Dr Nadine Houédé
email: nadine.houede@chu-nimes.fr

Philippe Pourquier

Targeting the genetic alterations of the PI3K-AKT-mTOR pathway: its potential use in the treatment of bladder cancers

Keywords: Urothelial bladder cancer, PI3K, AKT, PTEN, mTOR, TSC1 AMP-activated protein kinase, Bad: B cell leukemia/lymphoma-2 associated death agonist, Bcl-2: B cell leukemia-2, Bim: B cell leukemia/lymphoma-2 interacting mediator of cell death, CREB: cyclic AMP response element-binding protein, Deptor: DEP domain containing mTORinteracting protein, eIF-4E: eukaryotic translation initiation factor 4E, FKBP38: FK506 Binding Protein 38, Foxo3a: Forkhead box O3, GSK3: glycogen synthase kinase-3, Receptor Substrate 1, LKB1: Liver kinase B1, Mcl-

Urothelial carcinoma of the bladder is the most frequent tumor of the urinary tract and represents the fifth cause of death by cancer worldwide. The current first line chemotherapy is a combination of cipslatin and gemcitabine with median survival not exceeding 15 months. Vinflunine is the only drug approved by EMEA as second-line treatment and few progresses have been made for the past 20 years to increase the survival of metastatic patients, especially those who are not eligible for cisplatinbased regimen. The recent studies characterizing the genetic background of urothelial cancers of the bladder, revealed chromosomal alterations that are not seen at the same level in other types of cancers. This is especially the case for mutations of genes involved in the PI3K/AKT/mTOR signaling pathway that occupies a major place in the etiology of these tumors. Here, we describe the mutations leading to constitutive activation of the PI3K/AKT/mTOR pathway and discuss the potential use of the different classes of PI3K/AKT/mTOR inhibitors in the treatment of urothelial bladder cancers. Despite the recent pivotal study evidencing specific mutations of TSC1 in bladder cancer patients responding to everolimus and the encouraging resultsobtained with other derivatives than rapalogs, few clinical trials are ongoing in bladder cancers. Because of the genetic complexity of these tumors, the crosstalks of the PI3K/AKT/mTOR pathway with other pathways, and the small number of eligible patients, it will be of outmost importance to carefully choose the drugs or drug combinations to be further tested in the clinic.

Introduction

The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway (Fig. 1) is an essential pathway for various cellular processes including cell growth, cell survival, cell motility, angiogenesis, as well as cell metabolism [START_REF] Bartholomeusz | Targeting the PI3K signaling pathway in cancer therapy[END_REF][START_REF] Courtney | The PI3K pathway as drug target in human cancer[END_REF][START_REF] Knowles | Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer[END_REF][START_REF] Mccubrey | Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance[END_REF]McCubrey, Steelman, Chappell, Abrams, Montalto, et al., 2012;[START_REF] Populo | The mTOR Signalling Pathway in Human Cancer[END_REF][START_REF] Shaw | Ras, PI(3)K and mTOR signalling controls tumour cell growth[END_REF]. It is also involved in the resistance to "conventional" chemotherapeutic agents by modulating the expression of efflux pumps from the ABC transporters family (H. A. Burris, 3rd, 2013;[START_REF] Tazzari | Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts[END_REF]. Its constitutive activation, that is mainlydue to genetic alterations of key genes involved in this pathway,is observed in various types of cancers (see (P. [START_REF] Liu | Targeting the phosphoinositide 3-kinase pathway in cancer[END_REF]for review), including urothelial bladder cancers [START_REF] Bambury | Actionable mutations in muscle-invasive bladder cancer[END_REF][START_REF] Knowles | Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer[END_REF]. As such it representsan attractive targets for cancer therapy [START_REF] Chang | Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy[END_REF][START_REF] Liu | Targeting the phosphoinositide 3-kinase pathway in cancer[END_REF].Urothelial carcinoma of the bladder represents the most frequent tumor of the urinary tract and leads to a fatal issue for approximately 150,000 patients each year worldwide [START_REF] Ploeg | The present and future burden of urinary bladder cancer in the world[END_REF]. In the case of advanced metastatic forms of the disease,chemotherapies such ascombination of cisplatin with gemcitabine or combination of cisplatin with methothrexate, vinblastine and adriamycine (MVAC protocol) are used, with a 5-year survival rate not exceeding 15% [START_REF] Sio | Chemotherapeutic and targeted biological agents for metastatic bladder cancer: A comprehensive review[END_REF][START_REF] Von Der Maase | Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer[END_REF], which stresses the urgent need for alternative therapeutics. The growing number of studies reporting new alterations of this pathway leading to its constitutive activation, have led to the identification of new generations of tyrosine kinase inhibitors specifically targeting such mutations in varioustumor types with significant improvement in terms of tumor responses [START_REF] Bambury | Actionable mutations in muscle-invasive bladder cancer[END_REF][START_REF] Bartholomeusz | Targeting the PI3K signaling pathway in cancer therapy[END_REF][START_REF] Bitting | Targeting the PI3K/Akt/mTOR pathway in castration-resistant prostate cancer[END_REF]H. A. Burris, 3rd, 2013;McCubrey, Steelman, Chappell, Abrams, Montalto, et al., 2012). In this review, we will provide an update of the genomic alterations that have been specifically identified in bladder cancers. We will also discuss the use of inhibitors targeting the PI3K/AKT/mTOR pathway in patients with urothelial cancers, taking into account the known limitations resulting from feed-back loops and the delicate issue of cross-talks with other essential pathways regulating cell proliferation such as the RAS/RAF/MEK/ERK pathway [START_REF] Courtney | The PI3K pathway as drug target in human cancer[END_REF][START_REF] Markman | Targeting the PI3K/Akt/mTOR pathway-beyond rapalogs[END_REF][START_REF] Mccubrey | Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance[END_REF](Fig. 1). This latter aspect is certainly representing one of the major challenges of modern pharmacology regarding the rational choice for synergistic drug combinations.

The PI3K/AKT/mTOR pathway and its cross-talks with other pathways

The PI3K/AKT/mTOR pathway is initiated by the tyrosine phosphorylation of the intracellular domain ofreceptor tyrosine kinases (RTKs) resulting from interaction of RTKs with their respective ligands at the cell surface (Fig. 1). This induces the activation of class IA phosphatidylinositol 3-kinase (PI3K) as second messenger. Class IA PI3K is a heterodimeric kinase composed of two subunits: the catalytic subunit p110 and the regulatory subunit p85. There are four isoforms of p110 (, , ) encoded by PIK3CA, PIK3CB, PIK3CG, and PIK3CD genes, respectively, the delta isoforms being primarily found in lymphocytes [START_REF] Engelman | The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism[END_REF][START_REF] Katso | Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer[END_REF]. Similarly, the three isoforms p85,  and  are encoded by three distinct genes, PIK3R1, PIK3R2, and PIK3R3 [START_REF] Engelman | The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism[END_REF][START_REF] Katso | Cellular function of phosphoinositide 3-kinases: implications for development, homeostasis, and cancer[END_REF]. p85 contains aSrc homology domain (SH2) that allows recognition and binding to activated RTK phosphotyrosine. This binding can be direct but can alsobe mediated by adaptor proteins such as IRS-1 (Insulin Receptor Substrate 1) [START_REF] Carpenter | Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit[END_REF]. It results in the activation of the p110 catalytic subunit and its relocalization to the inner layer of the cytoplasmic phospholipid membrane where it phosphorylates the γ'-hydroxyl group of phosphatidylinositol-4,5biphosphate (PIP2) to generate phosphatidylinositol-3,4,5-triphosphate (PIP3) (Fig. 1) [START_REF] Cantley | The phosphoinositide 3-kinase pathway[END_REF].

PIP3 plays a pivotal role in further signaling of the pathway as it is a substrate of various protein kinases containing a pleckstrin homology (PH) domain such as the serine-threonine kinase AKT and the phosphoinositide-dependent kinase 1 (PDK1) [START_REF] Cantley | The phosphoinositide 3-kinase pathway[END_REF]. This interaction leads to the recruitment of AKT to the cell membrane where itis phosphorylatedon both threonine 308 by PDK1 [START_REF] Alessi | Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha[END_REF][START_REF] Stokoe | Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B[END_REF] and serine 473by the mTORC2 complex via a positive feed-back loop [START_REF] Sarbassov | Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex[END_REF], leading to its full activation.

Dephosphorylation of serine 473 can be achieved by the PH domain leucine-rich repeat protein phosphatase (PHLPP) resulting in cell growth inhibition [START_REF] Gao | PHLPP: a phosphatase that directly dephosphorylates Akt, promotes apoptosis, and suppresses tumor growth[END_REF]. PIP3 can also bind the tumor suppressor phosphatase and tensin homolog (PTEN) that dephosphorylates PIP3 to PIP2 (Fig. 1). As such, PTEN represents a key negative regulator of the PI3K/AKT/mTOR signaling [START_REF] Chalhoub | PTEN and the PI3-kinase pathway in cancer[END_REF].

Activated AKT can phosphorylatea myriad of downstream effectors regulating a variety of essential processes such as protein synthesis, cell metabolism, cell growth/proliferation,as well as cell survival and resistance to various exogenous stresses [START_REF] Hay | The Akt-mTOR tango and its relevance to cancer[END_REF]Steelman, Stadelman, et al., 2008).

Providing an exhaustive list of all these targets and describing the functional consequences of their phosphorylation in terms of activation or inhibition is beyond the scope of this review. Only AKT substrates that could be relevant for cell response to anticancer drugs will be cited. Among them, AKT modulates the activity of multiple transcription factors such as CREB (cyclic AMP response elementbinding protein),the phosphorylation of which increases the expression of antiapoptotic genes such as Bcl-2 or Mcl-1 [START_REF] Du | CREB is a regulatory target for the protein kinase Akt/PKB[END_REF]. Activated AKT alsoinhibits the transcriptional activity of Foxo3a (Forkhead box O3) leadingto reduced expression of several pro-apoptotic genes such as caspase-9, caspase-3, Bim, Bad, Noxa, or Puma [START_REF] You | FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal[END_REF] or the expression of p27 cyclin-dependent kinase inhibitor (p27

Kip1

) that is required for cell cycle arrest in G1 [START_REF] Liang | PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest[END_REF]. Itcan also result in increased expression of anti-apoptotic genes such as Bcl-2, XIAP, or NF-B [START_REF] Dan | Aktdependent regulation of NF-{kappa}B is controlled by mTOR and Raptor in association with IKK[END_REF]. The transcriptional activity of NF-B is also under the control of AKT that directly phosphorylates the inhibitor of B kinase (IKK). This leads to the phosphorylation of the inhibitory protein IB,inducing its degradation and the releaseof NF-B that translocates into the nucleuswhere itregulates the expression of its target genes [START_REF] Ozes | NF-kappaB activation by tumour necrosis factor requires the Akt serine-threonine kinase[END_REF]. This mechanism has been implicated in the modulation of ABBC1 expression, a membrane transporter (formerly called MRP1) playing a major role in drug resistanceto various anticancer agents [START_REF] Tazzari | Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts[END_REF].

AKT also inhibits the function of the tuberous sclerosis TSC1/2 complex. Tuberous sclerosis is a rare dominant autosomal disorder resulting from mutations in one of the two genes TSC1 (hamartin) or TSC2 (tuberin), leading to the occurrence of non-malignant tumors in different organs including brain, retina, kidney, heart, and lung [START_REF] Crino | The tuberous sclerosis complex[END_REF]. AKT phosphorylates TSC2 that leads to the disruption of its interaction with TSC1 [START_REF] Inoki | TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling[END_REF] and further blockage of the GAP (GTPase activating protein) function of the complex which in turn prevents the conversion of the active form of RHEB (Ras homolog enriched in brain) bound to GTP in its inactive form bound to GDP. Then, active RHEB phosphorylates mTOR (mammalian target of rapamycin), the third master player of this signaling pathway, at serine 2448 [START_REF] Inoki | Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling[END_REF]. The TOR gene was originally discovered by a genetic screen in yeast Saccharomyces cerevisiae looking for resistant clones to the fungicide rapamycin [START_REF] Heitman | Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast[END_REF], and its homologs were further characterized in many other species including mammals. mTOR is a high molecular weight serine/threonine kinase from the PIKK (PI3K-related protein kinase) family [START_REF] Zoncu | mTOR: from growth signal integration to cancer, diabetes and ageing[END_REF]. It is part of two complexes, mTORC1 and mTORC2 that differ from their composition and have distinct functional properties, starting with responsiveness to rapamycin treatment, mTORC1 being the most sensitive to the drug. In mTORC1, TOR associates with PRAS40 (proline-rich Akt substrate 40 kDa), mLST8 (mammalian Lethal-with-Sec-Thirteen 8), FKBP38 (FK506 Binding Protein 38), Raptor (Regulatory associated protein of mTOR), and Deptor (DEP domain containing mTOR-interacting protein) proteins whereas it interacts with mSIN1 (stress-activated protein kinase-interacting protein 1), mLST8, Rictor (Rapamycin-insensitive companion of mTOR), Protor, Deptor, and the heat shock protein Hsp70 in the mTORC2 complex (Fig. 1) [START_REF] Populo | The mTOR Signalling Pathway in Human Cancer[END_REF][START_REF] Zoncu | mTOR: from growth signal integration to cancer, diabetes and ageing[END_REF]. Raptor serves as a scaffold protein to recruit mTORC1 substrates [START_REF] Hara | Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action[END_REF][START_REF] Kim | mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery[END_REF].

Depending on the complex mLST8 regulates the interaction of mTOR with Raptor or Rictor [START_REF] Zeng | Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML[END_REF] and activates its kinase activity(D. H. [START_REF] Kim | GbetaL, a positive regulator of the rapamycin-sensitive pathway required for the nutrient-sensitive interaction between raptor and mTOR[END_REF]. Conversely, PRAS40 or Deptor have been described to exert a negative regulation on their respective complexes [START_REF] Peterson | DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival[END_REF][START_REF] Sancak | PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase[END_REF]. PRAS40 can be directly phosphorylated by AKTthat results in the inhibition of mTORC1 activation by Rheb [START_REF] Sancak | PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein kinase[END_REF]. Similarly, FKBP38 was also proposed to play an inhibitory role on mTORC1 [START_REF] Sato | Specific activation of mTORC1 by Rheb Gprotein in vitro involves enhanced recruitment of its substrate protein[END_REF]. Phosphorylation of AKT at serine 473 by mTORC2 depends on mSIN1 [START_REF] Frias | mSin1 is necessary for Akt/PKB phosphorylation, and its isoforms define three distinct mTORC2s[END_REF]and is also regulated by the heat-shock protein Hsp70 [START_REF] Martin | Hsp70 associates with Rictor and is required for mTORC2 formation and activity[END_REF].

As mentioned earlier, mTORC1 and mTORC2 complexes differently contribute to the global effect of PI3K/AKT/mTOR pathway activation on cell growth and the maintenance of cellular homeostasis.mTORC1 signaling is primarily mediated via the regulation of various factors involved in translation [START_REF] Populo | The mTOR Signalling Pathway in Human Cancer[END_REF][START_REF] Zoncu | mTOR: from growth signal integration to cancer, diabetes and ageing[END_REF]. Among them, mTORC1 phosphorylates the serine/threonine kinase S6K that leads to the phosphorylation of the 40S ribosomal protein S6 resulting in increased translation of S6K target genes [START_REF] Hornberger | mTOR is the rapamycin-sensitive kinase that confers mechanically-induced phosphorylation of the hydrophobic motif site Thr(389) in p70(S6k)[END_REF]. mTORC1 also phosphorylates 4E-BP1 favoring its dissociation from the eukaryotic translation initiation factor 4E (eIF-4E), leading to the abrogation of its inhibitory effect on translation initiation [START_REF] Pause | Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function[END_REF]. Regulation of translation by mTORC1 is highly sensitive to nutrient (amino-acids, glucose) levels and is tightly regulated by the two other kinases AMPK (AMP-activated protein kinase) and ULK1(unc-51-like kinase 1), the mammalian homolog of Atg1 that initiates the catabolic process of autophagy(J. [START_REF] Kim | AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1[END_REF][START_REF] Nazio | mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6[END_REF]. When enough nutrients are present, phosphorylation of ULK1 on S757 by mTORC1 prevents its interaction with AMPK and prevents autophagy (J. [START_REF] Kim | AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1[END_REF][START_REF] Nazio | mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6[END_REF]. In deprivation conditions,AMPK interacts with and phosphorylates ULK1 at other sites leading to the initiation of autophagy that is necessary for the restoration of nutrient levels [START_REF] Egan | The autophagy initiating kinase ULK1 is regulated via opposing phosphorylation by AMPK and mTOR[END_REF]. In parallel, AMPK phosphorylates TSC2 that inhibits mTORC1 activity, a mechanism that is further amplified by the phosphorylation of AMPK by the liver kinase B1 (LKB1) [START_REF] Inoki | TSC2 mediates cellular energy response to control cell growth and survival[END_REF].mTORC1 also plays an indirect role in angiogenesis by regulating the translation of HIF-1 (Hypoxia-inducible transcription factor 1) and the expression of its target genes encoding for essential angiogenic factors such as VEGF and PDGF [START_REF] Manning | Rheb fills a GAP between TSC and TOR[END_REF]. Importantly, mTORC1 is capable to activate PI3K by a negative feed-back loop implicating the activation of S6K that in turns inhibits IRS-1 phosphorylation at Ser302 [START_REF] Harrington | The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins[END_REF]) (Fig. 1).

Probably because of its "resistance" to rapamycin, fewer studies have investigated the functional roles of the mTORC2 complex, but a growing number of studies provide evidences for its role in the regulation of multiple processes and its cross-talk with mTORC1 complex. mTORC2 was first identified as a key regulator of cytoskeleton organization and cell shape [START_REF] Jacinto | Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive[END_REF], strengthening its potential role in cell migration, motility and tumor metastases (Zhou & Huang, 2011). PKC, a kinase from the AGC (protein kinase A/protein kinase G/Protein kinase C) family is involved in this mechanism [START_REF] Sarbassov | Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton[END_REF]), but it is probable that other mTORC2 effectors could also contribute to this regulation. Other AGC kinases are also substrates of TORC2, including AKT (see above) and the serum and glucocorticoid-induced kinase 1 (SGK1) [START_REF] Garcia-Martinez | mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum-and glucocorticoid-induced protein kinase 1 (SGK1)[END_REF], the phosphorylation of which leads to the regulation of sodium transport via the epithelial Na+ channel (ENaC) [START_REF] Bogusz | A novel N-terminal hydrophobic motif mediates constitutive degradation of serum-and glucocorticoid-induced kinase-1 by the ubiquitinproteasome pathway[END_REF]. Similarly to mTORC1, mTORC2 also regulates translationthrough its direct association with ribosomal proteins and the stabilization of newly synthesized polypeptides, including Akt [START_REF] Oh | mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide[END_REF]. This process is inhibited in the presence of dual inhibitors of mTORC1 and mTORC2 [START_REF] Carayol | Critical roles for mTORC2-and rapamycininsensitive mTORC1-complexes in growth and survival of BCR-ABL-expressing leukemic cells[END_REF][START_REF] Oh | mTORC2 can associate with ribosomes to promote cotranslational phosphorylation and stability of nascent Akt polypeptide[END_REF][START_REF] Yu | Biochemical, cellular, and in vivo activity of novel ATPcompetitive and selective inhibitors of the mammalian target of rapamycin[END_REF]. mTORC2 is also involved in metabolism and in autophagy, although these roles have not been as clearly established than for mTORC1 [START_REF] Oh | mTOR complex 2 signaling and functions[END_REF].

As shown in figure 1, RTKs activate both PI3K/AKT/mTOR and MAPK/ERK pathways that positively and negatively regulate each other. The molecular mechanisms involved in these cross-talks have been reviewed elsewhereand clearly demonstrate their importance in cell survival to inhibitors targeting either of these pathways (Carracedo, Baselga, & Pandolfi, 2008;[START_REF] Mendoza | The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation[END_REF]. It was demonstrated that ERK could phosphorylate TSC2 and suppresses its tumorsuppressive function [START_REF] Ma | Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis[END_REF]. Inhibition of mTORC1 was also shown to activate the MAPK/ERK pathway via a S6K-PI3K-Ras negative feedback loop [START_REF] Carracedo | Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer[END_REF]. This served as a rationale to test whether the effects of mTOR inhibitors could be potentiated by the addition of MEK1/2 inhibitors. Such a combination induced synergistic effects as compared to each inhibitor used separately in several in vitro and xenograft models (Carracedo, Baselga, et al., 2008).

By controlling such a wide range of ubiquitous cellular processes that are essential for the development and dissemination of cancer cells, it is not surprising that the PI3K/AKT/mTOR signaling pathway remainsone of the most attractive target for the development of new anticancer agents. This is of particular importance in the context of alterations of specific genes involved in this pathway. As it will be described further, these "actionable" mutations have been identified at much higher rates in bladder cancers as compared to other cancer types, further reinforcing the interest of using inhibitors of this pathway to kill tumor cells in a more selective way.

Bladder cancers

Epidemiology of bladder cancer

Badder cancer is the ninth most common cancer worldwide with approximately 380,000 new cases and 150,000 deaths in 2008 [START_REF] Jemal | Global cancer statistics[END_REF]. It is the second cancer of the genito-urinary tract,just behind prostate cancerand it occurs at a median age of 60 years old. The highest incidence rates are found in industrialized areas such as Europe and Northern America,but also occurin North African countries. Various risk factors with different impact on incidence have been associated with urothelial cancers. It is now admitted that smoking is the most important one, as tobacco smoke contains various carcinogens including-naphtylamine and PAH (Polycyclic Aromatic Hydrocarbons). aromatic amines are essentially metabolized by the liver and metabolites are excreted via the urinary tract, leading to their retention into the bladder where they could exert their carcinogenic effects.It is known that bladder cancer incidence is much higher in men(approximately 4-fold higher than in women) [START_REF] Fajkovic | Impact of gender on bladder cancer incidence, staging, and prognosis[END_REF], a difference that was also evidenced in animal models (rats and mice) treated with nitrosamine derivatives [START_REF] Bertram | Specific induction of bladder cancer in mice by butyl-(4hydroxybutyl)-nitrosamine and the effects of hormonal modifications on the sex difference in response[END_REF][START_REF] Okajima | Effects of sex hormones on development of urinary bladder tumours in rats induced by N-butyl-N-(4-hydroxybutyl) nitrosamine[END_REF]. This gender disparity was first attributed to the higher proportion of smokers among men during the past century, but it turned out that tobacco could not completely explain such a disparity. Indeed, it was shown that age-standardized incidence of bladder cancer risk in neversmokers was also higher in men [START_REF] Stenzl | Treatment of muscle-invasive and metastatic bladder cancer: update of the EAU guidelines[END_REF], indicating that other risk factors may also be involved. These include exposure to other carcinogens or aromatic amines (pesticides in particular) through environmental and occupational exposures [START_REF] Burger | Epidemiology and risk factors of urothelial bladder cancer[END_REF]. Implication of hormonal factors has also been demonstrated in animal models in whichsex difference of bladder cancer development could be abolished bycastrating males or by treating females with testosterone [START_REF] Bertram | Specific induction of bladder cancer in mice by butyl-(4hydroxybutyl)-nitrosamine and the effects of hormonal modifications on the sex difference in response[END_REF]. A link has also been established with several genetic factors such as the status of enzymes involved in the metabolism of carcinogens. This is especially the case for the NAT enzymes (N-acetlytransferase 1 and 2) for which slow acetylating activity is associated with increased bladder cancer risk in human [START_REF] Marcus | Cigarette smoking, N-acetyltransferase 2 acetylation status, and bladder cancer risk: a case-series meta-analysis of a gene-environment interaction[END_REF]Marcus, Vineis, & Rothman, 2000) and for GSTA1 and GSTM1 (glutathion S-transferases) for which low activity null-genotypes were found associated with bladder cancer risks in association with smoking [START_REF] Matic | GSTA1, GSTM1, GSTP1, and GSTT1 polymorphisms and susceptibility to smoking-related bladder cancer: a case-control study[END_REF]. The implication of liver enzymes involved in the metabolism of aromatic amines such as the UGT enzymes involved in the Oor N-glucuronidation, and the SULT enzymes involved in sulfatation, have also been hypothesized but need further confirmation.Ethnicity may also have an impact on bladder cancer risk, as several studies evidenced a disparity indisease-specific survival between blacks and whites or other ethnic groups, with a poorer survival for black patients [START_REF] Yee | Ethnic differences in bladder cancer survival[END_REF]. Socioeconomic status, medical conditions (including chronic inflammation, or endemic parasitosis with schistosomes that is observed in Northern part of Africa) or more generally access to care, are some variables that could possibly explain such a difference, though further investigations are needed to validate these hypotheses and reduce this disparity. This is of outmost importance sincethe management of bladder cancers represents a real economic burden with a lifetime cost ranging from $100,000 to $120,000, the major part of the expenses being associated with surveillance and treatment of recurrences [START_REF] Avritscher | Clinical model of lifetime cost of treating bladder cancer and associated complications[END_REF].

Staging of bladder tumors is performed according to the TNM classification. It includes non muscle-invasive tumorsthat are classified into three groups: pTa papillary tumors from variable grade and pTis high grade flat tumors, that do not show invasion of the lamina propria and pT1 papillary tumors from variable grades where the lamina propria is invaded. Other tumors are high grade muscle-invasive tumors and are classified as pT2, pT3 or pT4 tumors. More than 90% of tumors are conventional urothelial carcinoma, the other subtype being urothelial carcinoma with aberrantdifferentiation such as squamous/glandular differenciation, small cell carcinoma, sarcomatoid carcinoma or micropapillary carcinoma [START_REF] Black | The impact of variant histology on the outcome of bladder cancer treated with curative intent[END_REF][START_REF] Kantor | Epidemiological characteristics of squamous cell carcinoma and adenocarcinoma of the bladder[END_REF].At the time of diagnosis 75 to 85% of the tumors are non-muscle invasive tumors, among which 60 to 70% will recur within the first year whereas and only 10 to 20% will progress to muscle-invasive and/or metastatic forms of the disease. Transurethral resection of the bladder represents the treatment of choice for non muscle-invasive tumors. Itcan be associated with endovesical instillation of mitomycin C in the case of moderate risk of recurrence or of BCG (Calmette Guérin Bacillus) in the case of high risk of recurrence or resistance to Mitomycin C [START_REF] Babjuk | EAU guidelines on non-muscleinvasive urothelial carcinoma of the bladder: update 2013[END_REF]. Cystectomy is used as a curative treatment for non-metastatic forms of muscle-invasive tumors, for non urothelial tumors or tumors that are refractory to bladder-conservatory treatments. It could also be proposed in the case of high risk non muscle-invasive tumors and in some palliative situations [START_REF] Witjes | EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines[END_REF].Half of the patients with muscle-invasive tumors (classified as pT2, pT3 or pT4) will develop a metastatic disease within two years. Unfortunately, the median survival of patients with lymph node metastasis is of 18 months and of only 15 months for those with distant metastases(von der [START_REF] Von Der Maase | Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer[END_REF].

Chemotherapies of bladder cancers

Urothelial bladder cancers are,to some extent, sensitive to chemotherapy with objective response rates reaching 50%. However these tumors cannot be cured and relapse is invariably observed following first line chemotherapy. Unfortunately, no improvement of overall survival could be achievedwithin the past 20 years. For locally advanced or metastatic forms of urothelial carcinoma, the best treatment remains cisplatin-based chemotherapies witha median survival of 14 to 15 months for eligible patients [START_REF] Bamias | Systemic chemotherapy in inoperable or metastatic bladder cancer[END_REF][START_REF] Von Der Maase | Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer[END_REF].For noneligible patients, several carboplatin-based chemotherapies have been tested with similar results [START_REF] Sio | Chemotherapeutic and targeted biological agents for metastatic bladder cancer: A comprehensive review[END_REF]. Other single-agent chemotherapies,or combinations of gemcitabine with these agents, have been the object of multiple phase II clinical trials (C. C. [START_REF] Lin | Systemic therapy for metastatic urothelial carcinoma[END_REF][START_REF] Sio | Chemotherapeutic and targeted biological agents for metastatic bladder cancer: A comprehensive review[END_REF]. These chemotherapies included the main classes of anticancer agents with drugs such as taxanes (docetaxel, paclitaxel), alkylating agents (ifosfamide, oxaliplatine), antimetabolites (5-FU, permetrexed), the topoisomerase I inhibitor topotecan, tyrosine kinase inhibitors (lapatinib, sorafenib, gefitinib, sunitinib) or the proteasome inhibitor bortezomib (J. J. [START_REF] Kim | Recent advances in treatment of advanced urothelial carcinoma[END_REF][START_REF] Lin | Systemic therapy for metastatic urothelial carcinoma[END_REF][START_REF] Sio | Chemotherapeutic and targeted biological agents for metastatic bladder cancer: A comprehensive review[END_REF]. Despite some encouraging response rates as compared to standard chemotherapies, no significant change inoverall survival could be noticed(J. J. [START_REF] Kim | Recent advances in treatment of advanced urothelial carcinoma[END_REF][START_REF] Lin | Systemic therapy for metastatic urothelial carcinoma[END_REF][START_REF] Sio | Chemotherapeutic and targeted biological agents for metastatic bladder cancer: A comprehensive review[END_REF].A phase III trial with the vinca-alkaloïd vinflunineassociated with best supportive care showed increased overall survival (6.9 vs 4.3 months) when compared to best supportive care alone [START_REF] Bellmunt | Long-term survival results of a randomized phase III trial of vinflunine plus best supportive care versus best supportive care alone in advanced urothelial carcinoma patients after failure of platinum-based chemotherapy[END_REF]. Vinflunine is currently the unique drug being approved by EMEA as second line treatment of UC.

As mentioned earlier, new chemotherapies are still under evaluation and results from phase II trials are awaited to define their positioning as compared to already approved regimen(Tables2,3&www.clinicaltrials.gov). Concerning taxanes, encouraging tumor response were noticed for the nanoparticule albumin-bound derivative of paclitaxel, abraxane. The phase II trial with cabazitaxel, a derivative that is less susceptible than docetaxel to the efflux pump ABCB1 (formerly called P-glycoprotein), is still ongoing and the phase II trial comparing larotaxel to gemcitabine/cisplatine combination was discontinued prematurely. Clinical trials withEGFR-targeted therapies such as cetuximab or gefitinib were disappointing and responses obtained with VEGF signaling inhibitors bevacizumab or sunitinib, or with the mTOR inhibitoreverolimus (Fig. 5) were not better than second line treatment (J. J. [START_REF] Kim | Recent advances in treatment of advanced urothelial carcinoma[END_REF][START_REF] Lin | Systemic therapy for metastatic urothelial carcinoma[END_REF][START_REF] Sio | Chemotherapeutic and targeted biological agents for metastatic bladder cancer: A comprehensive review[END_REF]. However, in the case of everolimus, a recent report clearly showed that patients with specific alterations of the TSC1 gene displayed significantly prolonged survival [START_REF] Iyer | Genome sequencing identifies a basis for everolimus sensitivity[END_REF], emphasizing the importance of the genetic background of the tumors in the response to chemotherapies, especially therapies that target the PI3K/AKT/mTOR pathway.

Deciphering the genetic background of urothelial cancers has been the object of numerous studies using "standard" approaches such as direct sequencing, CGH or CGH arrays, FISH analyses, or IHC.

Despite some variations in the number of patients included in these studies and the disparity in tumor subtypes that were analyzed, it was striking to note the recurrent nature of these alterations, both in terms of chromosome gains or losses, or in terms of point mutations. Losses or deletions were observed for chromosomes1q, 3p, 4p, 8p, 11p, 9pq, 12p, 13q, 15q, 16q, or 17q, leading to the inactivation of numeroustumor suppressor genes that are present in these loci (Guo, et al., 2013;[START_REF] Iyer | Genome sequencing identifies a basis for everolimus sensitivity[END_REF][START_REF] Lindgren | Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma[END_REF][START_REF] Mhawech-Fauceglia | Genetic alterations in urothelial bladder carcinoma: an updated review[END_REF][START_REF] Network | Comprehensive molecular characterization of urothelial bladder carcinoma[END_REF][START_REF] Richter | Chromosomal imbalances are associated with a high risk of progression in early invasive (pT1) urinary bladder cancer[END_REF][START_REF] Simon | Patterns of chromosomal imbalances in muscle invasive bladder cancer[END_REF]. As an example, frequent loss of 3p11 is associated with the inactivation of the fragile histidine triad (FIHT) gene that is frequently observed in late stage bladder cancers and has been associated with poor survival [START_REF] Skopelitou | FHIT gene expression in human urinary bladder transitional cell carcinomas[END_REF].

Interestingly, inactivation of FIHT by hypermethylation of its promoter was also observed in 16% of bladder tumors [START_REF] Maruyama | Aberrant promoter methylation profile of bladder cancer and its relationship to clinicopathological features[END_REF]. Deletion of 9pq and 10q are associated with the loss of CDKN2A and PTEN functions, respectively. Similarly, deletion of 13q is associated with the inactivation of the RB1 tumor suppressor gene involved in cell cycle control [START_REF] Smith | Bladder cancer clinical guidelines panel summary report on the management of nonmuscle invasive bladder cancer (stages Ta, T1 and TIS). The American Urological Association[END_REF]. Loss of RB1 and PTEN are linked with the progression of in situ carcinomas towards more aggressive high grade tumors and with reduced overall survival(Castillo-Martin, Domingo-Domenech, Karni-Schmidt, Matos, & Cordon-Cardo, 2010). In terms of chromosome amplifications, gainsof 1q, 3pq, 5p, 6p, 7q, 8pq, 11q, 12q, 17q, and 20q have been observed (Guo, et al., 2013;[START_REF] Iyer | Genome sequencing identifies a basis for everolimus sensitivity[END_REF][START_REF] Mhawech-Fauceglia | Genetic alterations in urothelial bladder carcinoma: an updated review[END_REF][START_REF] Network | Comprehensive molecular characterization of urothelial bladder carcinoma[END_REF][START_REF] Prat | Detection of chromosomal imbalances in papillary bladder tumors by comparative genomic hybridization[END_REF][START_REF] Richter | Chromosomal imbalances are associated with a high risk of progression in early invasive (pT1) urinary bladder cancer[END_REF][START_REF] Simon | Patterns of chromosomal imbalances in muscle invasive bladder cancer[END_REF]and are often accompanied by the overexpression of oncogenes, some of them being specifically associated with a subset of aggressive tumors. Among them one could cite MYC, TRIO, oncogenes from the receptor protein tyrosine kinase (RTK) family such as EGFR1, ERBB2, FGFR1 or MET, the cyclins CCND1and CCNE1involved in G1/S transition, transcription factors such as PPARG or the RB1-interactantE2F3, the E3-ligase MDM2, genes involved in the methylation of histones such as SETDB1 or genes involved in apoptosis such as BCL2L1 [START_REF] Goebell | Bladder cancer or bladder cancers? Genetically distinct malignant conditions of the urothelium[END_REF][START_REF] Mhawech-Fauceglia | Genetic alterations in urothelial bladder carcinoma: an updated review[END_REF]. Along with chromosome copy number variations, a series of gene rearrangements andactivating point mutations have also been identified in genes already known to drive tumor progression such as TP53, FGFR3, RB1, BRAF or HRAS [START_REF] Lindgren | Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma[END_REF][START_REF] Williams | Oncogenic FGFR3 gene fusions in bladder cancer[END_REF]. While TP53 mutations are characteristic from invasive tumors, frequency of FGFR3 amplificationsand activating point mutations reaches is more elevated in low grade tumors than in high grade muscle-invasive tumors (80% vs 50%). This suggested the existence of two distinct pathways implicating either FGFR3 or TP53 for tumor development [START_REF] Goebell | Bladder cancer or bladder cancers? Genetically distinct malignant conditions of the urothelium[END_REF][START_REF] Lindgren | Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma[END_REF]. Activating fusions of FGFR3 with the TACC3 or BAIAP2L1 genes were also observed in some cases and conferred enhanced sensitivity to FGFR inhibitors [START_REF] Williams | Oncogenic FGFR3 gene fusions in bladder cancer[END_REF][START_REF] Wu | Identification of targetable FGFR gene fusions in diverse cancers[END_REF].

The PI3K/AKT/mTOR signaling pathway was also found to be alteredin a substantial number of bladder tumors (more than 40% of urothelial carcinoma). As it will be detailed further, these alterations correspond to mutations of several key genes regulating this pathway, including the p110and the p85subunits of PI3K (referred to as PIK3CA and PIK3R1, respectively), AKT, PTEN, TSC1, TSC2, or LKB1 (Table 1).

With the development of new generation sequencing and the use of integrative approaches, a more defined picture of the genomic landscape of urothelial tumors could be obtained by several groups analyzing independent cohorts of patients (Guo, et al., 2013;[START_REF] Iyer | Genome sequencing identifies a basis for everolimus sensitivity[END_REF][START_REF] Lindgren | Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma[END_REF][START_REF] Mhawech-Fauceglia | Genetic alterations in urothelial bladder carcinoma: an updated review[END_REF][START_REF] Network | Comprehensive molecular characterization of urothelial bladder carcinoma[END_REF]. Reassuringly, most alterations that were observed in former systematic studies were confirmed and converged towards a classification of bladder cancers that would rely more on the level of alterations and the combination of genes that are altered,rather thanon the pathological gradingof the tumors [START_REF] Lindgren | Integrated genomic and gene expression profiling identifies two major genomic circuits in urothelial carcinoma[END_REF][START_REF] Network | Comprehensive molecular characterization of urothelial bladder carcinoma[END_REF].

These studies also confirmed significant rate of alterations in key genes involved in theRTK-MAPK-RAS-RAF and the PI3K/AKT/mTOR pathways(more than 40% for both pathways), further reinforcing their status of potential "targetable" pathways in the treatment of bladder cancers. Interestingly, these comprehensive studies also identified mutations in genes that were not found to be mutated at a significant level in other tumor types [START_REF] Network | Comprehensive molecular characterization of urothelial bladder carcinoma[END_REF]. For instance, mutations in genes involved in chromatin remodeling such as UTX, MLL-MLL3, CREBBP-EP300, NCOR1, ARID1A orCHD6 genes were detected in more than half of UC patients analyzed [START_REF] Gui | Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder[END_REF]. The Cancer Genome Atlas

Research Network study analyzing a cohort of 131 patients with high grade muscle-invasive urothelial bladder carcinoma, reported recurrent mutations in a series of genes including TP53, FGFR3, NF2, ARID1A, KDM6A, MLL2 (also called KMT2D), CDKN1A, ERCC2, STAG2, RXRA, ELF3, NFE2L2

(formerly called NRF2), KLF5, TXNIP, FOXQ1, RHOB, FOXA1, PAIP1, BTG2, ZFP36L1, RHOA and CCND3 [START_REF] Gui | Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder[END_REF]Guo, et al., 2013;[START_REF] Network | Comprehensive molecular characterization of urothelial bladder carcinoma[END_REF][START_REF] Platt | Spectrum of phosphatidylinositol 3-kinase pathway gene alterations in bladder cancer[END_REF][START_REF] Sjodahl | A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in TSC2 and PIK3R1[END_REF], among which 9 (CDKN1A, ERCC2, RXRA, ELF3, KLF5, FOXQ1, RHOB, PAIP1, BTG2) were not found to be significantly altered in any other types of cancers [START_REF] Network | Comprehensive molecular characterization of urothelial bladder carcinoma[END_REF]. More recently, a truncating mutation associated with inactivation of the STAG2gene involved in chromosome segregation was identified in 36% of non-invasive bladder cancers that were not prone to recurrences, and in 16% of invasive tumors [START_REF] Solomon | Frequent truncating mutations of STAG2 in bladder cancer[END_REF]. Together, these results open new area of research in the identification of potential targetable mutations that could be used for the development of new treatments for urothelial carcinoma of the bladder.

Alterations of the PI3K/AKT/mTOR pathway as targets in urothelial carcinomas

As mentioned earlier, up to 40% of urothelial carcinomas of the bladder display constitutive activation of the PI3K/AKT/mTOR pathway [START_REF] Bambury | Actionable mutations in muscle-invasive bladder cancer[END_REF][START_REF] Ching | Expanding therapeutic targets in bladder cancer: the PI3K/Akt/mTOR pathway[END_REF]Knowles, et al., 2009) resulting from either inactivating deletions or mutations of tumor suppressor genes such as PTEN or TSC1, or activating amplifications or mutations of proto-oncogenes such as PIK3CA or AKT1. These alterations are found independently of the urothelial carcinoma subtype, which probably indicates their causal role in tumorigenesis. Table 1 provides an updated detailed inventory of the alterations inthe key genes involved in the PI3K/AKT/mTOR pathway that have been reported in bladder tumors from a variety of patient cohorts. Overall, it is remarkable to observe similar mutation rates across studies, despite a heterogeneity in the proportions of patients with non muscle-invasive vs muscle-invasive tumors that were included in these studies and regardless of the technique used to determine these alterations.It is also interesting to note the discrepancy in mutation rates among the different factors implicated in the pathway, as no alteration of mTOR could be observed while PIK3CA, the p110 subunit of PI3K showeda very high rate of mutations(around 20% for most studies) that is unique to bladder cancers. Mutations of PIK3CA are predominantly located within hotspots such as E542 and E545 residues within the helical domain, and H1047 in the kinase domain of the protein, mutations of other residues being observed at much lower frequency.It is interesting to note that these activating mutations can confer significant advantages in terms of proliferation, cell motility, cell migration, and resistance to anoikis when expressed in normal human urothelial cells [START_REF] Ross | PIK3CA mutation spectrum in urothelial carcinoma reflects cell context-dependent signaling and phenotypic outputs[END_REF]. Several mutations in PIK3R1 (the p85 subunit of PI3K) were also observed (Table 1). These mutations are predominantly located within the BH domain of interaction with PTEN, or in the SH2 domain of the protein that is involved in its interaction with p110, indicating they oncogenic role in urothelial carcinoma via the deregulation of p110 and PTEN functions. In the case of LKB1, a single study reported a missense mutation at serine 19 resulting in early stop codon for approximately 80% of the 50 patients included in the study [START_REF] Tigli | LKB1 mutations and their correlation with LKB1 and Rheb expression in bladder cancer[END_REF]. It remains to be seen whether this high mutation rate is linked to the specificity of this cohort, or whether this is a recurrent alteration in bladder cancers.

Loss of PTEN or reduced expression of PTEN represents the most frequent alteration that can be found in bladder cancers. Most studies investigating PTEN status reported loss of heterozygocity at significant rates, whereasinactivating point mutations are relatively rare (Table 1). PTEN deficiency is often associated with the activation of the PI3K/AKT/mTOR pathwaywhich explains why PTEN negative tumors showed better responses to mTOR inhibitors, a rationale that have been used to test these derivatives in bladder cancers [START_REF] Neshat | Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR[END_REF][START_REF] Podsypanina | An inhibitor of mTOR reduces neoplasia and normalizes p70/S6 kinase activity in Pten+/-mice[END_REF][START_REF] Shi | Enhanced sensitivity of multiple myeloma cells containing PTEN mutations to CCI-779[END_REF][START_REF] Steelman | Suppression of PTEN function increases breast cancer chemotherapeutic drug resistance while conferring sensitivity to mTOR inhibitors[END_REF].However, one should be cautious about the impact of PTEN loss, as a phase II trial investigating the mTOR inhibitor everolimus in patients with locally advanced or metastatic transitional cell carcinoma of the urothelial tract found an unexpected correlation between PTEN loss and resistance to the drug [START_REF] Seront | Phase II study of everolimus in patients with locally advanced or metastatic transitional cell carcinoma of the urothelial tract: clinical activity, molecular response, and biomarkers[END_REF]. In fact, PTEN loss was accompanied with AKT activation in response to mTOR inhibition by a negative feedback loop, the mechanism of which remains to be fully elucidated [START_REF] Seront | PTEN deficiency is associated with reduced sensitivity to mTOR inhibitor in human bladder cancer through the unhampered feedback loop driving PI3K/Akt activation[END_REF]. More recently, a retrospective study analyzing PTEN status (among other markers of the PI3K/AKT pathway) in patients with advanced endometrial carcinoma treated by everolimus concluded that expression of proteins of the PI3K/mTOR pathway could not predict response tothis mTOR inhibitor [START_REF] Tredan | Predicting everolimus treatment efficacy in patients with advanced endometrial carcinoma: a GINECO group study[END_REF]. Further studies are clearly needed to investigate the place of PTEN status in response to anticancer drugs targeting the PI3K/AKT/mTOR pathway in urothelial cancers.

AKT1 was also found to be mutated in bladder cancers, at a rate not exceeding 5% (Table 1), with a specific hotspot concerning the E17K substitution in the PH domain of the protein [START_REF] Carpten | A transforming mutation in the pleckstrin homology domain of AKT1 in cancer[END_REF]. This mutation results in constitutive activation of AKT1 and its localization to the plasma membrane independently of upstream signaling [START_REF] Carpten | A transforming mutation in the pleckstrin homology domain of AKT1 in cancer[END_REF]. It is also mutually exclusive from PIK3CA mutations and PTEN loss. As far as RHEB is concerned, only a single missense mutation (Y35C) could be detected in one study analyzing 99 tumors of patients with transitional cell carcinoma (Guo, et al., 2013).

As revealed by several studies TSC1 and TSC2 genes that are upstream of mTOR and control its activity via RHEB, were found to be mutated at significant rates in bladder cancers, regardless of the tumor status (Table 1). TSC1 displayed a wide spectrum of mutations including nonsense, missense, frameshift, and splicing mutations, as well as in-frame deletions.These inactivating mutations are distributed throughout the TSC1 gene and may in principle affect all the functions of the corresponding protein [START_REF] Network | Comprehensive molecular characterization of urothelial bladder carcinoma[END_REF]. In general the tumors bearing these mutations also showed a concomitant loss of the wild-type allele [START_REF] Platt | Spectrum of phosphatidylinositol 3-kinase pathway gene alterations in bladder cancer[END_REF]. LOH was also observed for TSC2, but conversely to TSC1, fewer inactivating point mutations could be observed (Table 1). In patients with TSC2 mutations, no concomitant TSC1 mutations could be observed [START_REF] Sjodahl | A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in TSC2 and PIK3R1[END_REF].

Drugs targeting the PI3K/AKT/mTOR signaling pathway

The drugs that are known to target the PI3K/AKT/mTOR pathway are detailed in figures 2-6. They can be classified in five categories depending on their relative specificity towards each target of the pathway. They include the PI3K inhibitors (Fig. 2), the PI3K/mTOR dual inhibitors (Fig. 3), the AKT inhibitors (Fig. 4), the mTOR inhibitors(Fig. 5) and the PDK1 inhibitors (Fig. 6). Table 2 gives a list of all these inhibitors depending on their targets and the actual stage of their development in various types of cancers.

PI3K inhibitors

Wortmannin and the quercetin derivative LY-294002 were the first derivatives to be used as PI3K

inhibitors, but because of their lack of specificity, they were only used as research tools and were not pushed further into clinic. Many other pan-isoforms PI3K inhibitors with various chemical structures have been identified and are tested in clinical trials [START_REF] Rodon | Development of PI3K inhibitors: lessons learned from early clinical trials[END_REF].

They include a series of wortmannin derivativessuch as the pegylated-17-hydroxywortmannin PWT-458that is more stable, the structural analog PX-866 that irreversibly inhibits PI3Ks [START_REF] Ihle | Molecular pharmacology and antitumor activity of PX-866, a novel inhibitor of phosphoinositide-3-kinase signaling[END_REF]and is in phase II clinical trial for various solid tumors. In phase I, PX-866 showed some partial responses and disease stabilizations when combined with docetaxel, but no significant association between PIK3CA status and the extentof PFS could be observed [START_REF] Bowles | A multicenter phase 1 study of PX-866 in combination with docetaxel in patients with advanced solid tumours[END_REF].For the peptide derivative of LY-294002, SF1126, in vitro and in vivo preclinical data showed promising activities in the treatment of multiple myelomas [START_REF] De | An integrin-targeted, pan-isoform, phosphoinositide-3 kinase inhibitor, SF1126, has activity against multiple myeloma in vivo[END_REF].GDC-0941 that inhibits PI3K and also targets HIF1, has been successfully used in combination with MEK inhibitors for the treatment of non-small cell lung cancers [START_REF] Zou | The novel dual PI3K/mTOR inhibitor GDC-0941 synergizes with the MEK inhibitor U0126 in non-small cell lung cancer cells[END_REF]. BAY 80-6946 also inhibits both PI3K  and  and showed peculiar efficiency in tumors with activated PI3K(N. Liu, et al., 2013). NVP-BKM120

(Buparlisib) [START_REF] Maira | Identification and characterization of NVP-BKM120, an orally available pan-class I PI3-kinase inhibitor[END_REF]has been tested in multiple advanced solid tumors [START_REF] Rodon | Phase I dose-escalation and -expansion study of buparlisib (BKM120), an oral pan-Class I PI3K inhibitor, in patients with advanced solid tumors[END_REF], including bladder cancer as second line treatment (Table 3). XL-147 was tested in patients with refractory advanced solid malignancies. As expected, it inhibits the PI3K/AKT/mTOR pathway, but was also found toinhibit the MEK/ERK pathway significantly [START_REF] Shapiro | Phase I safety, pharmacokinetic, and pharmacodynamic study of SAR245408 (XL147), an oral pan-class I PI3K inhibitor, in patients with advanced solid tumors[END_REF].Other derivatives specifically inhibit class I PI3K such as ZSTK474 or CH5132799. Preclinical data showed promising activity in solid tumors for ZSTK474 accompanied with anti-inflammatory effects [START_REF] Yaguchi | Antitumor activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibitor[END_REF]. CH5132799 had also an inhibitory activity towards PI3Kαmutants in vitro and showed activity in tumors harboring PIK3CA mutations [START_REF] Tanaka | The selective class I PI3K inhibitor CH5132799 targets human cancers harboring oncogenic PIK3CA mutations[END_REF]. Other compounds were reported to specifically target a single isoform of PI3K. For instance, PIK75 [START_REF] Knight | A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling[END_REF]and NVP-BYL719 inhibit p110α, NVP-BYL719 being the object of numerous trials in advanced tumors alone or in combinations with MEK inhibitors [START_REF] Furet | Discovery of NVP-BYL719 a potent and selective phosphatidylinositol-3 kinase alpha inhibitor selected for clinical evaluation[END_REF].GSK-2636771 and TGX221 specifically inhibit p110 . Of particular interest is the specific inhibitor of p110 , AS-252424, that prevents the growth of T24 bladder cancer cells induced by the stimulation of kinin receptors which is associated with the activation of the PI3K/AKT pathway [START_REF] Sgnaolin | Functional and molecular characterization of kinin B1 and B 2 receptors in human bladder cancer: implication of the PI3Kgamma pathway[END_REF]. AS-252424 is thus representing an attractive option for the treatment of bladder cancers with constitutive activation of PI3K. Finally, the IC87114 compound and its oral form derivative CAL-101 specifically target PI3K, an isoform that is specifically expressed in lymphocytes, providing a special interest to develop these derivatives in the treatment of hematological malignancies. CAL-101 inhibits the PI3K signaling by preventing AKT1 phosphorylation at threonine 308 [START_REF] Lannutti | CAL-101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the treatment of Bcell malignancies, inhibits PI3K signaling and cellular viability[END_REF] and provided approximately 30% partial responses in chronic lymphocytic leukemia. Recently, the characterization of GS-9820 as a new selective inhibitor of PI3Kemphasized the role of this PI3K isoform in the regulation of osteoclast morphology, actin cytoskeletalorganization, and resorptive activity [START_REF] Shugg | Effects of isoform-selective phosphatidylinositol 3-kinase inhibitors on osteoclasts: actions on cytoskeletal organization, survival, and resorption[END_REF].

Dual PI3K-mTOR inhibitors

The similarity between mTOR and PI3K ATP catalytic site has rendered possible to design compounds that could inhibit both kinases (including both mTORC1 and mTORC2 complexes), resulting in higher efficiency than rapamycin analogs alone, a phenomenon that could be explained by the negative feed-back function of mTOR,the inhibition of whichcan activate PI3K via S6K and the RAS/RAF/MEK/ERK pathway (Fig. 1) [START_REF] Engelman | Targeting PI3K signalling in cancer: opportunities, challenges and limitations[END_REF]. Therefore, inhibiting both mTOR and PI3K could prevent such a feed-back loop and enhance treatment efficacy. In this line, combination with MEK inhibitors could be of interest, though it is not known whether cumulative toxicities could be tolerable. Another interesting feature resides in the fact that dual inhibitors could also block the proliferation of cancer cells with PIK3CA activating mutations [START_REF] Brachmann | PI3K and mTOR inhibitors: a new generation of targeted anticancer agents[END_REF]. The first dual PI3K/mTOR inhibitor to be described is PI-103. It belongs to the pyridofuropyrimidine family and inhibits class I PI3K at nanomolar ranges and mTOR at submicromolar concentrations [START_REF] Raynaud | Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases[END_REF]. PI-103 was active against a wide range of cancer cell lines and induced tumor delay in xenograft models despite an extensive metabolization [START_REF] Raynaud | Pharmacologic characterization of a potent inhibitor of class I phosphatidylinositide 3-kinases[END_REF].PI-103 showed promising activity in AML and in glioblastoma and could potentiate the effects of radiotherapy and chemotherapy [START_REF] Prevo | Class I PI3 kinase inhibition by the pyridinylfuranopyrimidine inhibitor PI-103 enhances tumor radiosensitivity[END_REF][START_REF] Westhoff | The pyridinylfuranopyrimidine inhibitor, PI-103, chemosensitizes glioblastoma cells for apoptosis by inhibiting DNA repair[END_REF]. The PI-103

derivative WJD008 shows enhanced inhibitory activity towards p110 and mTOR and was found to suppress the growth of transformed cells harboring PIK3CA activating mutations [START_REF] Ponzo | The Met receptor tyrosine kinase and basal breast cancer[END_REF]. The imidazoquinoline derivativeNVP-BEZ235 inhibits all forms of PI3K and was active in several preclinical tumor models including breast cancer cells expressing the PIK3CA activating mutation H1047R [START_REF] Serra | NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations[END_REF].NVP-BEZ235 shows synergistic or additive effects with several cytotoxic agents in cancer cell lines or in xenograft models [START_REF] Baumann | The novel orally bioavailable inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin, NVP-BEZ235, inhibits growth and proliferation in multiple myeloma[END_REF][START_REF] Manara | NVP-BEZ235 as a new therapeutic option for sarcomas[END_REF]. Interestingly, NVP-BEZ235 and the new derivative NVP-BTG226 also enhanced radiosenstivity in cancer cell lines including the HRAS mutated T24 bladder cancer cell line [START_REF] Fokas | NVP-BEZ235 and NVP-BGT226, dual phosphatidylinositol 3kinase/mammalian target of rapamycin inhibitors, enhance tumor and endothelial cell radiosensitivity[END_REF]. Open label phase I with NVP-BEZ235showed activity in patients with activated PI3K despite a need for pharmacokinetic improvement (H. [START_REF] Burris | First-in-human phase I study of the oral PI3K inhibitor BEZ235 in patients (pts) with advanced solid tumors[END_REF]. Three morpholino 1,3,5-triazine PKI compounds, PKI-402, PKI-587 (also known as PF-0521238) and its orally available derivative PKI-179also showed potent inhibitory activity against class I PI3K (including mutant PIK3CA) and mTOR [START_REF] Mallon | Antitumor efficacy profile of PKI-402, a dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor[END_REF][START_REF] Venkatesan | PKI-179: an orally efficacious dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor[END_REF][START_REF] Yuan | PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity[END_REF]. These derivatives are active in various tumor cell models in vitro and in vivo and the results of clinical trials are awaited. PF-04691502 is an orally available dual PI3K/mTOR inhibitor that showed potent inhibition of PIK3CA-mutant and PTEN-null cell lines proliferation [START_REF] Yuan | PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity[END_REF]. Results of phase I study demonstrated PF-04691502 efficacy to decrease the phosphorylation of downstream effectors of PI3K in tumor tissue [START_REF] Britten | Phase I study of PF-04691502, a small-molecule, oral, dual inhibitor of PI3K and mTOR, in patients with advanced cancer[END_REF].XL-765 is a dual inhibitor that prevents AKT phosphorylation induced by PI3K/mTORC2 [START_REF] Prasad | Inhibition of PI3K/mTOR pathways in glioblastoma and implications for combination therapy with temozolomide[END_REF]. XL-765 has been developed as monotherapy or in combination with other chemotherapy for the treatment of a broad range of malignancies and is the object of many ongoing clinical trials. The morpholino thienopyrimidine derivatives GDC-0980 and its analog GNE-477 [START_REF] Heffron | Identification of GNE-477, a potent and efficacious dual PI3K/mTOR inhibitor[END_REF] represent another class of dual PI3K/mTOR inhibitors that are active in activated PI3K cancer cell models [START_REF] Wallin | GDC-0980 is a novel class I PI3K/mTOR kinase inhibitor with robust activity in cancer models driven by the PI3K pathway[END_REF].

Clinical trials are currently ongoing in advanced solid tumors and Non-Hodgkin's lymphoma. Similarly the two dual inhibitors GSK-2126458 and GSK-1059615are in clinical trials for advanced solid tumors and lymphoma.

AKT inhibitors

Few AKT inhibitors have been developed so far, probably because of the secondary effects linked to the multiple regulatory functions of AKT (Table 1). The alkylphosphocholine perifosine (KRX-0401) targets the pleckstrin homology domain of AKT, leading to the inhibition of PDK1-dependent activation of AKT and the inhibition of its translocation to the cell membrane [START_REF] Krawczyk | Perifosine--a new option in treatment of acute myeloid leukemia?[END_REF]. It is active in numerous cancer lines and potentiates radiotherapy and cell response to various chemotherapies. Perifosine has been the object of extensive number of clinical trials alone or in combination but the results of two phase III trials in colorectal cancer and multiple myeloma failed to show efficacy. The MK-2206 derivative is an allosteric inhibitor that also targets the pleckstrin homology domain of AKT. It is particularly active in a subset of colorectal cancer cells that are dependent on IGF-1R and was recently shown to exert apoptotic effects via two distinct mechanisms:

the induction of apoptosis inducing factor (AIF), and the inhibition of the cytoskeletal protein Ezrin [START_REF] Agarwal | Akt inhibitor MK-2206 promotes anti-tumor activity and cell death by modulation of AIF and Ezrin in colorectal cancer[END_REF]. MK-2206 is being tested alone or in combination in many types of solid tumors and hematological malignancies and provided some encouraging antitumoral activity. The mechanism of AKT inhibition by Triciribine (API-2) is not well defined, but preclinical studies clearly demonstrate its capability to inhibit the phosphorylation of all AKT members and the growth of tumor xenografts models overexpressing AKT [START_REF] Mirzoeva | Autophagy suppression promotes apoptotic cell death in response to inhibition of the PI3K-mTOR pathway in pancreatic adenocarcinoma[END_REF]. GSK-690693 is anATP-competitive specific inhibitor of AKT members, and has shown significant activity in various tumor models [START_REF] Rhodes | Characterization of an Akt kinase inhibitor with potent pharmacodynamic and antitumor activity[END_REF], but the clinical trials were terminated.GDC-0068 is a very selective ATP-competitive inhibitor of all AKT members. It showed robust anticancer activity in vitro and in xenograft models, and this activity was correlated with various markers of AKT activation (J. [START_REF] Lin | Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models[END_REF]. Furthermore, GDC-0068 enhanced cell response to other classical anticancer agents (J. [START_REF] Lin | Targeting activated Akt with GDC-0068, a novel selective Akt inhibitor that is efficacious in multiple tumor models[END_REF]. KP372-1 is an equal mixture of two isomers that inhibit AKT, PDK1 and FLT3 (Zeng, et al., 2006). By inhibiting the AKT downstream targets and the FLT3/PIM signaling, these small moleculeswere efficiently inhibiting AML cell growth while barely affecting normal hematopoietic cells (Zeng, et al., 2006).A-443654 is an indazole-pyridine basedhighly selective AKT inhibitor with a narrow therapeutic window [START_REF] Luo | Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo[END_REF] that exerts its cytotoxicity by interfering with mitotic progression and bipolar spindle formation via the inhibition of the promoter activity of Aurora Akinase (X. [START_REF] Liu | Akt inhibitor a-443654 interferes with mitotic progression by regulating aurora a kinase expression[END_REF].AZD-5363 inhibits all three isoforms of AKTs and P70S6K at the same concentration. It inhibits the growth of a wide range of cancer cell lines and sensitivity to the drug was correlated with the presence of activating PIK3CA mutations, or with PTEN loss or inactivating mutation [START_REF] Davies | Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background[END_REF].AZD5363 is active in tumor xenografts, as monotherapy or in combination with docetaxel and has entered several clinical trials for solid malignancies [START_REF] Davies | Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background[END_REF]. XL-418 was shown to inhibit both AKT and S6K but the only clinical trial in solid tumors with this molecule was suspended.

mTOR inhibitors

Rapamycin and its analogs (rapalogs) were the first selective mTOR inhibitors to enter into the clinic. They act as allosteric inhibitors of mTORC1 via their association to the FKBP12 protein, resulting in the dissociation of the complex and subsequent inhibition of mTOR signaling [START_REF] Lamming | Rapalogs and mTOR inhibitors as antiaging therapeutics[END_REF]. Everolimus and temsirolimus are approved by the FDA for the treatment of several cancers including renal cell carcinoma, lymphoma, astrocytoma and pancreatic cancers [START_REF] Benjamin | Rapamycin passes the torch: a new generation of mTOR inhibitors[END_REF]. The ridaforolimus is in clinical trial for the treatment of sarcomas [START_REF] Chawla | Phase II study of the mammalian target of rapamycin inhibitor ridaforolimus in patients with advanced bone and soft tissue sarcomas[END_REF]. It is interesting to note that sensitivity to rapalogs was associated with the presence of activating PIK3CA mutations, or with PTEN inactivation due to PTEN loss or the presence of missense mutations [START_REF] Carew | Mechanisms of mTOR inhibitor resistance in cancer therapy[END_REF]. Activity of rapalogs is also probably linked to their antiangiogenic effects as treatment of renal cancer cells induced a marked reduction of HIF-1 levels [START_REF] Mahalingam | Vorinostat enhances the activity of temsirolimus in renal cell carcinoma through suppression of survivin levels[END_REF].

Besides rapalogs, a variety of other mTOR inhibitors have been developed and can target both mTORC1 and mTORC2 complexes. This is the case for PP-242 and its derivative INK-128 that are potent and selective ATP competitor of both complexes and have shown antitumoral activity against a variety of cancer types. It is surmised that the greater effect of PP-242 over rapamycin in multiple myeloma cell lines could be attributed to the inhibition of mTORC2 [START_REF] Hoang | Targeting TORC2 in multiple myeloma with a new mTOR kinase inhibitor[END_REF]. INK-128 has entered clinical trials for advanced solid malignancies and multiple myelomas. OSI-027and

OXA-01 are other selective inhibitors of mTORC1 and mTORC2 complexes and inhibit the phosphorylation of Akt and of several downstream targets of mTORc1 such as S6K, S6 riboprotein, and 4EBP1 [START_REF] Bhagwat | Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin[END_REF][START_REF] Falcon | Reduced VEGF production, angiogenesis, and vascular regrowth contribute to the antitumor properties of dual mTORC1/mTORC2 inhibitors[END_REF]. Preclinical data demonstratedthat treatment with OSI-027 reduced cell proliferation in various cancer cell lines, including bladder cancer cells in which association with lapatinib was highly synergistic [START_REF] Becker | The combination of an mTORc1/TORc2 inhibitor with lapatinib is synergistic in bladder cancer in vitro[END_REF][START_REF] Bhagwat | Preclinical characterization of OSI-027, a potent and selective inhibitor of mTORC1 and mTORC2: distinct from rapamycin[END_REF].

Treatment with OXA-01 also showed significant anti-angiogenic effects and slowed vessel regrowth after discontinuation of VEGFR inhibition [START_REF] Falcon | Reduced VEGF production, angiogenesis, and vascular regrowth contribute to the antitumor properties of dual mTORC1/mTORC2 inhibitors[END_REF]. A phase I with OSI-027 in lymphoma or advanced solid tumors has been completed and showed some antitumoral activity [START_REF] Tan | First-in-human phase I study exploring three schedules of OSI-027, a novel small molecule TORC1/TORC2 inhibitor, in patients with advanced solid tumors and lymphoma[END_REF].

Palomid 529 is a pan mTOR inhibitor with strong antitumor effects associated with potent antiangiogenic effects, but is only tested in patients with age-related macular degeneration. The Ku0063794 derivative is a highly selective inhibitor of mTORC complexes and inhibited downstream effectors of mTOR such as AKT and S6 [START_REF] Pike | Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: the discovery of AZD8055 and AZD2014[END_REF]. The relatively low potency of Ku0063794 in cell models led to its optimization and the identification of the clinical derivatives AZD-8055 and AZD-2014 [START_REF] Pike | Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: the discovery of AZD8055 and AZD2014[END_REF]. These compounds retained a high selectivity towards mTOR but showedimproved aqueous solubility and cellular potency. The AZD-2014 was developed from AZD-8055 in order to improve the pharmacokinetic parameters of the compound especially to reduce its turnover in human hepatocytes [START_REF] Pike | Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: the discovery of AZD8055 and AZD2014[END_REF]. AZD-2014 shows a potent activity towards colon cancer cells in vitro and in vivo and seems to induce an autophagic death rather than apoptosis [START_REF] Huo | Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014[END_REF]. Both compounds are currently in clinical trials for advanced solid malignancies.

A series of pyrazolopyrimidine, WAY-600, WYE-132, WYE-687, and WYE-354 have also been tested

and specifically inhibited both mTORC complexes at nanomolar ranges in vitro, a selectivity that was retained in various cancer cell lines [START_REF] Yu | Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2[END_REF][START_REF] Yu | Biochemical, cellular, and in vivo activity of novel ATPcompetitive and selective inhibitors of the mammalian target of rapamycin[END_REF].These compounds were also active in xenograft models and may enter clinical trials.

Torin 1 is apyridinonequinoline derivative isolated from a screen to identify selective mTOR inhibitors [START_REF] Thoreen | An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1[END_REF]. Torin1 exerts its activity by interacting with the unique Trp2239 residue of mTOR within the catalytic site of the kinase [START_REF] Yang | mTOR kinase structure, mechanism and regulation[END_REF]. Torin2 is an orally available derivative of Torin1 with improved metabolic stability and plasma exposure (Q. [START_REF] Liu | 1-(3-(trifluoromethyl)phenyl)benzo[END_REF].

Torin2 inhibits mTORC1-dependent phosphorylation of S6K but, unlike Torin1, also targets the PIKKsATM, ATR and DNA-PK, sensitizing cells to ionizing radiations(Q. Liu, et al., 2013). Torin2 displayed a potent activity against a panel of lung cancer cell lines inducing both apoptosis and autophagy, and showed synergistic activity in combination with a MEK inhibitor (Q. Liu, et al., 2013).

PDK1 inhibitors

NVP-BAG956 is an imidazoquinoline derivative which targets PDK1 as well as PI3K. As compared to all other classes of inhibitors of the mTOR pathway, NVP-BAG956 was the most potent agent against T-ALL cell lines and potentiated the antileukemic activity of both nilotinib and RAD001 in vivo [START_REF] Weisberg | Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL-and mutant FLT3-expressing cells[END_REF]. OSU-03012 (AR-12) was synthesized based on the celecoxib backbone, but does not retain the inhibitory effect on COX2 [START_REF] Zhu | From the cyclooxygenase-2 inhibitor celecoxib to a novel class of 3phosphoinositide-dependent protein kinase-1 inhibitors[END_REF]. Though OSU-03012 can inhibit PDK1 and AKT signaling at specific concentrations, various studiessuggested that activation of ERstress proteins and modulation of HSP70 and HSP90 chaperones could also participate to its cellular toxicity in various cancer types. OSU-03012 showed synergistic effects with lapatinib in glioma and breast cancer cells [START_REF] Booth | OSU-03012 interacts with lapatinib to kill brain cancer cells[END_REF][START_REF] West | OSU-03012 sensitizes breast cancers to lapatinib-induced cell killing: a role for Nck1 but not Nck2[END_REF]. OSU-03012 is in clinical trial for lymphoma and solid tumors.

BX-912, BX-320, and BX-795 were synthesized from an amidopyrimidine backbone and identified as PDK1 selective inhibitors that could block the PDK1 activity in tumor cells and induce cell growth inhibition and/or apoptosis in vitro, BX-320 being also active in in vivo tumor models [START_REF] Feldman | Novel small molecule inhibitors of 3-phosphoinositide-dependent kinase-1[END_REF]. More recently, it was shown that BX-795 could also achieve a G2 block in PDK1-/-cells, suggesting that other targets could be responsible for its cellular toxicity [START_REF] Tamguney | Analysis of 3-phosphoinositidedependent kinase-1 signaling and function in ES cells[END_REF].Indeed BX-795 also inhibits Cdk1,Cdk2, and Aurora A, B and C with similar potencies [START_REF] Tamguney | Analysis of 3-phosphoinositidedependent kinase-1 signaling and function in ES cells[END_REF]. Nevertheless, these BX derivatives represent interesting optional treatments to be tested in the clinic. As compared to other inhibitors described above, the small molecule GSK-2334470 displayed a much higher selectivity towards PDK1 and showed a potent cytotoxicity towards glioblastoma and renal cancer cell lines [START_REF] Najafov | Characterization of GSK2334470, a novel and highly specific inhibitor of PDK1[END_REF].

5.The use of PI3K/AKT/mTOR inhibitors to treat bladder cancers

As already mentioned earlier, the plethora of genomic alterations of the PI3K/AKT/mTOR signaling pathway that were identified in bladder tumor samples emphasized the importance of this pathway in the onset and/or the progression of urothelial carcinoma, pointing towards its use as a potential target.

Preclinical studies showed the efficacy of rapamycin in inhibiting the growth of bladder cancer cells in vitro [START_REF] Fechner | Rapamycin inhibits in vitro growth and release of angiogenetic factors in human bladder cancer[END_REF]. Similar results were also observed for everolimus (RAD001) that could significantly inhibit the growth of nine bladder tumor cell line [START_REF] Mansure | Inhibition of mammalian target of rapamycin as a therapeutic strategy in the management of bladder cancer[END_REF], and in xenograft models, an effect that was accompanied with inhibition of the downstream effectors of mTOR such as inhibition of protein synthesis through the S6K and 4EBP1 pathways, and reduction in angiogenesis [START_REF] Chiong | Effects of mTOR inhibitor everolimus (RAD001) on bladder cancer cells[END_REF][START_REF] Mansure | Inhibition of mammalian target of rapamycin as a therapeutic strategy in the management of bladder cancer[END_REF]. These data represented a strong biological rationale to use inhibitors of the PI3K-AKT-mTOR pathway in bladder cancers. However, only few clinical trials have been performed, probably because of the low number of patients with metastatic disease and the relative fragility of these patients. mTOR inhibitors everolimus and temsirolimus that were approved in renal and breast cancers have been tested in small series of patients with advanced bladder cancers. These trials were performed in heavily pretreated patients who were refractory to platinum salts. The results were globally disappointing despite some long-term responses for several patients (Table 3). In one phase II trial, disease control was maintained up to 14 months and was even observed for patients with poor prognostic factors [START_REF] Seront | Phase II study of everolimus in patients with locally advanced or metastatic transitional cell carcinoma of the urothelial tract: clinical activity, molecular response, and biomarkers[END_REF]. Interestingly, more than half of non-responder patients showed no expression of PTEN and PTEN loss was only observed in non-responders patients, emphasizing the potential role of PTEN as a biomarker of tumor response to everolimus. Similar results were obtained in the other phase II trial with a median progression free survival of 2.6 months and a median overall survival of 8.3 months [START_REF] Milowsky | Phase II study of everolimus in metastatic urothelial cancer[END_REF]. In order to investigate the molecular basis for specific responses to everolimus, whole genome sequencing of 14 patients included in the phase II trial of Seront et al. were performed restrospectively and evidenced an inactivating mutation of the TSC1 gene [START_REF] Iyer | Genome sequencing identifies a basis for everolimus sensitivity[END_REF]. Further analysis of 5 patients with significant responses to everolimus revealed that 4 patients were also mutated for TSC1, response rate being different depending on the mutation [START_REF] Iyer | Genome sequencing identifies a basis for everolimus sensitivity[END_REF]. This discovery further reinforced the importance of the PI3K/AKT/mTOR signaling pathway as a potential attractive target for bladder cancer, and positioned everolimus as an interesting option for patients with inactivated TSC1. However, it is important to keep in mind that tumors with TSC1 mutations presented different genetic backgrounds that could also explain the differences in patients'

responses to everolimus, in particular mutations that were identified at significant rates as mentioned in section 3.3 [START_REF] Gui | Frequent mutations of chromatin remodeling genes in transitional cell carcinoma of the bladder[END_REF]Guo, et al., 2013;[START_REF] Network | Comprehensive molecular characterization of urothelial bladder carcinoma[END_REF][START_REF] Platt | Spectrum of phosphatidylinositol 3-kinase pathway gene alterations in bladder cancer[END_REF][START_REF] Sjodahl | A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in TSC2 and PIK3R1[END_REF]). To cite one example,activating mutations of NFE2L2, a transcription factor that regulates the anti-oxidant program in response to oxidative stress [START_REF] Shibata | Cancer related mutations in NRF2 impair its recognition by Keap1-Cul3 E3 ligase and promote malignancy[END_REF]was associated with enhanced expression of genes involved in ROS-induced detoxification that could in turn have an impact on everolimus response as mTOR pathway is also involved in the regulation of oxidative stress [START_REF] Darzynkiewicz | In search of antiaging modalities: Evaluation of mTOR-and ROS/DNA damage-signaling by cytometry[END_REF]. In this line, alterations leading to activating or inactivating mutations of other genes that were significantly altered in bladder cancer may also influence tumor response to everolimus or other mTOR inhibitors. Temsirolimus was also tested in a phase II trial including 15 patients but, conversely to other trials, no significant responses has been be observed, leading to the conclusion that temsirolimus seemed to have poor activity in metastatic transitional carcinoma of the urothelium that are resistant to platinum [START_REF] Gerullis | A phase II trial of temsirolimus in second-line metastatic urothelial cancer[END_REF].Other clinical trials targeting the PI3K/AKT/mTOR signaling pathway have been performed or are currently in progress (Table 3, http://clinicaltrials.gov). Paradoxically, these trials are mainly testing associations of mTOR inhibitors everolimus (and temsirolimus for a single trial) with existing cytotoxic agents such as cisplatin, gemcitabine, or paclitaxel. Most of these trials are in the recruiting phase. As of today, we could inventory only two trials with molecules targeting other factors of the PI3K/AKT/mTOR pathway than mTOR alone (Table 3, http://clinicaltrials.gov). One phase I trial evaluated the PI3K/mTOR dual inhibitors GSK2126458 in patients with advanced cancers. Interestingly, in patients with bladder cancers objective responses were observed in 1 out of 3 patients with PIK3CA mutations and in two patients out of 15 patients with wild-type PIK3CA [START_REF] Munster | PI3K kinase inhibitor GSK2126458 (GSK458): clinical activity in select patient (pt) populations defined by predictive markers (study P3K112826)[END_REF]. From these preliminary results, and the complex genetic landscape of bladder tumors that has recently been revealed by several independent studies on large cohorts using integrative approaches, it seems reasonable to postulate that the activity of PI3K inhibitors does not solely rely on PIK3CA status, but that otheralterations might also contribute to tumor response including the anti-angiogenic effects of these molecules.The second trial is a phase II trial (NCT01551030)investigating the specific PI3K inhibitor buparlisib (BKM-120) in metastatic transitional cell carcinoma of the urothelium. This trial is currently in its recruiting phase and results are awaited eagerly.

Conclusions

The recent studies characterizing the genetic background of urothelial cancers of the bladder, revealed the unicity of this model with alterations that are not seen at the same level in other types of cancers. This is especially the case for the PI3K/AKT/mTOR signaling pathway that occupies a major place in the etiology of these tumors due to its constitutive activation that was observed in a significant number of these tumors. Though no mutations of mTOR itself could be detected, mTOR inhibitors were the first inhibitors of this pathway to be used for the treatment of advanced refractory bladder cancers. However, it appeared that targeting mTOR was only beneficial for a subset of bladder cancer patients, because of negative feed-back loop mechanisms leading to further activation of PI3K. In this line, combinations of mTORC inhibitors with PI3K inhibitors or the use of dual PI3K/mTOR inhibitors may increase the number of effective responses. A pivotal study also demonstrated that other alterations of this pathway such as TSC1 mutations in the case of everolimus, may also play a crucial role in drug response to mTOR inhibitors, further reinforcing the notion that a global genetic mapping of the pathway should be taken into account to asses more precisely the potential clinical benefit of such targeted therapies. Even though a wide range of new specific inhibitors of PI3K, AKT or PDK1

have been characterized at the preclinical level for their potent anticancer activity in vitro or in xenograft models,it is paradoxical to note that only two new inhibitors, a PI3K inhibitor and a dual PI3K/mTOR inhibitor are currently in clinical trial for bladder cancers. This could certainly be explained by the low frequency of this cancer and/or the relative fragility of these patientsheavily treated patients, especially those who are refractory to cisplatin-based regimen. An additional level of complexity will have to be taken into account, as regulators of other key cellular pathways such as TP53, FGFR3, RAS, RAF, MEK, or ERK,were found to be altered in a significant number of urothelial tumors and couldthus also contribute to tumor response to chemotherapies. In this line, combinations of inhibitors of the PI3K/AKT/mTOR inhibitors with inhibitors of FGFR3 such as dovitinib, or with MEKinhibitors, may certainly represent alternative options to improvetumor control. Similar reasoning could be performed in the case of combinations with antiangiogenic derivatives that showed promising synergistic effects. With the future of personalized medicine, it is of outmost importance to further characterize at the molecular level each subtype of urothelial cancers in order to identify the best treatment options. The recent identification of significant rates of alterations in genes involved in chromatin remodeling and of epigenetic changesin advanced bladder cancers is another compelling evidence of the genetic complexity of these tumors, but opens a new area of research to identify new derivatives that could specifically target these tumor subtypes.These data emphasize the importance to carefully choose the most pertinent target(s) and the drugs or (drug combinations) to be further tested in the clinic and the necessity of cooperative efforts to conduct these trials.

Figure 1 :

 1 Figure 1: Schematic representation of the PI3K/AKT/mTOR signaling pathway and its cross-talk with the MAPK/MEK/ERK pathway. Activation of receptor tyrosine kinases (RTKs) by their respective ligands activates the recruitment of the PI3Ks and induces the phosphorylation of the p110 subunit that catalyzes the conversion of PIP2 to PIP3. Recruitment of PDK1 to the cell membrane induces AKT phosphorylation that triggers multiple downstream effectors,including TSC1 and TSC2 that inhibit RHEB, a suppressor of mTOR functions. mTOR is part of two separate complexes, mTORC1 and mTORC2. mTORC1 induces the activation of factors involved the regulation of translation as well as other factors involved in angiogenesis and autophagy. mTORC2 regulates AKT activity by a feed-back loop. The PI3K/AKT/mTOR pathway is regulated by the level of nutrients and shows cross-talks with other pathways regulating cell growth or survival such as the MPAK/MEK/ERK pathway (see Text for details). Interaction of rapamycin with FKBP12 leads to selective inhibition of the mTORC1 complex. Phosphorylations are indicated by red circles with Ps.

Figure 2 :Figure 3 :Figure 4 :Figure 5 :

 2345 Figure 2: Chemical structures of the main PI3K inhibitors

Table 1 :

 1 Mutations of the genes involved in the PI3K/AKT/mTOR signaling pathway found in urothelial bladder cancers. § The most frequent mutations and/or LOH are indicated in bold; indel: insertion or delation mutations; fs: frame-shift. * Missense mutations leading to early termination codon. ǂ Percentages in parentheses indicate the percentage of LOH for the corresponding gene.

Conflict of Interest:

NH is the principal investigator of the Phase II trial evaluating efficacy of Temsirolimus in second line therapy for patients with advanced bladder cancer (VESTOR -NCT01827943) sponsored by Pfizer.

PP: None

Gene

Mutations [START_REF] Ohtsu | Everolimus for previously treated advanced gastric cancer: results of the randomized, double-blind, phase III GRANITE-1 study[END_REF], [START_REF] Campone | Effect of visceral metastases on the efficacy and safety of everolimus in postmenopausal women with advanced breast cancer: subgroup analysis from the BOLERO-2 study[END_REF], [START_REF] Fazio | Everolimus plus octreotide long-acting repeatable in patients with advanced lung neuroendocrine tumors: analysis of the phase 3, randomized, placebo-controlled RADIANT-2 study[END_REF], [START_REF] Franz | Everolimus for astrocytomas in tuberous sclerosis -author's reply[END_REF], [START_REF] Pavel | Everolimus plus octreotide long-acting repeatable for the treatment of advanced neuroendocrine tumours associated with carcinoid syndrome (RADIANT-2): a randomised, placebo-controlled, phase 3 study[END_REF], [START_REF] Motzer | Phase 3 trial of everolimus for metastatic renal cell carcinoma : final results and analysis of prognostic factors[END_REF] Temsirolimus mTORC1 mRCC, Metastatic breast cancer, Mantel cell lymphoma III [START_REF] Hutson | Randomized phase III trial of temsirolimus versus sorafenib as second-line therapy after sunitinib in patients with metastatic renal cell carcinoma[END_REF], [START_REF] Rini | Randomized phase III trial of temsirolimus and bevacizumab versus interferon alfa and bevacizumab in metastatic renal cell carcinoma: INTORACT trial[END_REF], [START_REF] Wolff | Randomized phase III placebo-controlled trial of letrozole plus oral temsirolimus as first-line endocrine therapy in postmenopausal women with locally advanced or metastatic breast cancer[END_REF], [START_REF] Hess | Phase III study to evaluate temsirolimus compared with investigator's choice therapy for the treatment of relapsed or refractory mantle cell lymphoma[END_REF]