The Tuberous Sclerosis Complex (TSC) is a genetic disease characterized by growth of hamartomas in different organs including brain, kidney, lung, skin, and heart. These lesions are sources of morbidity and mortality in patients with TSC, as they may cause intractable epilepsy, autism, developmental delay, renal and pulmonary failure. Known causes of TSC are loss of function mutations in TSC1 and TSC2 genes. The majority of TSC lesions contain multiple cell types of the mesenchymal lineage, as in the case of angiomyolipomas, lymphangioleiomyomatosis and angiofibromas. A unique cell type named perivascular epithelioid cell (PEC) is constantly present in mesenchymal TSC lesions, such as angiomyolipomas and lymphangioleiomyomatosis, basing on morphological features and the common expression of melanocytic and myogenic markers. Therefore, these lesions are officially classified, along with other tumors, as PEComas. Their cell of origin and the molecular mechanisms underlying their pathogenesis remain poorly defined.

Here we generated a novel mosaic Tsc1 knockout mouse model which develops renal mesenchymal lesions recapitulating human Perivascular Epithelioid Cell tumor (PEComa) observed in TSC patients. We identified YAP, the transcriptional coactivator of Hippo pathway, was upregulated in both renal lesions of TSC mouse model and human angiomyolipoma samples in a mTOR-dependent manner.

Inhibition of YAP with genetic or pharmacological tools greatly attenuates the proliferation and survival of Tsc1 null cells in vivo and in vitro. Futhermore, we found YAP accumulation in TSC1/TSC2 deficient cells is due to impaired degradation of the protein through the autophagosome/lysosome system.

Thus the regulation of YAP by mTOR and autophagy is a novel mechanism of growth control, matching YAP activity with nutrient availability under growth permissive conditions. It may serve as a potential therapeutical target for TSC and other diseases with dysregulated mTOR activity.

Introduction

Tuberous Sclerosis Complex

Tuberous Sclerosis Complex (TSC) is a human genetic disease which is characterized by development of hamartomas in multiple organs including brain, kidney, lung, skin and heart. These benign tumors lead to a combination of symptoms such as seizures, developmental delay, autism, skin abnormality, renal and pulmonary failure. TSC is caused by loss of function mutations in two genes, TSC1 (9q34) and TSC2 (16p13). The prevalence of TSC is estimated to be 1 in 6,000 individuals [START_REF] Crino | The tuberous sclerosis complex[END_REF].

Clinical features

Brain

TSC patients can be affected by three types of tumors in the brain: cortical tuber, subependymal nodules, subepedymal gaint cell astrocytomas (SEGA). 80% of TSC patients develop cortical tubers, which is a developmental abnormality characterized by neuronal dys-lamination, dysmorphic neurons and enlarged astrocytes in the cortex. Subependymal nodules form along the walls of lateral and third ventricles featured by abnormal gial cells and multinucleated cells. SEGA are composed of proliferative astrocytes and gaint cells which usually arise near foramen of Monro, channels connecting lateral and third ventricles.

Kidney

55% to 75% of TSC patients develop renal angiomyolipomas (AML) which are composed of abnormal blood vessels, smooth muscle cells and adipocytes. Their occurrence are usually bilateral and multiple. Angiomyolipomas may cause spontaneous bleeding and hemorrhages when their size is larger than 3 cm in diameter. Another morbidity associated with angiomyolipomas is the chronic kidney disease and renal failure caused by progressive infiltration of angiomyolipomas in some cases.

Although the classic angiomyolipomas contain three components including abnormal vasculatures, proliferative smooth muscle cells and fat cells, the contribution of each part can be viable from case to case. In some patients, the angiomyolipomas are composed of mainly smooth muscle cells and vasculatures, thus named fat poor angiomyolipomas; while in other cases, adipocytes could be the dominate component. Through genetic studies, similar second-hit mutations were identified in all the three components from the same angiomyolipoma sample indicating they are derived from one progenitor cell [START_REF] El-Hashemite | Mutation in TSC2 and activation of mammalian target of rapamycin signalling pathway in renal angiomyolipoma[END_REF][START_REF] Karbowniczek | Renal angiomyolipomas from patients with sporadic lymphangiomyomatosis contain both neoplastic and nonneoplastic vascular structures[END_REF]. However, the cell of origin of angiomyolipomas remains elusive.

A distinctive cell type, perivascular epithelioid cell (PEC), is constantly found in angiomyolipomas, because of which angiomyolipomas are classified as PEComas (discussed below). Intriguingly, Perivascular epithelioid cells express both melanocytic (Hmb45 and/or Mart1) and myogenic (aSMA) markers, while its normal tissue counterpart is not yet identified. Since melanocyte is derived from neural crest during vertebrate development, it is postulated that PEComas are originated from neural crest cells [START_REF] Sarnat | Embryology of the neural crest: its inductive role in the neurocutaneous syndromes[END_REF]. Because angiomyolipomas are mesenchymal and the epithelial cells of kidneys are generated from embryonic mesoderm, it is also hypothesized that angiomyolipomas arise from abnormal mesenchymal-epithelial transition during early kidney development or aberrant epithelial-mesenchymal transition occurred in TSC1/2 null renal tubular cells during late kidney developmental stages. These different possibilities still need to be tested to identify the cell of origin of angiomyolipomas.

Epithelial lesions, such as cysts and renal cell carcinomas, are also associated with TSC with relatively low incidences of 20-30% and 2-3% respectively.

Lung

Lymphangioleiomyomatosis (LAM) are female-dominant pulmonary tumors associated with TSC which affect 26-39% of female patients. They are composed of proliferative smooth muscle cells which could infiltrate different pulmonary structures, such as airways, alveolar septa and vasculatures. Similar with angiomyolipomas, perivascular epithelioid cells are also identified in lymphangioleiomyomatosis, which express both melanocyitc and myogenic markers. Around 30-60% patients with sporadic LAM were also affected with angiomyolipomas. Interestingly, the same mutated loci were identified both in LAM cells and in angiomyolipomas cells from the same patients indicating they may arise from the same ancestor or LAM may be a result of metastasis of mesenchymal cells from renal angiomyolipomas or vice versa [START_REF] Carsillo | Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis[END_REF]. However, this hypothesis remains to be tested experimentally.

Skin

Dermatological symptoms are present in 90% of TSC patients including facial angiofibromas, periungual fibromas and hypomelanic macules. They are among the major diagnostic criteria of TSC and usually cause no problems.

Heart

Cardiac rhabdomyomas are associated with 50-70% TSC infants. A few of them may cause significant clinical problems. They usually disappear spontaneously in later adult life of the patients.

Perivascular Epthelioid Cell tumors (PEComas)

Most of the tumors associated with TSC are mesenchymal, such as angiomyolipomas, lymphangioleiomyomatosis, angiofibromas and rhabdomyomas. In renal angiomyolipomas and pulmonary lymphangioleiomyomatosis, a unique cell type, perivascular epithelioid cell (PEC), is constantly present in these tumors, because of which these tumor are classified as PEComas [START_REF] Martignoni | PEComas: the past, the present and the future[END_REF][START_REF] Pea | Perivascular epithelioid cell[END_REF]. Morphologically, PEC has epithelioid appearance characterized by clear to granular cytoplasm and round to oval central-located nuclei. They are usually located near vasculatures. Immunohistochemically, PEC shows expression of both myogenic and melanocytic markers. The cell of orgin of these tumors remains elusive and the molecular mechanism governing their initiation and progression is yet to be fully defined.

Genetics

Two TSC-determining genetic loci, TSC1 and TSC2, were identified by linkage analysis and positional cloning utilizing multigenerational family samples [START_REF] Kandt | Linkage of an important gene locus for tuberous sclerosis to a chromosome 16 marker for polycystic kidney disease[END_REF][START_REF] Van Slegtenhorst | Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34[END_REF]. TSC1 is located on chromosome 9q34 and TSC2 is located on chromosome 16p13. A wide spectrum of mutations were found in these two genes of TSC patients including allelic variants, missense mutations and large genomic deletions. In some cases, large genomic deletions in TSC2 also disrupt the adjacent PKD1 causing severe polycystic kidney disease.

The frequency of mutations in these two genes are different. Mutations in TSC1 were mapped in 15% of TSC patients, while TSC2 mutation were identified in 80-90% of TSC patients. Loss of heterozygosity (LOH) in these two genes were found in the majority of TSC associated tumors, indicating TSC1 and TSC2 are tumor suppressor genes according to Knudson's two-hit model [START_REF] Knudson | Mutation and cancer: statistical study of retinoblastoma[END_REF][START_REF] Sepp | Loss of heterozygosity in tuberous sclerosis hamartomas[END_REF].

Pathophysiology

TSC1 and TSC2, one protein complex

TSC1 encodes a 140 kDa protein which shows no homology with other proteins. The 200 kDa TSC2 protein was identified to harbor a GTPase activating protein (GAP) domain in its C-terminal region [START_REF] Wienecke | Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity[END_REF][START_REF] Xiao | The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis[END_REF]. This domain was found to be responsible for the tumor suppressor activity of TSC2 using cell culture and xenograft experiments [START_REF] Jin | Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region[END_REF]. Both of these two proteins are evolutionally conserved from yeast to human.

A functional link between TSC1 and TSC2 proteins was established in later studies which demonstrated that these two proteins are physically associated with each other [START_REF] Plank | Hamartin, the product of the tuberous sclerosis 1 (TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles[END_REF][START_REF] Van Slegtenhorst | Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products[END_REF]. TSC1 is essential in stabilizing TSC2 protein and preventing its degradation by ubiquitin-proteasome pathway [START_REF] Benvenuto | The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination[END_REF]. The interdependent property of this heterodimeric protein complex were further validated in fruitfly and yeast [START_REF] Gao | TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth[END_REF][START_REF] Matsumoto | Role of the Tsc1-Tsc2 complex in signaling and transport across the cell membrane in the fission yeast Schizosaccharomyces pombe[END_REF][START_REF] Potter | Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size[END_REF].

TSC1/TSC2 complex, a tumor suppressor in Insulin signaling pathway

Loss of function mutations of Tsc1 or Tsc2 in Drosophila leaded to massive tissue overgrowth which was characterized by increased cell proliferation and enlarged cell size. The phenotype of Tsc1-Tsc2 double mutant was similar to either Tsc1 or Tsc2 single mutant. In addition, overexpression of either Tsc1 or Tsc2 alone had no effect on tissue growth, while overexpression of both of them dramatically attenuated organ size through decreased cell proliferation and cell size. These observations implicated that Tsc1 and Tsc2 function together as a tumor suppressor regulating both cell proliferation and cell size during tissue growth [START_REF] Gao | TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth[END_REF][START_REF] Tapon | The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation[END_REF].

Insulin signaling was previously identified to be an important regulator of tissue growth during development in Drosophila [START_REF] Stocker | Genetic control of cell size[END_REF]. Tsc1 or Tsc2 mutant fruit flies showed similar phenotypes with Pten (phosphatase and tensin homologue deleted on chromosome 10) mutant, a negative regulator in insulin signaling [START_REF] Gao | Drosophila PTEN regulates cell growth and proliferation through PI3K-dependent and -independent pathways[END_REF][START_REF] Goberdhan | Drosophila tumor suppressor PTEN controls cell size and number by antagonizing the Chico/PI3-kinase signaling pathway[END_REF][START_REF] Huang | PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development[END_REF].

Overexpression of insulin receptor lead to lethality in Drosophila and this could be rescued by co-overexpression of Tsc1 and Tsc2 but not by overexpression of either Tsc1 or Tsc2 alone. Through extensive genetic epistasis studies in Drosophila, TSC1/TSC2 complex was positioned in insulin signaling pathway downstream of Insulin Receptor/PTEN/AKT (protein kinase B) and upstream of S6K (ribosomal protein S6 kinase) by 2001 [START_REF] Potter | Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size[END_REF].

TSC1/TSC2 complex, an inhibitor of mTOR through Rheb

Subsequent studies in Drosophila showed that the phenotypes of Tsc1 or Tsc2 mutants could be rescued by deletion of an upstream activator of S6K, TOR (target of rapamycin) which was later known to be a core kinase in insulin signaling pathway and master regulator of cell growth and metabolism [START_REF] Gao | Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling[END_REF]. Inhibition of mTOR (mammalian target of rapamycin) by rapamycin induced apoptosis and suppressed proliferation of TSC1 or TSC2 null cells in vitro and in mice [START_REF] Kenerson | Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors[END_REF].

Furthermore, a small G-protein Rheb (Ras homologue enriched in brain) was identified linking TSC1/TSC2 complex to mTOR. In Drosophila, Rheb was identified to be a positive regulator of tissue growth upstream of TOR and S6K, downstream of TSC1/TSC2 complex [START_REF] Stocker | Rheb is an essential regulator of S6K in controlling cell growth in Drosophila[END_REF]. Biochemical studies showed that TSC1/TSC2 complex could directly bind and inhibit Rheb GTPase activity through the GAP domain of TSC2 [START_REF] Inoki | Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling[END_REF]. GTP-bound thus activated Rheb could directly bind and activate mTOR kinase activity [START_REF] Long | Rheb binds and regulates the mTOR kinase[END_REF]. Thus, TSC1/TSC2 complex was identified to be a tumor suppressor through inhibiting mTOR activity. mTOR kinase functions in two distinct complexs regulating cell proliferation, survival and cell metabolism [START_REF] Laplante | mTOR signaling in growth control and disease[END_REF]. mTOR kinase complex 1 (mTORC1) is composed of mTOR, raptor (regulatory associated protein of mTOR), LST8 (lethal with SEC13 protein 8), PRAS40 (proline-rich Akt substrate 40 kDa), DEPTOR (DEP domain containing mTOR-interacting protein) and Tti/Tel2 complex while mTOR kinase complex 2 (mTORC2) consists of mTOR, rictor (rapamycininsensitive companion of mTOR), SIN1 (stress-activated protein kinase interacting protein 1), LST8, DEPTOR and Tti/Tel2. The primary role of TSC1/TSC2 complex is a negative regulator of mTORC1 through inhibiton of Rheb activity. TSC1/2-mTORC1 pathway senses and integrates a diversity of upstream signals including growth factors, amino acids, oxygen, energy status and stress to control cell growth (Huang and Manning, 2008).

TSC1/2-mTORC1 signaling

Growth factors activates mTORC1 through PI3K/Akt/TSC1-TSC2/Rheb signaling axis.

TSC2 is phosphorylated by Akt primarily on Serine939 and Theronine1462 in response to growth factor stimulation [START_REF] Inoki | TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling[END_REF]. Although this phosphorylation event do inhibit the activity of TSC1/TSC2 complex toward Rheb/mTORC1, the GAP activity of TSC2 is not altered upon phosphorylation by Akt.

By cellular fractionation studies, it was reported that more TSC2 was detected in the cytosolic fractions rather than membrane fractions following growth factor stimulation [START_REF] Cai | Activity of TSC2 is inhibited by AKTmediated phosphorylation and membrane partitioning[END_REF]. TSC1/TSC2 complex is later identified to detach from lysosomes and lose contact with Rheb, a portion of which is residing on the surface of lysosomes and activating mTORC1 there, upon phosphorylation by Akt in response to growth factors [START_REF] Menon | Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome[END_REF]. In addition, ERK and RSK are also demonstrated to directly phosphorylate TSC2 and promote mTORC1 activation in response to growth factor treatment [START_REF] Roux | Tumorpromoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase[END_REF].

Inflammatory cytokine tumor necrosis factor α (TNFα) activates mTORC1 signaling through TSC1. It was uncovered that the downstream kinase of TNFα signaling inhibitory κB kinase β (IKKβ) bind and phosphorylate TSC1 on Serine487 and Serine511, leading to disruption of TSC1/TSC2 complex and degradation of TSC1 [START_REF] Lee | IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway[END_REF].

Cellular energy stress, such as accumulation of AMP, rapidly inhibit mTORC1 activity through LKB1 (Liver kinase B1)/AMPK (AMP-dependent protein kinase) signaling pathway [START_REF] Shaw | The LKB1 tumor suppressor negatively regulates mTOR signaling[END_REF]. Activated AMPK phosphorylates and activates TSC2 on Threonine1271 and Serine1387. Low oxygen levels also lead to mTORC1 inactivation through transcriptional activation of REDD1 (regulated in DNA damage and development 1) by HIFα [START_REF] Brugarolas | Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex[END_REF]. REDD1 triggers the dephosphorylation and activation of TSC2 through removal of 14-3-3 proteins associated Serine939 and Theronine1462 and PP2A-dependent dephosphorylation of Akt on Thr308 (but not Ser473) [START_REF] Dennis | REDD1 enhances protein phosphatase 2A-mediated dephosphorylation of Akt to repress mTORC1 signaling[END_REF]. DNA damage inhibits mTORC1 signaling by activating TSC1/TSC2 complex through both AMPK and REDD1 [START_REF] Corradetti | Regulation of the TSC pathway by LKB1: evidence of a molecular link between tuberous sclerosis complex and Peutz-Jeghers syndrome[END_REF]. In addition, Wnt signaling has been shown to activate mTORC1 signaling by glycogen synthesis kinase 3β (GSK3β)-mediated phosphorylation of TSC2 [START_REF] Inoki | TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth[END_REF][START_REF] Mak | Aberrant beta-catenin signaling in tuberous sclerosis[END_REF]. Amino acids availability tightly regulates mTORC1 activity. It is mainly through heterodimeric GTPases RagA/B and RagC/D, which were anchored to lysosomes through Ragulator protein complex (Lamtor1-5) and v-ATPase [START_REF] Sancak | Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids[END_REF][START_REF] Sancak | The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1[END_REF][START_REF] Zoncu | mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase[END_REF]. With high amino acids levels, RagA/B is charged with GTP and RagC/D is bound to GDP, which facilitate mTORC1 lysosomal localization and activation. Several protein complex, such as GATOR1 (DEPC5, Nprl2, Nprl3), GATOR2 (Mios, Seh1l, WDR24, WDR59, Sec13) and Folliculin complex (Folliculin and FNIP1) which have GAP or GEF activities toward Rag GTPase, are identified to be involved in the activation of mTORC1 by amino acids [START_REF] Bar-Peled | A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1[END_REF][START_REF] Bar-Peled | Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1[END_REF]. In addition, the class III PI3K vacuolar protein sorting 34 (VPS34), the Ste20 related kinase mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) and inositol polyphosphate multikinase (IPMK) are linked to mTORC1 signaling activation by amino acids [START_REF] Byfield | hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase[END_REF][START_REF] Findlay | A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling[END_REF][START_REF] Kim | Amino acid signaling to mTOR mediated by inositol polyphosphate multikinase[END_REF]. Whether TSC1/TSC2 complex is involved in the amino acid-induced mTORC1 activation remains to be fully understood. In TSC2 null cells, amino acid withdraw leads to more than 50% decrease of mTORC1 signaling measured by phosphorylation of its downstream targets S6K and 4EBP1 [START_REF] Smith | The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses[END_REF], indicating that there are amino acids sensing pathways leading to mTORC1 activation independent of TSC1/TSC2 complex. It has been shown recently that upon amino acid withdraw, Rag GTPases release mTORC1 from lysosome surface and recruit TSC1/TSC2 to the lysosome to inhibit Rheb in the mean time. Without TSC2, cells can not fully release mTORC1 from lysosome [START_REF] Demetriades | Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2[END_REF]. This may partially explain the residue mTORC1 activity upon amino acid removal in TSC2 null cells. However, the direct molecular sensor of amino acids remain to be identified.

By phosphorylating specific effector proteins, mTORC1 regulates multiple downstream signaling pathways. mTORC1 promotes protein synthesis by phosphorylating S6 kinase 1 (S6K1) and translational regulators eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) [START_REF] Laplante | mTOR signaling in growth control and disease[END_REF]. mTORC1 directly phosphorylates Lipin1, leading to its cytoplasmic sequestration and facilitating sterol regulatory element-bindng protein 1/2 (SREBP1/2) function, to drive nucleotides, lipid synthesis and proteasome biogenesis [START_REF] Peterson | mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway[END_REF]Zhang et al., 2014b). mTORC1 regulates energy metabolism through activating the transcription and translation of hypoxia inducible factor 1α (HIF1α).

Through phosphorylating and inhibiting TFEB nuclear translocation, mTORC1 suppresses lysosome biogenesis [START_REF] Settembre | A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB[END_REF]. mTORC1 also directly phosphorylates a kinase complex, consisting of ULK1/Atg13/FIP200 (unc-51-like kinase 1/ mammalian autophagy-related gene 13/ focal adhesion kinase familyinteracting protein of 200 kDa), to inhibit the initiation of autophagy, which is an eukaryotic catabolic pathway that sequesters cellular organelles and proteins in double-membrane autophagosomes, delivers and degrades the cargos in lysosomes [START_REF] Xie | Autophagosome formation: core machinery and adaptations[END_REF].

TSC1 and TSC2 function beyond mTORC1

Although TSC1 and TSC2 have been shown to function in one complex inhibiting mTORC1 signaling pathway, it does not rule out the possibility that they may also function separately and independent of mTORC1. TSC1 was identified to interact with actin binding protein ezrin-radixin-moesin. Cells which are deficient of TSC1 showed decreased focal adhesion formation. Overexpression of TSC1 leaded to activation of small GTPase Rho which induced stress fiber assembly and focal adhesion formation [START_REF] Lamb | The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho[END_REF]. Futhermore, overexpression of TSC2 also activated GTPase Rho, but not Rac1 or cdc42, through which regulating cell adhesion and migration [START_REF] Astrinidis | Tuberin, the tuberous sclerosis complex 2 tumor suppressor gene product, regulates Rho activation, cell adhesion and migration[END_REF].

TSC1/TSC2 was demonstrated to interact with and promote the activity of mTORC2.

In TSC1 or TSC2 null cells, the level of phosphorylated Akt Serine473, a substrate of mTORC2, was decreased. This effect was also uncovered in tumor cells from TSC patients (Huang et al., 2008).

1.1.3.6 TBC1D7, the third partner of TSC1/TSC2 complex TBC1D7 is identified as the third partner of TSC1/TSC2 complex which is found to associate with TSC1/TSC2 complex ubiquitously [START_REF] Dibble | TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1[END_REF]. Disruption of TBC1D7 decreases the GTPase activating protein activity of TSC complex against Rheb and activates mTORC1 signaling. However, genetic sequencing studies did not find mutations in TBC1D7 locus of TSC patients.

Animal models of TSC

Various animal models have been created for detailed characterization of the cellular and molecular pathogenic process of TSC-associated tumors and for testing of potential therapeutic interventions.

The Eker rat

The Eker rat, with a spontaneous Tsc2 mutation, was first discovered and maintained by Dr. Eker. Homozygous pups with Tsc2 mutations died around embryonic day 11 (E11) and E12. Tsc2 heterozygous rats developed renal cystadenomas, uterine leiomyomas, splenic hemangiosarcomas and brain subependymal lesions. The renal tumors in Eker rats showed the activation of mTORC1 pathway and had been used to test the efficacy of mTOR inhibitor in treating TSC1/2-null tumor cells.

Tsc1+/-and Tsc2+/-mice

Tsc1 and Tsc2 knockout alleles were generated in 1999-2002 [START_REF] Kobayashi | Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 mutation in mice[END_REF][START_REF] Kobayashi | A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice[END_REF][START_REF] Kwiatkowski | A mouse model of TSC1 reveals sexdependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells[END_REF][START_REF] Onda | Tsc2(+/-) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background[END_REF]. Tsc1-/and Tsc2-/-embryos died at mid-gestation stage during development. Tsc1+/-and Tsc2+/-mice showed similar phenotypes characterized by development of cystadenomas in the kidney and hemangiomas in the liver. They were also found to have impaired spatial learning capacities. The tumor growth and neurocognitive defects in these model could be reversed by mTOR inhibition.

Tissue-specific Tsc1/2 knockout model, brain

An astrocyte specific Tsc1 knockout was generated by crossing a floxed allele of Tsc1 (Tsc1fl/fl) and GFAP-Cre allele [START_REF] Uhlmann | Astrocyte-specific TSC1 conditional knockout mice exhibit abnormal neuronal organization and seizures[END_REF]. These mice showed increased number of astrocytes in the brain and developed seizures at around 2 month old with a median survival of 12 weeks.

Meikle et al, reported a neuronal specific Tsc1 knockout mouse model using synapsin I-Cre [START_REF] Meikle | A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival[END_REF]. These mice developed seizures and displayed enlarged neurons in the cerebral cortex and hippocampus. They also displayed defects in neuronal myelination.

Another study targeted Tsc1 specifically in a portion of neuronal progenitor cells using doxycyclin inducible Nestin-Cre [START_REF] Goto | Regulable neural progenitorspecific Tsc1 loss yields giant cells with organellar dysfunction in a model of tuberous sclerosis complex[END_REF]. These mice developed severe seizures and premature death which can be rescued by mTOR inhibitor treatments. Interestingly these mice showed vacuolated enlarged cells similar with gaint cell observed in cortical tubers of TSC patients.

Two mouse models which develop human subependymal nodules (SEN) like lesions were generated using EMX1-Cre to target embryonic telencephalic neural stem cells and tamoxifen-inducible Nestin-Cre to target neuronal progenitor cells respectively [START_REF] Magri | Sustained activation of mTOR pathway in embryonic neural stem cells leads to development of tuberous sclerosis complex-associated lesions[END_REF][START_REF] Zhou | Tsc1 mutant neural stem/progenitor cells exhibit migration deficits and give rise to subependymal lesions in the lateral ventricle[END_REF]. These two studies reported for the first time that human TSC lesions in the lateral vertricle region were reproduced in mice and proved their neural stem cell origin. Deletion of Tsc1 in Purkinje cells of mouse cerebellum using L7-cre leaded to autismlike behavior of the mutant mice [START_REF] Tsai | Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice[END_REF]. mTOR inhibition by rapamycin reversed the behavioural defects.

Tissue-specific Tsc1/2 knockout model, kidney

Disrupting Tsc1 in a subset of epithelial tubular cells in kidney induced the formation of multiple cysts due to activation of mTORC1 since this phenomenon could be rescued by rapamycin treatment [START_REF] Zhou | Loss of Tsc1, but not Pten, in renal tubular cells causes polycystic kidney disease by activating mTORC1[END_REF].

Tissue-specific Tsc1/2 knockout model, heart

Targeting Tsc1 in cardiovascular tissue using smooth muscle protein-22 (SM22) Cre resulted in ventricular hypertrophy as a result of mTOR activation in Tsc1 null cells and a median survival of 24 days [START_REF] Malhowski | Smooth muscle protein-22-mediated deletion of Tsc1 results in cardiac hypertrophy that is mTORC1-mediated and reversed by rapamycin[END_REF]. Rapamycin treatment led to longer survival and decreased ventricular hypertrophy of the mutants.

Mouse models for mesenchymal tumors in kidney and lung

Mesenchymal tumors in the kidney and lung are major sources of morbidity and mortality of TSC adult patients. However, they have never been reproduced in currently available TSC animal models. Their cell of orgin is unknown and the molecular nature underlying the proliferation and differentiation during their initiation and progression processes remain to be de defined.

Treatment

The safety and efficacy of mTOR inhibitor (Everolimus) in treating TSC patients were tested first in a Phase I/II clinical trail on SEGA, later in two Phase III, randomized, double-blind and placebo-control clinical studies on SEGA, Angiomyolipoma and sporadic Lymphangioleiomysmatosis [START_REF] Bissler | Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2): a multicentre, randomised, doubleblind, placebo-controlled trial[END_REF][START_REF] Franz | Everolimus in the treatment of subependymal giant cell astrocytomas, angiomyolipomas, and pulmonary and skin lesions associated with tuberous sclerosis complex[END_REF]. Everolimus has been shown to be effective in reducing the tumor size and approved for the treatment of TSC-associated SEGA and Angiomyolipomas. However, the tumors started regrowth when the treatment was discontinued. Furthermore, long term administration of mTOR inhibitors have undesirable side effects, including dislipidemia, hyperglycaemia, stomatitis, skin rash, lung fibrosis, leucopoenia and anaemia.

Hippo-YAP signaling 1.2.1 Hippo signaling network

Hippo signaling pathway is another critical regulator of organ size during development in additional to the Insulin pathway, first indentified in Drosophila and conserved in mammals [START_REF] Halder | Hippo signaling: growth control and beyond[END_REF][START_REF] Pan | The hippo signaling pathway in development and cancer[END_REF][START_REF] Tapon | The Hippo pathway--from top to bottom and everything in between[END_REF][START_REF] Yu | The Hippo pathway: regulators and regulations[END_REF]. A kinase cascade is in the center of Hippo pathway which consist of the Ste20-like protein kinase Hippo (MST1/2 in mammals) and the NDR family protein kinase Warts (Lats1/2 in mammals), disruption of which lead to tissue overgrowth and tumorigenesis in Drosophila and mice. Warts (Lats1/2 in mammals) kinase in turn phosphorylates and inactivates transcriptional coactivator Yorkie (YAP/TAZ in mammals), the downstream effector of Hippo pathway.

YAP/TAZ transcriptional coactivators

YAP and its homolog TAZ are the major downstream effectors of Hippo pathway which shuttle between cytoplasm and nucleus. In nucleus, they function as transcriptional coactivators interacting with specific transcription factors to regulate the transcription of downstream target genes. YAP has been shown to be a master regulator of tissue growth. YAP null embryos die at embryonic day 8.5 [START_REF] Morin-Kensicki | Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65[END_REF], while TAZ null mice appear viable [START_REF] Hossain | Glomerulocystic kidney disease in mice with a targeted inactivation of Wwtr1[END_REF]. YAP and TAZ double mutant embryos cound not even survive the 16-cell morula stage during development. Overexpression of YAP in the mouse liver, intestine and skin lead to massive tissue overgrowth and tumorigenesis [START_REF] Camargo | YAP1 increases organ size and expands undifferentiated progenitor cells[END_REF][START_REF] Dong | Elucidation of a universal sizecontrol mechanism in Drosophila and mammals[END_REF][START_REF] Schlegelmilch | Yap1 acts downstream of alpha-catenin to control epidermal proliferation[END_REF]. YAP plays a critical role in promoting the anchorage-independent growth and epithelial to mesenchymal transition (EMT) of cancer cells in vitro [START_REF] Zhao | Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control[END_REF]. Nuclear localization of YAP have been observed in about 60% human hepatocelluar carcinomas, 15% human ovarian cancers and 65% human non-small-cell lung cancers [START_REF] Wang | Overexpression of yes-associated protein contributes to progression and poor prognosis of nonsmall-cell lung cancer[END_REF][START_REF] Xu | Yes-associated protein is an independent prognostic marker in hepatocellular carcinoma[END_REF]Zhang et al., 2011). Thus, YAP is identified to be an oncoprotein.

Several DNA-binding transcription factors have been shown to be associated with YAP, such as the Runt family member Runx2 [START_REF] Yagi | A WW domaincontaining yes-associated protein (YAP) is a novel transcriptional co-activator[END_REF], the p53 family member p73 [START_REF] Strano | Physical interaction with Yesassociated protein enhances p73 transcriptional activity[END_REF] and TEAD/TEF family transcription factors [START_REF] Vassilev | TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yesassociated protein localized in the cytoplasm[END_REF]. TAZ has also been found to interact with a panel of transcription factors including Runx2, thyroid transcription factor 1, PPARr, Tbx5, TEAD1, Pax3, and Smad2/3/4. Among these transcription factor, TEAD/TEF family transcription factors 1-4 (TEAD1-4, Sd in Drosophila) were demonstrated to be the critical mediators of the physiological functions of YAP/TAZ in Hippo pathway [START_REF] Chan | TEADs mediate nuclear retention of TAZ to promote oncogenic transformation[END_REF][START_REF] Ota | Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling[END_REF][START_REF] Zhang | Transcriptional output of the Salvador/warts/hippo pathway is controlled in distinct fashions in Drosophila melanogaster and mammalian cell lines[END_REF][START_REF] Zhao | TEAD mediates YAP-dependent gene induction and growth control[END_REF]. Multiple transcriptional downstream target genes of YAP/TAZ have been identified, such as connective tissue growth factor (CTGF), the EGF family member amphiregulatin (AREG), Cyr61 and IAP family member BIRC5.

Regulation of YAP/TAZ in mammals 1.2.3.1 Phosphorylation-dependent cytoplasmic sequestration

Phosphorylation of YAP on Serine127 by Lats1/2 kinase leads to its binding to 14-3-3 and cytoplasmic sequestration. This mechanism is conserved in TAZ and Yki. A number of upstream signals regulate YAP in this manner. Cell mechanical property changes such as disruption of stress fibres F-actin or actin contractility caused phosphorylation and nuclear export of YAP through RHO GTPase and Lats1/2 kinase [START_REF] Dupont | Role of YAP/TAZ in mechanotransduction[END_REF][START_REF] Fernandez | Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila[END_REF][START_REF] Sansores-Garcia | Modulating F-actin organization induces organ growth by affecting the Hippo pathway[END_REF][START_REF] Wada | Hippo pathway regulation by cell morphology and stress fibers[END_REF]. Extracellular cues such as lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P) were also found to modulate YAP phosphorylation and cytoplasmic retention through G protein coupled receptors (GPCR)/RHO GTPase/Lats1/2 [START_REF] Miller | Identification of serum-derived sphingosine-1-phosphate as a small molecule regulator of YAP[END_REF][START_REF] Yu | Regulation of the Hippo-YAP pathway by Gprotein-coupled receptor signaling[END_REF]. In skin keratinocytes, YAP was phosphorylated by an unknown kinase and sequestered in the cytosol through binding to 14-3-3 and α-catenin [START_REF] Schlegelmilch | Yap1 acts downstream of alpha-catenin to control epidermal proliferation[END_REF]. Extra centrosomes in tetraploid cells leaded to YAP phosphorylation and inhibition through Lats1/2 activation by modulating small GTPase Rac1 and RhoA [START_REF] Ganem | Cytokinesis failure triggers hippo tumor suppressor pathway activation[END_REF].

Additionally, a diversity of proteins modulate YAP phosphorylation and cytoplasmic retetion through regulating its upstream kinases including Merlin, Kibra, RASSFs, PP2A, TAO, MARK1/PAR1 and SIKs.

Phosphorylation-dependent degradation through ubiquitin-proteasome pathway

Lats1/2 kinase could also phosphorylates YAP on Serine381 which primes the subsequent phosphorylation of YAP by CK1delta/epsilon leading to the recruitment of the E3 ubiquitin ligase SCF(β-TRCP). β-TRCP promotes the ubiquitination of YAP and its degradation by the proteasome [START_REF] Zhao | A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP)[END_REF].

Phosphorylation-independent regulation

Angiomotin has been shown to directly bind to YAP and lead to its association with tight junctions [START_REF] Zhang | Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein[END_REF]. In addition, multiple proteins directly interact with YAP/TAZ to modulate their activities in different contexts including WW domainbinding protein 2 (WBP2) [START_REF] Chen | Characterization of the WW domain of human yes-associated protein and its polyproline-containing ligands[END_REF], multiple ankyrin repeats single KH domain-containing protein (MASK) [START_REF] Sidor | Mask proteins are cofactors of Yorkie/YAP in the Hippo pathway[END_REF], zona occludens protein 1 (ZO1), ZO2 [START_REF] Oka | Functional complexes between YAP2 and ZO-2 are PDZ domain-dependent, and regulate YAP2 nuclear localization and signalling[END_REF], homeodomain-interacting protein kinase 2 (HIPK2) [START_REF] Poon | Homeodomain-interacting protein kinase regulates Hippo pathway-dependent tissue growth[END_REF].

Crosstalk between two master regulators of organ size, mTOR signaling and Hippo-YAP signaling

Genetic studies have shown that the Tuberous Sclerosis Complex (TSC) 1/TSC2/mammalian Target Of Rapamycin (mTOR) and the Hippo/Yes-associated protein 1 (YAP) pathways are master regulators of organ size, which are often involved in tumorigenesis. The crosstalk between these signal transduction pathways in coordinating environmental cues, such as nutritional status and mechanical constraints, is crucial for tissue growth. YAP has been shown to regulate mTOR activity through modulating PTEN expression level by mir29 [START_REF] Tumaneng | YAP mediates crosstalk between the Hippo and PI(3)K-TOR pathways by suppressing PTEN via miR-29[END_REF]. EGF/PI3K/PDK1 regulates YAP dephosphorylation and nuclear localization in a Akt/mTOR independent mechanism [START_REF] Fan | Regulation of Hippo pathway by mitogenic growth factors via phosphoinositide 3-kinase and phosphoinositidedependent kinase-1[END_REF][START_REF] Strassburger | Insulin/IGF signaling drives cell proliferation in part via Yorkie/YAP[END_REF].

Whether and how mTOR regulate YAP remains elusive.

Results

Generation of mosaic-Tsc1KO mice which develop multisystem lesions showing similarities with human TSC.

Since TSC is a multiorgan mosaic disease with early manifestations in infancy and childhood, we generated mice that were homozygous for a floxed Tsc1 allele (Tsc1 fl/fl ), and carried a ubiquitously expressed and temporally regulated Cre transgenic allele (CAGGCre-ER TM ) (Hayashi and McMahon, 2002;[START_REF] Meikle | A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival[END_REF].

Depending on the dose of Tamoxyfen (TM), Cre-ER TM stochastically causes recombination of floxed alleles in a variable percent of cells in every tissue (Hayashi and McMahon, 2002), thus potentially targeting cells that give rise to the heterogeneous lesions. To circumvent the embryonic lethality of the whole body Tsc1 deletion, we injected a relatively low dose of TM (10 mg/kg) intraperitoneally to pregnant dams at embryonic day 15.5 (E15.5). Cre-ER TM -positive pups were viable at birth and reached young adulthood at the same Mendelian frequency as the control littermates. However the majority of Cre-ER TM -positive mice did not survive over 10 weeks of age. We therefore focused our analysis on 6 week-old mice before the onset of lethality. At this age, recombination of the Tsc1 allele was seen in every organ analysed at 30% -80% frequency, depending on the organ (Fig. 3a). These data indicate that generalized mosaicism for complete Tsc1 loss was achieved in these mice. Henceforth, these mutant mice are named mosaic-Tsc1KO.

Histological analysis of mosaic-Tsc1KO mice was performed in multiple tissues, with special attention to anatomical locations that are commonly affected in TSC. The cerebral cortex revealed a disorganization of the cortical layers and the presence of enlarged dysplastic neurons immunoreactive with antibodies against phosphorylated ribosomal protein S6 (rpS6), a marker of mTORC1 activity (Fig. 3b), as previously observed upon brain specific deletion of Tsc1 [START_REF] Meikle | A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival[END_REF][START_REF] Zhou | Tsc1 mutant neural stem/progenitor cells exhibit migration deficits and give rise to subependymal lesions in the lateral ventricle[END_REF]. Furthermore, the dermal layer of the skin was thickened while the hypodermal fat layer was severely reduced, as assessed by Trichrome staining (Fig. 3c). The vessels decorated by ICAM-1 antibodies were increased in numbers and abnormally enlarged in the dermal and muscular layers of the skin of mosaic-Tsc1KO mice. Macroscopically, the fur of the mutant mice was fairer than in their littermates (data not shown). Kidneys were enlarged and highly susceptible to haemorrhage (Fig. 4a). Kidney failure was underscored by a three fold increase of the plasma urea levels (Fig. 3d). In conclusion, the mosaic-Tsc1KO mice develop a multisystem and early-onset disease resembling different aspects of human TSC.

Mosaic-Tsc1KO mice develop renal mesenchymal lesions recapitulating PEComas associated with human TSC.

We investigated whether the stochastic Tsc1 deletion at E15.5 of embryogenesis targets cells giving rise to mesenchymal lesions, such as PEComas.

We examined the mutant kidneys in detail, as the observed haemorrhages might be due to vascular lesions, and haemorrhage is an important complication of TSC renal angiomyolipoma [START_REF] Bissler | Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2): a multicentre, randomised, doubleblind, placebo-controlled trial[END_REF]. Consistent with the phenotype in other TSC mutant animal models [START_REF] Onda | Tsc2(+/-) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background[END_REF][START_REF] Zhou | Loss of Tsc1, but not Pten, in renal tubular cells causes polycystic kidney disease by activating mTORC1[END_REF], the mosaic-Tsc1KO kidneys were polycystic, a sign of abnormal proliferation of tubular epithelial cells (Fig. 4b). However, immunostaining with Vimentin antibody, a marker of mesenchymal cells, revealed several lesions (Fig. 4b), appearing as small, well defined, fasciculated nodules (Fig. 4c). Cells within the lesions expressed additional mesenchymal markers, such as H-Caldesmon and α-smooth muscle actin (αSMA) (Fig. 5a and5b). These nodules were proliferative, as demonstrated by immunostaining with Ki67 antibodies, and displayed high mTORC1 signalling, as shown by phosphorylation of rpS6 (Fig. 4c). They were frequently observed near vessels (Fig. 4c), and/or contained disorganized vascular components highlighted by CD31-positive endothelial cells and PDGF receptor β-, αSMAdouble positive pericytes (Fig. 5a and5b). Importantly, these mesenchymal lesions also showed immunoreactivity with the HMB45 antibody recognizing the melanocytic marker Pmel (also named gp100), which is a pathognomonic feature of PEComas (Fig. 4d).

Moreover the mRNA levels of several melanocytic markers including Mart1, Cathepsin K, Pmel, Tyrp1 were sharply increased in mutant kidneys (Fig. 4e). Finally, immunoblot analysis of protein extracts confirmed the induction of both melanocytic (Mart1) and myogenic markers (αSMA), along with mTORC1 activation in mosaic-Tsc1KO kidneys (Fig. 4f). To monitor Cre recombinase activity within the lesions, the mosaic-Tsc1KO mice were crossed with transgenic mice carrying a flox-stop-flox-lacZ reporter at the Rosa26 locus. As shown in Fig. 5c, the epithelial cells lining the cysts and the majority of the cells in the mesenchymal lesions underwent Cremediated recombination and were therefore Tsc1 deficient.

To directly compare the mesenchymal lesions found in mosaic-Tsc1KO kidneys with human specimens, we screened samples of renal angiomyolipomas occurring in TSC patients. As shown in Fig. 4g, the majority of human angiomyolipomas were surrounded by a number of smaller lesions, so called microhamartomas (or microscopic angiomyolipomas), which shared the common expression of cathepsin K, αSMA and PmeI. It is likely that the small lesions represent early developmental stage of angiomyolipomas. Importantly, these microhamartomas exhibited a striking similarity with their murine counterparts in terms of morphology and immunophenotype. In conclusion, mosaic-Tsc1KO mouse kidneys develop renal mesenchymal lesions similar with PEComas observed in human TSC patients. However we did not observe any kidney angiomyolipomas in these mice. This may be due to the premature mortality seen in these mice, precluding further lesion development into full blown angiomyolipomas.

YAP is up-regulated by mTOR in renal lesions of mosaic-

Tsc1KO mice and human angiomyolipoma.

The phenotype of mosaic-Tsc1KO mouse kidneys consisted of enlarged organ size, polycystosis and microscopic PEComas. To gain mechanistic insights, we screened transcriptional outputs of developmental pathways involved in tissue growth, planar cell polarity, and multipotency. These included target genes of Notch, Wnt, Hedgehog and Hippo pathways (data not shown and Fig. 6a). Of note, the mosaic-Tsc1KO kidneys had a two to eight fold increase in mRNA levels of connective tissue growth factor (CTGF), amphiregulin (Areg), and Cyr61, all known transcriptional targets of the Hippo pathway (Tumaneng et al., 2012a). This stimulation was blunted by treating the mice every other day for two weeks with the rapamycin derivative Temsirolimus (5 mg/kg), suggesting a tight control of the Hippo pathway by mTOR in Tsc1 mutant kidneys. Next, we examined the levels of YAP, whose function is required for the transcriptional regulation of CTGF, Areg and Cyr61 downstream of the Hippo pathway. While YAP mRNA levels were not affected by Tsc1 deletion and mTOR inhibition (Fig. 6a), the protein amount tightly correlated with mTORC1 activity as assessed by phosphorylated rpS6 levels in kidneys (Fig. 6b). The increase in YAP levels was detected both in cytosol and nuclei (Figure 6c). TAZ, the homolog of YAP, was also up-regulated at the protein level in mosaic-Tsc1KO kidneys (Fig. 6b).

To localize the kidney cell compartment expressing YAP, immunostaining was performed (Fig. 6d). In adult floxed control littermates, YAP was found in scattered tubular epithelial cells of the kidney cortex. Strikingly, Tsc1 deletion caused a widespread expression of YAP in both tubular epithelial cells lining the cysts and in spindle shaped mesenchymal cells of the kidney cortex. Notably, in the mosaic-Tsc1KO mouse kidneys a large fraction of YAP was localized in the nuclei, consistent with an active function as a transcriptional regulator. To determine whether the activation of the YAP pathway also occurred in human TSC, first we examined YAP, CTGF, and Areg protein levels in TSC angiomyolipomas by immunoblotting (Fig. 6e). The activation of mTORC1, as assessed by 4EBP1 and rpS6 phosphorylation, and the increased YAP protein were seen in all samples examined. Importantly, the transcriptional targets CTGF and Areg

were also expressed at higher levels in angiomyolipomas. Since the levels of TAZ were more variable among the human samples analysed, we decided to focus our analysis on YAP in this study. Next, we analyzed YAP localization in the different cell types of human angiomyolipomas and in the adjacent kidney structures. As shown in Fig. 6f, high levels of YAP immunoreactivity were detected in all cell types that could be found in angiomyolipomas, i.e. adipocytes, smooth muscle cells and blood vessel components. In the adjacent kidney regions, YAP protein was almost undetectable, except for small YAP-positive cysts of epithelial origin lining the lesion. The YAP antibody also stained small human renal micro-hamartomas that closely resembled the mesenchymal lesions observed in mosaic-Tsc1KO mouse kidneys (Fig. 6g).

Finally, we examined other human TSC-related PEComas from different organs. In pulmonary lymphangioleiomyomatosis, hepatic angiomyolipomas, as in renal angiomyolipomas, YAP expression invariably correlated with mTORC1 activity in tumor cells (Fig. 7). After screening a total of ten PEComas, YAP was found to be consistently highly expressed in all the specimens, and may be considered as a novel marker. Interestingly, we detect YAP expression already at early stages of human PEComa development, suggesting a putative role in the origin of the lesion.

In addition, YAP upregulation and nuclear localization was also observed in TSCassociated brain tumor SEGA (Fig. 8).

YAP is required for the abnormal proliferation and survival of TSC1/2 null cells in vitro.

We then examined whether YAP contributed to the growth phenotype of TSC1/TSC2 deficient cells. We cultured mouse embryonic fibroblasts (MEFs) that were Tsc2 -/-and Tsc2 +/+ MEFs, which were in p53 -/-background [START_REF] Zhang | Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR[END_REF].

YAP protein levels were increased by three fold in the Tsc2 null cells (Fig. 9a). This effect was also observed in nuclear fractions (data not shown), and was paralleled by increased expression of the YAP target CTGF (Fig. 9a and9g). Thus the upregulation of the YAP pathway can also be observed in a cell autonomous manner upon TSC complex deficiency. This effect could not be ascribed to a relief of Hippo/Lats inhibition of YAP in Tsc2 null cells, because the amount of Ser112phosphorylated YAP, a site of the Lats1/2 kinases [START_REF] Zhao | A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP)[END_REF], parallelled the increase in total YAP protein in the mutant cells (Fig. 9a). Tsc2 deletion is known to provide a proliferative advantage over wild type controls in serum starvation conditions [START_REF] Zhang | Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR[END_REF]. After 24h of serum starvation, the phosphorylation of the mTORC1 substrate rpS6 and the YAP protein levels were shut off in controls while remaining high in Tsc2 -/-cells (Fig. 9b). As expected, cell counting over a three day-period after serum starvation indicated that the number of Tsc2 -/-cells steadily increased as opposed to wild type control (Fig. 9c). Importantly the proliferation of Tsc2 -/-cells was blunted by YAP knockdown (Fig. 9c and9d). Cell viability was also sensitive to YAP levels, as indicated by the drop in cell number at the 72 hour time point (Fig. 9c), and the increase in TUNEL positive apoptotic cells (Fig. 9e). The effect of YAP knockdown was mimicked by treatment with verteporfin, a compound that was reported to interfere with YAP binding to the TEAD1-4 transcription factors, and accordingly inhibited CTGF mRNA expression in Tsc2 -/-cells (Fig. 9f and9g) [START_REF] Liu-Chittenden | Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP[END_REF]. YAP levels were also increased after TSC1 and TSC2 knockdown by RNAi in human embryonic kidney 293 (HEK293) cells (Fig. 9h). The concomitant knockdown of YAP impaired the proliferative advantage of TSC1depleted HEK293 cells in serum starved conditions (Fig. 9i). Taken together, these data demonstrate that YAP activity is, at least partly, required to promote proliferation of TSC complex deficient cells in vitro. +/+ and Tsc2 -/-MEFs were cultured under serum starvation conditions and treated with DMSO or Verteporfin for a 72-hour period. (f) Cell number was counted. Three independent experiments were performed and analysed (Mean ± s.e.m., *p<0.05, **p<0.01). (g) Expression of Ctgf mRNA was assessed by qRT-PCR in different treatments. Three independent experiments were performed and analysed (Mean ± s.e.m., *p<0.05, **p<0.01, ***p<0.001). (h) Expression of indicated proteins was analysed by immunoblot in HEK293 cells transfected with siRNAs against TSC1, TSC2 or siCtrl. YAP protein level was normalized with β-actin. Four independent experiments were performed and analysed (Mean ± s.e.m., *p<0.05). (i) Percentage of BrdU positive HEK293 cells with indicated treatments under serum deprivation conditions for 24 hours was assessed by three-hour BrdU pulselabeling before harvest. Three independent experiments were performed and analysed (Mean ± s.e.m., **p<0.01, *** p<0.001).

Inhibition of YAP pharmacologically or genetically attenuates abnormal proliferation and induces apoptosis of Tsc1 null cells in vivo.

To assess the effects of YAP inhibition in vivo, mosaic-Tsc1KO and control mice were treated intraperitoneally with verteporfin (100 mg/kg) every other day for ten days [START_REF] Liu-Chittenden | Genetic and pharmacological disruption of the TEAD-YAP complex suppresses the oncogenic activity of YAP[END_REF]. The YAP inhibitor reduced the kidney to body weight ratio by 50% (Fig. 10a), and attenuated overall kidney size and polycystosis in mutant mice without affecting control kidneys (Fig. 10b). These effects were concomitant to a reduction in CTGF expression (Fig. 10c). The proliferation of the mesenchymal lesions was severely affected by verteporfin treatment, as indicated by Ki67 staining of α-SMA positive cells (Fig. 10d). Furthermore, verteporfin treatment led to apoptosis of both epithelial and mesenchymal cells in mosaic-Tsc1KO kidneys (Fig. 10e). To specifically affect YAP expression by genetic means, we crossed CAGGCre-ER TM ; Tsc1 fl/fl with Tsc1 fl/fl ;Yap fl/+ mice. The offspring mice were injected with TM at postnatal day 2 (P2), in order to generate mosaic-Tsc1KO carrying a mutant allele of Yap (mosaic-Tsc1KO;Yap +/-) . The heterozygous deletion of Yap was sufficient to reduce YAP protein levels in mosaic-Tsc1KO mice (Fig. 11c). This was accompanied by a sharp attenuation of the kidney phenotype as compared to mosaic-Tsc1KO mice, as assessed by the kidney to body weight ratio (Fig. 11a), kidney size and polycystosis (Fig. 11b), and mesenchymal cell proliferation (Fig. 10d and 11e). The residual lesions observed in mosaic-Tsc1KO;Yap +/-kidneys displayed rpS6 phosphorylation, suggesting efficient recombination (data not shown). Whole body heterozygous deletion of Yap (Yap +/-) did not inhibit kidney growth and proliferation (Fig. 11f, 11g and 11h), indicating that the reduction of YAP gene dosage was particularly effective in a background of Tsc1 loss of function. Thus strategies targeting YAP activity might be beneficial in treating TSC related kidney overgrowth and PEComa formation. +/+ and Yap +/-mice (N=3). The number of Ki67 positive cells were counted in ten distinct fields per each section. Two sections were analysed per each mouse.

YAP is accumulated in TSC deficient cells due to blockage of autophagy in a mTOR-dependent manner.

YAP is under tight control by a variety of upstream regulators (Tumaneng et al., 2012a). To gain mechanistic insights into the novel crosstalk between the TSC1/TSC2/mTORC1 and YAP, we asked which step of YAP expression was affected in Tsc2 -/-cells. Consistent with the data in mosaic-Tsc1KO kidneys (Fig. 6a), the comparable YAP mRNA amount in Tsc2 -/-and Tsc2 +/+ cells ruled out transcriptional effects (data not shown). We did not find evidence for a regulation at the step of translational initiation, as the recruitment of YAP mRNA in the polysomal fraction was also equivalent in the two genotypes (data not shown). A major node in YAP regulation is the phosphorylation on five serine residues by the Lats1/2 kinases that causes cytosolic retention and promotes protein degradation by the proteasome (Huang et al., 2005;[START_REF] Zhao | A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP)[END_REF]. However no major alteration in Lats1/2 kinase activity was detected in Tsc2 -/-cells and kidney tissues, as indicated by the Ser-112 YAP phosphorylation (Fig. 6a and data not shown). Accordingly, 8 hour-treatment with the proteasome inhibitor MG132, while leading to a significant accumulation of polyubiquitinated proteins, did not affect YAP levels in either Tsc2 -/-or control cells in growing conditions (data not shown). Intriguingly, the YAP phosphorylation by Lats1/2 kinases has been reported to induce a rapid degradation of YAP in high density, but not in low density culture conditions [START_REF] Zhao | A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP)[END_REF], the latter pertaining to the experimental setup used in this study. Thus, in growing cells with no contact inhibition and with high mTOR activity, YAP levels are controlled by an alternative mechanism. TSC1/2 mutant cells have impaired autophagic flux [START_REF] Parkhitko | Tumorigenesis in tuberous sclerosis complex is autophagy and p62/sequestosome 1 (SQSTM1)dependent[END_REF], as indicated by the reduced conversion of LC3-I to the cleaved and lipidated form LC3-II (Fig. 12c). We wondered whether an alteration in the lysosomal route of degradation might explain the YAP accumulation in Tsc2 -/-MEFs. The lysosomotropic drug and autophagy inhibitor chloroquine (CQ) led to the accumulation of LC3-II that normally undergoes lysosomal turnover. This effect was paralleled by the accumulation of YAP protein in control MEFs, while the amount of YAP was not further increased by chloroquine in Tsc2 -/-cells (Fig. 12a). In MEFs, YAP protein was localized in cytosol, nucleus and cytosolic vesicles, a fraction of which was also positive for the lysosomal marker Lamp2 (Fig. 12b). Impairment of lysosomal degradation by chloroquine treatment increased the amount of YAP detectable in the lysosomes, indicating that YAP is a cargo for lysosomal degradation. In Tsc2 -/-cells, the amount of YAP localized in the lysosomes was reduced as compared to control cells in both untreated and chloroquine-treated conditions (Fig. 12b and12c). Of note, interactome studies in Drosophila cells revealed a strong association of the Hippo pathway with endocytosis and vesicle trafficking complexes [START_REF] Kwon | The Hippo signaling pathway interactome[END_REF].

To analyze the crosstalk between mTOR kinase activity and YAP, we used torin1, a potent ATP competitive inhibitor of mTOR (Thoreen et al., 2009), as assessed by the dephosphorylation of the mTOR substate 4EBP1 (Fig. 12d and12e).

Torin1 treatment of Tsc2 -/-MEFs promoted LC3 lipidation, indicating an induction of the autophagic flux, which was accompanied by a reduction of YAP levels (Fig. 12d).

The effect of Torin1 was also observed in autophagy competent control MEFs (Fig. 12d and12e). However, in autophagy deficient Atg7 -/-MEFs, mTOR inhibition was largely unable to induce YAP degradation (Fig. 12e). Similar to Torin1 treatment, nutrient starvation also promoted YAP degradation concomitantly with the induction of autophagy, in a chloroquine-sensitive manner (Fig. 12f). Of note, ectopic Flag-YAP co-immunoprecipitated with the autophagy cargo receptor p62 and with the upstream kinase Lats1 in basal conditions (Fig. 12g). Upon autophagy induction by nutrient starvation together with chloroquine treatment to induce a late autophagy block, the interaction with p62 and Lats1 was respectively up-and down-regulated. Finally, the in vivo deletion of Atg7 in epithelial cells of kidney proximal tubules was sufficient to cause the accumulation of YAP in lysates from the whole mouse kidneys (Fig. 12h).

we extended these observations to two additional models of autophagy deletion in liver: the adenoviral-Cre mediated deletion of Atg7 in Atg7f/f mice and the deletion of Vps15 in Vps15f/f mice carrying an Albumin-Cre transgene (Vps15f/f; Alb-Cre) (Fig. 13). In conclusion, mTOR regulates YAP degradation through autophagy, which is dependent on ATG7. Our data favour a model in which the control of YAP lysosomal degradation by mTOR matches YAP activity with nutrient availability in growth permissive conditions.

Conclusion and discussion

In this study, we generated a mosaic Tsc1 mutant mouse model which developed renal mesenchymal lesions showing similarities with human PEComas of TSC patients. To gain insights into the pathogenesis of these lesions, we screened the transcriptional outputs of several signalling pathways, which govern proliferation, differentiation and maintenance of multipotency during development, including Notch, Wnt, Hedgehog and Hippo pathways. Strickingly, we revealed a transcriptional signature of the Hippo pathway in Tsc1 mutant kidneys. We identified that YAP was upregulated by mTOR in mouse and human TSC1/2 null cells. Inhibition of YAP either genetically or pharmacologically greatly attenuates the abnormal proliferation and induces apoptosis of TSC1/2 null cells, both in vitro and in PEComas of mosaic Tsc1 mutant mice. Finally, we demonstrated that YAP accumulation in TSC1/2 deficient cells was due to impaired degradation of the protein by autophagy in a mTOR-dependent manner. These results suggested YAP as a potential therapeutical target for TSC and other diseases with dysregulated mTOR activity.

The cell of origin of TSC-associated PEComas

Utilizing a strategy that targeting Tsc1 in a portion of cells randomly during development, we were able to reproduce human renal PEComa-like mesenchymal lesions in mice. These two counterparts show similar immunohistochemistry signatures characterized by expression of both myogenic and melanocytic lineage markers. Detailed characterization of this model raised several possibilities to explore the cell of orgin of TSC-associated PEComas.

Pericytes

Pericyte is the mural cell of the capillaries which closely associated with endothelial cells contributing to the integrity of small blood vessels. In renal PEComas of Tsc1 mouse mutant, we observed a small portion of degenerated pericytes labelled by PDGFRβ and αSMA. Intriguingly, pericytes have been shown to be able to differentiate into adipocytes and smooth muscle cells in vitro, which are two major components commonly observed in angiomyolipomas [START_REF] Armulik | Pericytes: developmental, physiological, and pathological perspectives, problems, and promises[END_REF].

Therefore, disruption of Tsc1/Tsc2 in pericytes specifically should be pursued to explore the possibility of pericytes as the cell of origin of TSC-associated PEComas.

Smooth muscle cells

In the Tsc1 null PEComas, a large portion of cells are αSMA positive or H-caldesmon positive smooth muscle cells. It is possible that they are the prime cell of origin of TSC-associated PEComas. This hypothesis could be test by targeting Tsc1/Tsc2 specifically in smooth muscle cells.

Neural crest cells

Neural Crest is a transient cell population rising during vertebrate development which migrate into multiple organs and differentiate into a diversity of cell types including melanocytes, smooth muscle cells, bone cells, peripheral neurons and glias [START_REF] Huang | Induction of the neural crest and the opportunities of life on the edge[END_REF]. Since PEComas express two linage markers, melanocytic and myogentic markers, It has been purposed that neural crest cells are potential progenitor cells that give rise to PEComas [START_REF] Lim | Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?[END_REF]. It is important to generate TSC mutants using neural crest specific Cre alleles to test this possibility.

Potential pharmacological strategies against YAP for TSC lesions

We identified that YAP was upregulated in PEComas of Tsc1 mutant mouse models and human PEComas associated with TSC, such as angiomyolipomas and lymphangioleiomyomatosis. Inhibition of YAP by genetic means or verteporfin, a compound that disrupts the interaction between YAP and its transcription factor TEAD, greatly suppresses the abnormal proliferation and survival of Tsc1/Tsc2 null cells in vitro and in vivo. These results indicate that YAP is a potential target for TSC therapy and this mouse model can be utilized to test the efficacy of multiple interventions modulating YAP activity for treatment of TSC (Fig. 14).

GPCR agonist/antagonist

A large variety of G protein coupled receptors (GPCR) have been recently identified as positive and negative up-stream regulators of the Hippo/YAP pathway [START_REF] Yu | Regulation of the Hippo-YAP pathway by Gprotein-coupled receptor signaling[END_REF]. Future studies should define the GPCR present in the different TSC lesions and test their activity on YAP. Of note, pharmacological agents acting on the angiotensin and adrenergic GPCR are well tolerated and have interesting actions that should be considered in this scenario. Angiotensin antagonists ameliorate renal injury in conditions of hypertension and fibrosis (Remuzzi et al., 2002). Propranolol, a β adrenergic antagonist, has striking efficacy against infantile hemangioma, possibly due to inhibition of angiogenesis and pericyte functions (Leaute-Labreze et al., 2008).

Whether these agents act on YAP and are effective against TSC PEComas, are remarkable possibilities to explore.

Statins

The lipid product of mevalonate pathway, geranylgeranyl pyrophosphate, plays an important role in the post-translational modification of small GTPases, such as Rho and Rheb. Statins, the inhibitors of the rate-limiting enzyme HMG-CoA in the mevalonate pathway which lead to down-regulation of mTOR complex 1 activity through inhibiting Rheb, can also induce YAP/TAZ phosphorylation and nuclear export by acting on Rho GTPase. Interestingly, Simvastatin has already been shown to inhibit the proliferation and survival of TSC2 null cells in vitro, while the efficacy of atorvastatin against TSC remains controversial. Therefore, the effectiveness and molecular mechanism underlying the roles of statins in treating TSC remain to be fully understood.

Peptides inhibiting interaction between YAP and TEAD

The mammalian Vestigial-like proteins VGLL4 have been identified as a transcriptional repressor of YAP through its competitive binding to TEAD1-4 through Tondu domains [START_REF] Jiao | A peptide mimicking VGLL4 function acts as a YAP antagonist therapy against gastric cancer[END_REF]Zhang et al., 2014a). Peptides mimicking VGLL4's Tondu domains attenuate YAP-dependent tumor growth in vitro and in vivo. It will be interesting to determine the activity of VGLL4 in TSC null cells and test the effect of Tondu domain like peptides in TSC mouse models.

Regulation of YAP degradation by autophagy

Autophagy is an evolutionally conserved catabolic pathway which recycles cellular proteins and organelles in lysosomes. There are three different forms of autophagy that has been described: macroautophagy, microautophagy and chaperone-mediated autophagy.

Macroautophagy (hereafter as autophagy) sequesters the cellular proteins and damaged organelles in double-membrane autophagosomes, delivers and degrades these cargos in lysosomes. Microautophagy directly engulfs cytosolic cargos like glycogen into lysosomes. Chaperone-mediated autophagy recognizes soluble cargos by chaperone Hsc70, delivers them to the receptor on the lysosomal membrane Lamp2A and translocates the cargos into the lysosomes.

We revealed that YAP protein accumulation in TSC1/2 null cells is due to blockage of macroautophagic degradation of YAP in a mTOR-dependent manner since YAP degradation by inhibition of mTOR is attenuated in ATG7 deficient cells in which autophagosome formation is disrupted. Furthermore, YAP is accumulated in ATG7 deficient renal tubular cells.

There are two ways for the cargos of autophagy, such as proteins or organelles, to be loaded onto double-membrane autophagosomes (Fig. 15). The first route is through directly interacting with ATG8, a protein family that bind to and required for the formation of autophagosomes. The second is by linking targets to ATG8 proteins through cargo receptors, such as p62, NBR1, NDP52, VCP and optineurin [START_REF] Stolz | Cargo recognition and trafficking in selective autophagy[END_REF].

In the future, it is important to identify the responsible cargo receptor that links YAP to autophagosomes and map the precise domain of YAP critical to this interaction.

Autophagy is dysregulated in many human diseases such as neurodegeneration disease and cancer and it will be interesting to explore the role of YAP in the pathogenesis of these settings. determined by counting BrdU-positive nuclei among at least 200 cells in distinct fields.

For in vivo studies, mice were injected intraperitoneally with BrdU (50 mg/kg) two hours before sacrifice or 24 hours before sacrifice (mosaic-Tsc1KO;Yap +/- experiments).

TUNEL assay

Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay were performed using In Situ Cell Death Detection Kit (Roche) according to manufacturer's instructions.

Temsirolimus and verteporfin treatments

Temsirolimus (LC Laboratories) was dissolved in 100% enthanol (25 mg/ml), aliquoted and stored at -20°C. 1 mg/ml working solution was prepared in 5% Tween 80, 5% PEG400 and 86% PBS before injection. Mice were injected intraperitoneally with vehicle or temsirolimus (5 mg/kg) every other day for two weeks. Verteporfin (Selleckchem) was dissolved in DMSO (100 mg/ml), aliquoted and stored at -80°C.

Working solution was prepared at 10 mg/ml in PBS freshly before use. Mice were administered intraperitoneally with vehicle or verteporfin solution at a dose of 100 mg/kg every other day for ten days. In vitro, cells were treated with DMSO or verteporfin with a dose of 4 µM.

Quantitative RT-PCR

Total RNA was extracted from tissue or cells using an RNAeasy Mini Kit (QIAGEN), following manufacturer's instructions. Single-strand cDNA was synthesized from 1 µg of total RNA with SuperScript II (Invitrogen) and random hexamer primers. Real-time quantitative PCR was performed on MX3005P instrument (Agilent) using a Brilliant III SYBR Green QPCR Master Mix (Agilent).

Relative amounts of the indicated mRNAs were determined by means of the 2 -∆∆CT method, normalizing with β-actin levels. The primer sequences used were mouse βactin 5'-GTGACGTTGACATCCGTAAAGA-3' and 5'-GCCGGACTCATCGTACTCC-3';

  1.2.3 Regulation of YAP/TAZ in mammals ....................................................... 1.2.3.1 Phosphorylation-dependent cytoplasmic sequestration .................... 1.2.3.2 Phosphorylation-dependent degradation through ubiquitin-proteasome pathway ......................................................................................................... 1.2.3.3 Phosphorylation-independent regulation........................................... 1.2.4 Crosstalk between two master regulators of organ size, mTOR signaling and Hippo-YAP signaling................................................................................... 2 Results.................................................................................................................. 2.1 Generation of mosaic-Tsc1KO mice which develop multisystem lesions showing similarities with human TSC.................................................................... 2.2 Mosaic-Tsc1KO mice develop renal mesenchymal lesions recapitulating PEComas associated with human TSC. ............................................................... 2.3 YAP is up-regulated by mTOR in renal lesions of mosaic-Tsc1KO mice and human angiomyolipoma. ....................................................................................... 2.4 YAP is required for the abnormal proliferation and survival of TSC1/2 null cells in vitro. .................................................................................................................. 2.5 Inhibition of YAP pharmacologically or genetically attenuates abnormal proliferation and induces apoptosis of Tsc1 null cells in vivo. ............................... 2.6 YAP is accumulated in TSC deficient cells due to blockage of autophagy in a mTOR-dependent manner. ................................................................................... 3 Conclusion and discussion ................................................................................ 3.1 The cell of origin of TSC-associated PEComas ............................................. 3.1.1 Pericytes.................................................................................................. 3.1.2 Smooth muscle cells................................................................................ 3.1.3 Neural crest cells ..................................................................................... 3.2 Potential pharmacological strategies against YAP for TSC lesions ............... 3.2.1 GPCR agonist/antagonist ........................................................................ 3.2.2 Statins ..................................................................................................... 3.2.3 Peptides inhibiting interaction between YAP and TEAD.......................... 3.3 Regulation of YAP degradation by autophagy ............................................... 4 Materials and Methods ........................................................................................ 5 References ........................................................................................................... 6 Publication ........................................................................................................... 7 Acknowledgements.............................................................................................

Figure 1 .

 1 Figure 1. TSC1/TSC2/TBC1D7-mTORC1 signaling pathway.

Figure 2 .

 2 Figure 2. Hippo-YAP signaling pathway.

Figure 3 .

 3 Figure 3. Mosaic-Tsc1KO mice develop multisystem lesions showing similarities with human TSC. (a) Genomic DNA was prepared from control (ctrl) and mosaic-Tsc1KO mice 1.5 months after tamoxifen injection, and the floxed/mutant alleles were detected by PCR in the indicated organs. (b) Expression of phospho-rpS6 in cerebral cortex sections from ctrl and mosaic-Tsc1KO mice was analyzed by immunohistochemical staining. (c) Skin samples of ctrl and mosaic-Tsc1KO mice were stained for ICAM-1 expression and using Trichrome. Arrows indicate ICAM-1 positive vessels. (d) Plasma urea concentrations were determined in ctrl and mosaic-Tsc1KO kidneys (Mean ± s.e.m., N=4 mice/group, *p<0.05). Scale bar, 100 µm. Three independent pregnant females were injected with Tamoxifen. For immunohistochemistry analysis, similar stainings were found in four different ctrl/mosaic-Tsc1KO mice (6 weeks of age, two sections per each mouse).

Figure 4 .

 4 Figure 4. Mosaic-Tsc1KO mice develop renal mesenchymal lesions recapitulating PEComas associated with human TSC. Kidneys from ctrl (N=3) and mosaic-Tsc1KO (N=3) mice were analyzed at 6 weeks of age. (a) Macroscopic photos and representative H&E staining. Arrows indicate hemorrhages. (b-d) Expression of vimentin (b, c), phospho-rpS6 and Ki67 (c) and Hmb45/Pmel (d) was assessed by immunohistochemistry. H&E staining was shown on left in panel c. Arrows in panel b indicate vimentin-positive micro-hamartomas in mosaic-Tsc1KO kidneys. For immunohistochemistry analysis, similar stainings were found in three different ctrl/mosaic-Tsc1KO mice (three sections per each mouse). (e) Expression of Mart1, Cathepsin K, Pmel and Tyrp1 mRNA was assessed by qRT-

Figure 5 .

 5 Figure 5. Mosaic-Tsc1KO mice develop renal H-caldesmon + mesenchymal lesions containing CD31 + endothelial cells and PDGFRβ + /αSMA + pericytes. (a, b) Kidney tissues were isolated from

Figure 6 .

 6 Figure 6. YAP is up-regulated by mTOR in renal lesions of mosaic-Tsc1KO mice and human angiomyolipoma. (a, b) Expression of indicated genes was determined by qRT-PCR (a) and immunoblot analysis (b) in kidney samples from Ctrl mice treated with placebo, mosaic-Tsc1KO mice treated with placebo or temsirolimus. Mice were injected intraperitoneally with vehicle or temsirolimus

Figure 7 .

 7 Figure 7. YAP levels correlates with rpS6 phosphorylation in human TSC-related PEComas. (a, b, c) Immunohistochemical analysis of rpS6 phosphorylation and YAP expression in human PEComa samples associated with TSC (a, renal angiomyolipomas; b, pulmonary lymphangioleiomyomatosis; c, hepatic angiomyolipoma) (N, normal tissue; T, Tumor). Scale bar, 100 µm.

Figure 8 .

 8 Figure 8. YAP upregulation and nuclear localizaiton in human TSC-related SEGA. Immunohistochemical analysis of YAP expression in human SEGA samples associated with TSC. Scale bar, 100 µm.

Figure 9 .

 9 Figure 9. YAP is required for the abnormal proliferation and survival of TSC1/2 null cells in vitro. (a, b) Expression of indicated proteins was analysed by immunoblot in Tsc2 +/+ and Tsc2 -/-MEFs (these MEFs were generated in p53 -/-background) cultured with or without serum for 24 hours. Similar results were observed in three independent experiments. (c-e) Tsc2 +/+ and Tsc2 -/-MEFs transfected with siCtrl or siYAP were cultured in serum starvation conditions for a 72-hour period. (c) Cell number was counted in different groups. Three independent experiments were performed and analysed (Mean ± s.e.m., **p<0.01). (d) Expression of YAP protein was analysed by immunoblot at 24-hour time point. Similar results were observed in three independent experiments. (e) Apoptotic cells were identified by TUNEL assay at 48-hour time point. The percentage of TUNEL positive cells was determined by counting TUNEL-positive nuclei among at least 500 cells in distinct fields of each experiment. Three independent experiments were performed and analysed (Mean ± s.e.m., *p<0.05, **p<0.01). (f, g) Tsc2 +/+ and Tsc2 -/-MEFs were cultured under serum starvation conditions and treated with DMSO or Verteporfin for a 72-hour period. (f) Cell number was counted. Three independent experiments were

Figure 10 .

 10 Figure 10. Verteporfin treatment inhibits proliferation and induces apoptosis of Tsc1 null cells in vivo. Mice were administered intraperitoneally with vehicle or verteporfin at a dose of 100 mg/kg every other day for ten days before sacrifice. Mice were sacrificed at 6 weeks of age. Three

Figure 11 .

 11 Figure 11. Heterozygous deletion of Yap compromises the proliferation of renal mesenchymal lesions in mosaic-Tsc1KO mice. Three independent tamoxifen injection experiments were performed and female mice of indicated genotypes were pooled for analysis. (a) Kidney/Body weight ratio of the indicated mice sacrificed at 6 weeks of age (Ctrl N=4, mosaic-Tsc1KO N=5, mosaic-Tsc1KO;Yap +/-N=3). (b) Representative pictures of hematoxylin and eosin staining of kidney sections from the indicated mice. (c) Expression of indicated proteins was analysed by immunoblot in mouse kidney samples from the indicated genotypes. (d) Percentage of Ki67 and αSMA double positive cells in αSMA positive kidney lesions from mice of the indicated genotypes. Immunofluoresence staining and counting were performed on three sagittal sections from different kidney regions for each mouse

Figure 12 .

 12 Figure 12. YAP is accumulated in TSC deficient cells due to blockage of autophagy. (a) Expression of indicated proteins was analysed by immunoblot in Tsc2 +/+ and Tsc2 -/-MEFs treated with or without choloroquine (CQ) for 8 hours. Similar results were observed in three independent experiments. (b, c) Localization of YAP and Lamp2 was analysed by immunofluorescence in Tsc2 +/+ and Tsc2 -/-MEFs treated with or without CQ (20µM) for 24 hours. Insets show part of the cells indicated with squares. 50 cells were counted for yellow punctas in each group. Three independent experiments were performed and analysed (mean ± s.e.m, *p<0.05, **p<0.01, Scale bar 10µm). (d, e)

Figure 14 .

 14 Figure 14. Potential pharmacological strategies against YAP for TSC treatment.

Figure 15 .

 15 Figure 15. Regulation of YAP degradation through macroautophagy.

  Data are shown as means ± s.e.m. Analysis were performed by unpaired, twotailed, Student's t test unless otherwise indicated. P<0.05 was considered statistically significant. supervision and guidance from my mentor Mario Pende throughout these four-year PhD studies. He taught me how to design experiments and how to analyse data. He trained me the way of performing scientific investigation, step by step logically and confidently. He showed me how to deliver a scientific speech and how to construct and describe an intriguing science story in words. I cannot forget his open, critical and constructive attitudes during our discussion on research progress nearly every other day. His grand scientific vision, collaborative and working-hard spirits lighten my future path to become a scientist. His much guidance and help during these four years provide me an opportunity to start an attractive scientific career. Thanks a lot, Mario! I would like to thank Chi Zhang, with whom I worked side by side during the last two years of my PhD and characterized the role of YAP in TSC-associated PEComas together. I want to thank Ganna Panasyuk, Caroline Treins and Ivan Nemazanyy who taught me how to work with mice and multiple experimental techniques especially in my orientation year. I appreciate the much help from Vonda Koka and Delphine Pion who provided me a lot of significant and helpful technical support. I thank Yves De Keyzer, Stephanie Puget, Patricia Dill, Gregory Bonfils, Cecilia Patitucci, Karim Nadra, Catherine Caillaud and Laura Rouviere for their helpful scientific suggestions. And I would like to thank Prof. Fabiola Terzi, Prof. Guido Martignoni, Prof. David J. Kwiatkowski and Prof. Stefano Fumagalli for their insightful comments and constructive help to this study.Finally, I would like to thank my wife Shu Zhang and our lovely baby Yacheng who provided me with warm supports to finish this study. I appreciate the strong support from my parents, families and friends whom I missed a lot during my PhD studies in Paris.

Abbreviation AML

Expression of indicated proteins was analysed by immunoblot in Tsc2

+/+ , Tsc2 -/-and Atg7 +/+ , Atg7

-/-MEFs culturing with or without Torin1 for 24 hours. Similar results were observed in three independent experiments. (f) Immunoblot analysis of YAP and LC3 in Hela cells with indicated treatments. YAP levels were normalized with β-actin. Similar results were observed in three independent experiments. (g) Immunoprecipitation analysis of interacting proteins of flag-tagged YAP in the indicated culturing conditions for 0.5 hour. Similar results were observed in three independent experiments. (h) Expression of indicated proteins was analysed by immunoblot in ctrl (N=3) and proximal tubules specific Atg7 knockout mouse kidney samples (N=3).

Materials and Methods

Mice

Tsc1 fl/fl and CAGGCre-ER mice were obtained from Jackson laboratories.

Yap fl/fl mice were previously described [START_REF] Xin | Regulation of insulin-like growth factor signaling by Yap governs cardiomyocyte proliferation and embryonic heart size[END_REF] indicated. Tamoxifen (Sigma) dissolved in corn oil (Sigma) (10 mg/ml) was injected intraperitoneally into pregnant females with a dose of 10 mg/kg at E15.5 (the day of vaginal plug observed was considered as E0.5). 1 mg/ml tamoxifen prepared in corn oil was used to inject subcutaneously the pups (10 mg/kg) at postnatal day 2 to generate mosaic-Tsc1KO;Yap +/-. Blood was collected using tubes with EDTA (KABE LABORTECHNIK), centrifuged at 500g for 3mins. Plasma was isolated and analysed for urea levels with Olympus AU400 instrument.

Human TSC samples

Human samples were retrieved from the files of the Department of Pathology and Diagnostics of the University of Verona and from Institut Bergonié of Bordeaux.

The frozen samples included five angiomyolipoma samples and two resected kidney samples as consequences of TSC-unrelated diseases, pyelonephritis or oncocytoma.

Paraffin embedded human PEComa samples included seven angiomyolipomas, two lymphaleiomyomatosis and one hepatic angiomyolipomas. Human studies were ethically approved by the Comité de Protection des Personnes (CPP Ile de France III)

with the reference file #AT144.

Cell culture and reagents

HEK293, Tsc2 +/+ ;p53 -/-and Tsc2 -/-;p53

Immunohistochemistry and immunofluorescence

Mouse tissues were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 4 µm. Sections were deparaffinized and hydrated before being incubated in citrate buffer (pH6) sub-boiling for 10 mins. Sections were blocked in 3%

Goat normal serum, 1% BSA and 0.1% Triton X-100 for one hour and then incubated with the following primary antibodies overnight at 4°C: anti-phospho-rpS6 (#5364), anti-Vimentin (#5741), anti-YAP (#4912), and anti-PDGFRβ (#3169) were from Cell signalling, anti-αSMA (Sigma #A2547), anti-Ki67 (Thermo Scientific #PA5-19462), anti-Pmel/HMB45 (DAKO #M0634), anti-ICAM1 (Santa Cruz #sc-107), anti-Hcaldesmon (#ab50016), anti-CD31 (#ab28364), anti-Pmel (#ab137078) and anti-Cathepsin K (#ab19027) were from Abcam. Then sections were incubated with biotinylated secondary antibodies, detected with Vectastain Elite ABC kit (Vector) and DAB chromogen system (DAKO). For immunofluorescence, Alexa488 or Alexa568 conjugated secondary antibodies (Molecular Probe) were used and DNA was stained by DAPI within the mounting medium (Vector). For immunocytofluorescence, cells were grown on chamber slides (Millipore), fixed in 100% cold methanol, blocked in 3% Goat normal serum, 1% BSA and 0.1% Triton X-100/PBS, and incubated with the following antibodies: anti-YAP (Santa Cruz #sc-101199) and anti-Lamp2 (Abcam #ab13524). Immunofluorescence slides were analysed by microscopy ZEISS ApoTome2. ImageJ software was used for quantification.

Immunobloting and immunoprecipitation

Cells and tissues were lysed in cell lysis buffer (Cell signalling #9803) with complete protease inhibitor cocktail and phosSTOP phosphatase inhibitor cocktail

BrdU labelling

Cells were incubated with BrdU (Sigma) (10 µg/ml) for three hours before harvest. Then cells were washed, fixed in 4% paraformaldehyde 15 mins, incubated in citrate buffer (pH6) at sub-boiling temperature and detected with an anti-BrdU antibody (Roche #11170376001). The percentage of BrdU incorporation was