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Targeting the mTOR pathway in hepatocellular carcinoma: Current state and future trends

  • Author Footnotes
    † These authors contributed equally to this work.
    Matthias S. Matter
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD, USA
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  • Author Footnotes
    † These authors contributed equally to this work.
    Thomas Decaens
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD, USA
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  • Jesper B. Andersen
    Affiliations
    Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD, USA
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  • Snorri S. Thorgeirsson
    Correspondence
    Corresponding author. Address: Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 4146A, Bethesda, MD 20892, USA. Tel.: +1 301 496 1935; fax: +1 301 496 0734.
    Affiliations
    Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD, USA
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  • Author Footnotes
    † These authors contributed equally to this work.
Open AccessPublished:December 04, 2013DOI:https://doi.org/10.1016/j.jhep.2013.11.031

      Summary

      Mechanistic target of rapamycin (mTOR) regulates cell growth, metabolism and aging in response to nutrients, cellular energy stage and growth factors. mTOR is frequently up-regulated in cancer including hepatocellular carcinoma (HCC) and is associated with bad prognosis, poorly differentiated tumors, and earlier recurrence. Blocking mTOR with rapamycin and first generation mTOR inhibitors, called rapalogs, has shown promising reduction of HCC tumor growth in preclinical models. Currently, rapamycin/rapalogs are used in several clinical trials for the treatment of advanced HCC, and as adjuvant therapy in HCC patients after liver transplantation and TACE. A second generation of mTOR pathway inhibitors has been developed recently and is being tested in various clinical trials of solid cancers, and has been used in preclinical HCC models. The results of series of clinical trials using mTOR inhibitors in HCC treatment will emerge in the near future.

      Keywords

      Introduction

      Target of rapamycin (TOR) is an evolutionary well conserved serine/threonine protein kinase that belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family. Mechanistic TOR (mTOR; originally called mammalian TOR) has a broad range of action and is involved in regulation of cell growth, aging and metabolism [
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling in growth control and disease.
      ]. mTOR can be divided into two structurally and functionally distinct complexes named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling in growth control and disease.
      ]. mTORC1 is composed of mTOR, mLST8, DEPTOR, RAPTOR, and PRAS40. mTORC2 consists of mTOR, mLST8, DEPTOR, PROTOR, RICTOR, and mSIN1 [
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling in growth control and disease.
      ]. mTORC1 is a nutrient and energy sensor at both cellular and whole-body levels [
      • Benjamin D.
      • Colombi M.
      • Moroni C.
      • Hall M.N.
      Rapamycin passes the torch: a new generation of mTOR inhibitors.
      ]. When nutrients are available, mTORC1 is activated and stimulates anabolic processes such as protein synthesis, lipogenesis, and energy metabolism, whereas autophagy and lysosome biogenesis is inhibited [
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling in growth control and disease.
      ] (for more details see Fig. 1). mTORC1 is activated by a myriad of inputs such as growth factors, energy status, proinflammatory cytokines, oxygen levels, amino acids, and the canonical Wnt pathway [
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling in growth control and disease.
      ] (Fig. 1). Growth factors, e.g., insulin and insulin-like growth factor 1 (IGF1), exert their action on mTORC1 through receptor tyrosine kinases (RTK) and the well-characterized PI3K-AKT and Ras-Raf-Mek-Erk signaling pathways. These pathways activate mTORC1 by phosphorylating and thereby inhibiting the tumor suppressor TSC1-TSC2 (tuberous sclerosis 1 and 2) complex. The TSC1-TSC2 complex is a key regulator of mTORC1 and functions as a GTPase-activating protein (GAP) that negatively regulates Rheb by converting it into its inactive GDP-bound state [
      • Inoki K.
      • Li Y.
      • Xu T.
      • Guan K.L.
      Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.
      ,
      • Tee A.R.
      • Fingar D.C.
      • Manning B.D.
      • Kwiatkowski D.J.
      • Cantley L.C.
      • Blenis J.
      Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling.
      ]. In contrast, down-regulation of mTORC1, is accomplished via activation of the TSC1-TSC2 complex by AMPK, LKB1, and REDD1 in situations of low energy (high AMP), low oxygen levels [
      • Inoki K.
      • Zhu T.
      • Guan K.L.
      TSC2 mediates cellular energy response to control cell growth and survival.
      ], and DNA damage [
      • DeYoung M.P.
      • Horak P.
      • Sofer A.
      • Sgroi D.
      • Ellisen L.W.
      Hypoxia regulates TSC1/2-mTOR signaling and tumor suppression through REDD1-mediated 14-3-3 shuttling.
      ].
      Figure thumbnail gr1
      Fig. 1Schematic overview of the mTOR signaling pathway with the most important factors and their action.
      Much less is known about the later discovered mTORC2 signaling pathway. mTORC2 is insensitive to nutrients but does respond to growth factors such as insulin in association with ribosomes [
      • Zinzalla V.
      • Stracka D.
      • Oppliger W.
      • Hall M.N.
      Activation of mTORC2 by association with the ribosome.
      ]. Besides its initial described role in actin cytoskeleton organization, mTORC2 also activates cell metabolism, survival, and growth. mTORC2-ribosome interaction is a likely conserved mechanism of mTORC2 activation that is physiologically relevant in both normal and cancer cells.

      Involvement of mTOR pathway in hepatocellular carcinoma (HCC)

      Given its importance in cell growth and metabolism it is not surprising that mTOR plays a pivotal role in HCC. mTORC1 and mTORC2 pathways, including pRPS6, p-AKT, IGF-1R, and RICTOR are up-regulated in 40–50% of HCCs [
      • Sieghart W.
      • Fuereder T.
      • Schmid K.
      • Cejka D.
      • Werzowa J.
      • Wrba F.
      • et al.
      Mammalian target of rapamycin pathway activity in hepatocellular carcinomas of patients undergoing liver transplantation.
      ,
      • Villanueva A.
      • Chiang D.Y.
      • Newell P.
      • Peix J.
      • Thung S.
      • Alsinet C.
      • et al.
      Pivotal role of mTOR signaling in hepatocellular carcinoma.
      ,
      • Sahin F.
      • Kannangai R.
      • Adegbola O.
      • Wang J.
      • Su G.
      • Torbenson M.
      mTOR and P70 S6 kinase expression in primary liver neoplasms.
      ]. A similar upregulation is observed in other common cancer types such as breast, colon, and lung carcinomas [
      • Menon S.
      • Manning B.D.
      Common corruption of the mTOR signaling network in human tumors.
      ]. Moreover an up-regulation is frequently observed in cholangiocarcinoma, the second most common primary cancer of the liver [
      • Andersen J.B.
      • Spee B.
      • Blechacz B.R.
      • Avital I.
      • Komuta M.
      • Barbour A.
      • et al.
      Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors.
      ]. Activation of the mTOR pathway in HCC is associated with less differentiated tumors, bad prognosis, and earlier recurrence independently of the underlying etiology of liver cancer [
      • Villanueva A.
      • Chiang D.Y.
      • Newell P.
      • Peix J.
      • Thung S.
      • Alsinet C.
      • et al.
      Pivotal role of mTOR signaling in hepatocellular carcinoma.
      ,
      • Baba H.A.
      • Wohlschlaeger J.
      • Cicinnati V.R.
      • Hilgard P.
      • Lang H.
      • Sotiropoulos G.C.
      • et al.
      Phosphorylation of p70S6 kinase predicts overall survival in patients with clear margin-resected hepatocellular carcinoma.
      ,
      • Zhou L.
      • Huang Y.
      • Li J.
      • Wang Z.
      The mTOR pathway is associated with the poor prognosis of human hepatocellular carcinoma.
      ]. Furthermore, it is associated with deregulation of EGF, IGF, and PTEN pathways [
      • Villanueva A.
      • Chiang D.Y.
      • Newell P.
      • Peix J.
      • Thung S.
      • Alsinet C.
      • et al.
      Pivotal role of mTOR signaling in hepatocellular carcinoma.
      ] and, as expected, with increased lipogenesis in the tumor [
      • Calvisi D.F.
      • Wang C.
      • Ho C.
      • Ladu S.
      • Lee S.A.
      • Mattu S.
      • et al.
      Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma.
      ]. Surprisingly, alterations in copy number or somatic mutations of PTEN, PIK3CA, and PIK3B were not identified as major mechanisms of mTOR pathway deregulation in HCC by PCR [
      • Villanueva A.
      • Chiang D.Y.
      • Newell P.
      • Peix J.
      • Thung S.
      • Alsinet C.
      • et al.
      Pivotal role of mTOR signaling in hepatocellular carcinoma.
      ]. In accordance, more recent studies using next-generation sequencing technique revealed a low frequency of mutations in the mTOR pathway including mTOR, PIK3CA, and PTEN among others [
      • Guichard C.
      • Amaddeo G.
      • Imbeaud S.
      • Ladeiro Y.
      • Pelletier L.
      • Maad I.B.
      • et al.
      Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.
      ,
      • Cleary S.P.
      • Jeck W.R.
      • Zhao X.
      • Chen K.
      • Selitsky S.R.
      • Savich G.L.
      • et al.
      Identification of driver genes in hepatocellular carcinoma by exome sequencing.
      ,
      • Kan Z.
      • Zheng H.
      • Liu X.
      • Li S.
      • Barber T.D.
      • Gong Z.
      • et al.
      Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma.
      ]. The most frequently mutated gene, found in one study in 9.6% of HCC was RPS6KA3, a serine/threonine kinase involved in regulating PI3K/RAS signaling [
      • Guichard C.
      • Amaddeo G.
      • Imbeaud S.
      • Ladeiro Y.
      • Pelletier L.
      • Maad I.B.
      • et al.
      Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma.
      ]. Therefore, mutations in the mTOR pathway is a rare event in HCC and activation of the mTOR pathway appears to result largely from ligand dependent receptor activation.
      Genomic studies in the past have identified multiple molecular classifications of HCC and demonstrated deregulated signaling pathways unique to subgroups of patients [
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ,
      • Chiang D.Y.
      • Villanueva A.
      • Hoshida Y.
      • Peix J.
      • Newell P.
      • Minguez B.
      • et al.
      Focal gains of VEGFA and molecular classification of hepatocellular carcinoma.
      ,
      • Lee J.S.
      • Chu I.S.
      • Heo J.
      • Calvisi D.F.
      • Sun Z.
      • Roskams T.
      • et al.
      Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling.
      ,
      • Lee J.S.
      • Heo J.
      • Libbrecht L.
      • Chu I.S.
      • Kaposi-Novak P.
      • Calvisi D.F.
      • et al.
      A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells.
      ,
      • Lee J.S.
      • Thorgeirsson S.S.
      Genome-scale profiling of gene expression in hepatocellular carcinoma: classification, survival prediction, and identification of therapeutic targets.
      ,
      • Nault J.C.
      • De Reynies A.
      • Villanueva A.
      • Calderaro J.
      • Rebouissou S.
      • Couchy G.
      • et al.
      A hepatocellular carcinoma 5-gene score associated with survival of patients after liver resection.
      ]. These studies indicated that the mTOR pathway and its upstream pathways PI3K and AKT occupy a central position in the network of deregulated signaling pathways in HCC. With the specific aim to identify driver genes associated with HCC prognosis, we have used an integrative approach combining data obtained from somatic copy number analysis and transcriptomics [
      • Woo H.G.
      • Park E.S.
      • Lee J.S.
      • Lee Y.H.
      • Ishikawa T.
      • Kim Y.J.
      • et al.
      Identification of potential driver genes in human liver carcinoma by genome wide screening.
      ]. Fifty driver genes were recognized and were linked to the mTOR, AMPK or EGFR pathways. In the molecular HCC classification by Boyault et al., 6 robust subgroups (G1-G6) were identified and the G1/G2 subgroup showed AKT activation with overexpression of IGF2, IGF1R, and GSK3β as well as PIK3CA, and AXIN1 mutations [
      • Boyault S.
      • Rickman D.S.
      • de Reynies A.
      • Balabaud C.
      • Rebouissou S.
      • Jeannot E.
      • et al.
      Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.
      ]. The G1/G2 patient subgroup was further confirmed in a large meta-analysis using integrative transcriptomics of 9 HCC data sets including a total of 603 patients [
      • Hoshida Y.
      • Nijman S.M.
      • Kobayashi M.
      • Chan J.A.
      • Brunet J.P.
      • Chiang D.Y.
      • et al.
      Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma.
      ]. This analysis assigned the patients into three subclasses (S1-S3), and the G1/G2 subgroup was enriched in the subclass S2, characterized again by activation of the upstream regulator of mTOR, AKT, in combination with MYC.
      Taken together, activation of mTOR plays a central role in HCC and blocking this pathway is an attractive strategy for HCC treatment. The main goal of this review is to offer the rationale for the use of mTOR inhibitors in HCC and provide an overview of the current and prospective clinical trials with mTOR inhibitors in HCC.

      Rapamycin and first generation mTOR inhibitors

      mTOR is targeted by rapamycin, a natural compound discovered from the bacterium Streptomyces hygroscopius more than 30 years ago. The two mTOR-containing complexes have different sensitivities to rapamycin. mTORC1 is inhibited by a complex formed by rapamycin and FKBP12 protein [
      • Heitman J.
      • Movva N.R.
      • Hall M.N.
      Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast.
      ]. In contrast, mTORC2 is generally resistant to rapamycin, however, in certain cell types, mTORC2 may show sensitivity after prolonged rapamycin treatment [
      • Sarbassov D.D.
      • Ali S.M.
      • Sengupta S.
      • Sheen J.H.
      • Hsu P.P.
      • Bagley A.F.
      • et al.
      Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.
      ]. Rapamycin (sirolimus) was first approved as an immunosuppressant for the prevention of graft rejection in kidney transplant recipients more than a decade ago [
      • Benjamin D.
      • Colombi M.
      • Moroni C.
      • Hall M.N.
      Rapamycin passes the torch: a new generation of mTOR inhibitors.
      ]. A few years later rapamycin obtained approval for its use as an anti-restenosis agent following balloon angioplasty in coronary arterial stents. The early success of rapamycin has encouraged the development of derivative compounds with improved bioavailability, called rapalogs: everolimus (RAD001), temsirolimus (CCI-779), and deforolimus (AP23573). Due to the important role of mTOR in cell growth and metabolism the primary interest shifted to anti-cancer therapy, and in 2007 temsirolimus (CCI-779) was approved for the treatment of renal cell carcinoma and shortly thereafter for mantle cell lymphoma. Meanwhile, everolimus (RAD001), has received approval for treatment of pancreatic neuroendocrine tumours, subependymal giant cell astrocytoma, renal cell carcinoma, and HER2-negative breast cancer in combination with Exemestane.
      In general, first generation mTOR inhibitors are well tolerated. The major toxicities include, stomatitis, headache, diarrhea, vomiting, and thrombocytopenia. Due to their role in metabolism they can cause hyperglycemia, hyperlipidemia, and hypophosphatemia. As for every immunosuppressive drug, the risk for infections is increased. Furthermore, reactivation of HBV is a serious complication and has been described in renal cell carcinoma patients under everolimus treatment [
      • Sezgin Goksu S.
      • Bilal S.
      • Coskun H.S.
      Hepatitis B reactivation related to everolimus.
      ,
      • Mizuno S.
      • Yamagishi Y.
      • Ebinuma H.
      • Nakamoto N.
      • Katahira M.
      • Sasaki A.
      • et al.
      Progressive liver failure induced by everolimus for renal cell carcinoma in a 58-year-old male hepatitis B virus carrier.
      ]. EASL Clinical practice Guidelines recommend, in which situation patients undergoing immunosuppressive therapy should receive prophylactic treatment against HBV-reactivation with a nucleoside analogue [
      • EASL clinical practice guidelines
      Management of chronic hepatitis B virus infection.
      ].
      Currently, neither rapamycin nor rapalogs have gained approval for HCC treatment. However, several clinical trials are ongoing or have recently been completed using rapamycin/rapalogs for the treatment of advanced HCC and as adjuvant therapy after transarterial chemoembolization (TACE) or after liver transplantation of HCC patients. Furthermore, several studies suggest that mTOR inhibition may even prevent HCC development in patients at risk. The application of mTOR inhibitors in these different settings will be discussed in more detail.

      Prevention of liver cancer by mTOR inhibition

      Several studies indicate that mTOR activation is involved in the initiation of liver cancer and plays a role in the malignant transition of hepatocytes to HCC. A gradual activation of the AKT/mTOR pathway was progressively induced from non-tumorous liver tissue toward the HCC thus supporting this concept [
      • Calvisi D.F.
      • Wang C.
      • Ho C.
      • Ladu S.
      • Lee S.A.
      • Mattu S.
      • et al.
      Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma.
      ]. Interestingly, increased mTOR activity conferred a preneoplastic phenotype to HepaRG, considered a terminally differentiated hepatic cell line [
      • Parent R.
      • Kolippakkam D.
      • Booth G.
      • Beretta L.
      Mammalian target of rapamycin activation impairs hepatocytic differentiation and targets genes moderating lipid homeostasis and hepatocellular growth.
      ]. Also, treatment with mTOR inhibitor everolimus prevented proliferation of hepatocytes suffering DNA damage in the fumarylacetoacetate hydrolase-deficient mouse model of chronic liver injury and HCC development [
      • Buitrago-Molina L.E.
      • Pothiraju D.
      • Lamle J.
      • Marhenke S.
      • Kossatz U.
      • Breuhahn K.
      • et al.
      Rapamycin delays tumor development in murine livers by inhibiting proliferation of hepatocytes with DNA damage.
      ]. In transgenic mice, mTOR activation by itself was shown to be sufficient for HCC development. Two independent studies analyzed liver-specific knockout of TSC1 and the resulting chronic activation of mTOR [
      • Menon S.
      • Yecies J.L.
      • Zhang H.H.
      • Howell J.J.
      • Nicholatos J.
      • Harputlugil E.
      • et al.
      Chronic activation of mTOR complex 1 is sufficient to cause hepatocellular carcinoma in mice.
      ,
      • Kenerson H.L.
      • Yeh M.M.
      • Kazami M.
      • Jiang X.
      • Riehle K.J.
      • McIntyre R.L.
      • et al.
      Akt and mTORC1 have different roles during liver tumorigenesis in mice.
      ]. Both studies demonstrated the development of liver tumors, however, only one was associated with inflammation [
      • Menon S.
      • Yecies J.L.
      • Zhang H.H.
      • Howell J.J.
      • Nicholatos J.
      • Harputlugil E.
      • et al.
      Chronic activation of mTOR complex 1 is sufficient to cause hepatocellular carcinoma in mice.
      ]. Likewise constant activation of mTOR in PTEN-deficient mice induced steatohepatitis and development of liver tumors [
      • Horie Y.
      • Suzuki A.
      • Kataoka E.
      • Sasaki T.
      • Hamada K.
      • Sasaki J.
      • et al.
      Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas.
      ]. mTOR inhibitors may also prevent HCC indirectly by reducing liver fibrosis, a risk factor for the development of HCC. Studies in rats have demonstrated that sirolimus and everolimus attenuated progression of fibrosis, in contrast to cyclosporine A and tacrolimus, two other immunosuppressive drugs [
      • Neef M.
      • Ledermann M.
      • Saegesser H.
      • Schneider V.
      • Reichen J.
      Low-dose oral rapamycin treatment reduces fibrogenesis, improves liver function, and prolongs survival in rats with established liver cirrhosis.
      ,
      • Patsenker E.
      • Schneider V.
      • Ledermann M.
      • Saegesser H.
      • Dorn C.
      • Hellerbrand C.
      • et al.
      Potent antifibrotic activity of mTOR inhibitors sirolimus and everolimus but not of cyclosporine A and tacrolimus in experimental liver fibrosis.
      ].
      Randomized prospective clinical trials have not been conducted to address the question if mTOR inhibitors prevent HCC. The evidence suggesting a potential HCC prevention is solely derived from epidemiological studies with metformin, a widely used anti-diabetic drug that reduces mTOR activity but importantly also ameliorates hyperinsulinemia, which is a risk factor for HCC [
      • Siegel A.B.
      • Zhu A.X.
      Metabolic syndrome and hepatocellular carcinoma: two growing epidemics with a potential link.
      ]. Metformin activates AMPK, which in turn suppresses the mTORC1 pathway (Fig. 2) [
      • Zakikhani M.
      • Dowling R.
      • Fantus I.G.
      • Sonenberg N.
      • Pollak M.
      Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells.
      ]. A recent study showed that metformin controlled gene expression at the level of mRNA translation to an extent comparable to that of canonical mTOR inhibitors and down regulated mRNAs, which encode for proliferation and tumor-promoting proteins via the mTORC1/4E-BP pathway [
      • Larsson O.
      • Morita M.
      • Topisirovic I.
      • Alain T.
      • Blouin M.J.
      • Pollak M.
      • et al.
      Distinct perturbation of the translatome by the antidiabetic drug metformin.
      ]. In addition, it was shown that metformin inhibited mTORC1 signaling independently of AMPK by suppressing Rag GTPase or activating REDD1 [
      • Ben Sahra I.
      • Regazzetti C.
      • Robert G.
      • Laurent K.
      • Le Marchand-Brustel Y.
      • Auberger P.
      Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1.
      ,
      • Kalender A.
      • Selvaraj A.
      • Kim S.Y.
      • Gulati P.
      • Brule S.
      • Viollet B.
      • et al.
      Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner.
      ]. A meta-analysis, including 5 clinical studies with more than 100,000 type 2 diabetes patients has been performed lately [
      • Zhang Z.J.
      • Zheng Z.J.
      • Shi R.
      • Su Q.
      • Jiang Q.
      • Kip K.E.
      Metformin for liver cancer prevention in patients with type 2 diabetes: a systematic review and meta-analysis.
      ]. This study showed an overall estimated 62% reduction in the risk of liver cancer if metformin was used as an anti-diabetic treatment instead of non-metformin treatment (e.g., sulfonylurea or insulin). Although there was considerable heterogeneity between the studies, the results are encouraging. Preventive clinical trials with metformin are already underway for different cancer types such as breast cancer, colon cancer, and esophagus cancer, however, for HCC they are lacking. Information of the potential preventive mechanism(s) of metformin on liver cancer is also limited. Recently, it was shown in vitro and in vivo, that metformin inhibited HCC cell growth through AMPK and LKB1 [
      • Chen H.P.
      • Shieh J.J.
      • Chang C.C.
      • Chen T.T.
      • Lin J.T.
      • Wu M.S.
      • et al.
      Metformin decreases hepatocellular carcinoma risk in a dose-dependent manner: population-based and in vitro studies.
      ,
      • Zheng L.
      • Yang W.
      • Wu F.
      • Wang C.
      • Yu L.
      • Tang L.
      • et al.
      Prognostic significance of AMPK activation and therapeutic effects of metformin in hepatocellular carcinoma.
      ]. Further elucidation of this mechanism(s) and defining criteria that identify individuals which are likely to benefit are therefore needed. Nevertheless, it has to be kept in mind that a few studies have shown that metformin can have tumor-promoting effects. Metformin enhanced growth of BRAF-mutant melanoma cells and ER-alpha negative breast cancer cell lines in vivo [
      • Phoenix K.N.
      • Vumbaca F.
      • Claffey K.P.
      Therapeutic metformin/AMPK activation promotes the angiogenic phenotype in the ERalpha negative MDA-MB-435 breast cancer model.
      ,
      • Martin M.J.
      • Hayward R.
      • Viros A.
      • Marais R.
      Metformin accelerates the growth of BRAF V600E-driven melanoma by upregulating VEGF-A.
      ]. Likewise, migration and invasion abilities of human pulmonary adenocarcinoma A549 cell lines increased under metformin treatment in vitro [
      • Wu N.
      • Gu H.J.
      • Li Q.
      Effects of antidiabetic drug metformin on the migration and invasion abilities of human pulmonary adenocarcinoma A549 cell line in vitro.
      ].
      Figure thumbnail gr2
      Fig. 2Schematic overview of the mTOR signaling pathway with the target position of drugs.
      Prevention of HCC by metformin and mTOR inhibitors may also involve autophagy, an important homeostatic cellular recycling mechanism, which has a dual role in carcinogenesis. Autophagy is important in limiting DNA damage caused by the accumulation of reactive oxygen species and damaged organelles through removal of dysfunctional proteins and organelles [
      • Janku F.
      • McConkey D.J.
      • Hong D.S.
      • Kurzrock R.
      Autophagy as a target for anticancer therapy.
      ]. Especially in the context of HCC development, which arises in 80% of the cases in the background of a chronic liver inflammation with constant DNA damage, induction of autophagy by mTOR inhibitors may prevent HCC development and recurrence. In contrast, increased autophagy may promote tumor growth, especially once tumor is established, as autophagy is also a pro-survival mechanism to cellular stress and further enhances chemoresistance [
      • Janku F.
      • McConkey D.J.
      • Hong D.S.
      • Kurzrock R.
      Autophagy as a target for anticancer therapy.
      ,
      • Cui J.
      • Gong Z.
      • Shen H.M.
      The role of autophagy in liver cancer: molecular mechanisms and potential therapeutic targets.
      ].

      Adjuvant therapy with mTOR inhibitors after liver transplantation or TACE

      For patients with non-resectable HCC and early stage disease, liver transplantation is recognized as the treatment of choice, with disease-free survival of 60–80% at 5 years [
      • Colombo M.
      • Raoul J.L.
      • Lencioni R.
      • Galle P.R.
      • Zucman-Rossi J.
      • Banares R.
      • et al.
      Multidisciplinary strategies to improve treatment outcomes in hepatocellular carcinoma: a European perspective.
      ]. Immunosuppressants, such as cyclosporine A, tacrolimus, and rapamycin are crucial for the prevention of graft rejection after transplantation. However, cyclosporine A and tacrolimus are known to induce tumor growth in preclinical studies [
      • Freise C.E.
      • Ferrell L.
      • Liu T.
      • Ascher N.L.
      • Roberts J.P.
      Effect of systemic cyclosporine on tumor recurrence after liver transplantation in a model of hepatocellular carcinoma.
      ,
      • Hojo M.
      • Morimoto T.
      • Maluccio M.
      • Asano T.
      • Morimoto K.
      • Lagman M.
      • et al.
      Cyclosporine induces cancer progression by a cell-autonomous mechanism.
      ]. On the contrary, rapamycin has additional anticancer function. Accordingly, a meta-analysis of 5 studies with a total of almost 3000 patients indicated that survival is significantly prolonged after liver transplantation of HCC patients if sirolimus is administered instead of non-sirolimus immunosuppressives [
      • Liang W.
      • Wang D.
      • Ling X.
      • Kao A.A.
      • Kong Y.
      • Shang Y.
      • et al.
      Sirolimus-based immunosuppression in liver transplantation for hepatocellular carcinoma: a meta-analysis.
      ]. Although these results indicate that sirolimus should be used as treatment of choice after liver transplantation of HCC patients, it has to be noted that none of the clinical studies conducted so far were randomized. Additional information regarding the efficacy of sirolimus to improve survival and prevent recurrence should be provided by three ongoing randomized controlled studies (Table 1). The largest trial is a multicenter, phase III study with over 500 patients (SiLVER) comparing sirolimus-based vs. sirolimus-free immunosuppression in patients undergoing liver transplantation for HCC. However, results are not expected before 2014.
      Table 1Summary of completed and ongoing clinical trials with first generation mTOR inhibitors in HCC.
      Recruiting: The study is currently recruiting participants.
      Active not recruiting: The study is ongoing but potential participants are not currently recruited.
      Completed: The study has ended normally, and participants are no longer being examined or treated.
      Terminated: The study has stopped recruiting participants early and will not start again.
      PR, partial response; CR, complete response; n.a., not available; R, randomized; NR, not randomized; MTD, maximal tolerated dose; DLT, dose limiting toxicity.
      TACE is the treatment of choice for intermediate HCC patients [
      • Colombo M.
      • Raoul J.L.
      • Lencioni R.
      • Galle P.R.
      • Zucman-Rossi J.
      • Banares R.
      • et al.
      Multidisciplinary strategies to improve treatment outcomes in hepatocellular carcinoma: a European perspective.
      ]. Following TACE treatment the levels of vascular endothelial growth factor (VEGF) have been shown to increase. VEGF promotes angiogenesis and is associated with bad prognosis. Therefore, TACE has been combined in clinical trials with other drugs that inhibit angiogenesis such as sorafenib, brivanib, or orantinib. Interestingly, everolimus has also been shown to reduce vessel formation in preclinical HCC models [
      • Semela D.
      • Piguet A.C.
      • Kolev M.
      • Schmitter K.
      • Hlushchuk R.
      • Djonov V.
      • et al.
      Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma.
      ] and two ongoing clinical trials are evaluating the effect of TACE in combination with everolimus (Table 1).

      mTOR inhibition in advanced HCC

      Sorafenib is the only approved drug for HCC treatment [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ,
      • Cheng A.L.
      • Kang Y.K.
      • Chen Z.
      • Tsao C.J.
      • Qin S.
      • Kim J.S.
      • et al.
      Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial.
      ]. However, the treatment effects are small, only selected patients are eligible for therapy, and side effects often limit applicability. Therefore, developing novel and effective therapies are urgently needed. Rapalogs have been shown to inhibit liver tumor growth in a large number of in vitro and in vivo pre-clinical studies [
      • Buitrago-Molina L.E.
      • Vogel A.
      mTor as a potential target for the prevention and treatment of hepatocellular carcinoma.
      ] and have encouraged clinical trials in HCC patients (Table 1, information retrieved form www.clinicaltrials.gov). Two phase I/II dose-finding studies of 39 and 28 patients have been completed, using everolimus as a first and second line single agent and resulted in dose recommendations of 7.5 mg/day and 10 mg/day respectively [
      • Zhu A.X.
      • Abrams T.A.
      • Miksad R.
      • Blaszkowsky L.S.
      • Meyerhardt J.A.
      • Zheng H.
      • et al.
      Phase 1/2 study of everolimus in advanced hepatocellular carcinoma.
      ,
      • Shiah H.S.
      • Chen C.Y.
      • Dai C.Y.
      • Hsiao C.F.
      • Lin Y.J.
      • Su W.C.
      • et al.
      Randomised clinical trial: comparison of two everolimus dosing schedules in patients with advanced hepatocellular carcinoma.
      ]. In both studies, complete response was not observed, however, one HCC patient in each study showed a partial response, and stable disease was observed for a short time in 71.4% and 40% of the patients. Three earlier clinical studies with 18 to 25 patients using rapamycin as a first line single agent provided interesting results with partial responses and even one complete response; however, the differences in treatment schedule prevent any firm conclusions [
      • Schoniger-Hekele M.
      • Muller C.
      Pilot study: rapamycin in advanced hepatocellular carcinoma.
      ,
      • Rizell M.
      • Andersson M.
      • Cahlin C.
      • Hafstrom L.
      • Olausson M.
      • Lindner P.
      Effects of the mTOR inhibitor sirolimus in patients with hepatocellular and cholangiocellular cancer.
      ,
      • Decaens T.
      • Luciani A.
      • Itti E.
      • Hulin A.
      • Roudot-Thoraval F.
      • Laurent A.
      • et al.
      Phase II study of sirolimus in treatment-naive patients with advanced hepatocellular carcinoma.
      ]. Overall, these preliminary results using rapamycin/rapalogs are encouraging; however, further studies with more patients are needed. Besides, one study using temsirolimus as a first line agent had to be terminated due to toxic events. The recent negative outcome of a multicenter randomized, double blind, phase III study dashed the hope to use mTOR inhibitors as a second line therapy for advanced HCC patients (press release). This study investigated the effect of everolimus or placebo in 546 patients with Child-Pugh A cirrhosis, whose disease progressed after treatment with or who were intolerant to sorafenib. It will be interesting to see if two other phase I/II studies using temsirolimus as a second line single agent obtain similar negative results.
      Because of resistance and compensatory activation of other signaling pathways the effect of rapalogs can be diminished. For example, after treatment with everolimus, an upregulation of MAPK was shown in tumor samples from breast cancer patients [
      • Carracedo A.
      • Ma L.
      • Teruya-Feldstein J.
      • Rojo F.
      • Salmena L.
      • Alimonti A.
      • et al.
      Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer.
      ]. As anticipated, combination of mTOR inhibition with a MAPK inhibitor (sorafenib) showed enhanced anti-tumoral effect in vitro and in vivo in cancer models including HCC [
      • Carracedo A.
      • Ma L.
      • Teruya-Feldstein J.
      • Rojo F.
      • Salmena L.
      • Alimonti A.
      • et al.
      Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer.
      ,
      • Piguet A.C.
      • Saar B.
      • Hlushchuk R.
      • St-Pierre M.V.
      • McSheehy P.M.
      • Radojevic V.
      • et al.
      Everolimus augments the effects of sorafenib in a syngeneic orthotopic model of hepatocellular carcinoma.
      ,
      • Newell P.
      • Toffanin S.
      • Villanueva A.
      • Chiang D.Y.
      • Minguez B.
      • Cabellos L.
      • et al.
      Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo.
      ,
      • Wang Z.
      • Zhou J.
      • Fan J.
      • Qiu S.J.
      • Yu Y.
      • Huang X.W.
      • et al.
      Effect of rapamycin alone and in combination with sorafenib in an orthotopic model of human hepatocellular carcinoma.
      ]. A combinatorial approach is currently performed in 9 clinical trials (Table 1). Rapalogs or rapamycin is complemented with multikinase inhibitor sorafenib, with VEGF inhibitor bevacizumab or with pasireotide, a somatostatin analog, which has been shown to inhibit tumor growth. Two phase I combination studies have been published recently [
      • Choo S.P.
      • Chowbay B.
      • Ng Q.S.
      • Thng C.H.
      • Lim C.
      • Hartono S.
      • et al.
      A phase 1 dose-finding and pharmacodynamic study of rapamycin in combination with bevacizumab in patients with unresectable hepatocellular carcinoma.
      ,
      • Kelley R.K.
      • Nimeiri H.S.
      • Munster P.N.
      • Vergo M.T.
      • Huang Y.
      • Li C.M.
      • et al.
      Temsirolimus combined with sorafenib in hepatocellular carcinoma: a phase I dose-finding trial with pharmacokinetic and biomarker correlates.
      ]. A combination of temsirolimus with sorafenib at the maximal tolerated dose (MTD; temsirolimus 10 mg weekly and sorafenib 200 mg twice daily), showed a partial response in 8% of the patients and a stable disease in 60% [
      • Kelley R.K.
      • Nimeiri H.S.
      • Munster P.N.
      • Vergo M.T.
      • Huang Y.
      • Li C.M.
      • et al.
      Temsirolimus combined with sorafenib in hepatocellular carcinoma: a phase I dose-finding trial with pharmacokinetic and biomarker correlates.
      ]. Although these results in 25 patients are promising, the median progression free survival was only 5.65 months, which is similar to outcomes from single agent sorafenib in the SHARP trial, though superior to outcomes observed in an Asia-Pacific clinical trial [
      • Llovet J.M.
      • Ricci S.
      • Mazzaferro V.
      • Hilgard P.
      • Gane E.
      • Blanc J.F.
      • et al.
      Sorafenib in advanced hepatocellular carcinoma.
      ,
      • Cheng A.L.
      • Kang Y.K.
      • Chen Z.
      • Tsao C.J.
      • Qin S.
      • Kim J.S.
      • et al.
      Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial.
      ]. In addition, a phase I study with rapamycin and bevacizumab of 24 patients at MTD (rapamycin 4 mg/day and bevacizumab 5 mg/kg every 14 days), reported a remarkable complete response in one patient that lasted 4.5 months, partial response in two patients, and stable disease in 14 patients [
      • Choo S.P.
      • Chowbay B.
      • Ng Q.S.
      • Thng C.H.
      • Lim C.
      • Hartono S.
      • et al.
      A phase 1 dose-finding and pharmacodynamic study of rapamycin in combination with bevacizumab in patients with unresectable hepatocellular carcinoma.
      ]. The ongoing clinical trials in phase I or II will reveal if a combinatorial approach improves efficacy in HCC treatment. The combination of drugs is certainly attractive, however, one drawback is that toxicities may increase, especially in cirrhotic patients and prevent that an efficacious dose can be applied. It is therefore important to perform pharmacokinetic studies in future clinical trials with dose escalation of both drugs (e.g., sorafenib and rapalogs), especially in cirrhotic patients.

      Second generation mTOR inhibitors

      We are currently awaiting the results of several clinical trials using rapalogs in HCC treatment; however, the success of rapalogs in cancer therapy in general has not been as impressive as initially hoped. Several possible reasons may account for the limited action of rapalogs. First, mTORC1 inhibition abrogates the negative feedback loop, which in turn activates PI3K-AKT with MAPK and RAS signaling and therefore may actually increase growth of cancer cells [
      • Carracedo A.
      • Ma L.
      • Teruya-Feldstein J.
      • Rojo F.
      • Salmena L.
      • Alimonti A.
      • et al.
      Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer.
      ]. Second, blocking of mTORC1 primarily leads to inhibition of cell growth and not cell death. Third, mTOR inhibition by rapalogs mainly results in inhibition of S6K, however, the second key substrate, 4E-BP1, is only insufficiently blocked [
      • Choo A.Y.
      • Yoon S.O.
      • Kim S.G.
      • Roux P.P.
      • Blenis J.
      Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation.
      ]. Finally, rapalogs do not inhibit mTORC2, which is often activated as part of the PI3K-mTORC2-AKT signaling axis [
      • Benjamin D.
      • Colombi M.
      • Moroni C.
      • Hall M.N.
      Rapamycin passes the torch: a new generation of mTOR inhibitors.
      ]. In order to overcome the shortcomings and resistance of rapalogs a second generation of mTOR inhibitors has been developed that functions as ATP-competitive inhibitors of mTOR and has several advantages over rapalogs. Unlike rapalogs, which inhibit only mTORC1, the ATP analogues block the phosphorylation of all known downstream targets of mTORC1 and mTORC2. Furthermore, because of the similarity between the kinase domains of mTOR and PI3Ks, some of these new compounds additionally inhibit PI3K, leading to a broad inhibitory action with blocking of the feedback activation of PI3K-AKT signaling described before. Second generation mTOR inhibitors can therefore be divided into mTORC1/2 inhibitors and mTOR/PI3K inhibitors (Fig. 2). In addition, a series of compounds have been developed that block upstream of the mTOR pathway such as AKT inhibitors and PI3K inhibitors.

      Translational genomics and development of second generation mTOR inhibitors

      Translational genomic analyses have been used to investigate the resistance to rapamycin. Jimenez et al. [
      • Jimenez R.H.
      • Boylan J.M.
      • Lee J.S.
      • Francesconi M.
      • Castellani G.
      • Sanders J.A.
      • et al.
      Rapamycin response in tumorigenic and non-tumorigenic hepatic cell lines.
      ] investigated rapamycin sensitivity or resistance in 13 HCC cell lines using drug-induced growth inhibition as the end point. The authors concluded that promoting or even maintaining effective drug sensitivity might be challenging, because of the molecular heterogeneity observed in the genomic profiles introduced in response to rapamycin, as well as determining (a) specific mechanism(s) of drug resistance. Furthermore, although the sensitivity to rapamycin was variable in all cell lines, the drug inhibited the phosphorylation of RPS6 and 4E-BP1, indicating that S6 and 4E-BP1 phosphorylation is not a useful marker for the antiproliferative effect of mTOR inhibitors. Interestingly, in an attempt to identify rapamycin sensitive genes a later study determined that many of the genes whose expression is altered by rapamycin are E-box containing and their regulation via mTOR was c-MYC independent [
      • Jimenez R.H.
      • Lee J.S.
      • Francesconi M.
      • Castellani G.
      • Neretti N.
      • Sanders J.A.
      • et al.
      Regulation of gene expression in hepatic cells by the mammalian Target of Rapamycin (mTOR).
      ]. Since the mTOR pathway is constitutively activated in a majority of diffuse large B-cell lymphoma this is an interesting disease model to study resistance mechanisms to mTOR inhibitors and to identify drugs, which may compliment the action of rapamycin [
      • Petrich A.M.
      • Leshchenko V.
      • Kuo P.Y.
      • Xia B.
      • Thirukonda V.K.
      • Ulahannan N.
      • et al.
      Akt inhibitors MK-2206 and nelfinavir overcome mTOR inhibitor resistance in diffuse large B-cell lymphoma.
      ]. The authors compared the genomic signatures of 4 rapamycin sensitive and 4 resistant cell lines, and found that the central mechanism involved in the resistance to rapamycin was controlled by AKT. Using the genomic signatures to explore the Connectivity Map database to identify drugs which may reverse drug resistance, PI3K/AKT and HDAC inhibitors were the most likely candidates to synergize with an mTOR inhibitor. Resistance mechanisms to rapamycin and its derivates also include epigenome based reprogramming (e.g., miRs). Long-term rapamycin treatment results in an increase of the miR-17-92 cluster and inhibition of this change restored the drug sensitivity [
      • Totary-Jain H.
      • Sanoudou D.
      • Ben-Dov I.Z.
      • Dautriche C.N.
      • Guarnieri P.
      • Marx S.O.
      • et al.
      Reprogramming of the microRNA transcriptome mediates resistance to rapamycin.
      ]. Likewise, in HCC the miR-216a/217 cluster is frequently upregulated, resulted in activation of the PI3K/AKT pathway and importantly resistance to sorafenib [
      • Xia H.
      • Ooi L.L.
      • Hui K.M.
      MicroRNA-216a/217-induced epithelial-mesenchymal transition targets PTEN and SMAD7 to promote drug resistance and recurrence of liver cancer.
      ].

      Preclinical studies of second mTOR, AKT, and PI3K inhibitors in HCC

      Several preclinical studies with second generation mTOR, AKT, and PI3K inhibitors were performed in HCC cell lines [
      • Kirstein M.M.
      • Boukouris A.E.
      • Pothiraju D.
      • Buitrago-Molina L.E.
      • Marhenke S.
      • Schutt J.
      • et al.
      Activity of the mTOR inhibitor RAD001, the dual mTOR and PI3-kinase inhibitor BEZ235 and the PI3-kinase inhibitor BKM120 in hepatocellular carcinoma.
      ,
      • Grabinski N.
      • Ewald F.
      • Hofmann B.T.
      • Staufer K.
      • Schumacher U.
      • Nashan B.
      • et al.
      Combined targeting of AKT and mTOR synergistically inhibits proliferation of hepatocellular carcinoma cells.
      ,
      • Masuda M.
      • Shimomura M.
      • Kobayashi K.
      • Kojima S.
      • Nakatsura T.
      Growth inhibition by NVP-BEZ235, a dual PI3K/mTOR inhibitor, in hepatocellular carcinoma cell lines.
      ], HCC xenograft mouse model [
      • Kirstein M.M.
      • Boukouris A.E.
      • Pothiraju D.
      • Buitrago-Molina L.E.
      • Marhenke S.
      • Schutt J.
      • et al.
      Activity of the mTOR inhibitor RAD001, the dual mTOR and PI3-kinase inhibitor BEZ235 and the PI3-kinase inhibitor BKM120 in hepatocellular carcinoma.
      ,
      • Masuda M.
      • Shimomura M.
      • Kobayashi K.
      • Kojima S.
      • Nakatsura T.
      Growth inhibition by NVP-BEZ235, a dual PI3K/mTOR inhibitor, in hepatocellular carcinoma cell lines.
      ], DEN-mouse HCC model [
      • Thomas H.E.
      • Mercer C.A.
      • Carnevalli L.S.
      • Park J.
      • Andersen J.B.
      • Conner E.A.
      • et al.
      mTOR inhibitors synergize on regression, reversal of gene expression, and autophagy in hepatocellular carcinoma.
      ] and in a diabetic rat model of HCC [
      • Evert M.
      • Calvisi D.F.
      • Evert K.
      • De Murtas V.
      • Gasparetti G.
      • Mattu S.
      • et al.
      V-AKT murine thymoma viral oncogene homolog/mammalian target of rapamycin activation induces a module of metabolic changes contributing to growth in insulin-induced hepatocarcinogenesis.
      ]. These studies have demonstrated that the second generation inhibitors were able to control HCC proliferation better than everolimus or sirolimus. It has also been demonstrated that combination of a PI3K inhibitor (BKM120) with cisplatin [
      • Kirstein M.M.
      • Boukouris A.E.
      • Pothiraju D.
      • Buitrago-Molina L.E.
      • Marhenke S.
      • Schutt J.
      • et al.
      Activity of the mTOR inhibitor RAD001, the dual mTOR and PI3-kinase inhibitor BEZ235 and the PI3-kinase inhibitor BKM120 in hepatocellular carcinoma.
      ] or a PI3K/mTOR dual inhibitor (BEZ235) with everolimus [
      • Thomas H.E.
      • Mercer C.A.
      • Carnevalli L.S.
      • Park J.
      • Andersen J.B.
      • Conner E.A.
      • et al.
      mTOR inhibitors synergize on regression, reversal of gene expression, and autophagy in hepatocellular carcinoma.
      ] could act synergistically with strong antitumor activity. The two drugs, everolimus and BEZ235 exerted tumor regression via inhibition of mTORC1 and mTORC2. This combined drug-effect was associated with an increase in autophagy independent of 4E-BP1. At low doses both drugs targeted mTORC1, however, inhibition of mTORC2 was enhanced by the drug combination. The cooperative drug-effect was further evident from the microarray analysis identifying a distinct set of genes, suggesting a phenotypic reversal similar to placebo-treated livers. Also, only the drug combination achieved significant inhibition of genes involved in cell cycle. These results have prompted a dose finding and a safety clinical trial of BEZ235 in combination with everolimus in patients with advanced solid tumors.
      Moreover, promising results may be expected from the combination of second generation mTOR, AKT or PI3K inhibitors with other drugs. This may be particularly important in cases of sorafenib resistance, which was recently demonstrated in a cancer stem cell subpopulation of HCC cells (i.e., label-retaining cancer cells), demonstrating sustained AKT and MAPK activation [
      • Xin H.W.
      • Ambe C.M.
      • Hari D.M.
      • Wiegand G.W.
      • Miller T.C.
      • Chen J.Q.
      • et al.
      Label-retaining liver cancer cells are relatively resistant to sorafenib.
      ]. Also, in a study comparing the HCC cell line HuH7 and sorafenib resistance HuH7 derivatives, the molecular alteration related to the acquired drug resistance included upregulation and activation of PI3K/AKT signaling [
      • Chen K.F.
      • Chen H.L.
      • Tai W.T.
      • Feng W.C.
      • Hsu C.H.
      • Chen P.J.
      • et al.
      Activation of phosphatidylinositol 3-kinase/Akt signaling pathway mediates acquired resistance to sorafenib in hepatocellular carcinoma cells.
      ]. The sorafenib resistance was overcome by either silencing AKT using RNAi or targeting the pathway using the novel allosteric AKT inhibitor (MK-2206). In agreement, the PI3K/mTOR inhibitors (PI103 and PKI-587) also augment the effect of sorafenib [
      • Gedaly R.
      • Angulo P.
      • Chen C.
      • Creasy K.T.
      • Spear B.T.
      • Hundley J.
      • et al.
      The role of PI3K/mTOR inhibition in combination with sorafenib in hepatocellular carcinoma treatment.
      ,
      • Gedaly R.
      • Angulo P.
      • Hundley J.
      • Daily M.F.
      • Chen C.
      • Evers B.M.
      PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.
      ]. Similarly, a recent study suggested that second-generation inhibitors (e.g., mTOR (AZD-8055), PI3K (BKM-120) or mTOR/PI3K (BEZ-235 and GDC-0980)) may be effective in sorafenib resistant HCC [
      • Serova M.
      • de Gramont A.
      • Tijeras-Raballand A.
      • Dos Santos C.
      • Riveiro M.E.
      • Slimane K.
      • et al.
      Benchmarking effects of mTOR, PI3K, and dual PI3K/mTOR inhibitors in hepatocellular and renal cell carcinoma models developing resistance to sunitinib and sorafenib.
      ]. It should be noted that this study unfortunately was performed in SK-HEP1 and its drug-resistant derivative (SK-Sora), which are derived from an HCC patient with ascites but are of endothelial origin. The combination of everolimus together with an AKT inhibitor (MK-2206), mTOR/PI3K inhibitor (BEZ235) or PI3K inhibitor (BKM120) showed improved anti-tumoral activity [
      • Kirstein M.M.
      • Boukouris A.E.
      • Pothiraju D.
      • Buitrago-Molina L.E.
      • Marhenke S.
      • Schutt J.
      • et al.
      Activity of the mTOR inhibitor RAD001, the dual mTOR and PI3-kinase inhibitor BEZ235 and the PI3-kinase inhibitor BKM120 in hepatocellular carcinoma.
      ,
      • Grabinski N.
      • Ewald F.
      • Hofmann B.T.
      • Staufer K.
      • Schumacher U.
      • Nashan B.
      • et al.
      Combined targeting of AKT and mTOR synergistically inhibits proliferation of hepatocellular carcinoma cells.
      ,
      • Thomas H.E.
      • Mercer C.A.
      • Carnevalli L.S.
      • Park J.
      • Andersen J.B.
      • Conner E.A.
      • et al.
      mTOR inhibitors synergize on regression, reversal of gene expression, and autophagy in hepatocellular carcinoma.
      ]. Further, improved efficacy was also achieved in vitro and in vivo with novel ATP-competitive mTOR kinase inhibitors together with histone deacetylase inhibitors [
      • Shao H.
      • Gao C.
      • Tang H.
      • Zhang H.
      • Roberts L.R.
      • Hylander B.L.
      • et al.
      Dual targeting of mTORC1/C2 complexes enhances histone deacetylase inhibitor-mediated anti-tumor efficacy in primary HCC cancer in vitro and in vivo.
      ].

      First results of clinical trials with new mTOR pathway inhibitors

      The results of several phase 1 clinical trials in advanced cancer (all types) are now available with this new generation of mTOR inhibitors. A phase 1 clinical trial with patients suffering from solid tumors and lymphoma using pan-mTOR inhibitor AZD8055 [
      • Naing A.
      • Aghajanian C.
      • Raymond E.
      • Olmos D.
      • Schwartz G.
      • Oelmann E.
      • et al.
      Safety, tolerability, pharmacokinetics and pharmacodynamics of AZD8055 in advanced solid tumours and lymphoma.
      ], demonstrated dose limiting toxicities (DLT) of grade 3 with an increase in transaminases and no RECIST objective response [
      • Naing A.
      • Aghajanian C.
      • Raymond E.
      • Olmos D.
      • Schwartz G.
      • Oelmann E.
      • et al.
      Safety, tolerability, pharmacokinetics and pharmacodynamics of AZD8055 in advanced solid tumours and lymphoma.
      ]. Accordingly, AZD8055 will not be further tested. It is interesting to note that altered liver function was not described with mTOR inhibitors such as temsirolimus or everolimus. It will be important to see if other pan-mTOR inhibitors such as OSI-027, AZD2014, INK128 or CC223 also alter liver function.
      Likewise, several dual mTOR-PI3K inhibitors are under evaluation and for 2 of them the phase 1 trial results have already been published. The first, BGT226 induced grade 3 diarrhea in 46% of patients at 125 mg, however, limiting the dose to 100 mg three times weekly resulted in insufficient inhibition of the PI3K pathway [
      • Markman B.
      • Tabernero J.
      • Krop I.
      • Shapiro G.I.
      • Siu L.
      • Chen L.C.
      • et al.
      Phase I safety, pharmacokinetic, and pharmacodynamic study of the oral phosphatidylinositol-3-kinase and mTOR inhibitor BGT226 in patients with advanced solid tumors.
      ]. Modeling based on pharmacokinetic data predicted that BGT226 dose of >4000 mg/day would be required to achieve efficacious plasma exposure, exceeding the safety dose range. The second, SF1126 is composed of the pan-PI3K and mTOR inhibitor LY294002 conjugated to an RGDS-targeting peptide to increase binding to integrins expressed on the tumor vasculature. In phase 1, SF1126 did not reach the maximum tolerated dose with a single dose limiting toxicity grade 3 diarrhea, reduced p-AKT and increased apoptosis [
      • Mahadevan D.
      • Chiorean E.G.
      • Harris W.B.
      • Von Hoff D.D.
      • Stejskal-Barnett A.
      • Qi W.
      • et al.
      Phase I pharmacokinetic and pharmacodynamic study of the pan-PI3K/mTORC vascular targeted pro-drug SF1126 in patients with advanced solid tumours and B-cell malignancies.
      ]. Further studies are planned in combination with rituximab in CD20+ B-cell malignancies. Other compounds such as BEZ235, XL765 (also known as SAR245409), GDC-0980, PF-04691502, and PF-05212384 (also known as PKI-587) have completed phase 1 trials but data are not yet published.
      Several new compounds have been designed to selectively inhibit PI3K with pan-PI3K targeting all class IA PI3Ks (BKM120, PX-866, XL147, GDC-0941, BAY80-6946, GSK2126458, CH5132799 and ATU027) or PI3K isoform-specific inhibitors (CAL101, BYL719, GSK2636771, and AZD6482). The phase 1 study with BKM120 [
      • Bendell J.C.
      • Rodon J.
      • Burris H.A.
      • de Jonge M.
      • Verweij J.
      • Birle D.
      • et al.
      Phase I, dose-escalation study of BKM120, an oral pan-Class I PI3K inhibitor, in patients with advanced solid tumors.
      ] has shown hyperglycemia, skin rash, and mood alteration as drug limiting toxicities. Once mood alterations were identified, selective serotonin reuptake inhibitors were prescribed, and no further mood alteration greater than grade 1 was seen. In the expansion study with the MTD (100 mg/d), most frequent grade 3/4 adverse effects (AEs) were increase transaminase (9.1%), asthenia (7.6%), and rash (6.1%). Anti-tumor activity is encouraging with 3 RECIST partial responses. A phase 1 study has also been completed for another compound (PX-866 [
      • Hong D.S.
      • Bowles D.W.
      • Falchook G.S.
      • Messersmith W.A.
      • George G.C.
      • O’Bryant C.L.
      • et al.
      A multicenter phase I trial of PX-866, an oral irreversible phosphatidylinositol 3-kinase inhibitor, in patients with advanced solid tumors.
      ]) showing that dose limiting toxicities consisted of grade 3 diarrhea and elevated AST. No RECIST response was seen in this trial. New trials in association with other drugs are currently ongoing.
      Finally, some compounds directly inhibit AKT with different mechanisms of action (plasma membrane disrupting agent, ATP-competitive and allosteric AKT inhibitors). Perifosine, a plasma membrane disrupting agent, has been extensively studied in phase I, II, and even phase III trials [
      • Van Ummersen L.
      • Binger K.
      • Volkman J.
      • Marnocha R.
      • Tutsch K.
      • Kolesar J.
      • et al.
      A phase I trial of perifosine (NSC 639966) on a loading dose/maintenance dose schedule in patients with advanced cancer.
      ,
      • Unger C.
      • Berdel W.
      • Hanauske A.R.
      • Sindermann H.
      • Engel J.
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      First-time-in-man and pharmacokinetic study of weekly oral perifosine in patients with solid tumours.
      ]. Despite promising first results with good tolerance and interesting anti-tumor activity, the latest phase III trial in metastatic refractory colorectal cancer was disappointing with no difference of overall survival or progression-free survival for perifosine plus capecitabine compared to capecitabine alone. In a small phase 1 trial with patient selection based on p-AKT positive tumor, triciribine phosphate monohydrate [
      • Garrett C.R.
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      • Wenham R.M.
      • Cubitt C.L.
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      • et al.
      Phase I pharmacokinetic and pharmacodynamic study of triciribine phosphate monohydrate, a small-molecule inhibitor of AKT phosphorylation, in adult subjects with solid tumors containing activated AKT.
      ] has shown no dose-limiting toxicities but a modest decrease of p-AKT after treatment. Response rates were not reported. Finally MK-2206, an allosteric AKT inhibitor, recently showed DLTs as mainly grade 3/4 skin rash [
      • Yap T.A.
      • Yan L.
      • Patnaik A.
      • Fearen I.
      • Olmos D.
      • Papadopoulos K.
      • et al.
      First-in-man clinical trial of the oral pan-AKT inhibitor MK-2206 in patients with advanced solid tumors.
      ]. In this trial no objective response was seen, however, tumor shrinkage was noted without reaching the level of the RECIST partial response. Of note, 9 patients have had paired tumor biopsy at baseline and at day 15. All of them demonstrated a decrease of p-AKT. Multiple drug combination and weekly schedule are now being tested to increase anti-tumor activity and minimize drug toxicity. Other compounds such as GSK690693, RX0201 (AKT antisense), PBI-05204, GSK2110183, GSK2141795, RG7440, GDC0068, and AZD5363 are under investigation or have completed phase 1 trials but the results are not yet published.

      Perspective on mTOR inhibitors in HCC therapy

      HCC is a very complex and heterogenous disease and progress in treatment of advanced HCC will likely occur slowly. One of the advantages of the first generation inhibitors (rapalogs) is the high specificity, the clinical approval and the comparatively few side-effects. Even if single agent therapies do not show the expected efficacy as has been revealed by the recent results from the EVOLVE study, rapalogs are still very attractive for use with other drugs. Moreover, sirolimus will most likely become first line treatment for HCC patients after liver transplantation.
      With regard to current available second generation mTOR inhibitors we can anticipate limitations in their use in HCC patients, although they are more effective than first generation inhibitors in preclinical studies. First, for patients with impaired liver function, the increase in transaminase is troublesome, as observed in several clinical trials with the mTOR inhibitor AZD8055 but also with PI3K/mTOR dual inhibitor BGT226. Second, the performed phase 1 clinical trials demonstrated modest monotherapy efficacy, which was related to adverse events limiting dose escalation, but also related to a cytostatic effect more than a cytolytic effect of these drugs. Therefore, improved efficacy may mainly be reached in combination with other drugs. Third, new generation mTOR inhibitors may stimulate autophagy more than first generation inhibitors, and may consequently cause tumor cell protection against chemotherapy-induced death.
      A strong focus should also be directed towards the discovery and validation of biomarkers, which predict tumor-response after therapy. Biomarkers for mTOR inhibitor efficacy have been evaluated in preclinical, but also in clinical studies [
      • Delbaldo C.
      • Albert S.
      • Dreyer C.
      • Sablin M.P.
      • Serova M.
      • Raymond E.
      • et al.
      Predictive biomarkers for the activity of mammalian target of rapamycin (mTOR) inhibitors.
      ,
      • Liao Y.M.
      • Sy A.
      • Yen Y.
      Markers for efficacy of mammalian target of rapamycin inhibitor.
      ]. Among others, these biomarkers include inactivation of PTEN, activating mutations of PI3KCA, expression levels of pS6, pS6K, S6K, and pAkt. Interestingly, a study demonstrated that human cancer cell lines carrying PI3KCA mutations were responsive to everolimus, except when KRAS mutations occurred concomitantly [
      • Di Nicolantonio F.
      • Arena S.
      • Tabernero J.
      • Grosso S.
      • Molinari F.
      • Macarulla T.
      • et al.
      Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus.
      ]. Similar results were obtained in a study with colon cancer cell lines [
      • Ducker G.S.
      • Atreya C.E.
      • Simko J.P.
      • Hom Y.K.
      • Matli M.R.
      • Benes C.H.
      • et al.
      Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors.
      ], and mTOR resistance was also shown in ovarian cancer cell lines, which overexpressed the apoptosis-inhibitory protein Bcl2 [
      • Aguirre D.
      • Boya P.
      • Bellet D.
      • Faivre S.
      • Troalen F.
      • Benard J.
      • et al.
      Bcl-2 and CCND1/CDK4 expression levels predict the cellular effects of mTOR inhibitors in human ovarian carcinoma.
      ]. Novel technologies such as next-generation sequencing may further path the way for the identification of new biomarkers, as has been shown in a recent study with bladder cancer patients, in which response to everolimus was clearly more effective in patients with a somatic mutation in the TSC1 complex [
      • Iyer G.
      • Hanrahan A.J.
      • Milowsky M.I.
      • Al-Ahmadie H.
      • Scott S.N.
      • Janakiraman M.
      • et al.
      Genome sequencing identifies a basis for everolimus sensitivity.
      ]. However, none of these markers have been confirmed in clinical trials. It is thus currently difficult to recommend any of these biomarkers for patient selection in clinical trials. In addition, it has to be emphasized, that substantial progress in the identification of biomarkers for HCC treatment is unlikely to occur, if tumor tissue is not taken before and after chemotherapy by means of a liver biopsy. Acquiring tumor tissue should therefore be mandatory for any phase II or phase III clinical study and will further help to develop novel therapies.
      Figure thumbnail fx2

      Financial support

      This work was supported by the Intramural Research Program of the NIH , National Cancer Institute, Center for Cancer Research . M.S.M. is supported by “ Schweizerische Stifung für Medizinisch-Biologische Stipendien ” ( PASMP-3_140071 ). T.D. is supported by the “Fondation Monahan” and “Prix Amgen pour la Recherche et l’Innovation en oncologie digestive 2011”.

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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