Isabelle Jourdain

Nathalie Spielewoy

James Thompson

Susheela Dhut

John R Yates

Takashi Toda
email: takashi.toda@cancer.org.uk

IDENTIFICATION OF A CONSERVED F-BOX PROTEIN 6 INTERACTOR ESSENTIAL FOR ENDOCYTOSIS AND CYTOKINESIS IN FISSION YEAST

Keywords: cytokinesis, endocytosis, F-box, fission yeast, HEAT repeats, MudPIT

The F-box domain is a degenerated motif consisting of ~40 amino acid residues that specifically bind Skp1, a core component of the SCF (Skp1-Cdc53/Cullin 1-F-box protein) ubiquitin ligase. Recent work, mainly performed in budding yeast, indicates that certain F-box proteins form non-SCF complexes together with Skp1 in the absence of cullins and play various roles in cell cycle and signalling pathways. However it is not established whether these non-SCF complexes are unique to budding yeast or common in other eukaryotes. Here using TAP purification coupled to MudPIT analysis (Multidimensional Protein Identification Technology), we have identified a novel conserved protein, Sip1 in fission yeast, as an interacting partner of an essential F-box protein Pof6. Sip1 is a large HEAT-repeats containing protein (217 kDa) and forms a complex with Pof6 and Skp1. This complex does not contain cullins, indicating that it is a novel non-SCF complex. Like Pof6 and Skp1, Sip1 is essential for cell viability and temperature sensitive sip1 mutants display cell division arrest as binucleate cells with septa. Sip1 localises to the nucleus and dynamic cytoplasmic dots, which turn out to be endocytic vesicles. Consistent with this, sip1 mutants are defective in endocytosis. Furthermore towards the end of cytokinesis, constriction of the actomyosin ring and dissociation of type II myosin and septum materials are substantially delayed in the absence of functional Sip1. These results indicate that the conserved Sip1 protein comprises a novel non-SCF F-box complex that plays an essential role in endocytosis, cytokinesis and cell division.

INTRODUCTION

Cytokinesis is a critical step in the cell life, as it couples the end of mitosis to the separation of the mother cell into two daughter cells. The main goal of cytokinesis is common to all organisms, and even if divergences exist, the fundamental sequences of events are universal [START_REF] Barr | Cytokinesis: placing and making the final cut[END_REF]. By the end of anaphase, the fission yeasts, like animals, positions an actomyosin ring (CAR, Contractile Actomyosin Ring) in the medial cortex region and constricts it to bring about partitioning of the cytoplasm [START_REF] Rajagopalan | Cytokinesis in fission yeast: a story of rings, rafts and walls[END_REF][START_REF] Guertin | Cytokinesis in eukaryotes[END_REF][START_REF] Goff | Controlling septation in fission yeast: finding the middle, and timing it right[END_REF][START_REF] Feierbach | Cytokinesis and the contractile ring in fission yeast[END_REF]. Concomitantly, the septum is deposited on the other side of the ring to separate the incipient daughter cells. Later a secondary septum is formed and the primary membrane is digested to allow the physical separation into two daughter cells. For these equatorial components including CAR, lipid rafts and septum to undergo assembly and dissolution in concert upon cytokinesis, membrane trafficking systems such as endocytosis, exocytosis and protein transport play vital roles. Accordingly not only actomyosin and glucans are essential in cytokinesis, but also a much larger set of conserved proteins are required for this process [START_REF] Rajagopalan | Cytokinesis in fission yeast: a story of rings, rafts and walls[END_REF][START_REF] Guertin | Cytokinesis in eukaryotes[END_REF][START_REF] Goff | Controlling septation in fission yeast: finding the middle, and timing it right[END_REF][START_REF] Feierbach | Cytokinesis and the contractile ring in fission yeast[END_REF]. For example, the anillin-like protein Mid1, in S. pombe and other organisms, defines the position of the CAR, and the protein phosphatase Clp1/Flp1, related to budding yeast Cdc14, coordinates cytokinesis with cell cycle progression. Being simple entities, fission yeast provides the significant advantage of combining genetic and biochemistry techniques; such features make them a powerful model by which to study cytokinesis. Consistently, a precise timing of the process has been reported [START_REF] Wu | Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis[END_REF] and a plethora of mutants with defects in cytokinesis and septation have been isolated [START_REF] Guertin | Cytokinesis in eukaryotes[END_REF].

The F-box proteins were discovered for their essential role in cell cycle progression [START_REF] Bai | SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box[END_REF]. The F-box proteins are defined by the presence of a degenerated stretch of 40 amino acids, the F-box domain representing Skp1-binding motif, normally in their amino-terminal portion, and contain a substrates-interacting domain in their carboxy-terminal region. They play the crucial role in recognising in a timely and specific manner a variety of substrates as an adaptor subunit of SCF (Skp1-Cdc53/Cullin 1-F-box protein) ubiquitin ligases [START_REF] Patton | Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis[END_REF]. Beside this well-recognised function, an increasing number of F-box proteins has been described in partnership with Skp1 as non-SCF complexes [START_REF] Hermand | F-box proteins: more than baits for the SCF?[END_REF]. For example, the budding yeast Rav2 F-box protein forms a complex with Skp1 and Rav1 and plays a role in vacuolar ATPase. It was recently shown that this complex appears to be conserved at least in fission yeast [10].

The S. pombe genome contains 18 genes encoding F-box proteins [START_REF] Hermand | F-box proteins: more than baits for the SCF?[END_REF][START_REF] Lehmann | Molecular interactions of fission yeast Skp1 and its role in the DNA damage checkpoint[END_REF]. Among the fission yeast F-box proteins, only two are essential, Pof1 and Pof6 [START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF][START_REF] Harrison | SCF Pof1 -ubiquitin and its target Zip1 transcription factor mediate cadmium response in fission yeast[END_REF]. While Pof1 follows the traditional pattern as a component of SCF Pof1 , a counterpart of the budding yeast SCF Met30 ubiquitin ligase [START_REF] Harrison | SCF Pof1 -ubiquitin and its target Zip1 transcription factor mediate cadmium response in fission yeast[END_REF], Pof6 seems to be less canonical. Pof6 was first identified as a Skp1 interactor and no binding to cullins was reported, bringing up the idea of a non-SCF complex [START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF]. In this study we isolated by TAP purification and MudPIT analysis a novel interactor of Pof6, named Sip1 (see below). We report characterisation of this novel type of the non-SCF complex that plays an essential role in endocytosis and cytokinesis.

EXPERIMENTAL Yeast strains and construction of sip1 temperature sensitive alleles

Yeast strains used in this study are described in Table I. Cells were grown in standard culture media, and standard yeast genetic methods were used. DNA fragments containing error-prone PCR amplified sip1 + -HA sequences with the G418 resistance A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. kan r gene were transformed into a wild type strain and 6 temperature sensitive (ts) alleles were isolated (see Supplementary methods for details).

TAP purifications

The TAP purifications were mainly prepared as described previously [START_REF] De Bruin | Cln3 activates G1-specific transcription via phosphorylation of the SBF bound repressor Whi5[END_REF]. For Pof6-TAP purification, 40 L of cells were grown in YE5S at 30°C and disrupted in liquid nitrogen. Proteins were solubilised in the lysis buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl, 5 mM EDTA, 10 % glycerol, 0.2 % NP-40, 1 mM NaF, 10 mM PMSF, adjusted to pH 8.0) and loaded for a two-step TAP purification. The eluted proteins were precipitated with TCA and subjected to MudPIT analysis (Multidimensional Protein Identification Technology, see Supplementary methods for experimental details). For Sip1-TAP purification, a total of 10 L of cells were cultured in YE5S at 30°C and disrupted in liquid nitrogen. The proteins were eluted in a single-step TAP purification and precipitated in TCA for further MudPIT analysis.

Coimmunoprecipitation experiments

Protein extracts were prepared in lysis buffer by breaking cells at 4°C with glass beads (4 times for 40 s) in a FastPrep FP120 apparatus (Savant. Co, MN, USA). The protein extracts were collected after 15 min of centrifugation at 10,000 x g at 4°C. The coimmunoprecipitations were performed as described previously [START_REF] Spielewoy | Regulation and recognition of SCF Grr1 targets in the glucose and amino acid signaling pathways[END_REF].

Synchronisation by centrifugal elutriation and FACS analysis

3 L of YE5S media were inoculated with a preculture overnight. Elutriation was performed with Avanti J-20 XP (Beckman Coulter, MN, USA) by the standard procedures. Concentrations and sizes of cells were measured with a Cell Counter (Sysmex KX-21N, Kobe, Japan). Septa were stained with calcofluor and the percentage of septated cells were calculated by counting over a total of 100 cells. The cell survivals were estimated by spreading a calculated number of cells on YE5S plates and incubating them at 30°C for 3-4 days. Standard methods for FACS analysis were followed using a FACScan machine (Becton Dickinson, CA, USA).

Fluorescence Microscopy

For all microscopy experiments, cells were grown and observed in minimum medium at 27 0 C, unless otherwise stated. Signals of GFP-Sip1 (expressed under the control of the thiamine-repressible nmt1 promoter) are essentially the same between in the presence (repressed) and absence (derepressed) of thiamine, though those in the absence of thiamine are stronger. For time-lapse analysis, therefore, derepressed conditions (-thiamine) were used as stronger signals were required for recording. Live cell imaging was performed in an imaging chamber (CoverWell, 20 mm diameter, 0.5 mm deep) (Molecular Probes) filled with 800 µl of 2% agarose in minimum medium and sealed with a 22x22 mm glass coverslip. FM4-64 (Molecular Probes Inc.) was dissolved in DMSO at a concentration of 1.64 mM and added to the cells at a final concentration of 1.89 µM. Cells were then incubated in the dark before being applied to the imaging chamber. Cells were imaged using an Olympus IX71 microscope with a 63X oil immersion lens, NA=1.4. Images were captured with a Coolsnap HQ digital CCD camera (Roper Scientific). Counts and measurements were made using Metamorph (Molecular Devices Corporation) and downloaded to Microsoft Excel for analysis. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

RESULTS

Identification of a novel interactor of the Pof6 F-box protein

To identify Pof6 interactors, we have performed a non-stringent Pof6-TAP purification in two steps from a large-scale culture (40 L) and coupled it to MudPIT analysis. A protein migrating at the size of Pof6 is specifically detected in the silver stain of Pof6-TAP elution (Figure 1A). Indeed the MudPIT data reveals a high coverage of Pof6 (19 unique peptides covered 56.2% the sequence) considering the size of the protein, 99.9 kDa (Supple. Figure S1A). As predicted, Skp1 appears in the elution with 58.4% coverage and a total of 30 peptides. The specificity of the interaction between Pof6 and Skp1 was confirmed by coimmunoprecipitations with both proteins (Figure 1B) as reported previously [START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF].

An uncharacterised ORF (SPBC27B12.08) was identified as one of the most abundant proteins pulled down in Pof6-TAP purification. Although the corresponding protein has the large size of 217 kDa, the peptide coverage reaches up to 60.6% of the sequence and a total of 792 peptides were isolated (Supple. Figure S1A). The specificity of the interaction between Pof6 and this novel protein was confirmed by coimmunoprecipitation (Figure 1C). We, consequently, named the protein according to that specific feature, Sip1 (pofSix interactor protein 1).

In a reciprocal experiment, we performed a single step Sip1-TAP purification from a 10 L culture to identify its binding partners (Figure 1D). One of the most abundant proteins identified by MudPIT analysis is Skp1 (Supple. Figure S1B). The peptides cover 66.5% of Skp1 sequence and a total of 35 peptides are present. The Fbox protein Pof6 is also detected as an interactor of Sip1 albeit with a much restricted coverage (6.1%). The difference between Pof6 coverage in Sip1-TAP elution and Sip1 coverage in Pof6-TAP elution may be explained by the amount of cells used for the purifications, the differences in the TAP purification protocols and the abundance/instability of each protein. To confirm the interactions observed by Sip1-TAP MudPIT analysis, Sip1-HA was immunoprecipitated. As shown in Figure 1E, Pof6 and Skp1 were specifically pulled down with Sip1-HA.

We then attempted to examine whether Pof6 and Sip1 could interact with each other in the absence of Skp1. For this purpose, an Skp1 shut-off strain was used. In this strain, the thiamine-repressible nmt1 promoter was inserted in front of the initiation codon of the genomic skp1 + gene together with HA epitope, by which the expression of HA-Skp1 could be shut off by adding thiamine to the medium [16]. Rather unexpectedly we found that when Skp1 levels were downregulated, both Sip1 and Pof6 became unstable, and accordingly we could barely detect either protein by immunoblotting (Figure 1F). Thus despite being not conclusive, this result indicates that Pof6 and Sip1 requires Skp1 for their stability, supporting the notion that these three proteins form a ternary complex in the cell.

It is of note that none of the two MudPIT datasets contains peptides for cullins (Supple. Figure S1) confirming the absence of interaction between Pof6 and the cullins as previously reported [START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF]. We made a careful visual inspection of the sequence corresponding to the Pof6 F-box domain and aligned it with different F-box domains present in SCF complexes [START_REF] Schulman | Insights into SCF ubiquitin ligases from the structure of the Skp1-Skp2 complex[END_REF][START_REF] Zheng | Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex[END_REF] (Supple. Figure 2A). The alignment shows that several residues important for the Skp1/F-box domain interface are present in Pof6 but the two residues involved in the Cul1/F-box domain core interface are not conserved; a highly conserved proline residue is replaced by threonine in Pof6 and an acidic residue is exchanged with asparagine. These residues have been shown to be specifically responsible for the interaction between Skp2 F-box protein and Cul1 [START_REF] Zheng | Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex[END_REF], substantiating our and previous results [START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF] showing that Pof6 does not interact with cullins.

The analysis of the Pof6 protein also reveals the presence of unusual domains in its carboxy-terminal region: two Sec10 domains and a CAAX sequence are present in the substrate-binding portion (Supple. Figure 2B). Such features suggest that there may be potential functional similarities between Pof6 and Rcy1 from budding yeast. Like Pof6, Rcy1 interacts with Skp1 and contains two Sec10 domains and a CAAX sequence in its carboxy-terminal region. In addition, it has been suggested that Rcy1 is part of a non-SCF complex [START_REF] Galan | Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast[END_REF]. These results collectively suggest that Pof6 does not belong to a conventional SCF complex but instead forms a novel complex with Sip1 and Skp1.

The sip1 + gene is essential in S. pombe and conserved amongst eukaryotes

To investigate Sip1 function, we asked whether the sip1 + gene is essential in S. pombe. We deleted by PCR one copy of the genes with a G418-resistant cassette in a diploid strain. This sip1::kan r /sip1 + strain was left to sporulate and the resulting tetrads were dissected to examine the viability of each haploid progeny. Only two out of four haploids grew into colonies after germination, and these viable colonies were sensitive to G418 (Figure 2A, left panel). We concluded that sip1 + , like pof6 + and skp1 + , is essential for cell viability. A close observation of the non-viable spores showed that germinations occur but not cell division (Figure 2A, right panel).

Homology search reveals that Sip1 contains several HEAT repeats (closed boxes in Figure 2B), which are implicated in protein-protein interactions and often found in subunits of multi-protein complexes [START_REF] Neuwald | HEAT repeats associated with condensins, cohesins, and other complexes involved in chromosome-related functions[END_REF]. These HEAT repeats are distributed widely along the whole Sip1 protein (Figure 2B). Interestingly, Sip1 protein appears to be conserved throughout the evolution from yeasts to human beings (Figure 2C). Homologies are observed in not only HEAT repeats but also other regions as well.

Sip1 plays an essential role in cytokinesis

To examine the function of Sip1, we produced ts alleles of sip1 + by mutagenic PCR (sip1-62, see Supplementary methods for experimental details). Centrifugal elutriation was performed to synchronise cell cultures in early G2. Wild type and the sip1-62 ts cells were grown at 25˚C, elutriated and then shifted to 36˚C. sip1-62 ts cells ceased division almost immediately and their viability decreased concomitantly with the increase of septated cells upon mitotic exit (40-60 min, Figure 3A). In addition unlike wild type cells, the percentage of septated cells did not decline afterwards once it reached a plateau. Consistent with this, the population of the binucleate cells closely followed the profile of the septated cells. These observations suggest that sip1 ts cells proceed through the G2 phase and enter mitosis to lose viability during cytokinesis. The DNA content of the synchronised cells showed that sip1-62 ts cells arrest with a 4C DNA content, indicating a completion of DNA replication in each separated nucleus (Figure 3B).

We further investigated the role of Sip1 in cell division by following the fate of the CAR and septum in sip1-62. The CAR was visualised using the type II myosin heavy chain protein, Myo2, tagged with GFP [START_REF] Wu | Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis[END_REF][START_REF] Mulvihill | Role of the two type II myosins, Myo2 and Myp2, in cytokinetic actomyosin ring formation and function in fission yeast[END_REF]. Cells were grown at the permissive temperature, shifted up to the restrictive temperature for 1 hr, stained with calcofluor and observed at 36 0 C over-time (Figure 4 and Supp Movies S1 and S2). In the sip1 mutant, the initiation timing of CAR and septum formation appeared very similar to that of wild type cells (Figure 4A andB). However, the CAR took nearly twice as much time to constrict. Although in the sip1 mutant, much variability was observed from one cell to the other in the timing of Myo2-GFP detachment, it was delayed ascribable to a prolonged CAR constriction process at the medial region (Figure 4B). In addition, septum materials remained at the equator. We could not even observe the execution of this process within the timeframe of experiments. Therefore Sip1, a newly identified conserved protein, plays an essential role in cytokinesis, in which it is required for CAR constriction and septum dissolution.

Sip1 localises to the nucleus and endocytic vesicles

To investigate the localisation of Sip1, we constructed a strain in which Sip1 was chromosomally tagged with GFP at its N-terminus and expressed under the nmt1 promoter. GFP-Sip1 fluorescence was detected in the nucleus as well as in cytoplasmic punctuate structures of various sizes and shapes (Figure 5). Time-lapse imaging showed that these dots were capable of fusing and fragmenting and were also highly motile (Figure 5A and Supple. Movie S3). Tracking of individual patches over time revealed two types of motile behaviour: a local, possibly, Brownian movement, and a long-range linear displacement (Figure 5A andB). Both movements were observed independently of the stage of the cell cycle (not shown). It is clear however that the GFP-Sip1 dots used the long-range movement to accumulate during nuclear separation at the position of the future division plane (Figure 5A-C). Threedimensional reconstructions of wide-field microscopy images showed that the GFP-Sip1 fluorescence lined up on both sides of the primary, calcofluor-stained septum. They did not comprise rings or plates, but instead remained as the form of dots (Supple. Movie S4).

To evaluate the possibility that the Sip1 patches represent Golgi vesicles, we constructed a strain containing GFP-Sip1 and mCherry-tagged Anp1 or Sec72, a cis-Golgi or trans-Golgi marker protein, respectively (kindly provided by A. Vjestica and S. Olifelenko) [START_REF] Vjestica | The actomyosin ring recruits early secretory compartments to the division site in fission yeast[END_REF]. GFP-Sip1 did not colocalise with Anp1-mCherry, and only very partially did so with Sec72-mCherry (Supple. Figure S3), indicating that Sip1 dots do not represent Golgi organelles. We next set out to examine whether GFP-Sip1 patches were stained with the amphiphilic dye FM4-64 [START_REF] Vida | A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast[END_REF]24]. In fission yeast, a 5-30 min incubation of the cells with this dye allows the visualization of endocytic vesicles that are being transported from the plasma membrane to the vacuolar membrane [START_REF] Gachet | Endocytosis in fission yeast is spatially associated with the actin cytoskeleton during polarised cell growth and cytokinesis[END_REF]. Under these conditions, we found that FM4-64 clearly stained GFP-Sip1-containing structures (Figure 6A). To test if Sip1 has any role in endocytosis, we incubated wild type and sip1-62 cells with FM4-64 for up to 90 min. Contrary to the situation in wild type cells where the dye was delivered to vacuoles by endocytosis, in the sip1-62 mutant, FM4-64 remained localising to the tips in growing cells, or to the equator in dividing cells, that is the sites where endocytosis should have occurred (Figure 6B). It is worth pointing out that the endocytosis defect was observed even at the permissive temperature, but was most strikingly obvious at the restrictive temperature. Taking all together, we conclude that Sip1 is a novel component of endocytic vesicles and essential for endocytosis and the completion of cytokinesis.

DISCUSSION

In this report, we identify a novel protein, Sip1, as a component of the Pof6/Skp1 complex in S. pombe. Immunoprecipitation experiments conform physical interactions between Sip1 and Pof6-Skp1. These three proteins appear to form a functional ternary complex, as in addition to coimmunoprecipitation data, we have found that protein stability of Sip1 and Pof6 is greatly compromised in the absence of Skp1, the phenomenon often observed in multi-subunit complexes. Given that a canonical SCF is a ubiquitin ligase in which stability of F-box proteins is often dependent upon structural integrity of a holo-SCF complex [START_REF] Hermand | F-box proteins: more than baits for the SCF?[END_REF], this instability might imply that the Skp1-Pof6-Sip1 complex is involved in ubiquitin-mediated reactions. Sip1 is essential and conserved amongst eukaryotes. This protein consists of a number of HEAT repeats, protein-protein interaction motifs, implying that Sip1 plays a scaffolding role in this ternary complex. As none of cullins have been identified in TAP-purification (this study and [START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF]), it is likely that Skp1, Pof6 and Sip1 constitute an essential non-SCF F-box complex.

Creation and analysis of a sip1-62 ts strain have unravelled an essential role for Sip1 in cytokinesis and endocytosis. It is worth mentioning that a ts skp1-3 allele and pof6-deleted haploid cells germinating from heterozygous diploids exhibited cytokinetic defects [START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF], reminiscent of the terminal phenotype of sip1-62 cells reported in this study. Sip1 localises to the nucleus and endocytic vesicles. Consistent with an interaction, Pof6 localises to the nucleus [START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF]. However, their cellular localisation is not completely parallel. In addition to the nucleus, Pof6 localises to cell tips during interphase and the CAR upon cytokinesis [START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF][START_REF] Matsuyama | ORFeome cloning and global analysis of protein localization in the fission yeast Schizosaccharomyces pombe[END_REF], not to endocytic vesicles. It should, however, be noted that GFP-Sip1 vesicles appear to constantly gather around the CAR during CAR formation, which might reflect a physical interaction between these two proteins.

One of the most important results presented in this study is the identification of Sip1 as an essential factor for endocytosis. Cytokinesis is a complex process, in which a number of intra-and extra-cellular signalling pathways are involved and converged [START_REF] Pollard | Progress towards understanding the mechanism of cytokinesis in fission yeast[END_REF] including membrane trafficking [START_REF] Albertson | Membrane traffic: a driving force in cytokinesis[END_REF]. Despite its importance, compared to our understanding of CAR formation and function, knowledge with regards to roles and mechanics of endocytosis in cytokinesis is still limited. Our study uncovers a novel component for endocytic vesicles, which will provide a tool by which to pursue the molecular pathways underlying endocytosis-mediated cytokinesis and cell separation.

Analysis of orthologues for Pof6 and Sip1 has been reported only in budding yeast. Like Pof6, F-box protein Rcy1 binds Skp1 and interestingly forms a non-SCF complex. Rcy1 participates in protein recycling from the endosomes to the Golgi and the plasma membrane [START_REF] Galan | Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast[END_REF]29]. However, unlike Pof6, Rcy1 is non-essential and these two proteins may not perform identical molecular functions [START_REF] Hermand | F-box proteins: more than baits for the SCF?[END_REF][START_REF] Hermand | Skp1 and the F-box protein Pof6 are essential for cell separation in fission yeast[END_REF]. So far we are not able to detect Pof6 orthologues by homology search in higher eukaryotes. The identification of functional counterparts of Pof6 in higher eukaryotes would be crucial to delineate their conserved roles. In the case of Sip1, its budding yeast orthologue is called Laa1. Like Rcy1 and unlike Sip1, Laa1 is non-essential [START_REF] Fernandez | Laa1p, a conserved AP-1 accessory protein important for AP-1 localization in yeast[END_REF]. Although no physical interaction between Rcy1 and Laa1 is known, similar to Rcy1, Laa1 is reportedly involved in clathrin-mediated transport between trans-Golgi network and endosomes, which displays some analogy to Sip1 functions.

In conclusion, we describe the first non-SCF complex in S. pombe. We show that the Skp1-Pof6-Sip1 complex plays an essential role in cell division. Further analyses point towards a precise role for this complex in endocytosis and cytokinesis. It would be now vital to explore a role of Sip1 in membrane trafficking and identify functional pathways in which the Sip1-Pof6-Skp1 complex is involved. Investigation of roles for Sip1 orthologues in higher eukaryotes would be the next phase of our research. Finally, besides Pof6, there are a number of F-box proteins in both fission yeast and other organisms, in which two critical proline and acidic residues are not A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. conserved [START_REF] Bai | SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box[END_REF]. We envision that non-SCF F-box complexes are more general than currently recognised and the elucidation of the biological functions of these novel complexes is a key challenge in the future. (B) DNA content analysis of wild type and sip1-62. Each strain was grown and treated as described in (A) and samples were taken every 20 min interval, followed by FACS analysis. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 25 Feb 2009 as manuscript BJ20081659 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081659

Biochemical

  Journal Immediate Publication. Published on 25 Feb 2009 as manuscript BJ20081659 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081659

  Biochemical Journal Immediate Publication. Published on 25 Feb 2009 as manuscript BJ20081659 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081659A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 25 Feb 2009 as manuscript BJ20081659 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081659A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 25 Feb 2009 as manuscript BJ20081659 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081659

Figure 1 :

 1 Figure 1: Identification of a novel interactor of Pof6 (A) TAP purifications of a non-tagged control strain (Table 1) and Pof6-TAP. Fractions corresponding to 1/100 of each elution (E) were loaded in parallel to 100 µg of total extract (TE) and silver-stained. Two bands migrating around 220 kDa and 100 kDa (arrows) may correspond to Sip1 and Pof6 respectively. (B) Coimmunoprecipitation of Pof6-TAP and Skp1. Proteins were immunoprecipitated (IP) with anti-TAP, anti-Skp1 or no antibodies (Ab). 100 µg of whole cell extract (WCE) were loaded and 2 mg of proteins were used for each IP. Pof6-TAP was immunodetected with anti-Pof6 antibodies and Skp1 with anti-Skp1 antibodies. (C) Coimmunoprecipitation of Pof6/Pof6-TAP and Sip1-HA. Extracts were immunoprecipitated with IgG Sepharose beads or anti-HA antibodies. Immunoprecipitates were blotted with anti-HA and anti-Pof6 antibodies. The asterisk indicates a non-specific band of the IgG. (D) Single step TAP purification of Sip1-TAP. 1/100 of the final elution (E) was loaded on the gel in parallel to 100 µg of total extract (TE) and silver-stained. A band around 75 kDa probably represents mixtures of heat shock 70 chaperone family (the coverage percentages of Ssa2, Ssc1 and Ssa1 are 80.2%, 68.8 % and 51.7 % respectively). (E) Coimmunoprecipitation of Sip1-HA, Pof6 and Skp1. Sip1-HA was immunoprecipitated with anti-HA antibodies and immuno-complex was blotted with individual antibodies. (F) Instability of Sip1 and Pof6 in the absence of Skp1. Whole cell extracts were prepared from a strain containing nmt1-HA-Skp1, Sip1-HA and Pof6-TAP in the absence (left, derepressed, + for Skp1) or presence (right, repressed, -for Skp1) of thiamine. Skp1 and Sip1 were immunodetected with anti-HA antibody, whilst Pof6-TAP was detected with anti-Pof6 antibody.

Figure 2 :

 2 Figure 2: sip1 + is an essential gene present amongst eukaryotes (A) Tetrad dissection of asci produced by a sip1::kan r /sip1 + diploid strain. Spores were grown for 3 days at 30ºC on rich media (left). Phase contrast micrographs showing the terminal morphology of germinating sip1-deleted spores (right). Bar indicates 5 µm. (B) Schematics of Sip1 protein domains. Sip1 protein is 1920 amino acids long and contains HEAT repeats (black). (C) Dendogram of Sip1 orthologues identified by YOGY search. Proteins are labelled with the UniProtKB nomenclature. The ClustalW alignment default parameters of MacVector 9.0 were used to create the tree. The evolutionary distance is indicated on each branch. S.p: Schizosaccharomyces pombe; D.m: Drosophila melanogaster; H.s: Homo sapiens; C.e: Caenorhabditis elegans; S.c: Saccharomyces cerevisiae.

Figure 3 :

 3 Figure 3: Characterisation of the sip1-62 mutant (A) Centrifugal elitriation. Wild type (top) or sip1-62 cells (bottom) were synchronised by elutriation at 25°C, shifted to 36°C (T0) and grown for 4 h. Samples were taken every 20 min to measure cell viability, septation index and cell numbers.(B) DNA content analysis of wild type and sip1-62. Each strain was grown and treated as described in (A) and samples were taken every 20 min interval, followed by FACS analysis.

Figure 4 :

 4 Figure 4: Cytokinesis is not completed in the sip1-62 mutant

(

  A) Time-lapse imaging of Myo2-GFP. sip1 + and sip1-62 cells containing Myo2-GFP (green) were stained with calcofluor (blue) in liquid cultures. These cells were shifted from 25 0 C to 36 0 C for 1 h and live imaging started. Pictures were acquired at 2 min intervals for up to 2 h. Every second picture of the cell medial plane is shown. Asterisks show the time when the septum appeared. Bar, 3µm. See also Supple. Movies S1 and S2. (B) Graphics showing the timing of cytokinetic events as they occur in sip1 + (blue) and sip1-62 (orange) cells. Cells were prepared as in (A). For each strain, results represent the mean of two independent experiments and error bars are SD of the average. A total of 39 cells for the wild type, and 31 cells for sip1-62 were counted. The time of septum dissolution in sip1-62 could not be accurately determined under our experimental conditions and is shown as an open diamond.septum; CAR, Cytokinetic Actomyosin Ring.

Figure 5 :

 5 Figure 5: Localisation tracking analysis of GFP-Sip1(A) Time-lapse imaging of cells expressing GFP-Sip1. The GFP fluorescence is detected in the nucleus and in punctuated structures, which accumulate at the medial plate of the cell. The time is indicated in min:sec and matches the time used in Supple. Movie S3. Red arrowheads, a patch being rapidly displaced towards the division plane. Note that this patch resulted from the fusion of at least 3 patches (e.g: compare times 12:30 and 14:00) (B) Trajectories of individual GFP-Sip1 patches movement. Each color represents a patch and each pattern represents the path followed by the patch over time. Open symbols (circles) indicate patches starting points. The path of the dot shown in (A) with the red arrowhead is shown in red in (B). Only patches that could unambiguously be followed for at least 4 min are shown. (C) Kymographs of a GFP-Sip1 cell stained with calcofluor. GFP-Sip1 gathers at the site of the division plane when the nuclei separate. As soon as the septum forms (blue arrowhead), patches line lateral to it. See also Supp Movie S4. N, Nucleus. Bar, 3µm.

Figure 6 .

 6 Figure 6. Sip1 localises to endocytic vesicles and is required for endocytosis (A) Localisation of GFP-Sip1 to endocytic vesicles. nmt1-GFP-Sip1 cells were stained for 5 min with FM4-64 and observed immediately before the dye was transported to the vacuoles. The enlargement (right panel) shows that both signals perfectly overlap. (B) Endocytosis defects in the sip1 mutant. Wild type (sip1+) and sip1-62 cells were stained for an extended time with FM4-64. Whilst in wild type, the dye localises to peripheries of individual vacuoles, in the mutant, this dye is not incorporated inside the cell and instead accumulates at the tips (plain arrowhead) and septum (open arrowhead). Bars, 5µm. Enlargement bar, 1 µm.

  IJ450 h -or + ade -leu1 kan r ::nmt1-GFP-sip1 + This study sec72 + -linker-mCherry::ura4 + Biochemical Journal Immediate Publication. Published on 25 Feb 2009 as manuscript BJ20081659 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081659 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Figure 1 :SFigure 2 :Figure 5 :Figure 6 :

 1256 Figure 1: Jourdain et al.

  sip1-62 sip1+ Biochemical Journal Immediate Publication. Published on 25 Feb 2009 as manuscript BJ20081659 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081659 A c c e p t e d M a n u s c r i p t

Table I : Strain list

 I

	Strain name Genotypes	Derivations
	513	h -leu1 ura4	Our stock
	972	h -	Our stock
	NSY001	h -pof6 + -2TAP-kan r	This study
	NSY065	h -leu1 ura4 sip1 + -3HA-kan r	This study
	NSY069	h -or + pof6 + -2TAP-hph r sip1 + -3HA-kan r	This study
	NSY110	h -/h + leu1/leu1 ura4/ura4 his7/his7 ade6-210/ade6-216	This study
		sip1::kan r /+
	NSY127	h -leu1 ura4 sip1 + -2TAP-kan r	This study
	NSY165	h -leu1 ura4 sip1-62-HA-kan r	This study
	NSY303	h-leu1 ura4 pof6 + -2TAP-hph sip1 + -3HA-kan r	This study
		hph r -nmt-HA-skp1 +
	NSY178	h -leu1 ura4 myo2

+ -GFP-kan r Our stock NSY191

h -or + sip1-62-HA-kan r myo2 + -GFP-kan r This study IJ449 h -or + ade -leu1 kan r ::nmt1-GFP-sip1 + This study anp1 + -linker-mCherry::ura4

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

We thank Dan Mulvihill for a Myo2-GFP strain, Aleksandar Vjestica and Snezhana Oliferenko for Golgi marker strains and Kathy Gould for TAP-tagging plasmids. We are grateful to Damien Hermand for exchanging unpublished information. We thank the Fermentation Unit of Cancer Research UK for preparations of large-scale fission yeast cultures. The work was supported by Cancer Research UK (T. T.) and National Institutes of Health grant P41RR11823. (J.R.Y.)

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.


Elizabeth Iorns

Christopher J Lord

Alan Ashworth
email: alana@icr.ac.uk

Parallel RNAi and compound screens identify the PDK1 pathway as a target for tamoxifen sensitisation

Keywords: Tamoxifen, RNA interference screen, chemical screen, PDK1, triciribine, tetrandrine

Tamoxifen is the most commonly used drug to treat breast cancer and acts by blocking estrogen receptor (ERα) signalling. Although highly effective, its usefulness is limited by the development of resistance. Given this, strategies that limit resistance by sensitising cells to tamoxifen may show utility in the clinic. To gain insight into how this might be achieved, we used chemical and genetic screens to identify targets and small molecule inhibitors that cause tamoxifen sensitisation. A high-throughput genetic screen, using an RNA interference library targeting 779 kinases and related proteins, identified the PDK1 signalling pathway as a strong determinant of sensitivity to multiple ERα antagonists including tamoxifen. A chemical screen using existing drugs and known kinase inhibitors also identified inhibitors of the PDK1 pathway, including triciribine and tetrandrine. Aside from identifying novel agents and targets for tamoxifen sensitisation, this approach also provides evidence that performing chemical and genetic screens in parallel may be useful.

Introduction

Approximately 70% of breast tumours express estrogen receptor α (ERα), and of these, most are fully dependent on estrogen signalling for their growth and can therefore be treated with anti-estrogen therapies. Tamoxifen, the most commonly used anti-estrogen therapy, has shown significant benefit in the treatment of ERα positive breast cancer [START_REF] Ebctcg | Tamoxifen for early breast cancer: an overview of the randomised trials. Early Breast Cancer Trialists' Collaborative Group[END_REF] and works by blocking ERα signalling in the breast. This ERα antagonism leads to the inhibition of cell cycle progression and arrest at the G 1 checkpoint [START_REF] Cariou | Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells[END_REF][START_REF] Musgrove | Cyclin D1 induction in breast cancer cells shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle[END_REF]. Despite its effectiveness, the utility of tamoxifen is limited by the development of drug resistance, which occurs in all patients with metastatic disease and approximately 40% of those with early onset breast cancer who are treated with this agent as an adjuvant to surgery [START_REF] Ring | Mechanisms of tamoxifen resistance[END_REF][START_REF] Jordan | Studies on the estrogen receptor in breast cancer"--20 years as a target for the treatment and prevention of cancer[END_REF].

Considerable efforts have been made to identify the determinants of tamoxifen resistance and sensitivity. Primarily, this has been performed on a gene-by-gene basis and while this has been informative, a complementary approach is to perform high throughput simultaneous analysis of many genes and pathways. This can be achieved genetically by the use of approaches such as RNA interference (RNAi) screening [START_REF] Iorns | Utilizing RNA interference to enhance cancer drug discovery[END_REF], which relies on the ability of short-interfering (si)RNA to silence gene function in a sequence-specific fashion. We have used such a high-throughput RNAi screen to identify CDK10 as a novel determinant of tamoxifen resistance [START_REF] Iorns | Identification of CDK10 as an Important Determinant of Resistance to Endocrine Therapy for Breast Cancer[END_REF]. In addition to genetic screens, a separate approach is to use libraries of chemical inhibitors to interrogate cellular phenotypes such as tamoxifen response. Given that chemical inhibition of proteins may sometimes produce differing phenotypes to those achieved by gene silencing or knockout [START_REF] Knight | Chemical genetics: where genetics and pharmacology meet[END_REF], the use of chemical screening has the potential to identify effects not achievable by genetic screening. Moreover, if the chemical libraries used include drugs in clinical use and with known pharmacokinetic and safety profiles, these screens offer the possibility of identifying effects that can be rapidly evaluated in phase II clinical trials, eliminating much of the toxicological and pharmacokinetic assessments required for novel compounds [START_REF] Dimasi | The price of innovation: new estimates of drug development costs[END_REF]. Additionally, the targets of existing drugs are often established, potentially allowing the mechanism of action to be determined more easily.

Given the relative benefits of genetic and chemical screens, we reasoned that parallel RNAi and inhibitor screens might be a useful approach to the identification of targets and inhibitors that cause sensitisation to tamoxifen. This proved to be the case and a number of novel targets were identified. Notably, multiple components of the PDK1 pathway were identified by the RNAi screen. Results from the chemical screen validated the results of the RNAi screen, therefore providing proof of the principle that genetic and chemical screens may indeed have complementary roles in target discovery.

Experimental

Cell lines, compounds and siRNA MCF7 cells were obtained from ATCC (USA) and maintained in phenol red free RPMI 1640 (Invitrogen), supplemented with 10% dextran charcoal treated FCS (10% (v/v)), 1nM estradiol, insulin, glutamine and antibiotics. 4OH tamoxifen and estradiol were obtained from Sigma, ICI 182780 was obtained from Tocris Bioscience UK, triciribine was obtained from BIOMOL International, Akti-1,2 was obtained from Calbiochem. MCF7 cells were transfected with SMARTpool siRNAs using Dharmafect 3 transfection reagent according to manufacturer's instructions (Dharmacon). The kinase siRNA library (siARRAY -targeting 779 known and putative human kinase genes including protein kinases, lipid kinases, adaptors and related genes) was obtained in ten 96 well plates from Dharmacon (USA). Each well in this library contained a SMARTpool of four distinct siRNA species targeting different sequences of the target transcript. The compound library encompassed the Prestwick Chemical Library® (Prestwick Chemicals) and the Screen-Well TM library (BIOMOL International).

Antibodies

Antibodies targeting the following epitopes were used: p21 CIP1 (556430, BD Biosciences, UK), phospho AKT Ser473 (4058, Cell Signalling, UK), AKT (9272, Cell Signalling, UK), and β-tubulin (T4026, Sigma, UK). All secondary antibodies used for western blot analysis were HRP conjugated.

siRNA screen method The RNAi screening methodology has been described previously [START_REF] Iorns | Identification of CDK10 as an Important Determinant of Resistance to Endocrine Therapy for Breast Cancer[END_REF]. In brief, MCF7 cells plated in 96 well plates were transfected 24 hours later with siRNA (final concentration 100 nM), using Dharmafect 3 (Dharmacon, USA) as per manufacturer's instructions. Twenty-four hours following transfection, cells were trypsinised and divided into four identical replica plates. At 48 hours following transfection, two replica plates were treated with 50 nM 4OH tamoxifen in media and two replica plates with 0.05% (v/v) ethanol vehicle in media. Media containing 4OH tamoxifen or vehicle was replenished after 48 and 96 hours, and cell viability was assessed after seven days of 4OH tamoxifen exposure using the CellTiter-Glo Luminescent Cell Viability Assay (Promega, USA) as per manufacturer's instructions. The luminescence reading for each well on a plate was expressed relative to the median luminescence value of all wells on the plate. The screen was completed in duplicate.

For each transfection the effect on cell growth and tamoxifen sensitivity were calculated. Cell growth: The effect of each individual siRNA SMARTpool on cell growth alone was calculated by dividing mean luminescence in the two replica wells treated with ethanol vehicle by mean luminescence of the replica wells transfected with siControl, and expressed as a percentage. Cell growth effect of siRNA (%) = [mean luminescence (2 replica wells with siRNA)/ mean luminescence (20 replica well treated siControl)] x 100. Tamoxifen sensitivity: Sensitivity to 4OH tamoxifen for each siRNA SMARTpool was assessed by calculating the surviving fraction following 4OH tamoxifen.

Surviving fraction = log 2 mean luminescence (2 replica wells with 4OH tamoxifen) -log 2 mean luminescence (2 replica wells with ethanol vehicle).

The surviving fractions were centred on the median surviving fraction of all 80 SMARTpools from one 96 well plate transfection, the results from all ten siRNA plates combined and results expressed as a Z score [START_REF] Abdi | Z-Scores[END_REF]. For the Z score the standard deviation of the screen was estimated from the standard deviation of all siControl wells. Validation of RNAi gene silencing was measured by real time quantitative RT-PCR (qPCR). MCF7 cells were transfected with siRNA, and RNA extracted 48 hours later with Trizol and phenol/chloroform extraction followed by isopropanol precipitation. cDNA was synthesized using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen) with oligo dT as per the manufacturer's instructions. Assay-on-Demand primer/probe sets were purchased from Applied Biosystems (Foster City, CA) details of which are available on request. Real-Time qPCR was performed on the 7900HT Fast Real-Time PCR System (Applied Biosystems), with endogenous control GAPDH. Standard curves were calculated for all reactions with serial dilutions of siControl transfected cells to calculate reaction efficiency. Gene expression was calculated relative to expression of GAPDH endogenous control, and adjusted relative to expression in siControl transfected cells.

Small molecule screening MCF7 cells were plated in 96 well plates. 24 hours later cells were treated with 10 µM BIOMOL and Prestwick compound libraries, half the plates in combination with 50 nM 4OH tamoxifen in media and half with 0.05% (v/v) ethanol vehicle in media. Media containing compounds, 4OH tamoxifen or vehicle was replenished after 48 and 96 hours, and cell viability was assessed after seven days 4OH tamoxifen exposure using CellTiter-Glo Luminescent Cell Viability Assay (Promega, USA) as per manufacturer's instructions. The luminescence reading for each well on a plate was expressed relative to the median luminescence value of all wells on the plate. The screen was completed in duplicate. For each replicate the effect on cell growth and tamoxifen sensitivity were calculated. Cell growth: The effect of each individual compound on cell growth alone was calculated by dividing luminescence in the well treated with ethanol vehicle by luminescence of the replica wells treated with DMSO control, and expressed as a percentage. Cell growth effect of compound (%) = [luminescence (well with compound)/ mean luminescence (10 replica wells treated DMSO control)] x 100. Tamoxifen sensitivity: Sensitivity to 4OH tamoxifen for each compound was assessed by calculating the surviving fraction following 4OH tamoxifen.

Surviving fraction = log 2 luminescence (well treated with 4OH tamoxifen) -log 2 luminescence (wells treated with ethanol vehicle).

The surviving fractions were centred on the median surviving fraction of all 80 compounds from each plate and results expressed as a Z score [START_REF] Abdi | Z-Scores[END_REF]. For the Z score the standard deviation of the screen was estimated from the standard deviation of all DMSO control wells.

Cell viability assays to measure drug sensitivity MCF7 cells were transfected with siRNA using Dharmafect 3 as per the manufacturer's instructions, divided 24 hours later into 96 well plates and exposed to various doses of drug from 48 hours post transfection. Cell viability was assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, USA) at nine days post transfection and surviving fraction for each dose of drug assessed by dividing the luminescence value of drug treated wells by the luminescence value of vehicle treated wells.

The median effect/combination index (CI) analysis [START_REF] Chou | Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies[END_REF] was used to determine synergy of combination exposures to inhibitors. Cell cultures were treated with each agent individually at its IC 50 concentration and at fixed multiples (two and four times) and fractions (0.50, 0.25 and 0.125) of the IC 50 concentrations. The inhibitors were also combined in these same dose-fixed ratios to determine CI. Synergy was defined as any CI value below 1. Experiments were performed in triplicate, and each experiment yielded CI values for each survival fraction. Experiments that yielded an average CI value of less Protein analysis MCF7 cells were transfected with siRNA using Dharmafect 3 as per the manufacturer's instructions. After 24 hours the media was refreshed with no estradiol, 1 nM estradiol, or 1 nM estradiol with 100 nM 4OH tamoxifen. Protein lysates were prepared after 48 hours using RIPA lysis buffer (50 nM Tris pH 8.0, 150 mM NaCl, 0.1% (w/v) SDS, 0.1% (v/v) DOC, 1% (v/v) TritonX-100, 50 mM NaF, 1 mM sodium vanadate and protease inhibitors). 100 µg of total cell lysate was loaded onto prefabricated 4-12% Bis-Tris gels (Invitrogen), with full range rainbow molecular weight marker (GE Healthcare, UK) as a size reference, and resolved by electrophoresis. Proteins were transferred to nitrocellulose membrane (Bio-Rad, USA), blocked and probed with primary antibody diluted 1 in 1000 in 1 x TBS-T with 5% (w/v) BSA overnight at 4°C. Secondary antibodies were diluted 1 in 5000 in 1 x TBS-T with 5% (w/v) marvel and incubated for one hour at room temperature. Protein bands were visualised using ECL (GE Healthcare, UK) and MR or XAR film (Kodak).

Fluorescence Activated Cell Scanning (FACS) analysis MCF7 cells were transfected with siRNA using Dharmafect 3 as per the manufacturer's instructions. After 24 hours the media was refreshed with 1 nM estradiol, or 1 nM estradiol with 100 nM 4OH tamoxifen. After 48 hours the cells were fixed in 70% (v/v) ice cold ethanol and stained with 4% (w/v) propidium iodide (PI) and 10% (w/v) RNase A in PBS. The sample readout was performed on the FACSCalibur (Becton Dickinson, USA) and the data was analysed using CellQuest Pro (Becton Dickinson, USA).

Results

We recently reported the results of a high throughput RNAi screen to identify determinants of tamoxifen sensitivity [START_REF] Iorns | Identification of CDK10 as an Important Determinant of Resistance to Endocrine Therapy for Breast Cancer[END_REF]. In this screen, we targeted 779 kinases and related proteins by RNAi and measured changes in the cellular response to tamoxifen, as assessed by cell viability. We elected to screen this protein family as kinases represent targets that are amenable to inhibitor development. Furthermore, we reasoned that this approach would increase the likelihood that agents already developed could be used to inhibit targets identified in our screen. In brief, the ERα positive, tamoxifen sensitive, human breast cancer cell line, MCF7, was transfected with short-interfering (si)RNA in a 96 well-plate format and then divided the day after transfection into four replica plates. Forty-eight hours after transfection, two replica plates were treated with 4OH tamoxifen (the active metabolite of tamoxifen) and two with vehicle (Figure 1A). The screen was repeated in duplicate and comparison of data from each screen revealed this approach to be highly reproducible. Data from the duplicate screens was combined in the final analysis (Figure 1B).

In our initial report, we focused upon RNAi effects that caused resistance to tamoxifen and established that CDK10 silencing could cause resistance, an effect most likely explained by an upregulation of MAPK signalling [START_REF] Iorns | Identification of CDK10 as an Important Determinant of Resistance to Endocrine Therapy for Breast Cancer[END_REF]. Here, we used the same screen data to identify tamoxifen sensitisation effects. The most sensitising siRNA SMARTpools are listed in Table 1. As a complementary approach to this RNAi identification of sensitisation effects, we also screened a library of 1200 drugs and kinase inhibitors to identify tamoxifen sensitisers. This chemical screen involved treating MCF7 cells with either 4OH tamoxifen or vehicle (EtOH) in combination with the compounds, for seven days before cell viability was assessed. The screen was repeated in duplicate and comparison of data from each screen revealed this approach to be highly reproducible. Data from the duplicate screens was combined in the final analysis (Figure 1C). Of the compounds causing sensitisation to tamoxifen, the 10 most significant results are listed 2. As a mark of the validity of the chemical screen, tamoxifen itself was identified as causing a sensitising effect.

By cross-referencing the results of the RNAi and compound screens we identified the PDK1 intracellular signalling pathway [START_REF] Mora | PDK1, the master regulator of AGC kinase signal transduction[END_REF] as an important target for tamoxifen sensitisation. Seven of the 20 most sensitising hits identified from the RNAi screen were components of the PDK1 signalling pathway as depicted in Figure 2A. In addition, all of the top 10 most sensitising hits identified from the compound screen, with the exception of betulin, significantly inhibited the PDK1 signalling pathway, as demonstrated by inhibition of phosphorylation of the well-characterised PDK1 substrate AKT (Figure 2B). The most potent inhibitor of PDK1 signalling identified from the compound screen, triciribine, was studied further along with PDK1 siRNA. We confirmed that PDK1 siRNA as well as triciribine inhibited AKT activation, as measured by phosphorylation (Figure 2C,D; Supplementary Tables 1,2). Furthermore, we confirmed that targeting the PDK1 pathway could sensitise to tamoxifen. This was achieved by performing cell survival assays with differing combinations of tamoxifen and an AKT inhibitor (Akti1,2) that is structurally unrelated to triciribine. To assess true synergy, the survival data was analysed using the median effect model [START_REF] Chou | Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF] that estimates the Combination Index (CIs) of two compounds. CIs of less than 1 are indicative of synergy and those greater than one indicate antagonistic activity [START_REF] Chou | Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF]. Tamoxifen and Akti1,2 combinations gave a mean CI of 0.41 (Figure 2E), indicating that inhibition of the PDK1 signalling pathway was synergistic with tamoxifen.

In the high-throughput RNAi screen we used pools of siRNA (SMARTpools) each consisting of four individual siRNA targeting one gene. To assess the possibility the tamoxifen sensitisation effect caused by PDK1 silencing in the high-throughput screen was the result of off-target effects [14], we confirmed gene silencing of PDK1 by the individual siRNA contained within the individual SMARTpool (Figure 3A). We also demonstrated that each of these siRNAs were able to elicit tamoxifen sensitisation. In contrast to the single dose of tamoxifen used in the parallel screens, we assessed the effect of individual PDK1 siRNAs over a range of tamoxifen doses (Figure 3B). This analysis indicated that the PDK1 siRNA effect observed in the screen was unlikely to be the result of an off target siRNA effect. We also validated the effect of triciribine over a range of tamoxifen doses (Figure 3C). This demonstrated that triciribine increased sensitivity to tamoxifen seven fold (DMSO SF 50 = 8.5 nM, Triciribine SF 50 = 1.2 nM). Finally we confirmed tamoxifen/triciribine synergy by use of the median effect model [START_REF] Chou | Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF]. Triciribine and tamoxifen gave a mean CI of 0.39, indicative of synergy.

Estrogen signalling can be targeted by pharmacological agents other than tamoxifen and we assessed the possibility that the tamoxifen sensitisation effects observed with triciribine and PDK1 silencing were also applicable to other forms of endocrine therapy. All four siRNA targeting PDK1 sensitised cells to ICI 182780 (fulvestrant), a drug that induces ERα degradation [START_REF] Jones | A new estrogen receptor antagonist--an overview of available data[END_REF] (Figure 4A). Furthermore, combinations of triciribine and ICI 182780 also showed clear synergy (Figure 4B). The action of aromatase inhibitors may be modelled by estrogen deprivation [START_REF] Santen | Adaptive hypersensitivity to estrogen: mechanisms and clinical relevance to aromatase inhibitor therapy in breast cancer treatment[END_REF] and either PDK1 siRNA or triciribine sensitised cells to estrogen deprivation (Figure 4C,D). Taken together with the observations using tamoxifen and ICI 182780, this suggested that suppression of the PDK1 pathway sensitises to inhibition of ERα signalling generally, rather than to tamoxifen specifically.

To understand the role of PDK1 in tamoxifen response, we examined whether the induction of cell cycle arrest at the G 1 checkpoint caused by tamoxifen [17] could be enhanced by PDK1 silencing. In the absence of tamoxifen, transfection of PDK1 siRNA did not grossly alter the cell cycle profile of cells, compared to cells transfected with a control siRNA (compare siRNA PDK1 G 0 /G 1 = 54.4 %, S = 6.0 %, G 2 /M = 14.8 % and 4E). Upon addition of tamoxifen, the G 0 /G 1 proportion of cells was modesty increased in control siRNA transfected cells, from 58.5 % to 63.7 %, consistent with arrest at the G 1 checkpoint (Figure 4E,F). In PDK1 siRNA transfected cells this tamoxifen-induced increase in G 0 /G 1 cells was more profound, rising from 54.4 % before tamoxifen treatment to 84.7 % after, suggesting that PDK1 silencing exacerbated tamoxifen-induced G 1 arrest. This increase in G 1 arrest was also mirrored by a decrease in cells in the G 2 /M phase of the cell cycle (Figure 4E,F). Tamoxifen is thought to induce cell cycle arrest by causing the upregulation of cyclin dependent kinase inhibitor proteins, including p21 CIP1 [START_REF] Cariou | Down-regulation of p21WAF1/CIP1 or p27Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells[END_REF][START_REF] Pestell | The cyclins and cyclin-dependent kinase inhibitors in hormonal regulation of proliferation and differentiation[END_REF]. p21 CIP1 protein levels are also known to be downregulated in an AKT phosphorylation-dependent mechanism [START_REF] Zhou | Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells[END_REF]20]. Consistent with these observations, we found that p21 CIP1 expression was significantly enhanced after tamoxifen treatment in PDK1 silenced cells (Figure 4G), suggesting a mechanism by which down-regulation of PDK1 could enhance G 1 arrest caused by tamoxifen.

In addition to triciribine, the chemical screen also identified sertraline, tetrandrine and methotrimeprazine maleate as non-toxic putative tamoxifen sensitisers (toxicity was defined as cell viability less than 0.5). To confirm the observations of the high-throughput screen and to establish the magnitude of the effect, dose response curves were performed over a range of 4OH tamoxifen concentrations. All of the compounds significantly increased sensitivity to tamoxifen (Supplementary Figure 1), confirming the results from the tamoxifen sensitisation drug screen. The most potent tamoxifen sensitiser was tetrandrine.

Tetrandrine, (1β)-6,6',7,12-teramethoxy-2,2'-dimethyl-berbaman, is derived from the root of Stephenia tetrandra, and has been used for cardiovascular diseases in traditional Chinese medicine for centuries [START_REF] Chen | Potential role of tetrandrine in cancer therapy[END_REF]. Tetrandrine has also been used in animal and human studies, showing little toxicity alongside both anti-inflammatory and antineoplastic activity [START_REF] Chen | Potential role of tetrandrine in cancer therapy[END_REF][START_REF] Xu | Combination of tetrandrine as a potential-reversing agent with daunorubicin, etoposide and cytarabine for the treatment of refractory and relapsed acute myelogenous leukemia[END_REF]. To confirm the observations of the high-throughput screen, we performed dose-response curves and demonstrated that tetrandrine did indeed sensitise cells to tamoxifen, causing a 9.9 fold increase in sensitivity (DMSO SF 50 = 14.8 nM, 10 µM tetrandrine SF 50 = 1.46 nM Figure 5A). As for triciribine, we used the median effect model to assess tamoxifen-tetradrine synergy, which, although moderate, was confirmed (mean CI 0.72, Figure 5B). Recent work has demonstrated that tetrandrine is able to induce G 1 arrest by AKT inhibition [START_REF] Chen | Involvement of PI3K/AKT/GSK3beta pathway in tetrandrine-induced G1 arrest and apoptosis[END_REF] and we also observed moderate AKT inhibition by tetrandrine (Figure 2B). Given this, and in light of our data from triciribine and PDK1 silencing, we assessed whether tetrandrine was able to exacerbate tamoxifen-induced G 1 arrest. Examination of cell cycle profiles supported this hypothesis (Figure 5C,D). In cells treated with the tetrandrine vehicle, DMSO, tamoxifen induced a modest G 1 arrest (G 1 proportion rises from 32.9 % to 37.1 %). Conversely tamoxifen plus tetrandrine produced a more pronounced G 1 arrest (G 1 proportion rises from 38.4 % to 54.6 %).

Discussion

Here, we have performed chemical and genetic screens which reveal the potential for targeting the PDK1 pathway as a means to sensitise cells to tamoxifen. PDK1 is activated by phosphoinositide-3 kinase (PI3K) and serves as a master regulator of the AGC (cAMP-dependent or cGMP-dependent protein kinases and protein kinase C) family of protein kinases [START_REF] Mora | PDK1, the master regulator of AGC kinase signal transduction[END_REF]. Interestingly, PDK1 is highly expressed in many human cancer cell lines [START_REF] Fry | Phosphoinositide 3-kinase signalling in breast cancer: how big a role might it play?[END_REF] and breast tumours [START_REF] Lin | Elevated phosphorylation and activation of PDK-1/AKT pathway in human breast cancer[END_REF], suggesting a role for PDK1 in breast cancer tumorigenesis. However, few studies have evaluated PDK1 as a potential target for cancer therapy. Significantly, both our compound and RNAi screens identified components of the PDK1 pathway as targets for tamoxifen sensitisation. Consistent with this, previous studies have shown that inhibition of the PDK1 activator, PI3K, can sensitise to endocrine therapies in both in vitro and in vivo models and that activation of the PDK1 substrate, AKT, causes resistance to tamoxifen in breast cancer models and tumours [START_REF] Sabnis | Inhibition of the phosphatidylinositol 3-kinase/Akt pathway improves response of longterm estrogen-deprived breast cancer xenografts to antiestrogens[END_REF][START_REF] Campbell | Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance[END_REF]. However, the PI3K inhibitors used, such as wortmannin or LY294002 have poor clinical potential and are unlikely to be developed for clinical use [START_REF] Lu | Preclinical testing of clinically applicable strategies for overcoming trastuzumab resistance caused by PTEN deficiency[END_REF]. Wortmannin has a short half-life [START_REF] Balciunaite | Early phosphoinositide 3-kinase activity is required for late activation of protein kinase Cepsilon in platelet-derived-growth-factorstimulated cells: evidence for signalling across a large temporal gap[END_REF] and LY294002 causes dermatitis in animal models [START_REF] Hu | In vivo and in vitro ovarian carcinoma growth inhibition by a phosphatidylinositol 3-kinase inhibitor (LY294002)[END_REF]. Furthermore, both act non-specifically on other PI3K family members resulting in considerable toxicity [START_REF] Finan | PI 3-kinase inhibition: a therapeutic target for respiratory disease[END_REF]. For these reasons neither compound has been used in clinical trials despite their anti-tumour activities in vitro and in vivo [START_REF] Cheng | The Akt/PKB pathway: molecular target for cancer drug discovery[END_REF]. The identification of triciribine from the compound screen is therefore significant as triciribine has previously shown promise as a potential anticancer drug in in vitro models [START_REF] Schweinsberg | Identification of the metabolites of an antitumor tricyclic nucleoside (NSC-154020)[END_REF] and preliminary clinical trials demonstrated that low concentrations of triciribine were nontoxic [START_REF] Feun | A phase II trial of tricyclic nucleoside phosphate in patients with advanced squamous cell carcinoma of the cervix. A Gynecologic Oncology Group Study[END_REF][START_REF] Hoffman | Phase I-II study: triciribine (tricyclic nucleoside phosphate) for metastatic breast cancer[END_REF], suggesting that combining low concentrations of triciribine with tamoxifen, fulvestrant or aromatase inhibitors in the clinic may be a relatively non-toxic approach for delaying the development of resistance. In addition to triciribine, tetrandrine was also identified as a potent sensitiser to tamoxifen. Tetrandrine has been used in animal and human studies with little toxicity and has shown both anti-inflammatory and antineoplastic activity [START_REF] Chen | Potential role of tetrandrine in cancer therapy[END_REF][START_REF] Xu | Combination of tetrandrine as a potential-reversing agent with daunorubicin, etoposide and cytarabine for the treatment of refractory and relapsed acute myelogenous leukemia[END_REF]. Previous studies have also identified tetrandrine as a sensitiser to a range of cancer therapies including doxorubicin [START_REF] He | Reduction of doxorubicin resistance by tetrandrine and dauricine in harringtonine-resistant human leukemia (HL60) cells[END_REF], vincristine and ionizing radiation [37], although the mechanisms underlying these effects remains unknown. Importantly, this is the first study to identify tetrandrine as a sensitiser to tamoxifen.

Our study verifies the importance of PI3K signalling in the modulation of tamoxifen sensitivity, and is the first to identify PDK1 as a target for tamoxifen sensitisation. We discovered that silencing of PDK1 increases sensitivity to multiple to endocrine therapies including tamoxifen, fulvestrant and estrogen deprivation. The observation that inhibition of PDK1 sensitises to a range of estrogen signalling inhibitors, suggests that the combination of PDK1 inhibitors with endocrine therapy merits investigation in the clinic. Although not currently in clinical use, PDK1 specific inhibitors have recently been developed and may be suitable for this purpose in the future [38].

Here we provide an example of how parallel RNAi and chemical screens performed in mammalian cells may complement each other [START_REF] Iorns | Utilizing RNA interference to enhance cancer drug discovery[END_REF]. Given the complexity of biochemical pathways and the number of protein-protein interactions now described for each of the proteins within a cell, deconvoluting the mechanism by which one RNAi screen hit controls a phenotype can be difficult. To this end, we have demonstrated that, by the use of a parallel chemical screen, it is possible to simultaneously validate data from an RNAi screen and suggest potential mechanisms by which RNAi hits determine a given cellular phenotype. It is also possible that RNAi screens could be used to partially deconvolute hits from parallel compound screens [START_REF] Iorns | Utilizing RNA interference to enhance cancer drug discovery[END_REF]. While in vitro small molecule screens are generally used to identify compounds that inhibit a validated protein target, the inhibitors identified may perform poorly in living cells. One solution is to perform small molecule inhibitor screens in cells, but this approach is also limited, as it requires the cellular targets of inhibitors to be identified, which can be challenging. By performing parallel RNAi and compound screens, small molecules and gene-specific siRNA reagents can be identified that cause similar cellular phenotypes, thus simplifying target identification. This proof-of-principle suggests that a combination RNAi/chemical approach in human cell lines could streamline the development of small molecules into drugs by improving compound target deconvolution [START_REF] Iorns | Utilizing RNA interference to enhance cancer drug discovery[END_REF]. Table 1. Results of tamoxifen sensitisation siRNA screen. List of the top 20 sensitising siRNA SMARTpools from siRNA screen displayed in Z score order with siRNA SMARTpool target gene, full name, tamoxifen sensitivity Z score, and the effect of siRNA on cell viability in vehicle alone plates.

Biochemical

  Journal Immediate Publication. Published on 03 Nov 2008 as manuscript BJ20081682 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081682 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

  Validation of gene silencing by siRNA Biochemical Journal Immediate Publication. Published on 03 Nov 2008 as manuscript BJ20081682 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081682 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical

  Journal Immediate Publication. Published on 03 Nov 2008 as manuscript BJ20081682 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081682 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society than 1 were repeated at least three times to allow for determination of confidence intervals for the CI values obtained.

  Biochemical Journal Immediate Publication. Published on 03 Nov 2008 as manuscript BJ20081682 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081682 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society in Table

Biochemical

  Journal Immediate Publication. Published on 03 Nov 2008 as manuscript BJ20081682 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081682 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society control siRNA G 0 /G 1 = 58.5 %, S = 4.2, G 2 /M = 15.7 %, Figure

37

  Chang, K. H., Chen, M. L., Chen, H. C., Huang, Y. W., Wu, T. Y. and Chen, Y. J. (1999) Enhancement of radiosensitivity in human glioblastoma U138MG cells by tetrandrine. Neoplasma. 46, 196-200 38 Torbett, N. E., Luna, A., Knight, Z. A., Houk, A., Moasser, M., Weiss, W., Shokat, K. M. and Stokoe, D. (2008) A chemical screen in diverse breast cancer cell lines reveals genetic enhancers and suppressors of sensitivity to PI3K isotype-selective inhibition. Biochem J Biochemical Journal Immediate Publication. Published on 03 Nov 2008 as manuscript BJ20081682 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081682 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical SocietyTables

FIGURE LEGENDS Figure 1 .

 1 FIGURE LEGENDS Figure 1. High throughput tamoxifen sensitisation screens. A. RNAi screen method. MCF7 cells plated in 96 well plates were transfected with siRNA. Each transfection plate contained 80 experimental siRNAs (SMARTpools of four different siRNA targeting the same gene (black circles)) supplemented with ten wells of non-targeting siControl (grey circles). Transfected cells were divided into four replica plates, half treated with ethanol vehicle alone and half with 4OH tamoxifen at 50 nM, the SF 80 of MCF7. Cell viability was assessed after seven days of 4OH tamoxifen exposure using CellTiter-Glo Luminescent Cell Viability Assay (Promega). B. Scatter plot of averaged Z scores from tamoxifen sensitisation siRNA screen carried out in duplicate. Black diamonds -siRNA SMARTpools targeting 779 kinase genes, Grey diamonds -siControl. C. Scatter plot of averaged Z scores from tamoxifen sensitisation compound screen carried out in duplicate. Black diamonds -1200 compounds, Grey diamonds -vehicle control (DMSO).

Figure 2 .

 2 Figure 2. Silencing of the PDK1 pathway increases sensitivity to tamoxifen. A. Diagram illustrating components of the PDK1 pathway identified in the top 20 most sensitising hits from the RNAi screen. B.All of the top 10 most sensitising hits from the chemical screen, with the exception of betulin, significantly inhibit phosphorylation of the PDK1 target protein, AKT. Following 24 hours of serum starvation, MCF7 cells were treated with 10µM compound or DMSO control for 30 minutes and then stimulated with 10% FBS to elicit AKT phosphorylation. Lysates were made 30 minutes following stimulation. A western blot using antibodies recognising phospho and total AKT is shown. C. PDK1 silenced cells have reduced AKT phosphorylation. MCF7 cells transfected 48 hours earlier with siRNA were treated with 1 nM estradiol (E2), or left untreated. Lysates were made 24 hours following treatment. A western blot using antibodies recognising phospho and total AKT is shown. D. Triciribine treated cells have reduced AKT phosphorylation. MCF7 cells were treated with 1nM estradiol (E2), or left untreated in combination with triciribine (Tri) or DMSO vehicle control. Lysates were made 24 hours following treatment. A western blot using antibodies recognising phospho and total AKT is shown. E. Combination indices of Akti-1,2 and 4OH tamoxifen treated MCF7 cells after seven days continuous exposure. Akti-1,2 is synergistic with 4OH tamoxifen (combination index < 1.0). Error bars represent the 95% confidence interval.

Figure 3 .

 3 Figure 3. Sensitisation screen hits, PDK1 siRNA, and triciribine, increase sensitivity to tamoxifen. A. Left panel. Real time quantitative PCR on cDNA prepared from MCF7 cells transfected with siRNA. Individual siRNAs 1, 2, 3, 4 and the PDK1 SMARTpool significantly reduced transcript expression of PDK1 compared to siControl transfected cells. * -p<0.05 compared to siControl (Student's t-test). Error bars represent the standard error of the mean (SEM). Right panel. Western blot analysis of lysates prepared from MCF7 cells transfected with PDK1 SMARTpool siRNA or siControl. Antibodies recognising PDK1 were used with β-tubulin as a loading control. PDK1 SMARTpool siRNA significantly reduced PDK1 protein expression compared to siControl transfected cells. B. Cell viability assay in MCF7 cells transfected with PDK1 SMARTpool, individual PDK1 siRNAs 2, 3 and 4, or siControl and treated with 4OH tamoxifen. PDK1 silenced cells have significantly increased sensitivity to 4OH tamoxifen. siControl SF 85 = 360 nM, PDK1 SMARTpool SF 85 = 19nM; a 19 fold increase in sensitivity. Error bars represent the SEM. C. Cell viability assay in MCF7 cells treated with triciribine or vehicle control (DMSO) in combination with 4OH tamoxifen. Triciribine treated cells are significantly more sensitive to 4OH tamoxifen than vehicle treated cells (Student's t-test p<0.05). DMSO SF 50 = 8.5 nM, Triciribine SF 50 = 1.2 nM; a seven fold increase in sensitivity. Error bars represent the SEM. D. Triciribine is synergistic with 4OH tamoxifen treatment. Combination indices of triciribine and 4OH tamoxifen treated MCF7 cells after seven days continuous exposure. Triciribine is synergistic with 4OH tamoxifen (combination index < 1.0). Error bars represent the 95% confidence interval.

Figure 4 .

 4 Figure 4. Tamoxifen sensitisation screen hits, PDK1 siRNA, and triciribine, increase sensitivity to multiple ERα inhibitors. A. Cell viability assay in MCF7 cells transfected with PDK1 SMARTpool, individual PDK1 siRNAs 2, 3 and 4, or siControl and treated with ICI 182780. PDK1 silenced cells have significantly increased sensitivity to ICI 182780. siControl SF 70 = 84 nM, PDK1 SMARTpool SF 70 = 8 nM; a 10.5 fold increase in sensitivity. Error bars represent the SEM. B. Triciribine is synergistic with ICI 182780 treatment. Combination indices of triciribine and ICI 182780 treated MCF7 cells after 7 days continuous exposure. Triciribine is synergistic with ICI 182780 (combination index < 1.0). Error bars represent the 95% confidence interval. C. Cell viability assay in MCF7 cells transfected with PDK1 SMARTpool, individual PDK1 siRNAs 2, 3 and 4, or siControl and deprived of estradiol. PDK1 silenced cells have significantly increased sensitivity to 4OH estrogen deprivation. * -p<0.05 compared to siControl (Student's ttest). Error bars represent the SEM. D. Survival fraction of triciribine and vehicle control (DMSO) treated MCF7 cells after 7 days continuous estrogen deprivation. Triciribine treated cells are significantly more sensitive to estrogen deprivation than vehicle treated cells. * -p<0.05 compared to siControl (Student's t-test). Error bars represent the SEM. E. Propidium iodide FACS plots. MCF7 cells were transfected with PDK1 SMARTpool siRNA or siControl and 48 hours later treated with 1 nM estrogen (E2), or 1 nM estrogen plus 100 nM 4OH tamoxifen (4OHT). 24 hours later, cell cycle profiles were assessed by propidium iodide FACS. Increased sensitivity to tamoxifen in PDK1 silenced cells is characterised by increased tamoxifen-induced G 1 arrest. F. Absolute differences were calculated by subtracting the % of cells in each phase of the cell cycle before 4OH tamoxifen treatment from the % of cells in each phase after 4OH tamoxifen treatment. Significantly larger differences in G 1 and G 2 /M phases after 4OH tamoxifen treatment were observed in PDK1 silenced cells, indicating increased tamoxifen-induced G 1 arrest. * -p<0.05 compared to siControl (Student's t-test). Error bars represent the SEM. G.PDK1 SMARTpool transfected cells have significantly higher induction of p21 CIP1 following tamoxifen treatment than siControl transfected cells. MCF7 cells transfected 48 hours earlier with siRNA were treated with 1 nM estradiol (E2), or 1 nM estradiol plus 100 nM 4OH tamoxifen (4OHT). Lysates were made 24 hours following treatment. A western blot using antibodies recognising p21 CIP1 and β-tubulin (as a loading control) is shown.

Figure 5 .

 5 Figure 5. Tamoxifen sensitisation by tetrandrine. A. Cell viability assay in MCF7 cells treated with tetrandrine or vehicle control (DMSO) in combination with 4OH tamoxifen. Tetrandrine treated cells are significantly more sensitive to 4OH tamoxifen than vehicle treated cells (Student's t-test p<0.05). B. Tetrandrine is synergistic with 4OH tamoxifen treatment. Combination indices of tetrandrine and 4OH tamoxifen treated MCF7 cells after seven days continuous exposure. Tetrandrine is synergistic with 4OH tamoxifen (combination index < 1.0). Error bars represent the 95% confidence interval. C.Propidium iodide FACS plots. MCF7 cells were treated with 10 µM tetrandrine or DMSO vehicle in combination with 1 nM estrogen (E2), or 1 nM estrogen plus 100 nM 4OH tamoxifen (4OHT). 24 hours later, cell cycle profiles were assessed by propidium iodide FACS. Increased sensitivity to tamoxifen in tetrandrine treated cells is characterised by increased tamoxifen-induced G 1 arrest. D. Absolute differences were calculated by subtracting the % of cells in each phase of the cell cycle before 4OH tamoxifen treatment from the % of cells in each phase after 4OH tamoxifen treatment. Significantly larger differences in G 1 phase after 4OH tamoxifen treatment were observed in tetrandrine treated cells, indicating increased tamoxifen-induced G 1 arrest. * -p<0.05 compared to DMSO (Student's t-test). Error bars represent the SEM.

Table 2 .

 2 Results of tamoxifen sensitisation compound screen. List of top 10 sensitising compounds from drug screen displayed in Z score order with library, compound name, tamoxifen sensitivity Z score, and the effect of the compound on cell viability in vehicle alone plates.

	Biochemical Journal Immediate Publication. Published on 03 Nov 2008 as manuscript BJ20081682
	siRNA	Full name	Tamoxifen	Cell
	SMARTpool CIT PRKCZ PDK1 FLJ23074 PRKDC ADAM9 PRKCA CALM3 PRKCL2 GRK7 AKT1 ABL1 ILK CD3E ACK1 PIM2 PIK3C2B IRAK3 PRKACB PIP5K1A A c c e p t e d M a n u s c r i p t sensitivity Z score viability Citron (rho-interacting, serine/threonine kinase 21) -8.40 0.54 protein kinase C, zeta -7.18 0.75 3-phosphoinositide dependent protein kinase-1 -6.19 0.35 Yeast Sps1/Ste20-related kinase 4 (S. cerevisiae) -5.56 0.70 protein kinase, DNA-activated, catalytic polypeptide -4.78 0.91 ADAM metallopeptidase domain 9 (meltrin gamma) -4.78 1.00 protein kinase C, alpha -4.78 0.73 calmodulin 3 (phosphorylase kinase, delta) -4.76 0.52 protein kinase N2 -4.73 1.07 G protein-coupled receptor kinase 7 -4.59 0.63 v-akt murine thymoma viral oncogene homolog 1 -4.57 0.63 v-abl Abelson murine leukemia viral oncogene homolog 1 -4.39 0.54 integrin-linked kinase -4.36 0.51 CD3e molecule, epsilon (CD3-TCR complex) -4.35 0.96 tyrosine kinase, non-receptor, 2 -4.33 0.81 pim-2 oncogene -4.26 0.81 phosphoinositide-3-kinase, class 2, beta polypeptide -4.21 0.88 interleukin-1 receptor-associated kinase 3 -4.18 0.66 protein kinase, cAMP-dependent, catalytic, beta -4.18 0.87 phosphatidylinositol-4-phosphate 5-kinase, type I,α -4.03 0.61	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081682

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Acknowledgements

We thank Breakthrough Breast Cancer for funding this work. EI was the recipient of a studentship from the Tertiary Education Commission of New Zealand. Biochemical Journal Immediate Publication. Published on 03 Nov 2008 as manuscript BJ20081682 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081682 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society