p97/VCP and under ER stress in human cells. We will then test the existence of a p97/VCP-RUVBL2-HDAC1 complex. And finally we will test the impact of this complex on the regulation of ER stress genes. The following article presents our findings.

Introduction

The Endoplasmic Reticulum (ER) is the first compartment of the secretory pathway and is responsible for the folding, maturation, quality control and transport of secreted or transmembrane proteins. It therefore plays a key role in the maintenance of cellular homeostasis (1). In response to the accumulation of improperly folded proteins, a well-characterized signaling pathway, the unfolded protein response (UPR) is triggered to restore ER homeostasis by increasing protein folding and clearance capacities (2). Activation of the UPR promotes the activity of a subset of transcription factors to induce the expression of stress response genes that mainly encode for proteins involved in normal ER functions. We recently uncovered a novel role for the AAA + ATPase (ATPases Associated with various cellular Activities) p97/VCP as a key player in the mediation of ER stress-induced transcriptional response (3), however the mechanisms underlying transcription regulation-mediated by this ATPase remains elusive. p97/VCP is an essential player in the control of protein homeostasis and its inactivation or loss of function have been implicated in numerous pathological states, including cancer (4). Most of p97/VCP functions were thus far linked to its segregase activity that is its capacity to disassemble and isolate a target substrate from membranes or large protein complexes, resulting in their further proteasomal degradation or recycling (5). Herein we show that p97/VCP interacts with RuvBL2-mSin3A-HDAC1/2 to regulate specific signaling complexes in a stress dependent manner. We show that p97/VCP-dependent complexes interact with specific transcription factors (ATF4, ATF6, XBP1s, USF2) and affect the expression of their respective target genes. Further analysis demonstrated that among the genes regulated by p97/VCP in an ER stress-dependent manner was GLI1, one of the main effector of Hedgehog pathway (Hh) which activation contributes to the development of several cancers (6,7).

Interestingly, the regulation of GLI1 was non-canonical and independent of the Hh ligand. Our results highlight a novel signaling pathway contributing to the regulation of ER stress and leading to the non-canonical activation of the Hh pathway by p97/VCP containing complexes.

Results

p97/VCP regulates HDACs expression upon ER stress

We previously reported a repressor role of the AAA+ ATPase RuvBL2 of ER stress genes under basal non-stress conditions (3). Upon ER stress RuvBL2 is specifically degraded by a p97/VCP dependent mechanism that in turn allows the transcription of ER stress genes. We found that in Cdc48.2 (C elegans ortholog of p97/VCP) knock-out worms, key transcriptional regulators accumulated at the protein level including HAD-1 (the orthologue of HDAC1 and HDAC2) (Fig.

1A)(3).

Further analysis of this interplay in C. elegans and in human cells using the STRING database, suggest that the interaction of p97/VCP with RuvBL2 might be at the center of a vast transcriptional network (Fig. 1B-C). To test if HDAC1 and HDAC2 were similarly regulated in human cells we transfected HeLa cells either with control (CTL) or p97/VCP siRNAs, and monitored the expression levels of several HDACs (HDAC1 to HDAC6) using immunoblotting (Fig. 1D-E). As observed in worms, both HDAC1 and HDAC2 accumulated in the p97/VCP knock-down cells. Similar, observations were made for HDAC6 which was not surprising as it was previously described to be regulated by p97/VCP (8), however no change in the expression of HDAC4 was observed, and HDAC3 and HDAC5 were expressed at undetectable levels in HeLa cells. Accumulation of HDAC1, HDAC2 and HDAC6 in p97/VCP-silenced cells was confirmed in other human cancer-derived cell lines including Huh7, U87, and U251 (Fig. S1, quantification not shown). Given the role of p97/VCP in ER stress regulation we next examined if the expression of HDACs could also be regulated in a stress dependent manner by and possibly inside a complex that comprise RuvBL2. To prove existence of this signaling complex, we first tested whether the expression of proteins was regulated by p97/VCP upon ER stress. Upon treatment with the pharmacological ER stressor Tunicamycin (Tun), we found that RuvBL2 was destabilized, as previously shown, whereas HDAC1 and HDAC2 were stabilized when compared to untreated cells (Fig. 2A-B). Under the same experimental conditions HDAC6 expression remained unchanged (Fig. 2A, quantification not shown). Moreover we noticed that ER stress induction (as evaluated by monitoring BiP protein expression level) tend to correlated with HDAC1 protein level (Fig. 2C). We have previously shown that RuvBL2 expression was dependent on p97/VCP (3) and here using a siRNA mediated knock-down approach under basal or ER stress conditions, we confirmed that HDAC1 expression also depended on p97/VCP (Figure 2D). Indeed in cells subjected to ER Together these results suggest that p97/VCP antagonizes ER stress-mediated regulation of HDAC1/2. to tunicamycin (5µg/mL) for 8h HeLa cells using a specific antibody and p97/VCP, RuvBL2 association was analyzed by immunoblotting (N=3). F. p97/VCP was immunoprecipitated from total protein extracts from untreated or exposed to tunicamycin (5µg/mL) for 8h HeLa cells using a specific antibody and mSin3A, HDAC1, RuvBL2, ING2 association was analyzed by immunoblotting (N=3). G. HDAC1 was immunoprecipitated from total protein extracts from HeLa cells treated with siRNA CTL or siRNA RuvBL2 using a specific antibody and mSin3A, p97/VCP, RuvBL2, ING2 association was analyzed by immunoblotting (N=3). H. RuvBL2 was immunoprecipitated from total protein extracts from HeLa cells treated with siRNA CTL or siRNA HDAC1 using a specific antibody, and mSin3A, p97/VCP, HDAC1 association was analyzed by immunoblotting (N=3). I. HDAC1 was immunoprecipitated from total protein extracts from HeLa cells treated with siRNA CTL or siRNA p97/VCP using a specific antibody and mSin3A, HDAC1, RuvBL2 association was analyzed by immunoblotting (N=3).

p97/VCP interacts with the mSin3-HDAC complex under ER stress conditions

Given the segregase role of p97/VCP, we then investigated whether this protein could interact directly with HDACs to control their expression. Using a co-immunoprecipitation approach, we found that p97/VCP co-immunoprecipitated with HDAC1, HDAC2 and HDAC6 (Fig. S2). The interaction between HDAC1 and p97/VCP was much stronger than that of HDAC2 or HDAC6 and as HDAC1 and HDAC2 protein levels were similar, this suggested a preferential binding of p97/VCP to HDAC1 in HeLa cells. These evidences lead us to hypothesize that a set ER stress genes could be regulated by a p97/VCP-RuvBL2-HDAC1 complex. As such we found that HDAC1 co-immunoprecipitated with both RuvBL2 and p97/VCP in HeLa cells and the interactions between p97/VCP and HDAC1 was stabilized upon ER stress (Fig 2E, quantification not shown).

Moreover, as p97/VCP interacted with both HDAC1 and HDAC2, we also tested whether it could co-immunoprecipitate with the mSin3A chromatin remodeler complex that contain both of these HDACs (9). p97/VCP co-immunoprecipitated with mSin3A, HDAC1 and ING2 another member of the mSin3a complex (10) (Fig. 2F). To verify if HDAC1, RuvBL2 and p97/VCP can be part of the same complex we used a siRNA-mediated knock-down approach, reasoning that if they are part of the same complex, silencing of one member should affect the stability of the whole complex.

Upon RuvBL2 knock down we found that HDAC1 and p97/VCP interaction was highly increased (Fig. 2G, quantification not shown). Upon HDAC1 silencing, p97/VCP and RuvBL2 interaction was enhanced and interestingly RuvBL2 appeared to form a complex with mSin3A thus suggesting that RuvBL2 not only interacted with HDAC1 in the β-Catenin repressor complex (11) but also interacted with the mSin3A complex (Fig 2H). Silencing p97/VCP also affected HDAC1/RuvBL2 interaction as less RuvBL2 was bound to HDAC1 in p97/VCP silenced cells (Fig 2I , quantification not shown). Together, these results suggested that HDAC1, RuvBL2 and p97/VCP can be found in the same complex, and that modulation of the expression level of one member of the complex affects the interaction of the two others. Moreover, p97/VCP and RuvBL2 were found associated with the mSin3A complex, a major chromatin remodeler further documenting the key role of the AAA+ ATPase in gene expression regulation. Given that p97/VCP have a plethora of cofactors, it is very likely that this protein can interact with signaling complexes other than mSin3A like RuvBL2 does to regulate gene expression.

The p97/VCP-RuvBL2-HDAC signaling complex controls ER stress target genes

To further characterize the role of p97/VCP-RuvBL2-HDAC1 in the regulation of ER stress gene expression, we measured the expression of IRE1a, Erdj4, BiP, Herpud, Edem, Orp150, Grp94 and Chop mRNA using real-time PCR in HeLa cells transfected with siRNA CTL, HDAC1, RuvBL2 or p97/VCP under basal or ER stress conditions (Fig. 3A-C and Fig. S3). Knock-down of the members of the complex did not affect the expression of the genes tested in a similar fashion.

Indeed HDAC1 knock-down had mild effect on IRE1, Erdj4 BiP and p97/VCP knock-down, described to lead to ER stress induction (19), exerted a stimulatory effect on the expression of most of the gene tested (IRE1, BiP, Erdj4, Herpud, Orp150, Grp94 and CHOP) mostly under basal conditions. Finally, RuvBL2 knock-down enhanced the induction of all genes tested but Grp94 under ER stress conditions. Although we demonstrated the existence of the p97/VCP-RuvBL2-HDAC1, these results indicate that the ER stress genes analyzed herein may not be regulated by the exact same mechanism. Interestingly, the ER stress genes whose expression was tested here are regulated by ATF4, ATF6 or XBP1s the 3 main ER stress transcription factors. We therefore hypothesized that the p97/VCP-HDAC1-RuvBL2 complex may directly control those transcription factors to regulate the transcription of their target genes. To test this, we evaluated the presence ATF4, ATF6, XBP1s in immune complexes containing p97/VCP-HDAC1-RuvBL2. We found that ATF6 and ATF4 co-immunoprecipitated with p97/VCP-HDAC1-RuvBL2 (Fig. 3D and Fig. S4)

but not with mSin3A whereas XBP1s co-immunoprecipitated with p97/VCP-HDAC1-RuvBL2 and mSin3A (Fig 3E). This result was confirmed by the fact that only XBP1s co-immunoprecipitated with ING2, another member of the mSin3A complex. These data suggested that ER stress-induced transcription factors are differentially incorporated in select transcription complexes: ATF6 and ATF4 are regulated a by p97/VCP-RuvBL2-HDAC1 but not mSin3A whereas XBP1s is regulated by the mSin3a-p97/VCP-RuvBL2 complex. Moreover, this result also indicated that RuvBL2 and HDAC1 interact in different complexes in the cell as suggested in Kim et al., 2006 (12). To further prove the role of p97/VCP in the regulation of ER stress-induced gene transcription, we used the pharmacological p97/VCP inhibitor (CB-5083, 13) and monitored the induction of the above referenced ER stress genes using real time PCR (Fig. 3F). As expected, a strong induction of all the ER stress target genes tested was observed that was even stronger than that caused by p97/VCP siRNA silencing (Fig. 3 A-C). We then evaluated the impact of p97/VCP pharmacological inhibition on the protein levels of the different members of the complexes using immunoblot (Fig.

3G, quantification not shown)

. Interestingly, all member of the mSin3A complex: mSin3A, HDAC1, HDAC2, ING2 showed a decreased of protein level whereas HDAC6 and RuvBL2 were stabilized upon CB-5083 treatment. Overall, these results indicate that genetic or pharmacological inhibition of p97/VCP, which cause a diminution in protein level or inhibition of ATPase activity respectively have different effects on the stability of the signaling complex and as a result on ER stress target gene expression. To identify additional genes that could be regulated by p97/VCP-dependent complexes, we tested the expression of 8 cancer relevant genes that were also deregulated at the transcriptional level in the p97/VCP-associated disease, inclusion body myopathy (14). To this end, the expression of BRCA1, EGFR, FGF2, FGF7, FGF12, GLI1, IGF2R and TGF mRNA was investigated in HeLa cells silenced for p97/VCP or not using real time PCR (Fig. 4A and Fig. S5). This showed that IGF2R and GLI1 were upregulated in p97/VCP silenced HeLa cells while other genes were not significantly altered (Fig. 4A). Mutation or loss of heterozygosity of IGF2R has been associated

with risk of hepatocellular carcinoma whereas GLI1 is one of the main transcription factors of the Hedgehog (Hh) pathway which activation has been implicated in the development of various cancers (6,7). The p97/VCP-dependent regulation of IGF2R and GLI1 expression was conserved in various cancer derived cell lines including Huh7 and U87 thus suggesting a common mechanism of regulation (Fig. S6). We then hypothesized that this transcriptional upregulation could be the result of the stabilization of a transcription factor and therefore we tested transcription factors described in the literature to regulate IGF2R and/or GLI1 (15). We found that in p97/VCP silenced cells USF2 expression was enhanced whereas that of HSF1 remained unchanged (Fig. 4B-C). To test if USF2 controls the expression of GLI1 and IGF2R in HeLa cells, we silenced USF2 and measured the expression of GLI1 and IGF2R mRNA using real time PCR (Fig. 4D and Fig. S6).

In USF2 knock-down cells the expression of GLI1 was decreased whereas that of IGF2R was unexpectedly increased (Fig. 4D and Fig. S7). These results suggest that USF2 stabilization in p97/VCP knock-down cells can explain GLI1 upregulation but not that of IGF2R. We then tested if, like other ER stress transcription factors (ATF4, ATF6, XBP1s), USF2 interacted with p97/VCPdependent complexes (Fig. 4E-F). Interestingly, USF2, as for XBP1s, was found to interact with p97/VCP, HDAC1, RuvBL2 and mSin3A (Fig 4F). Together these results suggest that the GLI1 gene is regulated by USF2, which is itself regulated by the p97/VCP-RuvBL2-HDAC1-mSin3A complex. H. Binding of USF2 to the GLI1 promoter (site 1) and change of Acetylated H3K14 (H3K14 ac) was tested by ChIP under basal conditions, or after 4h or 8h of tunicamycin (5µg/mL); ± SD; (N=2).

I. RT-qPCR analysis of three Hh genes (GLI1, Hhip, Patch1) in HeLa cells under basal conditions, or after 4h or 8h of tunicamycin (5µg/mL) ± SD; (N=3). J. RT-qPCR analysis of GLI1 in Hela cells treated with or without tunicamycin (5µg/mL) for 8h and with or without Visomegib (1µM) for 48h; ± SD; *, *** = P value < 0.05, 0.001 respectively (N=3).

ER stress induces the expression of GLI1 through a non-canonical mechanism

We showed that the p97/VCP-HDAC1-RuvBL2-mSin3a signaling complex regulates USF2 but because members of the complex are regulated upon ER stress, we then hypothesized that ER stress might affect USF2 stability or binding to the GLI1 promoter and therefore control GLI1 expression most likely in a Hh-independent manner. We thus tested this hypothesis using real time PCR and our result showed that beyond p97/VCP knock-down, tunicamycin-induced ER stress, HDAC1 knock-down under basal conditions or RuvBL2 upon ER stress also affected the expression GLI1 mRNA (Fig. 4G). These results suggest that all the members of the p97/VCP-HDAC1-RuvBL2 complex are important for the regulation of the GLI1 mRNA. To explain how ER stress induces GLI1 mRNA expression we performed Chromatin ImmunoPrecipitation (ChIP) on the GLI1 promoter where in silico analysis predicted a potential USF2 binding site. Our results showed that upon ER stress, USF2 was recruited to the GLI1 promoter (Fig 4H). Moreover, ER stress-mediated in our experimental system (Fig. S8). In the nucleus, p97/VCP is able to bind to the ubiquitinylated RuvBL2 and to induce its extraction from the repressor complexes thereby leading to their inactivation and the subsequent increase in acetylation. This mechanism would then allow the chromatin to be in an open state, a favorable condition for transcription activation. As GLI1 is one of the main effector of the Hh pathway, its activation upon ER stress might therefore lead to the non-canonical activation of the Hh pathway, which could play a role in adaptation to stress. Overall, in our model GLI1 and ER stress genes are turned off by deacetylation under basal conditions, but upon ER stress they are turned on through a p97/VCP-dependent mechanism that acts as a molecular switch by inactivating repressor complexes.

Although p97/VCP silencing lead to the stabilization of RuvBL2 and HDACs, it is also known to cause ER stress and to induce the expression of ER stress genes (19). In the context of our model, this suggests that either the remaining pool of p97/VCP is sufficient to induce ER stress genes and/or the cell can induce these genes by other mechanisms independent of p97/VCP-mediated extraction of ubiquitinylated-RuvBL2. As siRNA mediated knockdown (or pharmacological inhibition) are never fully effective it is conceivable that the remaining pool of p97/VCP is preferentially translocated to the nucleus where it could exert its functions towards RuvBL2 and thus induce transcription of ER stress genes. Additionally, in this scenario the lack of cytosolic p97/VCP could impair the degradation of RuvBL2 by the proteasome and explain its stabilization (Fig. S8). Example of such an alternative mechanism has been described in yeast as the degradation of most nuclear ubiquitinylated proteins is mediated by the ubiquitin protein ligase San1. Moreover, Gallagher et al. showed that although San1 and p97/VCP orthologue CDC-48 have common substrates, CDC-48 is not universally required for the degradation of ubiquitinylated nuclear proteins. Similarly, GLI1 is also induced upon p97/VCP silencing, although the mechanism described above might explain this phenotype, it is also possible that the accumulation of activators is responsible for GLI1 induction. Indeed, Smad2/3 are other transcription factors known to regulate GLI1 and they were recently demonstrated to interact with p97/VCP on chromatin regions corresponding to other genes (20). As a result it is probable that accumulation of USF2 and Smad2/3 is sufficient to induce GLI1 expression in p97/VCP-silenced cells. RuvBL2-mSin3A), the target genes are then accessible for the transcription machinery.

Overall our work shows that p97/VCP is a molecular switch able to induce ER stress gene expression by inhibiting repressor complex upon ER stress. We have discovered that this mechanism is not exclusive to ER stress genes. Indeed, ER stress also leads to the non-canonical activation GLI1 in a p97/VCP and the RuvBL2-mSin3a-HDAC1/2 complex-dependent manner and as a consequence activation of Hh genes. As p97/VCP inhibition was described to induce Epithelial to Mensenchymal Transition (EMT)-like phenotypes (21) and also to contribute to the activation of pro-oncogenic genes (GLI1, the present study), we have to take into careful consideration the use of p97/VCP pharmacological inhibitors in neoplastic diseases.

followed by washing 3 times in the lysis buffer. Labeled proteins were resolved on 10% polyacrylamide gels.

Statistical analysis -All data were analyzed by Student's t-test. The GraphPad Prism 5 software was used for statistical analysis.

Interacting Network -p97/VCP and RuvBL2/Reptin network were established using the STRING database and represent using the Cytoscape software.

Chromatin Immunoprecipitation -ChIP was conducted as previously described (22). Briefly, 4 and 8 hours after Tunicamycin treatment, HeLa cells (15x10 6 ) were cross-linked with 1% formaldehyde, followed by cell lysis. DNA was sheared using a Bioruptor 300 (Diagenode, Denville, NJ) to fragment DNA to ~600 bp. Aliquots of the sheared chromatin were then immunoprecipitated using magnetic beads and a H3K14Ac antibody (Millipore) or USF2 (Santa Cruz) or a normal rabbit IgG (Abcam). Following immunoprecipitation, cross-links were removed, and immunoprecipitated DNA was purified using spin columns and subsequently amplified by This model is supported by Baumeister and colleagues findings (77a, 77b) which have shown that under basal conditions, BiP one of the main ER stress gene and a target of ATF6, is repressed by HDAC1 which is bound to the ERSE1 sequence. Upon ER stress HDAC1 is removed and as a result transcription of BiP is activated (77a, 77b). Interestingly, HDAC1 binds to the same sequence as XBP1s and ATF6, strengthening the idea that these repressor complex are recruited by their interaction with ER stress transcription factors. Moreover, HDAC1 but not HDAC2 knock down induces BiP expression suggesting that this gene is not regulated by mSin3A complex which contains both HDACs. This result is consistent with our finding that ATF6 interact with HDAC1 but not mSin3A and strengthens the idea that certain ER stress genes and transcription factors are regulated by different RuvBL2-HDAC1 complexes (78).

ER stress activate the Hedgehog pathway mediated by the p97/VCP induction of GLI1

Another key finding of our work is the fact that GLI1, a major transcription factor of the Hedgehog pathway which has been involved in cancer progression is regulated by p97/VCP and ER stress (Figure 16)(80). We showed that USF2 a transcription factor responsible for the regulation of the GLI1 gene ( 81) is also regulated by a RuvBL2-mSin3A-HDAC complex. In basal conditions, the deacetylation repressor complex is probably recruited to the Gli1 gene by association with USF2. Upon ER stress removal of ubiquitinylated RuvBL2, inhibits the repressor complex, resulting in an increase in acetylation and ultimately activation of Gli1.

Figure 16: Model of the regulation of Hedgehog genes mediated by p97/VCP-RuvBL2-HDAC1

during ER stress -Under basal conditions, GLI1 is repressed by a repressor complex: RuvBL2-HDAC1-mSin3A that is recruited by USF2. Upon ER stress p97/VCP promotes the degradation of ubiquitinylated RuvBL2 thereby inactivating this complex and thus activating transcription of GLI1. GLI1 then promotes the transcription of its target Hh genes.

We then tested the impact this induction of Gli1 and we found that all the Hh target genes tested are indeed induced upon ER stress. Overall our data indicates that ER stress leads to a noncanonical activation of the Hh pathway mediated by p97/VCP-RuvBL2-mSin3a-HDAC.

Interestingly, p97/VCP silencing induces the expression of GLI1 a possible explanation is that in p97/VCP knockdown cell several activators of GLI1 accumulate. Indeed, Smad2/3 two transcription factors that are described to regulate GLI1 were also shown to be interacting with p97/VCP directly at the chromatin (57). Therefore in p97/VCP knockdown cells Smad2/3 and USF2 accumulation might be sufficient to cause induce GLI1 expression.

p97/VCP to regulate gene expression of distant pathways ?

We showed that p97/VCP is key to regulate GLI1 and ER stress genes in ER stress conditions. This regulation relies on the disruption of chromatin remodeler complex (RuvBL2-HDAC1 and RuvBL2-mSin3A-HDAC1) that target acetylated histones to repress their target genes. In fact this mechanism does not seem to be exclusive to ER stress as other reports have linked p97/VCP to the regulation of histone marks: Koike et al. showed that PolyQ cytosolic accumulation caused p97/VCP translocation to the nucleus and change in global acetylation of histones (62). Additionally, a report in yeast suggests the requirement of CDC-48 to regulate ubiquitinylation of H2B during replication (61). Moreover, p97/VCP gene regulation does not seem limited to ER stress and Hh genes as p97/VCP was shown to regulate Heat Shock genes by forming an inactive complex with HSF1 and HDAC6 in basal conditions (40). In other pathways, p97/VCP regulates IKB the repressor of NFKB in the nucleus to regulate genes involve in cell survival and CDC-48 and its cofactor UBX7 were shown to regulate HIF1 and thereby genes involved in the response to hypoxia (59,60). Overall our results and these reports suggest that p97/VCP does not control transcription in one fashion, it seems to affect different histone marks but also regulates a variety of transcription factors involved in different pathways. As the result the extent of the genes it regulate in normal and cancer cells in a given condition is very hard to assess.

p97/VCP as a therapeutic target to treat cancer ? p97/VCP is found overexpressed in many cancers and it is suggested that it contributes to oncogenesis by helping to cope with the proteotoxic stress that exist in fast dividing cancer cells (66). In our study, we have showed that p97/VCP induces gene expression by targeting chromatin modifier complexes. We have also shown that these complexes repressed their target genes by interacting with transcription factors including ATF6, ATF4, XBP1s and USF2. Given that the mSin3A complex regulated many genes by interacting with transcription factors it is likely that some of these genes are regulated by p97/VCP during ER stress. This mechanism could in part explain the complex transcriptional reprogramming that was observed during the UPR. And such transcriptional reprogramming events have been linked to the acquisition of malignant traits such as inflammation, immunogenicity or angiogenesis in cancers (26). Moreover, p97/VCP overexpression in cancer cells might have an important impact on gene expression as it may affect the stability of the complexes it control and thereby the expression of their target genes.

Although the impact of pharmacological inhibition of p97/VCP in cancer cells is hard to assess as it is involved in various cellular processes including cancer relevant processes (transcription, autophagy, protein homeostasis, DNA damage) it has shown some encouraging results. Indeed, in hepatocellular carcinoma, Sorafenib a kinase inhibitor the only treatment available was shown to prevent phosphorylation, cause ER stress and ultimately cancer cell death (71). When combined with Dbeq a specific p97/VCP inhibitor the effects were even stronger.

More recently, NMS-783 and CB-5083 two p97/VCP inhibitors were also shown to induce apoptosis mediated by prolonged ER stress in cancer cell lines and xenografted mice, suggesting that p97/VCP is valid target for cancer treatment (72,73). However a recent report suggest that p97/VCP inhibition mediated by pharmacological inhibitor or siRNA induce Epithelial-Mesenchymal Transition like phenotype in cancer cell lines (81). Moreover, in our study p97/VCP protein level were reduced to 20-30% after siRNA treatment and caused prolonged ER stress but was not sufficient to induce apoptosis. It is also noteworthy that in p97/VCP silenced cell we noticed induction two oncogenes: GLI1 and IGF2R. In conclusion, although p97/VCP pharmacological inhibition has shown some encouraging results, it may also promote EMT or induction of oncogenes and therefore be an unsafe therapeutic strategy.

Fig. 2 .

 2 Fig.2. Normal vs. cancer cell secretory pathway control and biological outcomes. In normal cells, the secretory pathway adapts to fluctuation of environmental stresses and intracellular needs, through a complex molecular signaling pathway: the unfolded protein response (UPR). This adaptation program is triggered by three ER transmembrane sensors, namely IRE1, PERK and ATF6. The cell secretory proteins needs vary depending on cell type, differentiation state or on the physiological context. In cancer cells, the secretory pathway is subjected to a strong environmental pressure due to environmental stress factors, such as hypoxia, oxidative stress or chemotherapies, and to oncogenic pressure (e.g. Myc/aneuploidy stimulation of transcription). Furthermore, in these cells, the increase in secretion demand is also dependent on a substantial requirement of energy and amino acid supply. The integration of both intrinsic and extrinsic challenges results in disturbance of the ER homeostasis causing the UPR to be constitutively active in these cells. Disturbance of the secretory pathway will, as a consequence, lead to the modulation of the secretion of proteins important for cancer features such as growth factors and their associated receptors, extracellular matrix proteins, matrix metalloproteinases, inflammatory factors, integrins, immunogenic factors or pro-angiogenic factors.

Figure 9 :

 9 Figure 9: Representation of p97/VCP/CDC-48 cofactors and their functions -To date more than 40 cofactors are known for p97/VCP/CDC-48. This diagram shows in each pathway what cofactors are involved and their interacting partners (adapted from 32).

  processes require p97/VCP dependent extraction of ubiquitin modified proteins such as: transcription, cell cycle progression, DNA damage response and DNA replication. -Cell cycle progression and DNA replication -Aurora B kinase extraction by p97 at the end of mitosis from the chromatin of Xenopus laevis was the first case that reported extraction of

Figure 11 :

 11 Figure 11: Schematic representation of CDC48 nuclear import mechanism -Phosphorylation of CDC-48 at the penultimate tyrosine changes its tridimensional structure to reveal a nuclear import sequence (adapted from 44).

--Figure 12 :

 12 Figure 12: Schematic representation of the role of p97 during transcriptional coupled NER (REF) -UV light can cause DNA lesions such as abnormal DNA dimers which can stall the RNA pol II. The stalled RNA pol II is ubiquitinylated by CUL3 (E3) to which p97 and its cofactors can bind. CDC-48 then extracts the RNA pol II from the lesion site to the proteasome for degradation thus allowing access to the NER machinery for reparation (adapted from 51).

Figure 13 :

 13 Figure 13: Schematic representation of the role of p97/VCP during Double Strand Breaks -RNF8 and RNF168 are recruited at the DSB site and ubiquitinylate the chromatin associated proteins including L3MBTL1 which interact with the di-methyl H4K20. Then, p97/VCP and its cofactors UFD1/NPL4 which can bind to ubiquitin are recruited at the DBS site to extract L3MBTL1 thereby allowing binding at the same histone mark of the repair factor 53BP1 (adapted from 54).

  several pathway are affected by p97/VCP mutation including regulation of actin cytoskeleton, autophagy or lysosome. Moreover, several reports across human, yeast, worm or fruit fly have documented the molecular mechanism by which p97 can regulate gene expression. These reports suggest that there are two main mechanisms by which p97 can regulate gene expression: either by regulating the stability of transcription factors or by inducing change in histone marks. p97 regulation of transcription factors occurs at different locations in the cell. At the ER membrane p97/VCP is required for the release of Nrf1 and the proteolytic activation of Mga2p

  responsible for multisystem proteinopathy (MSP) a degenerative disorder which can manifest clinically as Amyotrophic Lateral Sclerosis (ALS), Inclusion Body Myopathy (IBM), Paget's disease of the bone (PDB), Frontotemporal Dementia (FTD), or as a combination of these disorders (REF). The three later phenotypes are often referred to as IBMPFD (Inclusion Body Myopathy with Paget disease and Frontotemporal Dementia). Is it noteworthy, that more recently patients with MSP due to p97/VCP mutations have displayed additional symptoms such as

  5 µL of RNAimax by pipetting up and down (13778030, Thermo Fisher Scientific) and in the second tube 150µL of OptiMEM was mixed with 3.5 µL of siRNA (CTL, p97/VCP, HDAC1, RuvBL2 or USF2) at [20µM] by pipetting up and down. Note that the manufacturer recommend to use a final concentration from [10 nM] to [100 nM] and we found that the [40 nM] final concentration was the best for the efficiency of the silencing and the survivability of the cells. After 5 mins the two tubes were mixed and after 15 mins incubation at RT, 250 µL of the mixture was drop in droplets to the corresponding well before being mixed by gently rocking the plate back and forth. After 48h the cells were ready to be harvest for either protein or RNA extraction. Note that the efficiency of the silencing was tested by immunoblotting and qPCR. List of siRNAs used in this study: , U87, U251, Panc1 cells were cultured in 6 wells plates for RNA extraction. Total RNAs were extracted using 1mL of Trizol reagent (15596018, Invitrogen) and the manufacturer recommendation. For the Reverse Transcription 2µg of mRNA were used with the Maxima Reverse Transcriptase (ER0741, Life technologies) following the manufacturer recommendation. For quantitative RT-PCR (RT-qPCR) all reactions were conducted using the SYBR qPCR Premix Ex Taq (TAKRR420W, Ozyme) in the QuantStudio 5 thermocycler (Applied Biosystem). Each PCR was performed in technical triplicates in 384-well plate. Each well contain 5µL of SYBR qPCR Premix Ex Taq, 0.4 µL of Forward and Reverse primer at [10 µM], 2.6 µL au water and 2µL of cDNA diluted to the 1:200 for a total volume of 10µL. The cycle of amplification consists of the following steps: Pre-run at 95°C for 30 secs, 40 cycles of amplification (95°C for 5 secs, 60°C for 34 secs) and 95°C for 15 secs and 60° for 1 min for the dissociation step. For analysis each sample was normalized to Gapdh and/or Actin expression level.List of the qPCR primers used in this study:

  RNAi clones instead reproducibly increased (average Z-score >1.5 or one of the two individual Z-scores >3) GFP fluorescence 1.5-fold above the fluorescence intensity measured with cdc-48.2 (-/-) ; ckb-2p::gfp worms fed with an empty vector and treated with tunicamycin (P<0.05).

2 .

 2 RNAi clones could activate gene transcription specifically under ER stress independently of cdc-48.If a targeted gene acts exclusively in the same genetic pathway as cdc-48.2, then its knockdown by RNAi should not increase ckb-2p::gfp transcription in a WT background, nor have an additive effect with the cdc-48.2 (-/-) mutation on ckb-2p::gfp transcription. We quantified and compared ckb-2p::gfp fluorescence intensities in both WT and cdc-48.2 (-/-) mutant worms fed with RNAi and exposed to tunicamycin. Twenty-seven RNAi clones increased fluorescence intensities in WT more than in cdc-48.2 (-/-) worms (fold change ≥1.4, Fig.1G). Nine other clones showed higher fluorescence in mutant worms compared to WT (fold change ≥1.4, Fig.1I), similar to cdc-48.1 RNAi. The corresponding 36 genes (27+9) were therefore not considered as strict suppressor of cdc-48.2 and were not further analyzed. We thus identified 64 suppressor RNAi clones that did not show any synthetic enhancement phenotype in cdc-48.2(-/-) relative to WT (fold-change <1.4 and P<0.05, Fig.1H). Taken together, these results identify genes controlling ckb-2p::gfp expression upon ER stress in a CDC-48 dependent fashion, and may provide mechanistic insight for the role of CDC-48 in ER stress induced gene expression (Fig.

  duplicate. Fluorescence average value for each plate was calculated and used to calculate the individual RNAi fold change. Plates showing no fluorescence induction in the positive control, no fluorescence decrease in negative control, or a high fluorescence mean were discarded and retested.

Figure 1 : 2

 12 Figure 1: RNAi screening identifies cdc-48.2 genetics interactors in the ckb-2 transcriptional response to ER stress. (A) cdc-48.2 is required to activate ckb-2p::gfp transcription in response to tunicamycin. Images of adult worms (left) expressing gfp under the control of the ckb-2 gene promoter in WT (upper panels) and in cdc-48.2 (-/-) mutants (lower panels) exposed to tunicamycin (5 μg/ml) or DMSO for 16 hours. (Scale bar: 50 μm, obj.: 10x). (B) Significant changes in fluorescence intensities were quantified using flow cytometry. L1 larvae (ckb-2p::gfp and cdc-48.2 (-/-) ; ckb-2::pgfp larvae) were fed with bacteria expressing the L4440 empty vector or ire-1 RNAi in liquid culture and exposed to tunicamycin (0.5 μg/ml) or DMSO for 16 hours. F0 was defined as the fluorescence intensity obtained in ckb-2p::gfp worms fed with the empty vector and treated with DMSO. (Mean ± s.e.m, N=8, 200 worms/experiment). P values were calculated using multiple t-test corrected using the Holm-Sidak method **P<0.001; *P<0.01. (C) Genomewide RNAi screen identifies suppressors and enhancers of cdc-48.2 (-/-) in ckb-2p::gfp transcription. Volcano plots present results obtained using C. elegans ORFeome library. (D) Retesting of RNAi clones from fisrt round. Classification of ER stress dependence of the 177 suppressor RNAi clones able to restore ckb-2p::gfp transcription in cdc-48.2 (-/-) mutant background. cdc-48.2 (-/-) ; ckb-2p::gfp synchronized L1 larvae were fed with the dsRNA expressing bacteria in liquid culture, treated with tunicamycin (0.5 μg/ml) or DMSO for 16 hours and fluorescence intensities were measured by flow cytometry. Tunicamycin dependent RNAi clones were defined as those that significantly increased fluorescence ratio following tunicamycin treatment ((E); Tunicamycin/DMSO F/F0 fold change>1.5). Tunicamycin independent RNAi clones were defined as those increasing ckb-2p::gfp fluorescence ratio in both conditions ((F); Tunicamycin/DMSO F/F0 fold change <1.5, P>0.05). Fluorescence ratios obtained with ruvb-2 RNAi are shown in magenta. Fluorescence ratios obtained with ckb-2p::gfp worms fed with the empty vector and treated with tunicamycin (2.38±0.18) or DMSO (1.05±0.2) are shown in cyan. F0 was defined as the fluorescence intensity obtained in cdc-48.2 (-/-) ; ckb-2p::gfp worms fed with the empty vector and treated with tunicamycin or DMSO, respectively. (Mean ± s.e.m, N=5). Identification of ER stress dependent RNAi clones targeting genes involved in the same genetic pathway as cdc-48.2 to increase ckb-2p::gfp transcription. Fluorescence ratio were determined on cdc-48.2 (-/-) ; ckb-2::gfp and ckb-2::gfp worms fed with the suppressor RNAi clones and treated

Figure 2 :

 2 Figure 2: Identification of RUVB2 as a candidate CDC-48 target. (A) List of RNAi clones suppressing the cdc-48.2 (-/-) phenotype. (B) Graph representing identified peptide number identified in function of peptide quantity ratio. cdc-48.2 (-/-) ; ckb-2::gfp and ckb-2::gfp synchronized L1 larvae were grown to the L4 stage and exposed to tunicamycin (5 μg/ml) for 16h on plates. Proteins (60 μg) were separated on a 10% SDS gel. A coomassie blue staining image representative of the SDS gel is shown on the left (1: cdc-48.2 (-/-) ; ckb-2::gfp, 2: ckb-2::gfp). Gel lanes were cut into slices before proteins were in-gel digested. Peptides were then identified and quantified by label-free LC-MS/MS mass spectrometry. Peptides that were more (magenta) or less (cyan) abundant in the cdc-48.2 (-/-) ; ckb-2::gfp than in ckb-2::gfp worms were defined as those having a ratio above 1.5 or below 0.5, respectively. N=3. (C) Graph representing peptide quantity ratio ((cdc-48.2 (-/-) ; ckb-2::gfp)/(ckb-2::gfp)) for the 93 proteins that are more abundant in cdc-48.2 (-/-) mutant background compared to WT background. (Mean ± s.e.m, N=3).

Figure 3 : 2

 32 Figure 3: RUVB-2 is a transcriptional repressor inactivated by CDC-48 upon ER stress. (A) Images of cdc-48.2 (-/-) ; ckb-2::gfp adult worms fed with either the L4440 empty vector (upper panel) or ruvb-2 RNAi (lower panel) and treated with tunicamycin (5 μg/ml) or DMSO for 16 hours on NGM agar plates. (Scale bar: 50 μm, obj: 10x). (B) Fluorescence was quantified by flow cytometry on ckb-2::gfp, cdc-48.1 (-/-) ; ckb-2::gfp and cdc-48.2 (-/-) ; ckb-2::gfp worms fed with ruvb-2 RNAi or empty vector starting at the L1 stage in liquid culture and exposed to tunicamycin (0.5 μg/ml) or DMSO for 16 hours. Fluorescence (F) was normalized to the basal fluorescence obtained with empty vector and DMSO in the WT background (F0). (Mean ± SD, N=5) ***P < 0.001. (C) Fluorescence (F) was quantified by flow cytometry on ckb-2::gfp and

Figure 4 :

 4 Figure 4: p97/CDC-48-mediated degradation of Reptin upon ER stress. (A) Reptin, Pontin, calnexin and quantification by immunoblot. Values are expressed as a percentage of the initial protein abundance in total HuH7 cell lysate before addition of tunicamycin (5 g/ml), (Mean ± SD, N=5). *P<0.05; **P<0.01. (B) Immunoblot analysis of Reptin in total protein extracts from HuH7 cells exposed to tunicamycin (5 μg/ml) for 0 to 2 hours. Protein levels were normalized to Calnexin (mean ± SD, N=3). *P<0.05; **P<0.01. (C) HuH7 cells expressing FLAG tagged Reptin were treated either with the p97/CDC-48 inhibitor DBeQ (20 μM, D), the ER stress inducer tunicamycin (2 g/ml; T) or both for 4 hours. FLAG tagged Reptin was immunoprecipitated from total protein extracts using anti-FLAG antibodies and p97/CDC-48 association was analyzed by immunoblot. (D) HuH7 cells were treated either with DBeQ (20 μM, D), (2 g/ml; T) or both for 4 hours. P97/CDC-48 was immunoprecipitated from total protein

Figure 5 :

 5 Figure 5: Reptin silencing enhances ATF6 and XBP1s activation. (A) GRP78 and GRP94 expression was detected using anti-KDEL antibodies (top blot) in HuH7 cells treated or not with tunicamycin and/or doxycycline (Dox) to induce reptin silencing (bottom blot). Expression of p97 was also monitored (middle blot). (B) eIF2 phosphorylation was monitored using specific antibodies (top blot) and reported to the total expression (bottom blot) in HuH7 cells treated or not with tunicamycin and/or doxycline (Dox). (C) ATF6 activation was monitored in the same experimental conditions using antibodies agains the N-terminal domain of ATF6. (D) Expression of unspliced XBP1 mRNA as determined by RT-PCR and expression of the XBP1s ligase RTCB as determined by immunoblot using anti RTCB antibodies in HuH7 cells treated or not with tunicamycin and/or doxycycline (Dox). (E) XBP-1 mRNA splicing as determined by RT-PCR under basal conditions or upon tunicamycin treatment (5 g/ml for 16 hours) in HuH7 cells subjected or not to doxycycline-induced Reptin silencing. Three independent experiments were performed and a representative image is shown. (F) HuH7 cells were treated either with DBeQ (20 μM, D), tunicamycin (2 g/ml; T) or both for 4 hours. XBP-1 mRNA splicing was determined by RT-PCR (Mean ± SD, N = 3). P values were calculated using multiple t-test corrected using the Holm-Sidak method. *P < 0.05; **P < 0.01. (G) HuH7 cells were treated with tunicamycin (2 g/ml; T) for 1 hour. XBP1s was immunoprecipitated and the complex was immunoblotted with anti Reptin antibodies (top blot). Total cell lysate (TCL) was immunoblotted with anti Reptin (middle blot) or anti XBP1s (bottom blot).

Figure 1 :

 1 Figure 1: Evolutionary conserved regulation of HDACs by p97/VCP. A. Graph representing peptide quantity ratio ((cdc-48.2 (-/-) ; ckb-2 ::gfp)/( ckb-2::gfp)) for the 93 proteins that are more abundant in cdc-48.2 (-/-) mutant background compared to WT background (Mean s.e.m, see (3) for more details. B. RuvBL2 and Cdc-48 interacting network in C. elegans. C. RuvBL2 and p97/VCP interacting network in H. sapiens. D. HDAC1, HDAC2, HDAC4, HDAC6, p97/VCP (Knock Down control), Tubulin (loading control) by immunoblot in HeLa cells treated with siRNA CTL or siRNA VCP for 48h (N=3). E. Quantification of D, ± SD, * = P value < 0.05.

Figure 1 :

 1 Figure 1:

  stress and treated with sip97/VCP (Fig 2D lane 4), HDAC1 was further stabilized compared to cells subjected to stressed cells treated with siCTL (Fig 2D lane 3, quantification not shown).

Figure 2 :

 2 Figure 2: A p97/VCP-RuvBL2-HDAC1 complex is differentially regulated in basal and stress conditions. A. Grp94, BiP (ER stress control), HDAC6, HDAC2, HDAC1, RuvBL2, Actin (loading control) by immunoblot in untreated or exposed to tunicamycin (5µg/mL) for 8h HeLa cells (N=3). B. Quantification of A, ± SD, * = P value < 0.05. C. Correlation between BiP protein level after tunicamycin induction and HDAC1 protein level. D. Grp94, BiP (ER stress control), p97/VCP (Knock Down control), HDAC1, Tubulin (loading control) by immunoblot in HeLa cells treated with siRNA CTL or siRNA p97/VCP and exposed or not to tunicamycin (5µg/mL) for 8h (N=3). E. HDAC1 was immunoprecipitated from total protein extracts from untreated or exposed

Figure 3 :

 3 Figure 3: p97/VCP-RuvBL2-HDAC1 regulates ER stress genes through regulation of their transcription factors A. RT-qPCR analysis of two ATF6 targets (IRE1, Erdj4) in siRNA CTL, siRNA HDAC1, siRNA RuvBL2 or siRNA p97/VCP conditions with or without tunicamycin treatment (5µg/mL for 8h); ± SD; *, **, *** = P value < 0.05, 0.005, 0.001 respectively (N=3). B.Same experiment as in A for two XBP1s targets (BiP, Herpud1) C. Same experiment as in A for two ATF6 and XBP1s cooperative targets (Edem1, Orp150). D. ATF6 was immunoprecipitated from total protein extracts from HeLa cells exposed to tunicamycin (5µg/mL) for 8h using a specific antibody and mSin3A, p97/VCP, HDAC1, RuvBL2 association was analyzed by immunoblotting (N=3). E. XBP1s was immunoprecipitated from total protein extracts from HeLa cells exposed to tunicamycin (5µg/mL) for 8h using specific a antibody and mSin3A, p97/VCP, HDAC1, RuvBL2, ING2 association was analyzed by immunoblotting (N=3). F. RT-qPCR analysis of six ER stress genes (IRE1, Erdj4, BiP, Herpud, Edem, Orp150) target of ATF6 and/or XBP1s in HeLa cells treated with 1 µM of CB-5083 for 16h; ± SD; *, **, *** = P value < 0.05, 0.005, 0.001 respectively (N=3). G. mSin3a, HDAC6, p97/VCP, Grp94, BiP, HDAC1, HDAC2, RuvBL2, ING2, Tubulin (loading control) by immunoblot in untreated or exposed to 1 µM of CB-5083 for 16h HeLa cells (N=3).

Figure 3 :

 3 Figure 3:

Figure 4 :

 4 Figure 4: GLI1 is regulated by p97/VCP and ER stress. A. RT-qPCR analysis of IGF2R and GLI1 in HeLa cells treated with siRNA CTL or siRNA p97/VCP; ± SD; *, ** = P value < 0.05, 0.005 respectively (N=3). B. p97/VCP (Knock Down control), HSF1, USF2, Tubulin (loading control) by immunoblot in HeLa cells treated with siRNA CTL or siRNA p97/VCP (N=3). C.Quantification of B; ± SD; * = P value < 0.05. D. RT-qPCR analysis of GLI1 in HeLa cells treated with siRNA CTL or siRNA USF2; ± SD; ** = P value < 0.005 (N=3). E. p97/VCP or USF2 were immunoprecipitated from total protein extracts from HeLa using specific antibodies and p97/VCP, USF2 association was analyzed by immunoblotting (N=3). F. USF2 was immunoprecipitated from total protein extracts from HeLa using a specific antibody and mSin3a, p97/VCP, HDAC1, RuvBL2, ING2 association was analyzed by immunoblotting (N=3). G. RT-qPCR analysis of GLI1 in siRNA CTL, siRNA HDAC1, siRNA RuvBL2 or siRNA p97/VCP conditions with or without tunicamycin treatment (5µg/mL for 8h); ± SD; *, ** = P value < 0.05, 0.005 respectively (N=3).

Figure 5 :

 5 Figure 5: Model of GLI1 and ER stress genes regulation mediated by p97/VCP. Under basal conditions, RuvBL2-mSin3A-HDAC1 repress the expression of GLI1 and ER stress genes. Upon ER stress p97/VCP act as a molecular switch and inhibits the repressor complexes by promoting degradation of ubiquitinylated RuvBL2 thus activating GLI1 and ER stress genes. Non-canonical activation of GLI1 leads to the expression of its target Hedgehog genes. Upon ER stress, p97/VCP can bound to ubiquitinylated RuvBL2 to extract repressor complexes (RuvBL2-HDAC1 or

  quantitative PCR. PCR primers were designed to amplify regions of the GLI1 promoter containing potential USF2 binding sites. The sequences of the primers are as follows: sense, TGAGGGAGGATGCTTAGGGG; antisense, GGTCAAGAGATTGAGACCATCC. Samples for quantitative SYBR PCR were performed in triplicate using the C1000 Thermal Cycler (Bio-Rad, Hercules, CA). Results are represented as Percentage of Input.

Figure S3 :

 S3 Figure S3: RT-qPCR analysis of Grp94 target of XBP1s and CHOP target of ATF4 in siRNA CTL, siRNA HDAC1, siRNA RuvBL2 or siRNA p97/VCP conditions with or without tunicamycin treatment (5µg/mL for 8h), *, *** = P value < 0.05, 0.001 respectively (N=3).

Figure S4 :

 S4 Figure S4: ATF4 was immunoprecipitated from total protein extracts from HeLa cells HeLa cells exposed to tunicamycin (5µg/mL) for 8h using a specific antibody and mSin3A, p97/VCP, HDAC1, RuvBL2 association was analyzed by immunoblotting (N=3).

Figure S5 :

 S5 Figure S5: RT-qPCR analysis of BRCA1, EGFR, FGF2, FGF12 and p97/VCP in Huh7 cells treated siRNA CTL or siRNA p97/VCP (Knock Down control); ± SD; *** = P value < 0.001 (N=3).

Figure S6 :

 S6 Figure S6: RT-qPCR analysis of IGF2R and GLI1 in U87 and Huh7 cells treated siRNA CTL or siRNA p97/VCP; ± SD; * = P value < 0.05 (N=3).

Figure S7 :

 S7 Figure S7: RT-qPCR analysis of IGF2R in Hela cells treated siRNA CTL or siRNA USF2; ± SD; *** = P value < 0.001 (N=3).

Figure S8 :

 S8 Figure S8: Immunohistochemistry staining of endogenous p97/VCP in Huh7 cells under basal conditions or after 6h of tunicamycin (5µg/mL) and quantification of cells with nuclear staining exclusively; ± SD; ** = P value < 0.005 (N=3).

  La protéine p97/VCP est un membre de la famille des ATPase AAA + et joue un rôle majeur dans de nombreux processus cellulaires tel que le contrôle de l'homéostasie protéique ou de fonctions associées à la chromatine (transcription, réplication, dommage à l'ADN, progression du cycle cellulaire). De plus, la protéine p97/VCP est impliquée dans un nombre croissant de maladies dont les cancers où il a été montré qu'elle contribue à l'homéostasie protéique et l'adaptation au stress oncogéniques. En effet, l'expression de la protéine p97/VCP est augmentée dans de nombreux cancers et dans certains cas corrèle avec une récurrence de la tumeur et un mauvais pronostique pour les patients. Cependant, le mécanisme moléculaire précis par lequel la protéine Le cancer est aujourd'hui la première cause de mortalité en France avec près de 150 000 decès par an représentant un enjeu de santé publique majeur. Les cancers gastrointestinaux représentent une des causes les plus courantes de cancer en France et dans le monde : près d'un quart des cancers. Alors que chaque année le taux de survie des cancers s'améliore, les cancers gastrointestinaux sont parmis les plus mortel, avec notamment les cancers du foie, du pancréas qui présentent une survie à 5 ans après diagnostic inférieur à 10%. Il y a donc une réelle nécessité d'étudier ces types de cancers notamment le cancer du foie auquel mon équipe s'intéresse afin de trouver des traitements plus efficace.

	Oligosaccharyl transferase Paget disease and Frontotemporal Dementia Protein disulfide isomerase RESUME PFD PDI RESUME SUBSTANTIEL
	PERK	Protein kinase (PKR)-like ER kinase
	PTM	Post Translational Modification
	QC	Quality control
	RER	Rough ER
	RIDD	RNA regulated IRE1 dependent decay
	RNA	Ribonucleic acid
	RuvBL2	RuvB Like AAA ATPase 2
	RyR	Ryanodine receptor
	S1P	Site-1 protease
	S2P	Site-2 protease
	SER	Smooth ER
	SERCA	Sarcoplasmic/endoplasmic reticulum calcium ATPase
	Sin3A	Sin3 Transcription Regulator Family Member A
	SNP	Single nucleotide polymorphism
	SPARC	Secreted protein acidic and rich in cysteine
	Sp1	Stimulating protein 1
	SREBP1/2 Sterol regulatory element binding protein 1 and 2
	SRP	Signal recognition particle
	σ1R	Sigma-1 receptor
	TP53	Tumor protein p53
	TRAP	Translocon associated protein
	uORF	Upstream open reading frames
	UPR	Unfolded protein response
	UPRE	UPR response element
	UPS	Ubiquitin/proteasome system
	VCP	Valosin-containing protein
	VDAC	Voltage dependant anion channel
	XBP-1	X-box binding protein 1
	XBP1s	Spliced form of XBP1
	Mots clefs: AAA+ ATPase, p97/VCP, Stress du Réticulum Endoplasmic, Homeostasie Protéique,
	Contrôle de l'expression des genes
	Chemistry Oncogenesis Stress Signaling, INSERM unité 1242, Centre Eugène Marquis, Avenue
	de la bataille Flrandres Dunkerque, 35042 Rennes cedex

p97/VCP régule l'homéostasie protéique des cellules tumorales reste incertain. Pour remédier à cela, nous avons démontré un rôle de la protéine p97/VCP dans le contrôle de l'expression des gènes lors du stress du Réticulum Endoplasmique (RE). Nous avons trouvé que en conditions basales, la protéine RuvBL2 fait partie d'un complexe remodeleur de la chromatine qui contient les protéines HDAC1 et mSin3A et agit comme un répresseur des gènes de stress du RE. De plus, nous avons identifié le gène Gli1, un effecteur connu de la voie de signalisation Hedgehog comme cible de la protéine p97/VCP et du complexe RuvBL2-HDAC1-mSin3A. Ainsi en condition de stress du RE, la voie de signalisation Hedgehog qui a été impliqué dans le développement de cancers est activée. Globalement, nos travaux indiquent que p97/VCP agit comme un interrupteur moléculaire pour inactiver le complexe répresseur RuvBL2-HDAC1 en condition de stress du RE et ainsi activer les gènes de stress du RE et de la voie de signalisation Hedhehog de façon non-canonique.

La protéine p97 est connue pour être impliquée dans de nombreuses pathologies neurologiques et des cancers. En effet, la surexpression de cette protéine a été associée à un mauvais pronostique de survie dans un très grand nombre de cancers : poumon, prostate, oesophage, colon, pancréas et foie. Bien que l'implication directe de la protéine p97 dans le développement et la progression du cancer du foie ait déjà été prouvée, les mécanismes moléculaires par lesquelles la protéine p97 contribue à la progression et au développement de ce cancer reste peu claire. Nos récents travaux ont permis de montrer que le Sorafenib, le seul traitement qu'il existe aujourd'hui contre le cancer du foie agissait sur cette protéine. In vitro le Sorafenib provoque la mort des cellules cancéreuse et nous avons montré que cet effet était augmenté en associant le Sorafenib à des molécules inhibitrices de la protéine p97, ouvrant la voie à de possibles nouveaux traitements. En effet, nous avons des preuves que la protéine p97 participerait au développement du cancer du foie en régulant de nombreux autres gènes dont certains oncogènes. Nous avons cette année découverte une nouvelle façon pour la protéine p97 de réguler à distance certain autres gènes notamment les gènes de stress du Réticulum Endoplasmique. Cette régulation impliquerait la translocation de p97 au noyau en condition de stress du Réticulum Endoplasmique et son association avec des complexes

  48, an AAA + ATPase involved ER stress response, in the regulation of ER stress-

associated UPR ER gene transcription. C. elegans expresses two p97/CDC-48 homologs, cdc-48.1 and cdc-48.2, which share similar functions in ERAD. While simultaneous silencing of both cdc-48.1 and cdc-48.2 leads to ER stress, UPR ER gene activation and lethality [3]. Inactivation of either cdc-48.1 or cdc-48.2 is viable but abolishes the transcriptional activation of UPR ER genes in response to ER stress

Table S1 : qPCR primer list

 S1

	GENE	FORWARD PRIMER 5'-3'	REVERSE PRIMER 5'-3'
	P97/VCP CCATCCGGAAAGGAGACATTT	GTCTGGAGCAACAATGCAATAAG
	GAPDH GACCTGACCTGCCGTCTAGAAAAA ACCACCCTGTTGCTGTAGCCAAAT
	GLI1	AGGGAGTGCAGCCAATACAG	ATTGGCCGGAGTTGATGTAG
	USF2	TTCGGCGACCACAACATCCAG	CAGTCACCTGGACTACGCGGT
	IRE1A	GCCACCCTGCAAGAGTATGT	ATGTTGAGGGAGTGGAGGTG
	BIP	TGTTGGAAGATTCTGATTTGAAGA TCACTCGAATACCATTCACAT
	EDEM	AGTCATCAACTCCAGCTCCAA	AACCATCTGGTCAATCTGTCG
	ERDJ4	TGGTGGTTCCAGTAGACAAAGG	CTTCGTTGAGTGACAGTCCTGC
	HERPUD TCCTCCTCCTGACGTTGTAAA	TGTTCGCXATCTAGTACATCC
	ORP150 GAAGATGCAGAGCCCATTTC	TCTGCTCCAGGACCTCCTAA
	RUVBL2 AAGTCCCGGAGATCCGTGAT	CGACCGGCAATCTTCCCTTC
	CHOP	AAGGCACTGAGCGTATCATGT	TGAAGATACACTTCCTTCTTGAACA

Table S2 : siRNA list GENE SEQUENCE 5' -3'

 S2

	P97/VCP	(GAAUAGAGUUGUUCGGAAU)TT
	HDAC1	(CAGCGACUGUUUGAGAACC)TT
	RUVBL2 (GAGAUCCAGAUUGAUCGACCAGCAA)TT
	USF2	(CCUCCACUUGGAAACGGUA)TT

remodeleurs de la chromatine. En effet en condition de stress du Réticulum Endoplasmique la protéine p97 s'associerait de façon préférentiel avec les protéines RuvBL2 et HDAC1 et entrainerait leur déstabilisation et par conséquence l'activation transcriptionnel des gènes qu'ils répriment. Il y a donc une réelle nécessité d'étudier la façon dont la protéine p97 participe à la régulation de ces gènes pour comprendre comment elle promouvoit le développement du cancer. Il est fort probable que le rôle et l'implication de la protéine p97 dans le cancer continue de croître dans les années à venir.Cette protéine pourrait en effet devenir une cible thérapeutique très intéressante. Ainsi, la découverte de molécules régulant cette protéine pourrait permettre en association avec des traitements courants de soigner le cancer du foie, mais aussi s'appliquer à d'autres cancers et pathologies.

In this study, I contribute to show that HDAC1/2 are regulated by p97/VCP and under ER stress. I showed that p97/VCP, RUVBL2 and HDAC1 are interacting together in repressor complexes. Moreover, these repressor complexes interact selectively with the major ER stress transcription factors to repress their target genes. Finally, I contribute to show that p97/VCP role of gene expression control extend to other genes including the oncogenes IGF2R and GLI1.

Acknowledgements

We apologize to colleagues whose work was not cited in this review due to space limitation. This work was funded by grants from Institut National du Cancer (INCa) and Ligue Contre le Cancer to EC. ND was supported by a post-doctoral fellowship from Fondation de France.

Acknowledgements

-We thank Dr. R. Pedeux for help with the post-translational modifications of reptin, E Attebi and K Rebora for help with C. elegans screens, Drs E. Snapp and F. Schoenen for the ERSE::Tomato construct and DBeC, respectively, Dr. S. Mitani for the cdc-48.1(tm544) and cdc-48.2(tm659) strains. Acknowledgements This work was supported by grants from the Institut National du Cancer (INCa, PLBIO 2012, 2014) and la Ligue Contre le Cancer to EC. KB was funded by a grant from La Ligue Contre le Cancer.

Conflict of interest

The authors declare that there are no conflicts of interest.

ARTICLE 2

Abstract

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR ER ) to restore ER homeostasis. The AAA + ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR ER genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in C. elegans, we have identified RUVB-2, a AAA + ATPase, as a novel repressor of a subset of UPR ER genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR ER genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.

Conflict of interest -

The authors declare no conflict of interest.

Abstract

Upon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), an adaptive pathway named the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The UPR controls the expression of stress genes to increase ER folding and clearance capacities.

Although the molecular events triggered by the UPR are well described, the transcriptional mechanism underlying the regulation of ER stress target genes cannot completely account for the transcriptional outputs observed. Here, we have found that p97/VCP, an AAA+ ATPase known to modulate ER stress target genes expression, dynamically interacts with RuvBL2 and the mSin3A-HDAC1/2 complex. These complexes selectively interact with the major ER stress transcription factors ATF4, ATF6, and XBP1s to control the transcription of their respective target genes.

Furthermore we have identified USF2 a regulator of the GLI1 gene as a novel target of the p97/VCP-RuvBL2-mSin3A-HDAC1/2 complex. GLI1 is a known effector of the Hedgehog (Hh) signaling and is transcriptionally repressed by a RuvBL2-mSin3A-HDAC1/2 complex under basal conditions. Remarkably, upon ER stress GLI1 transcription is induced through a mechanism requiring p97/VCP-mediated extraction and degradation of RuvBL2 in an Hh-independent manner. Overall, our work demonstrates that p97/VCP controls the activation of Hh signaling upon ER stress in a ligand independent fashion and defines the interplay between the newly identified p97/VCP-mSin3A-HDAC1/2 complex and the transcription factor USF2 as an essential player in this phenomenon. USF2 recruitment to the GLI1 promoter was accompanied by an increase in acetylation of histone 3 lysine 14 (acetyl H3K14) a histone mark generally associated with transcriptional activation (16,Fig 4H). These results suggest that under basal conditions the removal or lack of acetylation at the GLI1 gene cause its repression, however upon ER stress the GLI1 promoter is acetylated and USF2 recruited and as a consequence the gene is activated. Therefore our next step was to evaluate whether GLI1 target genes in the Hh pathway were also induced by ER stress (Fig 4I induction during ER stress is independent of the Hh ligand. Together these results suggest GLI1 is regulated at least in part by acetylation and that ER stress or change in protein level of the member of the complex p97/VCP-RuvBL2-HDAC1 are sufficient to induce its expression independently of the ligand. As a possible consequence of GLI1 overexpression during ER stress we found that its target genes in the Hh pathway (Hhip, Patch1) are also induce.

Discussion

In this study we have shown that p97/VCP can interact in a stress dependent manner with signaling complexes that include HDAC1, RuvBL2 and transcription factors to control gene expression of ER stress genes and GLI1. These results have allowed us to propose a model (Fig. 5) in which under basal conditions, GLI1 and ER stress genes are repressed by deacetylation mediated by chromatin remodeler complexes that contain RuvBL2-HDAC1 with or without mSin3A.

Deacetylation of the promoter renders it inaccessible for the transcription machinery and therefore prevents the transcription of the target genes. The fact that mSin3A can interact with transcription factors to be recruited at specific chromatin site is well established (17). This suggest that under basal conditions these repressor complexes could be recruited at GLI1 or ER stress gene promoters by interacting with the corresponding transcription factors: USF2, ATF4, ATF6 or XBP1s.

Moreover, under basal conditions p97/VCP is mainly located in the cytosol, however like described by Wang et al. (18) upon ER stress it translocate to the nucleus, an observation that we also found

Materials and Methods

Antibodies, Cell lines and Reagent -Antibody against B-Actin and Tubulin were from Sigma (A2228, T5168), antibody against p97/VCP were from Proteintech (60316-1) and Progen (65278), antibody against RuvBL2 and ATF6 were from Abcam (ab137834, ab37149), antibody against HDAC1, HDAC2, HDAC3, HDAC4, HDAC5 and HDAC6 were from cell signaling (9928), antibody against USF2, mSin3A and ATF4 were from Santa Cruz (sc-862, sc-5299,sc-200), antibody against HSF1 and KDEL were from Enzo life science (ADI SPA 901 F, ADI SPA 827), antibody against ING2 and XBP1s were homemade. Tunicamycin (Tun) was from Calbiochem (EMD Biosciences, Inc., Darmstadt, Germany). Vismodegib (Vis) was from Selleckchem (GDC-0449). CB-5083 was a kind gift of Dr Holger Auner. All cell lines used in this study (HeLa, U87, U251, Panc1, HuH7) were cultured in DMEM from life technologies (41965) supplemented with 10% FBS from Sigma-Aldrich (12003C). All cell cultures were maintained in a 37°C incubator containing 5% CO2.

Real Time-Quantitative PCR -Total RNA was extracted using Trizol reagent (Invitrogen). For the Reverse Transcription 2µg of mRNA were used with the Maxima Reverse Transcriptase from Life technologies (ER0741). For quantitative RT-PCR (RT-qPCR) all reactions were conducted using the SYBR qPCR Premix Ex Taq from Ozyme (TAKRR420W) and the QuantStudio 5 thermocycler (Life technologies). For analysis each sample was normalized to Gapdh and/or Actin mRNA. All primers used are listed in the Table S1 Small interfering RNA (siRNA) -All siRNA used in this study are listed in the Table S2. siRNAs were transfected into the cells by using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocols. Cells were used for the experiments 48h post-transfection.

Co-immunoprecipitation assay

DISCUSSION p97/VCP is a molecular switch that activates transcription of ER stress genes during ER stress

It is well established that p97/VCP is an important player in the regulation of protein homeostasis and particularly during ER stress as it contributes to the degradation of terminally misfolded proteins from the ER in the ERAD pathway (23). However, our work has highlighted a novel role for the AAA+ ATPase p97/VCP during ER stress in the regulation of ER stress gene expression. We have demonstrated that p97/VCP/CDC-48 is required in C. elegans and Human for the induction of certain ER stress genes during ER stress. This induction is induced by the degradation of ubiquitylated RuvBL2 another AAA+ ATPase that acts as a repressor of ER stress genes under basal conditions. RuvBL2's repressor role relies on its interaction with a chromatin remodeler complex that contains HDAC1 and/or mSin3A. Our result suggest that these chromatin remodeler complexes might be recruited at their target sites by interacting with ER stress transcription factors (XBP1s, ATF6, ATF4) that are expressed at low levels in basal conditions. Interestingly, XBP1s interacts with RuvBL2-HDAC1-mSin3A whereas ATF6 and ATF4 interact with RuvBL2-HDAC1 only. In coherence, with Kim and colleagues findings this result suggests that RuvBL2 can interact with HDAC1 in different complexes (78). Overall these results have allowed us to propose a model (Figure 15).

Figure 15: Model of the regulation of ER stress genes mediated by p97/VCP-RuvBL2-HDAC1 -In basal conditions, RuvBL2 and chromatin remodeler complexes are recruited at ER stress genes mediated by their association with ER stress transcription factors. RuvBL2 and HDAC1 and/or mSin3A prevent acetylation of ER stress genes thereby the chromatin is in a condensed "OFF" state. Upon ER stress induction, p97/VCP act as a molecular switch to turn "ON" ER stress by removing ubiquitinylated RuvBL2. The removal of RuvBL2 causes the inactivation or dissociation of the repressor complex and allows the fixation of transcription factors and cofactors required for the activation of these ER stress genes.

Perspectives

Although our work has unraveled some of the molecular mechanisms that occur before and after the induction of ER stress such as the recruitment of USF2 at the Gli1 promoter, the change in acetylation at the Gli1 promoter, the stronger interaction between p97/VCP and RuvBL2 we are still failing to have a dynamic vision of the events that occur and in particular at the chromatin. For instance, how and when p97/VCP shuttles to the nucleus is still unclear, in yeast it is thought that phosphorylation of the penultimate tyrosine is responsible for the nuclear import of CDC-48 (44) whereas in human the first study reported that residues 612 to 614 and their modifications are crucial for p97/VCP nuclear translocation (62) and a second study has shown that the N domain (the first 200 AA) are sufficient to be translocated to the nucleus (45).

We are currently investigating the timeframe by which p97/VCP translocates to the nucleus and the relevance of its tyrosine phosphorylation in this process. Moreover, which cofactors are required if any for p97/VCP translocation to nucleus and which are involved in the recognition of ubiquitinylated RuvBL2 remain to be examined. However, it is noteworthy that most but not all p97/VCP chromatin associated processes seem to involve Ufd1-Npl4 (31). Another limit of our model is the fact that we do not know yet how p97/VCP extraction/degradation of RuvBL2 affects the other members of the complex. Is the removal of RuvBL2 causing the dissociation of the complex from the chromatin or if p97/VCP also mediates their extraction? In fact, cullin3 a cofactor of p97/VCP was shown to interact with HDAC1 (83) therefore there is a possibility that p97/VCP mediates extraction of both RuvBL2 and HDAC1 from the chromatin to inactivate the complex. Finally, as p97/VCP is mostly found overexpressed in cancers (67), it would be interesting overexpressing it in our cells lines and tested the impact on gene expression, histone marks, protein stability as this could help us understand the physiology of cancer cells.

APENDIX

Beyond my study of the role of p97/VCP in the regulation of ER stress genes, I have also participated to another project of our group: the characterization of the functional impact of IRE1mutations found in Glioblastoma tumours.

In order to characterize the impact of the different IRE1 mutants, we thought to test if these mutations were affecting p53 (Figure 17). Using western blot, we have compared the protein levels of p53 in U87 WT cells or U87 expressing an IRE1 mutants (e.g: S769F, Q780*, P336L, A414T). No change was noticed in the expression of p53 in all mutants except in P336L expressing cells where p53 was increased by 15 fold. (Figure 17A). We confirmed by PCR that this was not due to an increase of mRNA level (Figure 17B). We also confirmed that p53 was not mutated by sequencing (Data not shown). Overall these results suggest that p53 elevated expression in IRE1a P336L expressing cells is not due to a mutation or a transcriptional event but most likely to a post translational event.

Figure 17: Analyse of p53 expression and mutation in U87 expressing IRE1 mutants -A) Analysis of p53 protein level in U87 WT, U87 control (Empty vector -EV) or U87 expressing IRE1a mutants, CANX use as a loading control. B) Analysis of p53 mRNA expression in U87 control (EV) or in cells expressing the IRE1 P336L variant.

Antagonistic IRE1 RNase functions dictate glioblastoma tumor development

Stéphanie Lhomond 1,17 , Tony Avril 2,3, ¶ , Nicolas Dejeans 1,17, ¶ , Mari McMahon 2,3,4 , Raphaël Pineau 1,2,3 , Olga Papadodima 5 , Konstantinos Voutetakis 5,6 , Marianthi Logotheti 5,7 , Néstor Pallares-Lupon 1 , Kathleen Schmit 1 , Pierre-Jean Le Reste 2,8 , Amandine Etchevery 9 , Jean Mosser 9 , Kim Barroso 1,2,3 , Elodie Vauléon 2,3 , Marion Maurel 2,3,4 , Gwénaële Jégou 2,3 , Afshin Samali 4 , John B. Patterson 10 , Olivier Pluquet 11 , Claudio Hetz 12,13,14,15,16 , Véronique Quillien 2,3 , Arisotelis Chatziioannou 5,6 and Eric Chevet 1,2,3* ABSTRACT Abnormal proteostasis control is emerging as a major hallmark of cancer, driving tumor aggressiveness. Genetic and pharmacological evidence suggest that the endoplasmic reticulum (ER), a major site for protein folding and quality control, plays a critical role in cancer development. This has been applied to glioblastoma multiform (GBM), the most lethal primary brain cancer with an overall survival of 15 months and no effective treatment. The ER stress sensor IRE1alpha contributes to GBM progression, impacting tissue invasion and tumor vascularization. IRE1alpha is an RNase that signals by catalyzing the splicing of the mRNA encoding the transcription factor XBP1, in addition to regulate the stability of certain miRNAs and mRNAs through a process known as Regulated IRE1 Dependent Decay (RIDD). Somatic mutations in the IRE1alpha gene have been identified in GBM and other forms of cancer. Here we investigated the contribution of IRE1alpha signaling to GBM based on the systematic comparison of mutant forms identified in cancer, and demonstrated a relevant role to the disease. We also identified a novel mutation associated with GBM with functional consequences to tumor formation. Taking advantage of the specific signaling outputs of the RNase domain of IRE1alpha engaged by distinct GBM-related mutations, we defined specific signaling signatures that were confronted to human GBM transcriptomes. This approach allowed us to demonstrate the antagonistic roles of XBP1 mRNA splicing and RIDD on tumor outcomes. This study provides the first demonstration of a dual role of IRE1alpha downstream signaling in cancer and opens a new therapeutic window to impair tumor progression.