Subset Of Human Hepatocellular Carcinomas Research Articles

Hepatocytic p62 suppresses ductular reaction and tumorigenesis in mouse livers with mTORC1 activation and defective autophagy

Either activation of mTORC1 due to loss of Tsc1 (tuberous sclerosis complex 1) or defective hepatic autophagy due to loss of Atg5 leads to spontaneous liver tumorigenesis in mice. The purpose of this study was to investigate the mechanisms by which autophagy contributes to the hepatic metabolic changes and tumorigenesis mediated by mTORC1 activation. Atg5 Flox/Flox (Atg5F/F) and Tsc1F/F mice were crossed with albumin-Cre mice to generate liver-specific Atg5 knockout (L-Atg5 KO), L-Tsc1 KO and L-Atg5/Tsc1 double KO (DKO) mice. These mice were crossed with p62/Sqstm1F/F (p62) and whole body Nrf2 KO mice to generate L-Atg5/Tsc1/p62 and L-Atg5/Tsc1-Nrf2 triple KO mice. These mice were housed for various periods up to 12 months, and blood and liver tissues were harvested for biochemical and histological analysis RESULTS: Deletion of Atg5 in L-Tsc1 KO mice inhibited liver tumorigenesis but increased mortality and was accompanied by drastically enhanced hepatic ductular reaction (DR), hepatocyte degeneration and metabolic reprogramming. Deletion of p62 reversed DR, hepatocyte degeneration and metabolic reprogramming as well as the mortality of L-Atg5/Tsc1 DKO mice, but unexpectedly promoted liver tumorigenesis via activation of a group of oncogenic signaling pathways. Nrf2 ablation markedly improved DR with increased hepatocyte population and improved metabolic reprogramming and survival of the L-Atg5/Tsc1 DKO mice without tumor formation. Decreased p62 and increased mTOR activity were also observed in a subset of human hepatocellular carcinomas. These results reveal previously undescribed functions of hepatic p62 in suppressing tumorigenesis and regulating liver cell repopulation and metabolic reprogramming resulting from persistent mTORC1 activation and defective autophagy. Metabolic liver disease and viral hepatitis are common chronic liver diseases and risk factors of hepatocellular carcinoma, which are often associated with impaired hepatic autophagy and increased mTOR activation. Using multiple genetically engineered mouse models of defective hepatic autophagy and persistent mTOR activation, we dissected the complex mechanisms behind this observation. Our results uncovered an unexpected novel tumor suppressor function of p62/Sqstm1, which regulated liver cell repopulation, ductular reaction and metabolic reprogramming in liver tumorigenesis.

Alpelisib combination treatment as novel targeted therapy against hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common primary cancer with an unsatisfactory long-term survival. Gain of function mutations of PIK3CA occur in a subset of human HCC. Alpelisib, a selective PIK3CA inhibitor, has been approved by the FDA to treat PIK3CA mutant breast cancers. In this manuscript, we evaluated the therapeutic efficacy of alpelisib, either alone or in combination, for the treatment of HCC. We tested alpelisib in mouse HCC induced by hydrodynamic injection of c-Met/PIK3CA(H1047R) (c-Met/H1047R), c-Met/PIK3CA(E545K) (c-Met/E545K), and c-Met/sgPten gene combinations. Alpelisib slowed down the growth of c-Met/H1047R and c-Met/E545K HCC but was ineffective in c-Met/sgPten HCC. Mechanistically, alpelisib inhibited p-ERK and p-AKT in c-Met/H1047R and c-Met/E545K HCC progression but did not affect the mTOR pathway or genes involved in cell proliferation. In human HCC cell lines transfected with PIK3CA(H1047R), alpelisib synergized with the mTOR inhibitor MLN0128 or the CDK4/6 inhibitor palbociclib to suppress HCC cell growth. In c-Met/H1047R mice, alpelisib/MLN0128 or alpelisib/palbociclib combination therapy caused tumor regression. Our study demonstrates that alpelisib is effective for treating PIK3CA-mutated HCC by inhibiting MAPK and AKT cascades. Furthermore, combining alpelisib with mTOR or CDK4/6 inhibitors has a synergistic efficacy against PIK3CA-mutated HCC, providing novel opportunities for precision medicine against HCC.

Low incidence of hepatocellular carcinoma in mice and cats treated with systemic adeno-associated viral vectors

Adeno-associated viral (AAV) vectors have emerged as the preferred platform for in vivo gene transfer because of their combined efficacy and safety. However, insertional mutagenesis with the subsequent development of hepatocellular carcinomas (HCCs) has been recurrently noted in newborn mice treated with high doses of AAV, and more recently, the association of wild-type AAV integrations in a subset of human HCCs has been documented. Here, we address, in a comprehensive, prospective study, the long-term risk of tumorigenicity in young adult mice following delivery of single-stranded AAVs targeting liver. HCC incidence in mice treated with therapeutic and reporter AAVs was low, in contrast to what has been previously documented in mice treated as newborns with higher doses of AAV. Specifically, HCCs developed in 6 out 76 of AAV-treated mice, and a pathogenic integration of AAV was found in only one tumor. Also, no evidence of liver tumorigenesis was found in juvenile AAV-treated mucopolysaccharidosis type VI (MPS VI) cats followed as long as 8 years after vector administration. Together, our results support the low risk of tumorigenesis associated with AAV-mediated gene transfer targeting juvenile/young adult livers, although constant monitoring of subjects enrolled in AAV clinical trial is advisable.

Cooperation Between MYC and β-Catenin in Liver Tumorigenesis Requires Yap/Taz.

Activation of MYC and catenin beta-1 (CTNNB1, encoding β-catenin) can co-occur in liver cancer, but how these oncogenes cooperate in tumorigenesis remains unclear. We generated a mouse model allowing conditional activation of MYC and WNT/β-catenin signaling (through either β-catenin activation or loss of APC - adenomatous polyposis coli) upon expression of CRE recombinase in the liver and monitored their effects on hepatocyte proliferation, apoptosis, gene expression profiles, and tumorigenesis. Activation of WNT/β-catenin signaling strongly accelerated MYC-driven carcinogenesis in the liver. Both pathways also cooperated in promoting cellular transformation in vitro, demonstrating their cell-autonomous action. Short-term induction of MYC and β-catenin in hepatocytes, followed by RNA-sequencing profiling, allowed the identification of a "Myc/β-catenin signature," composed of a discrete set of Myc-activated genes whose expression increased in the presence of active β-catenin. Notably, this signature enriched for targets of Yes-associated protein (Yap) and transcriptional coactivator with PDZ-binding motif (Taz), two transcriptional coactivators known to be activated by WNT/β-catenin signaling and to cooperate with MYC in mitogenic activation and liver transformation. Consistent with these regulatory connections, Yap/Taz accumulated upon Myc/β-catenin activation and were required not only for the ensuing proliferative response, but also for tumor cell growth and survival. Finally, the Myc/β-catenin signature was enriched in a subset of human hepatocellular carcinomas characterized by comparatively poor prognosis. Myc and β-catenin show a strong cooperative action in liver carcinogenesis, with Yap and Taz serving as mediators of this effect. These findings warrant efforts toward therapeutic targeting of Yap/Taz in aggressive liver tumors marked by elevated Myc/β-catenin activity.

C3HeB/FeJ Mice mimic many aspects of gene expression and pathobiological features of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a deadly cancer, underscoring the need for relevant preclinical models. Male C3HeB/FeJ mice model spontaneous HCC with some hepatocarcinogenesis susceptibility loci corresponding to syntenic regions of human chromosomes altered in HCC. We tested other properties of C3HeB/FeJ tumors for similarity to human HCC. C3HeB/FeJ tumors were grossly visible at 4 months of age, with prevalence and size increasing until about 11 months of age. Histologic features shared with human HCC include hepatosteatosis, tumor progression from dysplasia to poorly differentiated, vascular invasion, and trabecular, oncocytic, vacuolar, and clear cell variants. More tumor cells displayed cytoplasmic APE1 staining versus normal liver. Ultrasound effectively detected and monitored tumors, with 85.7% sensitivity. Over 5000 genes were differentially expressed based on the GSE62232 and GSE63898 human HCC datasets. Of these, 158 and 198 genes, respectively, were also differentially expressed in C3HeB/FeJ. Common cancer pathways, cell cycle, p53 signaling and other molecular aspects, were shared between human and mouse differentially expressed genes. We established eigengenes that distinguish HCC from normal liver in the C3HeB/FeJ model and a subset of human HCC. These features extend the relevance and improve the utility of the C3HeB/FeJ line for HCC studies.

Loss of Pten synergizes with c-Met to promote hepatocellular carcinoma development via mTORC2 pathway

Hepatocellular carcinoma (HCC) is a deadly malignancy with limited treatment options. Activation of the AKT/mTOR cascade is one of the most frequent events along hepatocarcinogenesis. mTOR is a serine/threonine kinase and presents in two distinct complexes: mTORC1 and mTORC2. While mTORC1 has been extensively studied in HCC, the functional contribution of mTORC2 during hepatocarcinogenesis has not been well characterized, especially in vivo. Pten expression is one of the major mechanisms leading to the aberrant activation of the AKT/mTOR signaling. Here, we show that concomitant downregulation of Pten and upregulation of c-Met occurs in a subset of human HCC, mainly characterized by poor prognosis. Using CRISPR-based gene editing in combination with hydrodynamic injection, Pten was deleted in a subset of mouse hepatocytes (sgPten). We found that loss of Pten synergizes with overexpression of c-Met to promote HCC development in mice (sgPten/c-Met). At the molecular level, sgPten/c-Met liver tumor tissues display increased AKT and mTOR signaling. Using Rictor conditional knockout mice, we demonstrate that sgPten/c-Met-driven HCC development strictly depends on an intact mTORC2 complex. Our findings therefore support the critical role of mTORC2 in hepatocarcinogenesis. sgPten/c-Met mouse model represents a novel valuable system that can be used for the development of targeted therapy against this deadly malignancy.

Oncogenic potential of histone-variant H2A.Z.1 and its regulatory role in cell cycle and epithelial-mesenchymal transition in liver cancer

H2A.Z is a highly conserved H2A variant, and two distinct H2A.Z isoforms, H2A.Z.1 and H2A.Z.2, have been identified as products of two non-allelic genes, H2AFZ and H2AFV. H2A.Z has been reported to be overexpressed in breast, prostate and bladder cancers, but most studies did not clearly distinguish between isoforms. One recent study reported a unique role for the H2A.Z isoform H2A.Z.2 as a driver of malignant melanoma. Here we first report that H2A.Z.1 plays a pivotal role in the liver tumorigenesis by selectively regulating key molecules in cell cycle and epithelial-mesenchymal transition (EMT). H2AFZ expression was significantly overexpressed in a large cohort of hepatocellular carcinoma (HCC) patients, and high expression of H2AFZ was significantly associated with their poor prognosis. H2A.Z.1 overexpression was demonstrated in a subset of human HCC and cell lines. H2A.Z.1 knockdown suppressed HCC cell growth by transcriptional deregulation of cell cycle proteins and caused apoptotic cell death of HCC cells. We also observed that H2A.Z.1 knockdown reduced the metastatic potential of HCC cells by selectively modulating epithelial-mesenchymal transition regulatory proteins such as E-cadherin and fibronectin. In addition, H2A.Z.1 knockdown reduced the in vivo tumor growth rate in a mouse xenograft model. In conclusion, our findings suggest the oncogenic potential of H2A.Z.1 in liver tumorigenesis and that it plays established role in accelerating cell cycle transition and EMT during hepatocarcinogenesis. This makes H2A.Z.1 a promising target in liver cancer therapy.

Glutamine depletion by crisantaspase hinders the growth of human hepatocellular carcinoma xenografts.

Background:A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO).Methods:Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1.Results:Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro.Conclusions:The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.

MicroRNA‐494 within an oncogenic microRNA megacluster regulates G 1 /S transition in liver tumorigenesis through suppression of mutated in colorectal cancer

Hepatocellular carcinoma (HCC) is associated with poor survival for patients and few effective treatment options, raising the need for novel therapeutic strategies. MicroRNAs (miRNAs) play important roles in tumor development and show deregulated patterns of expression in HCC. Because of the liver’s unique affinity for small nucleic acids, miRNA-based therapy has been proposed in the treatment of liver disease. Thus, there is an urgent need to identify and characterize aberrantly expressed miRNAs in HCC. In our study, we profiled miRNA expression changes in de novo liver tumors driven by MYC and/or RAS, two canonical oncogenes activated in a majority of human HCCs. We identified an up-regulated miRNA megacluster comprised of 53 miRNAs on mouse chromosome 12qF1 (human homolog 14q32). This miRNA megacluster is up-regulated in all three transgenic liver models and in a subset of human HCCs. An unbiased functional analysis of all miRNAs within this cluster was performed. We found that miR-494 is overexpressed in human HCC and aids in transformation by regulating the G1/S cell cycle transition through targeting of the Mutated in Colorectal Cancer tumor suppressor. miR-494 inhibition in human HCC cell lines decreases cellular transformation, and anti-miR-494 treatment of primary MYC-driven liver tumor formation significantly diminishes tumor size. Conclusion: Our findings identify a new therapeutic target (miR-494) for the treatment of HCC. (Hepatology 2014;58:202–215)

MicroRNA-29c functions as a tumor suppressor by direct targeting oncogenic SIRT1 in hepatocellular carcinoma

Mammalian sirtuin 1 (SIRT1) has connected to an ever widening circle of activities that encompass cellular stress resistance, energy metabolism and tumorigenesis. However, underlying mechanisms leading to oncogenic SIRT1 overexpression are less understood. In this study, we identified SIRT1 regulatory microRNA (miRNA) and its function in hepatocellular carcinoma (HCC). Aberrant SIRT1 overexpression was demonstrated in a subset of human HCCs. SIRT1 knockdown suppressed HCC cell growth by transcriptional deregulation of cell cycle proteins. This led to hypophosphorylation of pRb, which inactivated E2F/DP1 target gene transcription, and thereby caused significant increase of HCC cells to remain in the G1/S phase. A comprehensive miRNA profiling analysis indentified five putative endogenous miRNAs that are significantly downregulated in HCC. Ectopic expression of miRNA mimics evidenced miR-29c to suppress SIRT1 in HCC cells. Notably, ectopic miR-29c expression repressed cancer cell growth and proliferation, and it recapitulated SIRT1 knockdown effects in HCC cells. In addition, miR-29c expression was downregulated in a large cohort of HCC patients, and low expression of miR-29c was significantly associated with poor prognosis of HCC patients. Taken together, we demonstrated that miR-29c suppresses oncogenic SIRT1 by way of binding to 3'-untranslated region of SIRT1 mRNA causing translational inhibition in liver cancer cells. The loss or suppression of miR-29c may cause aberrant SIRT1 overexpression and promotes liver tumorigenesis. Overall, we suggest that miR-29c functions as a tumor suppressor by regulating abnormal SIRT1 activity in liver.

HDAC1 Inactivation Induces Mitotic Defect and Caspase-Independent Autophagic Cell Death in Liver Cancer

Histone deacetylases (HDACs) are known to play a central role in the regulation of several cellular properties interlinked with the development and progression of cancer. Recently, HDAC1 has been reported to be overexpressed in hepatocellular carcinoma (HCC), but its biological roles in hepatocarcinogenesis remain to be elucidated. In this study, we demonstrated overexpression of HDAC1 in a subset of human HCCs and liver cancer cell lines. HDAC1 inactivation resulted in regression of tumor cell growth and activation of caspase-independent autophagic cell death, via LC3B-II activation pathway in Hep3B cells. In cell cycle regulation, HDAC1 inactivation selectively induced both p21WAF1/Cip1 and p27Kip1 expressions, and simultaneously suppressed the expression of cyclin D1 and CDK2. Consequently, HDAC1 inactivation led to the hypophosphorylation of pRb in G1/S transition, and thereby inactivated E2F/DP1 transcription activity. In addition, we demonstrated that HDAC1 suppresses p21WAF1/Cip1 transcriptional activity through Sp1-binding sites in the p21WAF1/Cip1 promoter. Furthermore, sustained suppression of HDAC1 attenuated in vitro colony formation and in vivo tumor growth in a mouse xenograft model. Taken together, we suggest the aberrant regulation of HDAC1 in HCC and its epigenetic regulation of gene transcription of autophagy and cell cycle components. Overexpression of HDAC1 may play a pivotal role through the systemic regulation of mitotic effectors in the development of HCC, providing a particularly relevant potential target in cancer therapy.

Abstract 1068: Downregulation of PH domain leucine-rich repeat protein phosphatases (PHLPPs) identifies a subgroup of human hepatocellular and colorectal carcinomas with elevated levels of activated AKT and low apoptosis rate

Abstract Dysregulation of the phosphoinositide-3-kinase (PI3K)/AKT axis has been implicated in human hepatic and colorectal carcinogenesis. Although point mutations of the p110 alpha subunit of PI3K (PIK3CA), the upstream inducer of AKT, have been previously detected in these tumor types, the molecular mechanisms responsible for unrestrained activity of AKT remain largely unknown. Since it has been recently shown that PH domain leucine-rich repeat protein phosphatase 1 and 2 (PHLPP1 and PHLPP2) proteins are able to directly dephosphorylate AKT in vivo, we assessed the expression pattern of both PHLPP isoforms in a collection of human liver and colorectal cancer tissues and corresponding non-neoplastic surrounding counterparts using western blot, reverse-transcription real-time PCR, and immunohistochemical analyses. In liver specimens, we found that the expression of PHLPP1 and PHLPP2 was significantly decreased in a subset of human hepatocellular carcinomas (HCCP) (22/40, 55%; 15/40, 37.5%, respectively) characterized by a clinically aggressive phenotype (patient's survival shorter than 3 years following partial liver resection), whereas strong expression of the two proteins was detected in non-neoplastic surrounding liver tissues. Downregulation of PHLPP1 (36/50, 72%) and PHLPP2 (38/50, 76%) was frequent in colorectal tumors, whereas a remarkable upregulation of PHLPP1 and PHLPP2 occurred in the respective, adjacent normal colonic mucosa. In both liver and colorectal tumors, PHLPP1 and PHLPP2 levels inversely correlated with those of activated AKT and directly with the apoptosis rate. In human Hep3B and PLC HCC cell lines, displaying low PHLPP1 and PHLPP2 levels, overexpression of either PHLPP1 or PHLPP2 gene decreased the rate of cell proliferation and induced apoptosis. These effects were paralleled by decrease in AKT activation, upregulation of BAD, BAX, FOXO1, and active caspase 9 pro-apoptotic proteins, and downregulation of XIAP and Bcl-2 anti-apoptotic proteins. Conversely, silencing of either PHLPP1 or PHLPP2 gene by siRNA in the WRL-68 hepatoblast cell line (displaying high levels of PHLPP1 and PHLPP2) resulted in increased cell proliferation, reduced apoptosis, downregulation of BAD, BAX, FOXO1, and active caspase 9, and upregulation of XIAP and Bcl-2 proteins. In the same cell line, overexpression of a constitutively active form of AKT induced downregulation of PHLPP1 and PHLPP2, implying a negative modulation by AKT over PHLPP1 and PHLPP2 expression. Altogether, our results suggest that PHLPP1 and PHLPP2 phosphatases play a tumor suppressive role both in human hepatic and colorectal carcinogenesis via their ability to suppress AKT activation and to induce apoptosis Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1068. doi:10.1158/1538-7445.AM2011-1068

C/EBPα Is Up-regulated in a Subset of Hepatocellular Carcinomas and Plays a Role in Cell Growth and Proliferation

C/EBPalpha (cebpa) is a putative tumor suppressor. However, initial results indicated that cebpa was up-regulated in a subset of human hepatocellular carcinomas (HCCs). The regulation and function of C/EBPalpha was investigated in HCC cell lines to clarify its role in liver carcinogenesis. The regulation of C/EBPalpha expression was studied by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemistry, methylation-specific PCR, and chromatin immunoprecipitation assays. C/EBPalpha expression was knocked-down by small interfering RNA or short hairpin RNA. Functional assays included colony formation, methylthiotetrazole, bromodeoxyuridine incorporation, and luciferase-reporter assays. Cebpa was up-regulated at least 2-fold in a subset (approximately 55%) of human HCCs compared with adjacent nontumor tissues. None of the up-regulated samples were positive for hepatitis C infection. The HCC cell lines Hep3B and Huh7 expressed high, PLC/PRF/5 intermediate, HepG2 and HCC-M low levels of C/EBPalpha, recapitulating the pattern of expression observed in HCCs. No mutations were detected in the CEBPA gene in HCCs and cell lines. C/EBPalpha was localized to the nucleus and functional in Hep3B and Huh7 cells; knocking-down its expression reduced target-gene expression, colony formation, and cell growth, associated with a decrease in cyclin A and CDK4 concentrations and E2F transcriptional activity. Epigenetic mechanisms including DNA methylation, and the binding of acetylated histone H3 to the CEBPA promoter-regulated cebpa expression in the HCC cells. C/EBPalpha is up-regulated in a subset of HCCs and has growth-promoting activities in HCC cells. Novel oncogenic mechanisms involving C/EBPalpha may be amenable to epigenetic regulation to improve treatment outcomes.

Human homolog of NOTUM, overexpressed in hepatocellular carcinoma, is regulated transcriptionally by β‐catenin/TCF

The Drosophila Notum gene, which is regulated by the Wingless pathway, encodes a secreted hydrolase that modifies heparan sulfate proteoglycans. In comparative analysis of the gene expression profiles in primary human hepatocellular carcinomas (HCC) and normal organs, we observed that the human ortholog of Drosophila Notum was overexpressed markedly in a subset of HCC, but expressed rarely in adult normal tissues. Immunoblotting confirmed the overexpression of NOTUM protein in 12 of 40 primary HCC cases (30%). High levels of NOTUM protein were significantly associated with intracellular (nuclear or cytoplasmic) accumulation of beta-catenin protein: all 10 HCC with high intracellular beta-catenin also had high NOTUM expression, whereas only 2 of 30 cases (6.7%) without intracellular beta-catenin had high NOTUM expression (P < 0.00001). NOTUM expression in HepG2 cells was downregulated significantly by induction of a dominant-negative mutant of TCF4, a beta-catenin partner. In vivo binding of the beta-catenin/TCF complex to the NOTUM promoter was demonstrated by chromatin immunoprecipitation in HepG2 and SW480 cells, where canonical Wnt signaling is activated constitutively. These findings provide evidence that NOTUM is a novel target of beta-catenin/TCF4 and is upregulated in Wnt/beta-catenin signaling-activated HCC.

Human and mouse hepatocellular adenoma and carcinoma display similar tumorigenesis pathway alterations

Distinct pathways of genomic progression to benign and malignant tumors of the liver. Tward AD, Jones KD, Yant S, Cheung ST, Fan ST, Chen X, Kay MA, Wang R, Bishop JM. We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of β-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1α. Prompted by these findings, we uncovered a coincidence between activation of the protein–tyrosine kinase encoded by MET and activating mutations of β-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated β-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated β-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for pre-clinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway. [Abstract reproduced by permission of Proc Natl Acad Sci USA 2007;104:14771–14776]

Prognostic Value of MET, RON and Histoprognostic Factors for Urothelial Carcinoma in the Upper Urinary Tract

RON (recepteur d'origine Nantais) (Santa Cruz Technology, Santa Cruz, California) and c-met (Dako, Glostrup, Denmark) are members of the c-met proto-oncogene family. c-met encodes a receptor tyrosine kinase and has a role in oncogenesis. RON has a role in cell transformation and epithelial tumorigenesis. Over expression of the 2 genes has been demonstrated in human bladder cancer. We explored whether over expression of the 2 proteins has a role in the tumorigenesis and defined histoprognostic factors of poor clinical outcomes in patients with these tumors. We reviewed the records of 42 patients with upper urinary tract urothelial carcinoma. A total of 24 tumors were localized in the renal pelvis and 18 were in the ureter. Immunohistochemical staining for RON and c-met was performed using tissue microarrays. Patient age was 46 to 100 years (mean 70.6). Of the patients 23 (54%) died of disease. Over expression of c-met was associated with a higher risk of embolism (p = 0.0002), while over expression of RON was not significantly associated with emboli (p = 0.5). Univariate analysis showed that relapse was significantly associated with ureteral localization (p = 0.02), vascular invasion (p = 0.003), and high grade (p = 0.04) and high stage (0.02) urothelial carcinoma. The association with vascular invasion, and high grade and high stage urothelial carcinoma was also statistically significant (p <0.0001). Notably superficial tumors showed an important relapse rate (p = 0.003). Independent prognostic factors of relapse in upper urinary tract urothelial carcinoma are ureteral localization, vascular invasion, high grade and high stage. c-met seems to influence the development of vascular invasion via an unknown mechanism. Nevertheless, to our knowledge an association between c-met over expression and aggressive clinical behavior in upper urinary tract carcinomas has not been previously reported.

Distinct pathways of genomic progression to benign and malignant tumors of the liver

We used several of the genetic lesions commonly associated with human liver tumors to reconstruct genetic progression to hepatocellular carcinoma and adenoma in mouse models. We initiated tumorigenesis with a transgene of the protooncogene MET or by hydrodynamic transfection of MET in combination with other genes into the livers of adult animals. Hepatocellular carcinoma in both instances arose from cooperation between MET and constitutively active versions of beta-catenin. In contrast, adenomas were produced by cooperation between MET and defective signaling through the transcription factor HNF1alpha. Prompted by these findings, we uncovered a coincidence between activation of the protein-tyrosine kinase encoded by MET and activating mutations of beta-catenin in a subset of human hepatocellular carcinomas. Inactivation of MET transgenes led to regression of hepatocellular carcinomas despite the persistence of activated beta-catenin. The tumors eventually recurred in the absence of MET expression, however, presumably after the occurrence of one or more events that cooperated with activated beta-catenin in lieu of MET. These results offer insight into hepatic tumorigenesis, provide mouse models that should be useful in the further study of hepatic tumorigenesis and for preclinical testing, and identify a subset of human hepatocellular carcinomas that may be susceptible to combination therapy directed against Met and the Wnt signaling pathway.

Met-thodology

Met-thodology

Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype

Identification of specific gene expression signatures characteristic of oncogenic pathways is an important step toward molecular classification of human malignancies. Aberrant activation of the Met signaling pathway is frequently associated with tumor progression and metastasis. In this study, we defined the Met-dependent gene expression signature using global gene expression profiling of WT and Met-deficient primary mouse hepatocytes. Newly identified transcriptional targets of the Met pathway included genes involved in the regulation of oxidative stress responses as well as cell motility, cytoskeletal organization, and angiogenesis. To assess the importance of a Met-regulated gene expression signature, a comparative functional genomic approach was applied to 242 human hepatocellular carcinomas (HCCs) and 7 metastatic liver lesions. Cluster analysis revealed that a subset of human HCCs and all liver metastases shared the Met-induced expression signature. Furthermore, the presence of the Met signature showed significant correlation with increased vascular invasion rate and microvessel density as well as with decreased mean survival time of HCC patients. We conclude that the genetically defined gene expression signatures in combination with comparative functional genomics constitute an attractive paradigm for defining both the function of oncogenic pathways and the clinically relevant subgroups of human cancers.

Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation

Oval cells are hepatocytic precursors that proliferate in late-stage cirrhosis and that give rise to a subset of human hepatocellular carcinomas. Although liver regeneration typically occurs through replication of existing hepatocytes, oval cells proliferate only when hepatocyte proliferation is inhibited. Transforming growth factor-beta (TGF-beta) is a key inhibitory cytokine for hepatocytes, both in vitro and in vivo. Because TGF-beta levels are elevated in chronic liver injury when oval cells arise, we hypothesized that oval cells may be less responsive to the growth inhibitory effects of this cytokine. To examine TGF-beta signaling in vivo in oval cells, we analyzed livers of rats fed a choline-deficient, ethionine-supplemented (CDE) diet for phospho-Smad2. Phospho-Smad2 was detected in more than 80% of hepatocytes, but staining was substantially reduced in oval cells. Ki67 staining, in contrast, was significantly more common in oval cells than hepatocytes. To understand the inverse relationship between TGF-beta signaling and proliferation in oval cells and hepatocytes, we examined TGF-beta signaling in vitro. TGF-beta caused marked growth inhibition in primary hepatocytes and the AML12 hepatocyte cell line. Two oval cell lines, LE/2 and LE/6, were less responsive. The greater sensitivity of the hepatocytes to TGF-beta-induced growth inhibition may result from the absence of Smad6 in these cells. Our results indicate that oval cells, both in vivo and in vitro, are less sensitive to TGF-beta-induced growth inhibition than hepatocytes. These findings further suggest an underlying mechanism for the proliferation of oval cells in an environment inhibitory to hepatocytic proliferation.