Swan Thung

Gene expression in fixed tissues and outcome in hepatocellular carcinoma.

BACKGROUNDnIt is a challenge to identify patients who, after undergoing potentially curative treatment for hepatocellular carcinoma, are at greatest risk for recurrence. Such high-risk patients could receive novel interventional measures. An obstacle to the development of genome-based predictors of outcome in patients with hepatocellular carcinoma has been the lack of a means to carry out genomewide expression profiling of fixed, as opposed to frozen, tissue.nnnMETHODSnWe aimed to demonstrate the feasibility of gene-expression profiling of more than 6000 human genes in formalin-fixed, paraffin-embedded tissues. We applied the method to tissues from 307 patients with hepatocellular carcinoma, from four series of patients, to discover and validate a gene-expression signature associated with survival.nnnRESULTSnThe expression-profiling method for formalin-fixed, paraffin-embedded tissue was highly effective: samples from 90% of the patients yielded data of high quality, including samples that had been archived for more than 24 years. Gene-expression profiles of tumor tissue failed to yield a significant association with survival. In contrast, profiles of the surrounding nontumoral liver tissue were highly correlated with survival in a training set of tissue samples from 82 Japanese patients, and the signature was validated in tissues from an independent group of 225 patients from the United States and Europe (P=0.04).nnnCONCLUSIONSnWe have demonstrated the feasibility of genomewide expression profiling of formalin-fixed, paraffin-embedded tissues and have shown that a reproducible gene-expression signature correlated with survival is present in liver tissue adjacent to the tumor in patients with hepatocellular carcinoma.

Pivotal Role of mTOR Signaling in Hepatocellular Carcinoma

BACKGROUND & AIMSnThe advent of targeted therapies in hepatocellular carcinoma (HCC) has underscored the importance of pathway characterization to identify novel molecular targets for treatment. We evaluated mTOR signaling in human HCC, as well as the antitumoral effect of a dual-level blockade of the mTOR pathway.nnnMETHODSnThe mTOR pathway was assessed using integrated data from mutation analysis (direct sequencing), DNA copy number changes (SNP-array), messenger RNA levels (quantitative reverse-transcription polymerase chain reaction and gene expression microarray), and protein activation (immunostaining) in 351 human samples [HCC (n = 314) and nontumoral tissue (n = 37)]. Effects of dual blockade of mTOR signaling using a rapamycin analogue (everolimus) and an epidermal/vascular endothelial growth factor receptor inhibitor (AEE788) were evaluated in liver cancer cell lines and in a xenograft model.nnnRESULTSnAberrant mTOR signaling (p-RPS6) was present in half of the cases, associated with insulin-like growth factor pathway activation, epidermal growth factor up-regulation, and PTEN dysregulation. PTEN and PI3KCA-B mutations were rare events. Chromosomal gains in RICTOR (25% of patients) and positive p-RPS6 staining correlated with recurrence. RICTOR-specific siRNA down-regulation reduced tumor cell viability in vitro. Blockage of mTOR signaling with everolimus in vitro and in a xenograft model decelerated tumor growth and increased survival. This effect was enhanced in vivo after epidermal growth factor blockade.nnnCONCLUSIONSnMTOR signaling has a critical role in the pathogenesis of HCC, with evidence for the role of RICTOR in hepato-oncogenesis. MTOR blockade with everolimus is effective in vivo. These findings establish a rationale for targeting the mTOR pathway in clinical trials in HCC.

Genome-wide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma†

Although HCC is the third‐leading cause of cancer‐related deaths worldwide, there is only an elemental understanding of its molecular pathogenesis. In western countries, HCV infection is the main etiology underlying this cancers accelerating incidence. To characterize the molecular events of the hepatocarcinogenic process, and to identify new biomarkers for early HCC, the gene expression profiles of 75 tissue samples were analyzed representing the stepwise carcinogenic process from preneoplastic lesions (cirrhosis and dysplasia) to HCC, including 4 neoplastic stages (very early HCC to metastatic tumors) from patients with HCV infection. We identified gene signatures that accurately reflect the pathological progression of disease at each stage. Eight genes distinguish between control and cirrhosis, 24 between cirrhosis and dysplasia, 93 between dysplasia and early HCC, and 9 between early and advanced HCC. Using quantitative real‐time reverse‐transcription PCR, we validated several novel molecular tissue markers for early HCC diagnosis, specifically induction of abnormal spindle‐like, microcephaly‐associated protein, hyaluronan‐mediated motility receptor, primase 1, erythropoietin, and neuregulin 1. In addition, pathway analysis revealed dysregulation of the Notch and Toll‐like receptor pathways in cirrhosis, followed by deregulation of several components of the Jak/STAT pathway in early carcinogenesis, then upregulation of genes involved in DNA replication and repair and cell cycle in late cancerous stages. Conclusion: These findings provide a comprehensive molecular portrait of genomic changes in progressive HCV‐related HCC. (HEPATOLOGY 2007;45:938–947.)

Hepatic ‘stem cell’ malignancies in adults: four cases

Aims:u2002 Combined hepatocellular/cholangiocarcinomas have been explained by some investigators as bidirectional differentiation of neoplastic progenitor cell populations. The presence of hepatic progenitor cells has now been confirmed in humans, though whether they can give rise to malignant tumours has not been confirmed. We report four cases of small tumours identified in livers with features of chronic hepatitis which may suggest a role for malignant transformation of hepatic stem cells in hepatic malignancies.

Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo ☆

BACKGROUND/AIMSnThe success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models.nnnMETHODSnGene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts.nnnRESULTSnDifferent mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone.nnnCONCLUSIONSnRas activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.

IGF activation in a molecular subclass of hepatocellular carcinoma and pre-clinical efficacy of IGF-1R blockage

BACKGROUND & AIMSnIGF signaling has a relevant role in a variety of human malignancies. We analyzed the underlying molecular mechanisms of IGF signaling activation in early hepatocellular carcinoma (HCC; BCLC class 0 or A) and assessed novel targeted therapies blocking this pathway.nnnMETHODSnAn integrative molecular dissection of the axis was conducted in a cohort of 104 HCCs analyzing gene and miRNA expression, structural aberrations, and protein activation. The therapeutic potential of a selective IGF-1R inhibitor, the monoclonal antibody A12, was assessed in vitro and in a xenograft model of HCC.nnnRESULTSnActivation of the IGF axis was observed in 21% of early HCCs. Several molecular aberrations were identified, such as overexpression of IGF2 -resulting from reactivation of fetal promoters P3 and P4-, IGFBP3 downregulation and allelic losses of IGF2R (25% of cases). A gene signature defining IGF-1R activation was developed. Overall, activation of IGF signaling in HCC was significantly associated with mTOR signaling (p=0.035) and was clearly enriched in the Proliferation subclass of the molecular classification of HCC (p=0.001). We also found an inverse correlation between IGF activation and miR-100/miR-216 levels (FDR<0.05). In vitro studies showed that A12-induced abrogation of IGF-1R activation and downstream signaling significantly decreased cell viability and proliferation. In vivo, A12 delayed tumor growth and prolonged survival, reducing proliferation rates and inducing apoptosis.nnnCONCLUSIONSnIntegrative genomic analysis showed enrichment of activation of IGF signaling in the Proliferation subclass of HCC. Effective blockage of IGF signaling with A12 provides the rationale for testing this therapy in clinical trials.

Notch Signaling Is Activated in Human Hepatocellular Carcinoma and Induces Tumor Formation in Mice

BACKGROUND & AIMSnThe Notch signaling pathway is activated in leukemia and solid tumors (such as lung cancer), but little is known about its role in liver cancer.nnnMETHODSnThe intracellular domain of Notch was conditionally expressed in hepatoblasts and their progeny (hepatocytes and cholangiocytes) in mice. This was achieved through Cre expression under the control of an albumin and α-fetoprotein (AFP) enhancer and promoter (AFP-Notch intracellular domain [NICD]). We used comparative functional genomics to integrate transcriptome data from AFP-NICD mice and human hepatocellular carcinoma (HCC) samples (n = 683). A Notch gene signature was generated using the nearest template prediction method.nnnRESULTSnAFP-NICD mice developed HCC with 100% penetrance when they were 12 months old. Activation of Notch signaling correlated with activation of 3 promoters of insulin-like growth factor 2; these processes appeared to contribute to hepatocarcinogenesis. Comparative functional genomic analysis identified a signature of Notch activation in 30% of HCC samples from patients. These samples had altered expression in Notch pathway genes and activation of insulin-like growth factor signaling, despite a low frequency of mutations in regions of NOTCH1 associated with cancer. Blocking Notch signaling in liver cancer cells with the Notch activation signature using γ-secretase inhibitors or by expressing a dominant negative form of mastermind-like 1 reduced their proliferation in vitro.nnnCONCLUSIONSnNotch signaling is activated in human HCC samples and promotes formation of liver tumors in mice. The Notch signature is a biomarker of response to Notch inhibition in vitro.

MicroRNA-Based Classification of Hepatocellular Carcinoma and Oncogenic Role of miR-517a

BACKGROUND & AIMSnHepatocellular carcinoma (HCC) is a heterogeneous tumor that develops via activation of multiple pathways and molecular alterations. It has been a challenge to identify molecular classes of HCC and design treatment strategies for each specific subtype. MicroRNAs (miRNAs) are involved in HCC pathogenesis, and their expression profiles have been used to classify cancers. We analyzed miRNA expression in human HCC samples to identify molecular subclasses and oncogenic miRNAs.nnnMETHODSnWe performed miRNA profiling of 89 HCC samples using a ligation-mediated amplification method. Subclasses were identified by unsupervised clustering analysis. We identified molecular features specific for each subclass using expression pattern (Affymetrix U133 2.0; Affymetrix, Santa Clara, CA), DNA change (Affymetrix STY Mapping Array), mutation (CTNNB1), and immunohistochemical (phosphor[p]-protein kinase B, p-insulin growth factor-IR, p-S6, p-epidermal growth factor receptor, β-catenin) analyses. The roles of selected miRNAs were investigated in cell lines and in an orthotopic model of HCC.nnnRESULTSnWe identified 3 main clusters of HCCs: the wingless-type MMTV integration site (32 of 89; 36%), interferon-related (29 of 89; 33%), and proliferation (28 of 89; 31%) subclasses. A subset of patients with tumors in the proliferation subclass (8 of 89; 9%) overexpressed a family of poorly characterized miRNAs from chr19q13.42. Expression of miR-517a and miR-520c (from ch19q13.42) increased proliferation, migration, and invasion of HCC cells in vitro. MiR-517a promoted tumorigenesis and metastatic dissemination in vivo.nnnCONCLUSIONSnWe propose miRNA-based classification of 3 subclasses of HCC. Among the proliferation class, miR-517a is an oncogenic miRNA that promotes tumor progression. There is rationale for developing therapies that target miR-517a for patients with HCC.

Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib.

Purpose: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with active Wnt signaling. Underlying biologic mechanisms remain unclear and no drug targeting this pathway has been approved to date. We aimed to characterize Wnt-pathway aberrations in HCC patients, and to investigate sorafenib as a potential Wnt modulator in experimental models of liver cancer. Experimental Design: The Wnt-pathway was assessed using mRNA (642 HCCs and 21 liver cancer cell lines) and miRNA expression data (89 HCCs), immunohistochemistry (108 HCCs), and CTNNB1-mutation data (91 HCCs). Effects of sorafenib on Wnt signaling were evaluated in four liver cancer cell lines with active Wnt signaling and a tumor xenograft model. Results: Evidence for Wnt activation was observed for 315 (49.1%) cases, and was further classified as CTNNB1 class (138 cases [21.5%]) or Wnt-TGFβ class (177 cases [27.6%]). CTNNB1 class was characterized by upregulation of liver-specific Wnt-targets, nuclear β-catenin and glutamine-synthetase immunostaining, and enrichment of CTNNB1-mutation-signature, whereas Wnt-TGFβ class was characterized by dysregulation of classical Wnt-targets and the absence of nuclear β-catenin. Sorafenib decreased Wnt signaling and β-catenin protein in HepG2 (CTNNB1 class), SNU387 (Wnt-TGFβ class), SNU398 (CTNNB1-mutation), and Huh7 (lithium-chloride-pathway activation) cell lines. In addition, sorafenib attenuated expression of liver-related Wnt-targets GLUL, LGR5, and TBX3. The suppressive effect on CTNNB1 class–specific Wnt-pathway activation was validated in vivo using HepG2 xenografts in nude mice, accompanied by decreased tumor volume and increased survival of treated animals. Conclusions: Distinct dysregulation of Wnt-pathway constituents characterize two different Wnt-related molecular classes (CTNNB1 and Wnt-TGFβ), accounting for half of all HCC patients. Sorafenib modulates β-catenin/Wnt signaling in experimental models that harbor the CTNNB1 class signature. Clin Cancer Res; 18(18); 4997–5007. ©2012 AACR.

UHRF1 overexpression drives DNA hypomethylation and hepatocellular carcinoma.

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential regulator of DNA methylation that is highly expressed in many cancers. Here, we use transgenic zebrafish, cultured cells, and human tumors to demonstrate that UHRF1 is an oncogene. UHRF1 overexpression in zebrafish hepatocytes destabilizes and delocalizes Dnmt1 and causes DNA hypomethylation and Tp53-mediated senescence. Hepatocellular carcinoma (HCC) emerges when senescence is bypassed. tp53 mutation both alleviates senescence and accelerates tumor onset. Human HCCs recapitulate this paradigm, as UHRF1 overexpression defines a subclass of aggressive HCCs characterized by genomic instability, TP53 mutation, and abrogation of the TP53-mediated senescence program. We propose that UHRF1 overexpression is a mechanism underlying DNA hypomethylation in cancer cells and that senescence is a primary means of restricting tumorigenesis due to epigenetic disruption.