Elizabeth A. Conner

Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, accounting for an estimated 600,000 deaths annually. Aberrant methylation, consisting of DNA hypomethylation and/or promoter gene CpG hypermethylation, is implicated in the development of a variety of solid tumors, including HCC. We analyzed the global levels of DNA methylation as well as the methylation status of 105 putative tumor suppressor genes and found that the extent of genome-wide hypomethylation and CpG hypermethylation correlates with biological features and clinical outcome of HCC patients. We identified activation of Ras and downstream Ras effectors (ERK, AKT, and RAL) due to epigenetic silencing of inhibitors of the Ras pathway in all HCC. Further, selective inactivation of SPRY1 and -2, DAB2, and SOCS4 and -5 genes and inhibitors of angiogenesis (BNIP3, BNIP3L, IGFBP3, and EGLN2) was associated with poor prognosis. Importantly, several epigenetically silenced putative tumor suppressor genes found in HCC were also inactivated in the nontumorous liver. Our results assign both therapeutic and chemopreventive significance to methylation patterns in human HCC and open the possibility of using molecular targets, including those identified in this study, to effectively inhibit HCC development and progression.

Genomic and Genetic Characterization of Cholangiocarcinoma Identifies Therapeutic Targets for Tyrosine Kinase Inhibitors

BACKGROUND & AIMS Cholangiocarcinoma is a heterogeneous disease with a poor outcome that accounts for 5%-10% of primary liver cancers. We characterized its genomic and genetic features and associated these with patient responses to therapy. METHODS We profiled the transcriptomes from 104 surgically resected cholangiocarcinoma samples collected from patients in Australia, Europe, and the United States; epithelial and stromal compartments from 23 tumors were laser capture microdissected. We analyzed mutations in KRAS, epidermal growth factor receptor (EGFR), and BRAF in samples from 69 tumors. Changes in gene expression were validated by immunoblotting and immunohistochemistry; integrative genomics combined data from the patients with data from 7 human cholangiocarcinoma cell lines, which were then exposed to trastuzumab and lapatinib. RESULTS Patients were classified into 2 subclasses, based on 5-year survival rate (72% vs 30%; χ(2) = 11.61; P < .0007), time to recurrence (13.7 vs 22.7 months; P < .001), and the absence or presence of KRAS mutations (24.6%), respectively. Class comparison identified 4 survival subgroups (SGI-IV; χ(2) = 8.34; P < .03); SGIII was characterized by genes associated with proteasomal activity and the worst prognosis. The tumor epithelium was defined by deregulation of the HER2 network and frequent overexpression of EGFR, the hepatocyte growth factor receptor (MET), pRPS6, and Ki67, whereas stroma was enriched in inflammatory cytokines. Lapatinib, an inhibitor of HER2 and EGFR, was more effective in inhibiting growth of cholangiocarcinoma cell lines than trastuzumab. CONCLUSIONS We provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. The group of patients with the worst prognosis was characterized by transcriptional enrichment of genes that regulate proteasome activity, indicating new therapeutic targets.

Central Role of c-Myc during Malignant Conversion in Human Hepatocarcinogenesis

Hepatocarcinogenesis is a multistage process in which precursor lesions progress into early hepatocellular carcinomas (eHCC) by sequential accumulation of multiple genetic and epigenetic alterations. To decode the molecular events during early stages of liver carcinogenesis, we performed gene expression profiling on cirrhotic (regenerative) and dysplastic nodules (DN), as well as eHCC. Although considerable heterogeneity was observed at the regenerative and dysplastic stages, overall, 460 differentially expressed genes were detected between DN and eHCC. Functional analysis of the significant gene set identified the MYC oncogene as a plausible driver gene for malignant conversion of the DNs. In addition, gene set enrichment analysis revealed global activation of the MYC up-regulated gene set in eHCC versus dysplasia. Presence of the MYC signature significantly correlated with increased expression of CSN5, as well as with higher overall transcription rate of genes located in the 8q chromosome region. Furthermore, a classifier constructed from MYC target genes could robustly discriminate eHCC from high-grade and low-grade DNs. In conclusion, our study identified unique expression patterns associated with the transition of high-grade DNs into eHCC and showed that activation of the MYC transcription signature is strongly associated with the malignant conversion of preneoplastic liver lesions.

Dual functions of E2F-1 in a transgenic mouse model of liver carcinogenesis

Deregulation of E2F transcriptional control has been implicated in oncogenic transformation. Consistent with this idea, we recently demonstrated that during hepatocarcinogenesis in c-myc/TGFα double transgenic mice, there is increased expression of E2F-1 and E2F-2, as well as induction of putative E2F target genes. Therefore, we generated transgenic mice expressing E2F-1 under the control of the albumin enhancer/promoter to test the hypothesis that E2F family members may contribute to liver tumor development. Overexpression of E2F-1 resulted in mild but persistent increases in cell proliferation and death during postnatal liver growth, and no increases in hepatic regenerative growth in response to partial hepatectomy. Nevertheless, from 2 months postnatally E2F-1 transgenic mice exhibited prominent hepatic histological abnormalities including preneoplastic foci adjacent to portal tracts and pericentral large cell dysplasia. From 6 to 8 months onward, there was an abrupt increase in the number of neoplastic nodules (‘adenomas’) with 100% incidence by 10 months. Some adenomas showed evidence of malignant transformation, and two of six mice killed at 12 months showed trabecular hepatocellular carcinoma. Endogenous c-myc was up-regulated in the early stages of E2F-1 hepatocarcinogenesis, whereas p53 was overexpressed in the tumors, suggesting that both E2F-1-mediated proliferation and apoptosis are operative but at different stages of hepatocarcinogenesis. In conclusion, E2F-1 overexpression in the liver causes dysplasia and tumors and suggests a cooperation between E2F-1 and c-myc oncogenes during liver oncogenesis.

HEPATOCYTE GROWTH FACTOR/C-MET SIGNALING IS REQUIRED FOR STEM-CELL-MEDIATED LIVER REGENERATION IN MICE

Hepatocyte growth factor (HGF)/c‐Met supports a pleiotrophic signal transduction pathway that controls stem cell homeostasis. Here, we directly addressed the role of c‐Met in stem‐cell–mediated liver regeneration by utilizing mice harboring c‐met floxed alleles and Alb‐Cre or Mx1‐Cre transgenes. To activate oval cells, the hepatic stem cell (HSC) progeny, we used a model of liver injury induced by diet containing the porphyrinogenic agent, 3,5‐diethocarbonyl‐1,4‐dihydrocollidine (DDC). Deletion of c‐met in oval cells was confirmed in both models by polymerase chain reaction analysis of fluorescence‐activated cell‐sorted epithelial cell adhesion molecule (EpCam)‐positive cells. Loss of c‐Met receptor decreased the sphere‐forming capacity of oval cells in vitro as well as reduced oval cell pool, impaired migration, and decreased hepatocytic differentiation in vivo, as demonstrated by double immunofluorescence using oval‐ (A6 and EpCam) and hepatocyte‐specific (i.e. hepatocyte nuclear factor 4‐alpha) antibodies. Furthermore, lack of c‐Met had a profound effect on tissue remodeling and overall composition of HSC niche, which was associated with greatly reduced matrix metalloproteinase (MMP)9 activity and decreased expression of stromal‐cell–derived factor 1. Using a combination of double immunofluorescence of cell‐type–specific markers with MMP9 and gelatin zymography on the isolated cell populations, we identified macrophages as a major source of MMP9 in DDC‐treated livers. The Mx1‐Cre‐driven c‐met deletion caused the greatest phenotypic impact on HSCs response, as compared to the selective inactivation in the epithelial cell lineages achieved in c‐Metfl/fl; Alb‐Cre+/− mice. However, in both models, genetic loss of c‐met triggered a similar cascade of events, leading to the failure of HSC mobilization and death of the mice. Conclusion: These results establish a direct contribution of c‐Met in the regulation of HSC response and support a unique role for HGF/c‐Met as an essential growth‐factor–signaling pathway for regeneration of diseased liver. (HEPATOLOGY 2012)

Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer

BACKGROUND & AIMS Aberrant activation of the RAS pathway is ubiquitous in human hepatocarcinogenesis, but the molecular mechanisms leading to RAS induction in the absence of RAS mutations remain under-investigated. We defined the role of Ras GTPase activating proteins (GAPs) in the constitutive activity of Ras signaling during human hepatocarcinogenesis. METHODS The mutation status of RAS genes and RAS effectors was assessed in a collection of human hepatocellular carcinomas (HCC). Levels of RAS GAPs (RASA1-4, RASAL1, nGAP, SYNGAP1, DAB2IP, and NF1) and the RASAL1 upstream inducer PITX1 were determined by real-time RT-PCR and immunoblotting. The promoter and genomic status of RASAL1, DAB2IP, NF1, and PITX1 were assessed by methylation assays and microsatellite analysis. Effects of RASAL1, DAB2IP, and PITX1 on HCC growth were evaluated by transfection and siRNA analyses of HCC cell lines. RESULTS In the absence of Ras mutations, downregulation of at least one RAS GAP (RASAL1, DAB2IP, or NF1) was found in all HCC samples. Low levels of DAB2IP and PITX1 were detected mostly in a HCC subclass from patients with poor survival, indicating that these proteins control tumor aggressiveness. In HCC cells, reactivation of RASAL1, DAB2IP, and PITX1 inhibited proliferation and induced apoptosis, whereas their silencing increased proliferation and resistance to apoptosis. CONCLUSIONS Selective suppression of RASAL1, DAB2IP, or NF1 RAS GAPs results in unrestrained activation of Ras signaling in the presence of wild-type RAS in HCC.

Intact signaling by transforming growth factor β is not required for termination of liver regeneration in mice

Transforming growth factor β (TGF‐β) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF‐β signaling in liver regeneration in vivo, the TGF‐β type II receptor gene (Tgfbr2) was selectively deleted in hepatocytes by crossing “floxed” Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver‐specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF‐β1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7‐fold higher in R2LivKO mice compared with controls. Accelerated S‐phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion, TGF‐β regulates G1 to S phase transition of hepatocytes, but intact signaling by TGF‐β is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF‐β pathway is abolished, and may be a principal factor in the termination of liver regeneration. (HEPATOLOGY 2004.)

Loss of Hepatocyte Growth Factor/c-Met Signaling Pathway Accelerates Early Stages of N-nitrosodiethylamine-Induced Hepatocarcinogenesis

Hepatocyte growth factor (HGF) has been reported to have both positive and negative effects on carcinogenesis. Here, we show that the loss of c-Met signaling in hepatocytes enhanced rather than suppressed the early stages of chemical hepatocarcinogenesis. c-Met conditional knockout mice (c-metfl/fl, AlbCre+/-; MetLivKO) treated with N-nitrosodiethylamine developed significantly more and bigger tumors and with a shorter latency compared with control (w/w, AlbCre+/-; Cre-Ctrl) mice. Accelerated tumor development was associated with increased rate of cell proliferation and prolonged activation of epidermal growth factor receptor (EGFR) signaling. MetLivKO livers treated with N-nitrosodiethylamine also displayed elevated lipid peroxidation, decreased ratio of reduced glutathione to oxidized glutathione, and up-regulation of superoxide dismutase 1 and heat shock protein 70, all consistent with increased oxidative stress. Likewise, gene expression profiling done at 3 and 5 months after N-nitrosodiethylamine treatment revealed up-regulation of genes associated with cell proliferation and stress responses in c-Met mutant livers. The negative effects of c-Met deficiency were reversed by chronic p.o. administration of antioxidant N-acetyl-L-cysteine. N-acetyl-L-cysteine blocked the EGFR activation and reduced the N-nitrosodiethylamine-initiated hepatocarcinogenesis to the levels of Cre-Ctrl mice. These results argue that intact HGF/c-Met signaling is essential for maintaining normal redox homeostasis in the liver and has tumor suppressor effect(s) during the early stages of N-nitrosodiethylamine-induced hepatocarcinogenesis.

An Integrated Genomic and Epigenomic Approach Predicts Therapeutic Response to Zebularine in Human Liver Cancer

Drug-induced transcription and epigenetic changes can predict whether liver cancer can be successfully treated with an epigenome-targeting drug. A Crystal Ball for Cancer Therapy If you ask most first-year medical students, they are not likely to list fortune-telling as a required skill for a successful clinician. Yet, predicting how different patients will respond to therapy could save time, money, and lives. Andersen et al. peer into their crystal ball and provide an approach for determining whether patients with liver cancer will respond to treatment or not. Liver cancer causes more than half a million deaths annually. Although early cases may respond to treatment, it is often diagnosed at later stages when symptoms become more severe. Only a subset of patients with advanced-stage liver cancer is eligible for the most successful current therapy, sorafenib, leaving a large number of patients with few options. Epigenetic changes, or changes in gene expression not due to differences in DNA sequence, contribute to the initiation and progression of liver cancer. These changes are reversible, and epigenetic modifier drugs such as zebularine can reactivate tumor suppressor genes and decrease tumor cell growth. Andersen et al. characterized the epigenetic changes induced by zebularine in liver cancer cell lines. They found a distinct signature that identified two groups of cells—those sensitive and those resistant to zebularine. In an animal xenograft model, zebularine treatment of liver tumors with the “sensitive” profile resulted in increased survival and decreased metastasis to the lungs. Moreover, the zebularine-sensitive signature predicted prognosis of patients with liver cancer with an accuracy ranging from 84 to 96%. Therefore, this study not only provides a proof of principle that epigenetic modification may be a viable therapy for liver cancer but also gives us a glimpse into the future, allowing clinicians to predict clinical outcome for liver cancer patients, as well as the success or failure of this emerging treatment regimen. Epigenomic changes such as aberrant hypermethylation and subsequent atypical gene silencing are characteristic features of human cancer. Here, we report a comprehensive characterization of epigenomic modulation caused by zebularine, an effective DNA methylation inhibitor, in human liver cancer. Using transcriptomic and epigenomic profiling, we identified a zebularine response signature that classified liver cancer cell lines into two major subtypes with different drug responses. In drug-sensitive cell lines, zebularine caused inhibition of proliferation coupled with increased apoptosis, whereas drug-resistant cell lines showed up-regulation of oncogenic networks (for example, E2F1, MYC, and TNF) that drive liver cancer growth in vitro and in preclinical mouse models. Assessment of zebularine-based therapy in xenograft mouse models demonstrated potent therapeutic effects against tumors established from zebularine-sensitive but not zebularine-resistant liver cancer cells, leading to increased survival and decreased pulmonary metastasis. Integration of the zebularine gene expression and demethylation response signatures allowed differentiation of patients with hepatocellular carcinoma according to their survival and disease recurrence. This integrated signature identified a subclass of patients within the poor-survivor group that is likely to benefit from therapeutic agents that target the cancer epigenome.

Hepatocyte-specific c-Met Deletion Disrupts Redox Homeostasis and Sensitizes to Fas-mediated Apoptosis

The hepatocyte growth factor and its receptor c-Met direct a pleiotropic signal transduction pathway that controls cell survival. We previously demonstrated that mice lacking c-Met (Met-KO) in hepatocytes were hypersensitive to Fas-induced liver injury. In this study, we used primary hepatocytes isolated from Met-KO and control (Cre-Ctrl) mice to address more directly the protective effects of c-Met signaling. Loss of c-Met function increased sensitivity to Fas-mediated apoptosis. Hepatocyte growth factor suppressed apoptosis in Cre-Ctrl but not Met-KO hepatocytes concurrently with up-regulation of NF-κB and major antiapoptotic proteins Bcl-2 and Bcl-xL. Intriguingly, Met-KO hepatocytes exhibited intrinsic activation of NF-κBas well as Bcl-2 and Bcl-xL. Furthermore, unchallenged Met-KO cells displayed oxidative stress as evidenced by overproduction of reactive oxygen species, which was associated with greater NADPH and Rac1 activities, was blocked by the known NADPH oxidase inhibitors, and was paralleled by increased lipid peroxidation and reduced glutathione (GSH) content. N-Acetylcysteine, an antioxidant and GSH precursor, significantly reduced Jo2-induced cell death. Conversely, the GSH-depleting agent buthionine sulfoximine completely abolished the protective effects of N-acetylcysteine in Met-KO hepatocytes. In conclusion, genetic inactivation of c-Met in mouse hepatocytes caused defects in redox regulation, which may account for the increased sensitivity to Fas-induced apoptosis and adaptive up-regulation of NF-κB survival signaling. These data provide evidence that intact c-Met signaling is a critical factor in the protection against excessive generation of endogenous reactive oxygen species.