Human HCCs Research Articles

286 ALTERED LYSOPHOSPHATIDIC ACID RECEPTOR EXPRESSION IN LIVER REGENERATION AND HEPATOCELLULAR CARCINOMA (HCC)

pulsed DC±thymosin, splenocytes obtained frommice injected with TCL-pulsed DC exhibited E:T ratio-dependent cytotoxicity against MH134 cells. Moreover, tumor growth was significantly suppressed in mice co-treated with TCL-pulsed DC and thymosin as compared to those with thymosin or TCL-pulsed DC alone. Conclusion: Combined treatment with TCL-pulsed DC and thymosin 1-a was found to enhance anti-tumor efficacy in a murine HCC model. Therefore, anti-tumor efficacy of DC-based, active immunotherapy might be potentiated by adding thymosin 1-a in human HCCs.

The transcriptional regulator YAP induces Notch-pathway activity in hepatocytes and hepatocellular carcinoma cells

Aims: The Hippo-pathway regulates organ size and homeostasis. Studies revealed that deletion of Hippo-pathway kinases (e.g. Mst1/2) or overexpression of the transcriptional co-activator YAP lead to liver cancer; however, the underlying molecular mechanism have not identified so far. We therefore aimed to define possible effector mechanisms mediating YAP-dependent oncogenic activity in liver cancer. Methods: SiRNA-knockdown, expression profiling, and functional assays were performed using HCC cell lines. Gene expression and protein interaction were analyzed by immunoblotting or RT-PCR and co-immunoprecipitation, respectively. YAP transgenic (YAPLAP) and Mst1/2 knock-out mice as well as human HCCs were examined by IHC, immunoblotting, and RT-PCR. Results: Expression profiling after YAP inhibition in HCC cells and of YAPLAP liver tissues identified the Notch-pathway ligand Jag1 as potential target gene for the Hippo pathway. Expression of cleaved Notch receptor (NICD) and Notch target gene Hes1 were reduced in HCC cells after YAP inhibition. SiRNA-knockdown of Jag1 but not of Jag2 as well as inhibition of Notch signalling by γ-secretase phenocopied the biological effects detected after YAP inhibition (e.g., decreased viability). Overexpression of YAP induced tumor cell proliferation, whereas additional Jag1 inhibition partly compensated the effects. Expression of YAP/Jag1 and YAP/Hes1 significantly correlated in human HCCs. YAPLAP-mice developed dysplastic foci with increased Jag1 expression and elevated proliferation. Furthermore, time-resolved analysis showed a simultaneous increase of Jag1 and Hes1 levels after doxycylin-dependent YAP expression in isolated primary hepatocytes. Accordingly, Mst1/2-/- mice-derived HCCs showed high YAP, Jag1, and Hes1 levels. Conclusion: These data demonstrate that activation of YAP induces Notch-pathway activity by Jag1 expression and that Notch-signalling partly facilitates the oncogenic effects of YAP in vitro and in vivo.

Transcriptional regulation of miR-224 upregulated in human HCCs by NFκB inflammatory pathways

miR-224 is up-regulated in human HCCs as compared to both paired peri-tumoral cirrhotic tissues and cirrhotic livers without HCC. Here, we have cloned the miR-224 regulatory region and characterized its transcriptional regulation by the NFκB-dependent inflammatory pathways. Mature miRNA expression was evaluated by a 2 step stem-loop real-time RT-PCR. The recruitment of polymerase II and transcription factors on the pre-miR-224 promoter has been assessed by ChIPSeq and ChIP. We found miR-224 levels strongly up-regulated in both peri-tumoral cirrhotic livers and HCC samples as compared to normal livers. In silico analysis of the putative miR-224 promoter revealed multiple NFκB sites. We showed that LTα and TNFα activate transcription from the miR-224 promoter and of endogenous miR-224 expression in HCC cell lines, whereas the expression of miR-224 target API5 was reduced. Exogenously expressed p65/RelA activates the miR-224 promoter and a dominant negative form of IκBα (IκBSR) represses it. ChIP analysis showed that p65/NFκB is recruited on the miR-224 promoter and that its binding sharply increases after exposure to LPS, TNFα, and LTα. Altogether these findings link the inflammatory signals to NFκB-mediated activation of miR-224 expression. An antago-miR specific for miR-224 blocked LPS and LTα stimulated HCC cells migration and invasion. Conversely, the IKK inhibitor BMS-345541 blocks pre-miR-224-induced cellular migration and invasion. Our results identify p65/NFκB as a direct transcriptional regulator of miR-224 expression and link miR-224 up-regulation with the activation of the LPS, LTα, and TNFα inflammatory pathways and cell migration/invasion in HCC.

Abstract 1443: RhoE is underexpressed in hepatocellular carcinoma and suppresses cell motility and invasiveness

Abstract Hepatocellular carcinoma (HCC) is a major malignancy worldwide and is the second commonest fatal cancer in Southeast Asia and Mainland China including Hong Kong, due to the high prevalence of hepatitis B viral infection. Despite definite improvements in the outcome of patients with HCC, the overall prognosis of this cancer is still unsatisfactory because of late presentation and frequent tumor recurrence after surgical resection. In this regard, knowledge of the molecular and cellular targets underlying the development and progression of liver cancer may provide novel opportunities for therapeutic interventions for this cancer. As part of our continual study on the Rho/ROCK pathway in HCC, in this current study, we aimed to find out the role of RhoE (also known as Rnd3), a member of the Rho family of small GTP-binding proteins, in HCC. We have found that RhoE was frequently underexpressed in 43/71 (60.6%) of our human HCCs at mRNA level using qPCR, as compared with their corresponding non-tumorous livers (p <0.001). Moreover, RhoE knockdown in HCC cells by lentiviral infection of shRhoE enhanced cell migration and invasion in vitro. Using transwell migration assay, we showed that stable shRNA knockdown of RhoE in SMMC7721 and BEL7402 HCC cell lines resulted in faster migration of the cells than their corresponding non-target controls. Moreover, stable RhoE knockdown clones also invaded through the matrigel faster in matrigel invasion assay. Previous studies have indicated that phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr850 was essential for phosphorylation of myosin II and crucial for cell contractility. To further delineate the roles of RhoE on actomyosin contractility, the MYPT1 phosphorylation levels in the Rho stable knockdown transfectants in SMMC, BEL and Hep3B HCC cells were assessed by Western blotting and found to be increased as compared with their non-target control. This increased phosphorylation was reversed by ROCK inhibitor Y27632. Furthermore, the ROCK inhibitor Y27632 was able to suppress the accelerated cell migration brought by the knockdown of RhoE in SMMC, suggesting that RhoE may exert its effects by inhibiting the activity of ROCK1. Since MYPT1 is specifically phosphorylated by ROCK at Thr850, the finding suggests that RhoE interacts with and suppresses the activity of ROCK1 in HCC cells. To conclude, our data suggest that RhoE in HCC suppresses both cell motility and invasiveness of HCC cells and that RhoE may exert its effects by inhibiting the activity of ROCK1. (This study was funded by Hong Kong Research Grants Council CRF grants (RGC CRF HKU1/06C and HKU 7/CRG/09) 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 1443. doi:10.1158/1538-7445.AM2011-1443

Abstract 1074: Dishevelled 3 (Dvl3) is upregulated and enhances cell migration and invasion in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the most common fatal malignancies in the world and is particularly prevalent in China and Southeast Asia. Although the risk factors are well known, the molecular mechanisms underlying hepatocarcinogenesis is unclear. Alteration of Wnt signaling pathway is one of the key events in HCC development and so we performed a low-density array (LDA) analysis on 38 pairs of HCC samples and their corresponding non-tumorous livers to examine the expression profiles of different Wnt signaling components. We found that, among the genes studied, Dishevelled 3 (Dvl3) was significantly overexpressed in HCCs as compared with the corresponding non-tumorous liver tissues. In addition, Dvl3 overexpression in HCC was significantly associated with presence of venous invasion (P = .021). The overexpression result was confirmed by expansion of the sample size using quantitative real-time PCR (q-PCR). Next, we investigated the effects of Dvl3 on the migratory and invasive abilities of HCC cells. We established stable shRNA Dvl3-knockdown clones in PLC/PRF/5 HCC cells and found that knockdown of Dvl3 suppressed the migratory and invasive abilities of HCC cells using transwell migration and invasion assays, respectively, suggesting an enhancing role of Dvl3 in HCC cell movement and cell invasion. Although cell proliferation assay in vitro showed no significant difference between the stable Dvl3 knockdown and non-target control PLC/PRF/5 cells, in vivo study in SCID mice showed that the stable Dvl3-knockdown cells resulted in a lower incidence of tumors formed and a smaller tumor size. Furthermore, since Dvl3 is an upstream regulator of the Wnt/β-catenin pathway, using the TOPFOP luciferase reporter assay, we found that transient transfection of Dvl3 in PLC/PRF/5 and BEL-7402 HCC cell lines enhanced the β-catenin-dependent transcriptional activity. In conclusion, upregulation of Dvl3 was frequent in human HCCs. Dvl3 enhanced cell migration and invasion in vitro and tumorigenicity in vivo in SCID mice, and activated canonical β-catenin pathway. It may play an oncogenic role in HCC development. (This study was funded by Hong Kong Research Grants Council CRF grants (RGC CRF HKU1/06C and HKU 7/CRG/09).) 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 1074. doi:10.1158/1538-7445.AM2011-1074

Abstract 2450: Resistance of CD44+ hepatocellular carcinoma cancer stem cellsto immune effector responses

Abstract Hepatocellular carcinoma (HCC) is a common cause of cancer-related deaths worldwide. There is little therapy for advanced disease and recurrence rates can be as high as 50% following resection. Evidence in several tumor types suggests that cancer stem cells (CSCs) contribute to tumorigenicity, therapeutic resistance and recurrence, however the HCC CSC phenotype remains to be completely defined. HCC recurrence is influenced by the degree of immune response in the tumor microenvironment. Therefore, we aimed to characterize HCC-CSCs phenotype and investigate the interaction between CSCs and immune cell subsets. To identify a dominant CSC population in HCC, we examined several putative CSC markers, including CD44, CD90, CD133 and EpCAM on primary human resected HCCs (n=6), HCC xenografts (n=2), and HCC cell lines (n=3) using flow cytometry. Cells from all sources consistently showed discrete populations of CD44+ cells (2-40%), while other common CSC markers showed significant heterogeneity. CD44+ cells isolated from HCC xenografts or HepG2 cells by fluorescence-activated cell sorting (FACS) demonstrated enhanced tumorigenic potential with as little as 100 cells implanted into NOD-SCID/IL-2Rgnull mice compared to no tumor from 100 CD44− cells. CD44+ cells were also able to produce heterogenic tumor mass in vivo. Moreover, CD44+ cells demonstrated increased sphere forming capacity in serum free media (2 fold, p<0.05), and increased resistance to the chemotherapeutic agents cisplatin and doxorubicin in vitro. Additionally, CD44+ HCC cells were resistant to effector cytokines such as IFNγ and TNFα and effector responses during co-culture with anti-CD3/anti-CD28 activated T cells from healthy donor PBMCs. CD44+ HCC cells had 2-fold increased expression of B7-H1, a co-inhibitory molecule for T cells, when compared to non-CSCs during exposure to activated T cells or effector cytokine IFNγ, suggesting a possible mechanism of resistance to immune effectors in the HCC microenvironment. In conclusion, CD44 discriminates for the HCC CSC as evidenced by CD44+ subset demonstrating enhanced tumorigenicity, sphere forming capacity and resistance to chemotherapeutic agents. CD44+ CSC are also resistant to activated T cells and effector cytokines suggesting that microenvironmental factors related to immune response may be important for HCC recurrence or progression. 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 2450. doi:10.1158/1538-7445.AM2011-2450

Abstract 909: Activation of NF-κB and its downstream targets play a key role in the dramatic induction of hepatocarcinogenesis in metallothionein null mice

Abstract Hepatocellular Carcinoma (HCC) is one of the most prevalent cancers in the world and the third leading cause of cancer-related death. Although hepatitis C and fatty liver disease appear to be the main risk factors in most western countries other factors including alcohol, tobacco, diabetes, and other metabolic disorders can also increase the likelihood of HCC development. HCC is believed to develop through a tumor promoting mechanism involving inflammation and oxidative stress. We have previously shown that metallothionein (MT), a ubiquitously expressed free radical scavenger, is dramatically downregulated in human and rodent HCCs. To test the hypothesis that MT protects the liver from carcinogenesis by relieving oxidative stress, MT knockout (MTKO) mice were injected with Diethylnitrosamine (DEN), a potent liver carcinogen. When compared to wild-type DEN treated mice, the MTKO groups exhibited significantly higher (5-fold) incidence and an accelerated rate of hepatocarcinogenesis. Gene expression analysis at the preneoplastic stage revealed a dramatic increase in the levels of Serpine-1 in the MTKO mice, which was validated by real-time PCR. Serpine-1, a tissue glycoprotein and an active suppressor of fibrinolysis through the inhibition of tissue plasminogen activator and urokinase plasminogen activator, is also believed to increase cell migration and angiogenesis. Real-time PCR analysis of the pro-inflammatory cytokines known to induce Serpine-1 revealed substantial upregulation of TNF-α, IL-6, and IL-1β in the MTKO/DEN mice compared to the WT/DEN and untreated controls. Electrophoretic mobility shift assay confirmed differential activation of NF-κB, a downstream effector of pro-inflammatory cytokines and inducer of Serpine-1 expression, in the MTKO/DEN mice at the preneoplastic stage. These data demonstrate that Serpine-1 and NF- κB act in concert at the early stages of hepatocarcinogenesis when free radical scavenging is compromised. These data show the protective role of MT against HCC induction in a mouse model and provide the rationale for an alternate therapeutic strategy in HCC therapy. 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 909. doi:10.1158/1538-7445.AM2011-909

1121 MIR-224 IS A DIRECT TARGET OF HBX AND MODULATES HBV REPLICATION

Background: miR224 is frequently up-regulated in human HCCs, peri-tumoral cirrhotic tissues and cirrhotic livers without HCCs. We have recently shown that miR224 expression in the liver is induced by the NFkB-dependent inflammatory pathways (i.e. LTa, and TNFa) that are activated in a large proportion of chronic viral hepatitis, cirrhosis and HCC patients. Differently from other HCCrelated miRNAs that are over-expressed in several other cancers and are considered as bona fide onco-miRs (i.e. miR-21, miR-221, miR-222), miR-224 is apparently more HCC specific, suggesting a possible role of miR224 in liver physiopathology and/or chronic hepatitis infection before HCC development. Aim: To characterize the transcriptional regulation of miR224 by HBx and its effects on HBV replication. Methods: In vivo recruitment of HBx, transcription factors and chromatin remodeling enzymes was analyzed in ChIP-Seq and conventional ChIP experiments. miR224(pr) luciferase reporter constructs were generated and used to assess the ability of HBx to modulate miR224 expression. Endogenous miR224 expression and the impact of miR224 on HBV replication were assessed in HBV replicating HepG2 cells. Results: In a genome wide search of HBx cellular targets by ChIP-Seq we found that HBx protein binds in vivo to the miR224 regulatory region. HBx binding is accompanied by the co-recruitment of p65/NFkB, a loss of Pol II occupancy, the recruitment of the DNMT3a methyltransferase and reduced H4 histone acetylation. Accordingly, miR224 levels were reduced in HBV replicating cells and HBx repressed the miR224(pr) in luciferase reporter assays. pre-miR224 overexpression resulted in reduced HBV pgRNA levels and a 50% reduction in HBV replication. In silico analysis revealed the presence of several miR224 seed sequences on the HBV genome that were conserved across HBV genotypes, suggesting a direct effect of miR224 on the HBV pgRNA. Conclusions: Our results identify a functional regulatory loop between HBx, miR224 and HBV replication where HBx repression of miR224 expression relieves the negative effects of miR224 on HBV replication. These results are compatible with the low HBV replication observed in HCC tissues and support the hypothesis that the loss of the described regulatory loop might occur at the time of transformation or HCC progression.

1072 THE IMMUNOPHENOTYPE OF LIVER STEM CELLS AND ITS RELATIONSHIP TO HCC DEVELOPMENT ON TOP OF CHRONIC HCV; POSSIBLE PROGNOSTIC SIGNIFICANCE

Background and Aims: Identification of the exact role/significance of different types of liver stem cell markers was investigated. Methods: Sixty core liver biopsies representing chronic HCV, mixed schistosomiasis and chronic HCV, cirrhosis and HCC on top of HCV cirrhosis were stained immunohistochemically with monoclonal antibodies to stem cell markers CK 19, CK 34, OV6 and PCNA. Results: CD 34 was higher in HCC as compared to each of cirrhosis (p = 0.001), hepatitis (p = 0.001) and mixed groups (p = 0.001). Cirrhotics showed higher numbers as compared to HCV (p =0.001) and mixed groups (p = 0.001). No differences were noted between cirrhotics and HCCs. It correlated positively with PCNA/LI in HCV (r = 0.486, p = 0.022), cirrhotics (r = 0.723, p = 0.001) and HCCs (r = 0.871, p = 0.001). No correlations were observed in the combined group. OV 6 positivity was higher in the mixed group as compared to the HCV group (p =0.045). It was totally absent from HCC cases. In the mixed group, it correlated with PCNA/LI (r = 0.982, p = 0.001). It correlated with CK 19 in HCV (r = 0.546, p = 0.009), and cirrhotics (r = 0.868, p = 0.001). CK 19 was higher in the cirrhosis group, it correlated significantly with each of OV 6 in the HCV group and with CD 34 (r = 0.524, p = 0.037).in the cirrhosis group. No CK 19 positivity was detected in HCC group. In the combined group, it correlated with portal infiltrate (r = 0.866, p = 0.026) and spotty necrosis (r = 0.866, p = 0.026). In the cirrhosis group: it correlated with portal infiltrate (r = 0.501, p = 0.048) stage (r = 0.570, p = 0.021) Conclusions: In human HCCs occurring on top of chronic HCV infection, we propose that the immunophenotype of HPCs, whether they express OV6 or CD 34 could be considered as a prognostic sign; OV6 expression is associated with regenerative activity, while CD 34 expression is associated with a high probability of HCC development.

Independent phenotype of binuclear hepatocytes and cellular localization of UbD

Mice fed DDC (0.1%) for 10 weeks, and then withdrawn from the drug for 1 month, retain the ability to form Mallory–Denk bodies (MDBs) when the drug is refed for 7 days. The number of liver cells that form MDBs increased and partially replaced normal liver cells, at the end of 7 days of refeeding DDC. The MDBs that formed were associated with increased expression of UbD (also called FAT10) in the Mallory–Denk body forming cells. UbD is over expressed in 70% of human HCCs, but its cellular localization is not well established. UbD belongs to the UbL family (ubiquitin-like), and can be linked to others proteins with their 2 C-terminal glycine to lysine. By Western Blot, UbD was found to be covalently linked with proteins. We performed immunohistochemistry on tissue from mouse liver and found that UbD was located in the cytoplasm and in one or two nuclei of the same hepatocyte. However, in primary cell culture, UbD formed speckles within the cytoplasm of the liver cell. A similar pattern of cytoplasmic localization was observed in the Hepa 1–6 cell lines, which over expressed UbD fused with GFP at the C-Terminal. The localization and the control of UbD localization remain unclear. The identification of proteins that interact with UbD and the post translational modification of UbD would help to determine the regulation of this localization and function.

Abstract 4095: Aberrant expression of epigenetically regulated microRNAs in liver cancer

Abstract Liver cancer (hepatocellular carcinoma, HCC) is a major malignancy worldwide. Despite definite improvements in the outcome of patients with HCC, the overall prognosis of this cancer is still unsatisfactory because of late presentation and frequent tumor recurrence after surgical resection. New diagnostic and adjuvant treatment modalities are much awaited. MicroRNAs (miRNAs) are a class of small (∼22 nt) single-stranded noncoding RNAs widely expressed in eukaryotic cells. miRNAs play an important role in modulating gene expression by promoting mRNA degradation or translational repression. There is mounting evidence that miRNAs are implicated in almost all major physiological processes, including cell proliferation, differentiation, apoptosis, and human carcinogenesis. Epigenetic alterations including aberrant DNA methylation and histone modification are important in regulating chromatin structure and gene expression. In this study, we investigated the role of epigenetic alterations in miRNA deregulation in human HCC. miRNA expression changes of HCC cell lines upon treatment with DNA demethylating agent (5-Aza-dC) and histone deacetylase inhibitor (Trichostatin A, TSA) were compared with stem-loop RT-qPCR based TaqMan low density array. In total, 98 miRNAs were remarkably up-regulated upon 5-Aza-dC/TSA treatment. Among these, 35 miRNA were commonly upregulated by treatment with the epigenetic inhibitors in both cell lines. The majority of these miRNAs was either associated with promoter CpG islands or located on imprinted regions. Expression analysis revealed that these epigenetically regulated miRNAs were frequently deregulated in human HCCs, and their expression pattern was able to distinguish primary HCCs from their corresponding non-tumorous livers. Interestingly, CpG island-associated miRNAs tended to be down-regulated in human HCCs, whereas, miRNAs located on imprinted regions were frequently overexpressed in human HCC samples. Our findings suggest that aberrant epigenetic silencing and loss of imprinting may contribute to miRNA deregulation in human HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4095.

Abstract B40: Derangement HNF4α gene expression as a candidate marker of hepatocellular carcinoma progression

Abstract Hepatocellular carcinoma (HCC) is one of the world's most common cancers. The development of malignant phenotype is considered to be a multistep process that results from the accumulation of genetic alterations. Mechanisms of epithelial carcinogenesis are determined by the specific properties of tumor progenitor tissue. According to our investigations, HCC progression is associated with the dysfunction of hepatocyte nuclear factors (HNFs), which control liver-specific gene expression, and particularly HNF4α, a nuclear receptor playing a key role in hepatic differentiation. This gene is transcribed from two distinct promoters, which activity results in producing two groups of isoforms and reflects the level of cell differentiation. HNF4αP2 isoforms are synthesized in embryonic liver and normally disappear after birth, while HNF4αP1 become dominant in mature hepatocytes. Reexpression of HNF4αP1 reversed the malignant phenotype of dedifferentiated mouse HCC variants. Studies on the collection of chemically induced mouse HCCs of independent origin revealed strict correlation of HNF4α expression with tumor differentiation state. Using semiquantitative RT-PCR we have analyzed the expression of 46 genes in 21 samples of human HCCs. 17 of studied cases were not associated with Hepatitis viruses infections, while 4 of patients were infected with hepatitis B virus. The most frequent event revealed in HCC samples compared to corresponding non-tumor tissues was the complete loss (in 7 of 17 non-hepatitis HCCs) or significant decrease (in 5 of 17 non-hepatitis HCCs) of HNF4αP1 transcription. Decrease of HNF4α expression appeared to be a poor prognostic factor: patients with highly expressed HNF4αP1 had 100% 3-years survival, and 83% of them didn't have any disease progression, while only 33% of patients with low HNF4αP1 expression showed no disease progression. HCCs which were associated with hepatitis infection didn't show relevant alterations in HNF4αP1 expression. We suppose that hepatitis-associated hepatocarcinogenesis might develop through HNF4α1-independent mechanism. Intracellular localization and protein expression level of HNF4α isoforms on the paraffine sections of human HCCs was investigated immunohistochemically using specific mouse monoclonal antibodies for HNF4αP1 (K9218; R&D systems; 1:6000) or HNF4αP2 (H6939; R&D systems; 1:3000) groups of isoforms. HNF4α isoforms immunoreactivity was detectable in all studied tumors except severely dedifferentiated variants. Intense nuclear staining for HNF4αP2 was observed in hepatocytes, but nuclear staining for HNF4αP1 showed decreased intensity. We haven't revealed synthesis of any HNF4α isoforms in dedifferentiated tumors. All this data corroborate with RT-PCR analysis. We suppose that HCC progression is accompanied with activation of HNF4αP2 isoform, decreased synthesis of HNF4αP1 isoform while in severely dedifferentiated tumors expression of both groups of HNF4α isoforms is repressed. We assume that induction of HNF4αP2 expression might serve a marker of early hepatocarcinogenesis, while decrease and subsequent loss of HNF4αP1 and lately HNF4αP2 marks HCC progression. This provides a new data important for further development of approaches for HCC diagnostics and prognosis. Expanded analysis of human HCCs archival samples and further investigation of correlation between tumor differentiation state and patients postoperative survival rate with the pattern of HNF4α isoforms expression would allow to estimate the usefulness of the analysis of HNF4α isoforms expression for the long-term outlook for this tumor type. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B40

Epigenetic sylencing and transcriptional regulation of miRNAs modulated in human HCCs

Epigenetic sylencing and transcriptional regulation of miRNAs modulated in human HCCs

Expression and biological significance of c-FLIP in human hepatocellular carcinomas

Backgroundc-FLIP can be considered as a tumor-progression factor in regard to its anti-apoptotic functions. In the present study, we intended to investigate the expression of c-FLIP in human HCC tissues, and its relation with drug-induced cell apoptosis through the specific inhibition of c-FLIP expression by siRNA in 7721 cells.Methodsc-FLIP expression was quantified immunohistochemically in HCC tissues(eighty-six cases), and corresponding noncancerous tissues (fifty-seven cases). Patients with HCC were followed up for cancer recurrence. Then, the c-FLIP gene was silenced with specific siRNA in 7721 HCC cells. c-FLIP expression was detected by RT-PCR, Western Blot and immunocytochemical staining. The cellular viability and cell apoptosis were assayed in vitro with cells treated with doxorubicin.ResultsPositive immunostaining was detected for c-FLIP in 83.72% (72/86) human HCC tissues, 14.81% (4/27) hepatic cirrhosis, 11.11% (2/18) hepatic hemangioma tissues, and absent in normal hepatic tissues. The overexpression(more than 50%) of c-FLIP in HCC adversely affected the recurrence-free survival. Through c-FLIP gene silencing with siRNA, the expressions of c-FLIP mRNA and protein were remarkably down-regulated in 7721 HCC cells. And doxorubicin showed apparent inhibition on cell proliferations, and induced more apoptosis.ConclusionThese results indicate that c-FLIP is frequently expressed in human HCCs, and its overexpression implied a lesser probability of recurrence-free survival. The specific silencing of c-FLIP gene can apparently up-regulate drug-induced HCC cell apoptosis, and may have therapeutic potential for the treatment of human HCC.

Tissue factor pathway inhibitor‐2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma†

In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI-2 was frequently silenced in human HCC and HCC cell lines. TFPI-2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI-2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI-2 messenger RNA expression. In addition, TFPI-2 expression in HCC cell lines can be robustly restored by combined treatment with 5-Aza-dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI-2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI-2 significantly suppressed the proliferation and invasiveness of HCC cells. Our findings suggest that TFPI-2 is a candidate tumor suppressor gene in human HCC.

P21-Activated Protein Kinase Is Overexpressed in Hepatocellular Carcinoma and Enhances Cancer Metastasis Involving c-Jun NH2-Terminal Kinase Activation and Paxillin Phosphorylation

Hepatocellular carcinoma (HCC) is one of the major malignancies in the world. The prognosis of HCC is poor, due to frequent intrahepatic metastasis and tumor recurrence. P21-activated protein kinase (Pak1), a main downstream effector of small Rho GTPases, Rac1 and Cdc42, plays an important role in the regulation of cell morphogenesis, motility, mitosis, and angiogenesis. Here, we show that Pak1 gene was overexpressed in human HCCs. Overexpression of Pak1 in human HCCs was associated with more aggressive tumor behavior in terms of more metastatic phenotype and more advanced tumor stages. In addition, HCC cell line stably expressing Pak1 displayed increased cell motility rates and, conversely, knockdown of endogenous Pak1 expression by small interfering RNA reduced the migration rates of HCC cells. In an established metastatic HCC cell line, we found that Pak1 was overexpressed compared with its primary HCC cell line and this overexpression was associated with higher cell motility. Importantly, we found that c-Jun NH(2)-terminal kinase (JNK) was activated in HCC cell lines overexpressing Pak1. Inhibition of the JNK activity by chemical inhibitor significantly reduced the migration rates of HCC cells via attenuation of paxillin phosphorylation at Ser(178). In conclusion, our results document that Pak1 is overexpressed in HCCs and plays an important role in the metastasis of HCC. The mechanism by which Pak1 induces cancer metastasis may involve activation of JNK and phosphorylation of paxillin.

Chromosome alterations in human hepatocellular carcinomas correlate with aetiology and histological grade – results of an explorative CGH meta-analysis

All available comparative genomic hybridisation (CGH) analyses (n=31, until 12/2003) of human hepatocellular carcinomas (HCCs; n=785) and premalignant dysplastic nodules (DNs; n=30) were compiled and correlated with clinical and histological parameters. The most prominent amplifications of genomic material were present in 1q (57.1%), 8q (46.6%), 6p (22.3%), and 17q (22.2%), while losses were most prevalent in 8p (38%), 16q (35.9%), 4q (34.3%), 17p (32.1%), and 13q (26.2%). Deletions of 4q, 16q, 13q, and 8p positively correlated with hepatitis B virus aetiology, while losses of 8p were more frequently found in hepatitis C virus-negative cases. In poorly differentiated HCCs, 13q and 4q were significantly under-represented. Moreover, gains of 1q were positively correlated with the occurrence of all other high-frequency alterations in HCCs. In DNs, amplifications were most frequently present in 1q and 8q, while deletions occurred in 8p, 17p, 5p, 13q, 14q, and 16q. In conclusion, aetiology and dedifferentiation correlate with specific genomic alterations in human HCCs. Gains of 1q appear to be rather early events that may predispose to further chromosomal abnormalities. Thus, explorative CGH meta-analysis generates novel and testable hypotheses regarding the cause and functional significance of genomic alterations in human HCCs.

Analyses of central mediators indicating structural integrity of TGFβ-signaling in human HCCs

The altered expression pattern of the Epithelial Cell Adhesion Molecule (Ep-CAM) and the Carcinoembryonic Antigen (CEA) on tumor cells of epithelial origin as compared to normal epithelia may permit T cells to preferentially recognize and lyse these tumor cells. The binding affinity for human leucocyte antigen A2.1 (HLAA*0201) and the capacity to form stable peptide-major histocompatibility complex (MHC) interactions with this molecule were tested for 410 Ep-CAM-derived sequences, including an overlapping set of 9 amino-acid-long peptides, and 73 CEA-derived peptides fulfilling the HLAA*0201 motif. Peptides with a high binding affinity and a low peptide-MHC dissociation rate were subsequently tested for their immunogenicity in HLAA*0201Kb transgenic mice. One Ep-CAM-derived peptide and 1 CEA-derived peptide were able to reproducibly induce peptide-specific cytotoxic T cells (CTL) in these mice. This indicates that EpCAM and CEA are potential target antigens for CTL-mediated immunotherapy of epithelial cancers.

853 P38 MAPK induced G1 cell cycle arrest through up-regulation of P21CIP1 in TGF-B-treated human HCCS

853 P38 MAPK induced G1 cell cycle arrest through up-regulation of P21CIP1 in TGF-B-treated human HCCS

Loss of holocytochrome c-type synthetase causes the male lethality of X-linked dominant microphthalmia with linear skin defects (MLS) syndrome.

Girls with MLS syndrome have microphthalmia with linear skin defects of face and neck, sclerocornea, corpus callosum agenesis and other brain anomalies. This X-linked dominant, male-lethal condition is associated with heterozygous deletions of a critical region in Xp22.31, from the 5' untranslated region of MID1 at the telomeric boundary to the ARHGAP6 gene at the centromeric boundary. HCCS, encoding human holocytochrome c-type synthetase, is the only gene located entirely inside the critical region. Because single gene analysis is not feasible in MLS patients (all have deletions), we generated a deletion of the equivalent region in the mouse to study the molecular basis of this syndrome. This deletion inactivates mouse Hccs, whose homologs in lower organisms (cytochrome c or c1 heme lyases) are essential for function of cytochrome c or c1 in the mitochondrial respiratory chain. Ubiquitous deletions generated in vivo lead to lethality of hemizygous, homozygous and heterozygous embryos early in development. This lethality is rescued by expression of the human HCCS gene from a transgenic BAC, resulting in viable homozygous, heterozygous and hemizygous deleted mice with no apparent phenotype. In the presence of the HCCS transgene, the deletion is easily transmitted to subsequent generations. We did obtain a single heterozygous deleted female that does not express human HCCS, which is analogous to the low prevalence of the heterozygous MLS deletion in humans. Through the study of these genetically engineered mice we demonstrate that loss of HCCS causes the male lethality of MLS syndrome.