1. Co-activation Of PIK3CA And Yap Promotes Development Of Hepatocellular And Cholangiocellular Tumors In Mouse And Human Liver 2. Inactivation Of Fatty Acid Synthase Impairs Hepatocarcinogenesis Driven By AKT In Mice And Humans

Object:Primary liver cancer is one of the most common malignancies in adults and a leading cause of cancer related deaths worldwide. Hepatocellular carcinoma(HCC) and intrahepatic cholangiocarcinoma(ICC) are the major types of primary liver cancer, accounting for almost 90% and ~10% of all liver tumors, respectively. Another livertumor entity, known as mixed HCC/CCA, can also occur, although at significantly lower frequency. Due to its increasing incidence and related poor survival, innovative therapeutic options for liver cancer patients are necessary. For this purpose, a deeper knowledge of the molecular mechanisms underlying liver cancer development is highly required. Yap is a major downstream effector of the Hippo pathway, an evolutionally well-conserved potent regulator of organ size, tissue regeneration, stem cell self-renewal, and tumor development. Recently, numerous studies have detected overexpression of Yap in a variety of human tumor types, including colorectal, ovarian, and lung cancer. In HCC, Yap has been identified as a driver oncogene and an independent factor in predicting poor disease-free and overall survival. The phosphatidylinositol 3-kinase/mammalian target of rapamycin(PI3K/m TOR) pathway is aberrantly activated in variable types of cancers, including in 30-50% of HCC cases. This signaling cascade plays a pivotal role in many cellular processes, including growth, proliferation, survival, autophagy, metabolism, and cytoskeletal organization. PI3 Ks are heterodimeric lipid kinases composed of p110 catalytic subunits and p85 regulatory subunits, interacting with phosphatidylinositol-3-phosphate at the membrane and catalyzing the phosphorylation of AKT. Mutant forms of PIK3 CA, which encodes the p110 a catalytic subunit, have been found in various human cancers, including colon, breast, lung cancer, and HCC, leading to increased lipid kinase activity and oncogenesis. Both Yap and PI3K/m TOR signaling pathways are potent inducers of hepatocarcinogenesis, and a previous study showed that activation of Yap and PI3K/AKT/m TOR signaling correlated positively in HCC. However, whether the two pathways functionally interact along hepatocarcinogenesis has not been investigated to date, especially in vivo.Here, we studied whether activation of PI3 K and Yap cascades cooperated to promote development of liver tumors in mice, and investigated the underlying molecular mechanism for hepatic carcinogenesis. In addition, we also examined their expression in human liver cancer samples characterized by hepatocellularand/or cholangiocellular features.Methods:We ovexpressed Yap S127 A,PIK3CAH1047R,or co-expressed Yap S127 A and PIK3CAH1047 R, Yap S127AS94 A and PIK3CAH1047 R, or Yap WT and PIK3CAH1047 R in mice livers by hydrodynamic transfection, and observed their effect on liver tumor formation. And then, the liver tissues were harvested; real-time PCR, western blot, histologic and immunohistochemical analyses were performed to describe the molecular characterization and elucidate the underlying molecular mechanisms. Moreover, we analyzed a collection of human HCC(n=54), ICC(n=42), and mixed HCC/CCA(n=16) by immunohistochemistry for PIK3 CA and Yap staining to evaluate the possible relationship between PI3 K and Yap pathways in human liver cancers. In addition, the PIK3 CA specific inhibitor PIK75, and the disruptor of Yap-TEAD interaction Verteporfin, were applied either alone or in combination in HLF and SK/Hep1 HCC cell lines as well as in the EGI1 CCA cell line to assess the importance of the PI3 K and Yap on the in vitro growth of human HCC and CCA cell lines.Results: 1、Overexpression of Yap S127 A alone did not result in any liver anomaly even after 22.5 weeks post injection, whereas overexpression of PIK3CAH1047R(which will be referred to as PIK3 CA mouse) resulted in the occurrence of lipid-rich hepatocytes forming clusters throughout the liver parenchyma. However, no tumors developed up to 40 weeks post injection in PIK3 CA mice. In striking contrast, co-expression of PIK3CAH1047 R and Yap S127A(which will be referred to as PIK3CA/Yap mouse) led to rapid liver tumor development within 10-13 weeks post injection. 2、At the histological level, ~80% of the liver parenchyma from PIK3CA/Yap mice was occupied by tumor lesions, with the remaining liver tissue consisting of lipid-rich hepatocytes(morphologically identical to those detected in PIK3 CA mice) and normal liver tissue. Of note, three distinct tumor types were detected in PIK3CA/Yap mice:(i) pure HCC;(ii) pure CCA;(iii) mixed HCC/CCA.3、Combined injection of PIK3CAH1047 R and Yap WT, or PIK3CAH1047 R and Yap S127AS94 A resulted in the complete abrogation of tumor development in mice 13 weeks post injection. 4、Real Time RT-PCR and western blot showed that upregulation of liver tumor specific marker α-fetoprotein(AFP), Glypican 3(GPC3), and Ep CAM in PI3K/Yap tumors; tumor cell metabolism regulators including hexokinase2(HK2) and pyruvate kinase isozyme M2(PKM2), cell cycle regulatory genes, cyclin B1, cyclin D1, cyclin E1 and c-Myc and cancer proliferative marker, Ki67 were all were upregulated in PI3K/Yap tumors. 5、Immunohistochemistry results showed that p-AKT,p-m TOR and the key downstream molecular of PI3K/AKT/m TOR cascade p-RPS6,ACAC,FASN p-NDRG1, Notch2 and and its target SOX9, p-ERK1/2 were all positively expressed. 6、In HCC, upregulation of PIK3 CA and nuclear accumulation of Yap were detected in 23(42.6%) and 38(70.4%) specimens, respectively. Importantly 17 of 23(73.9%) HCC specimens showing upregulation of PIK3 CA concomitantly exhibited nuclear localization of the Yap protein. In ICC, PIK3 CA levels were induced in 24 of 42(57.1%) samples, whereas all specimens displayed immunoreactivity for Yap. Finally, PIK3 CA levels were elevated in 11 of 16(68.8%) mixed HCC/CCA, while Yap nuclear translocation was detected in 14 of the latter specimens(87.8%). Simultaneous upregulation of PIK3 CA and nuclear localization of Yap occurred in 10 of 16(62.5%) mixed HCC/CCA. No association between the staining patterns of PIK3 CA and Yap and clinicopathological features of the HCC, ICC, and mixed HCC/CCA patients, including etiology, presence of cirrhosis, α-fetoprotein levels, tumor size, and tumor grading was found. 7、Treatment with the two inhibitors alone resulted in a strong decrease of proliferation and induction of apoptosis in HCC and CCA cell lines. A further reduction of proliferation was detected in the three cell lines when the two drugswere administered combinatorially, whereas no additive effects on apoptosis were observed.Conclusion: 1、Co-activation of PIK3 CA and Yap promotes development of hepatocellular and cholangiocellular tumors in mouse and human liver. 2、PIK3CA/Yap tumors were characterized by activation of the AKT/m TOR, ERK/MAPK, and Notch pathways. 3、Combined suppression of PIK3 CA and Yap pathways is highly detrimental for the growth of human HCC and CCA cell lines. Combined suppression of the PI3K/AKT/m TOR and Yap pathways might be an innovative and effective therapeutic approach for the treatment of human liver tumors characterized by the aberrant activation of the Yap and PI3K/AKT/m TOR pathways.Object: Deregulated fatty acid biosynthesis, also known as de novo lipogenesis, is a key aberration in cancer. It provides rapidly proliferating cancer cells with a continuous supply of lipids and lipid precursors that are needed for membrane production, energy generation, and lipid-based post-transcriptional modifications of proteins. At the molecular level, de novo lipogenesis is characterized by an upregulation in tumor cells of lipogenic enzymes, including adenosine triphosphate citrate lyase(ACLY), acetyl-Co A carboxylase(ACAC), fatty acid synthase(FASN), and stearoyl-Co A desaturase 1(SCD1). FASN, the enzyme responsible for the production of long chain fatty acids from acetyl-co A and malonyl-Co A, is the most investigated lipogenic protein in cancer. FASNlevels are elevated in many tumor types, where they significantly correlate with cancer biological aggressiveness and unfavorable prognosis. In addition, upregulation of FASN occurs in preneoplastic and pre-invasive lesions of various organs. Also, FASN blockade triggers tumor growth restraint and massive apoptosis in numerous in vitro and in vivo models. Furthermore, FASN overexpression induces the development of prostate intraepithelial neoplasia in transgenic mice, thus acting as a bona fide oncogene in prostate cancer. Similarly, overexpression of FASN induces a cancer-like phenotype in non-tumorous breast cell lines. In hepatocellular carcinoma(HCC), aberrant expression of lipogenic enzymes including FASN has been linked both to tumor development and progression. For instance, overexpression of FASN occurs in liver preneoplastic lesions from rat models of chemically- and hormonally-induced hepatocarcinogenesis. Similarly, sustained lipogenesis and FASN upregulation characterize human liver clear cell foci, whose preneoplastic nature has been hypothesized. Also, levels of FASN and other lipogenic proteins as well as polymorphisms in lipogenic genes are associated with poor outcome in HCC patients. In addition, FASN suppression has been shown to be detrimental for HCC growth in vitro. Virtually all functional studies on FASN in HCC have been performed in HCC cell lines so far.Despite this body of evidence, key questions about FASN in HCC remain unanswered. Thus, it is unknown whether FASN contributes to liver tumor development and/or progression in vivo.Methods: FASN was overexpressed in the mouse liver, either alone or in combination with activated N-Ras, c-Met, or SCD1, via hydrodynamic injection to determine its oncogenic potential in vivo. Activated AKT was overexpressed via hydrodynamic injection in livers of conditional FASN or Rictor knockout mice to investigate whether AKT-driven hepatic steatosis and tumor development depends on FASN or Rictor. FASN was suppressed in human hepatoma cell lines via specific small interfering RNA or chemical inhibitor to investigate the molecular mechanisms associated with hepatocarcinogenesis regulated by FASN. Finally, levels of FASN, p-AKT, and Rictorwere analyzed in a collection of human HCC specimens by immunohistochemistry.Results: 1. Overexpression of FASN, either alone or in combination with other genes associated with hepatocarcinogenesis, did not induce histological liver alterations. 2. Genetic ablation of FASN resulted in the complete inhibition AKT-driven liver steatosis and hepatocarcinogenesis, and downregulation of phosphorylated/activated AKT in the mouse liver. 3. In human HCC cell lines, FASN silencing via specific small interfering RNA(si RNA)orchemical inhibitor C75 led to a decline in cell proliferation and a rise in apoptosis, which were paralleled by a decrease in the levels of phosphorylated/activated AKT, an event controlled by the mammalian target of rapamycin complex 2(m TORC2). 4. Mechanically, downregulation of AKT phosphorylation/activation following FASN inactivation was associated with a strong inhibition of rapamycin-insensitive companion of m TOR(Rictor), the major component of m TORC2, at post-transcriptional levelvia ELOVL5-independent mechanisms 5. Genetic ablation of Rictor impaired AKT-driven liver steatosis and hepatocarcinogenesis, and downregulation of phosphorylated/activated AKT in the mouse liver. Overexpression of Rictor could significantly decrease the growth restraint induced by FASN knockdown in HCC cells 6. Higher immunolabeling for FASN, p-AKT, and Rictor was found in 81.8%, 60.2%, and 21.6% of HCC specimens, respectively, when compared with surrounding nontumorous livers.Importantly, all HCC specimens showing p-AKTand Rictor overexpression also exhibited elevated FASN levels.Also, all samples showing induction of Rictor displayed elevatedp-AKT. Furthermore, 61.1%, 66.03% and 63.1% liver tumors displaying induction of FASN, p-AKT and Rictor, respectively, belonged to the HCC subset with poorer outcome, linking the overexpression of these proteins to a dismal prognosis in HCC. No association between FASN, p-AKT and Rictorstaining patterns and other clinicopathological features of the patients was detected.Conclusion: 1. FASN is not a driver oncogeneper se in the mouse liver. 2. FASN is necessary for AKT-driven hepatocarcinogenesis. 3. FASN is involved in the control of AKT activation via regulation of Rictorat post-transcriptional level. 4. Pharmacological blockade of FASN might be highly useful in the treatment of human HCC characterized by activation of the AKT pathway.