Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. We sequenced and analyzed the whole genomes of 27 HCCs, 25 of which were associated with hepatitis B or C virus infections, including two sets of multicentric tumors. Although no common somatic mutations were identified in the multicentric tumor pairs, their whole-genome substitution patterns were similar, suggesting that these tumors developed from independent mutations, although their shared etiological backgrounds may have strongly influenced their somatic mutation patterns. Statistical and functional analyses yielded a list of recurrently mutated genes. Multiple chromatin regulators, including ARID1A, ARID1B, ARID2, MLL and MLL3, were mutated in ∼50% of the tumors. Hepatitis B virus genome integration in the TERT locus was frequently observed in a high clonal proportion. Our whole-genome sequencing analysis of HCCs identified the influence of etiological background on somatic mutation patterns and subsequent carcinogenesis, as well as recurrent mutations in chromatin regulators in HCCs.

Epithelial-mesenchymal transition (EMT), a crucial event in cancer progression and embryonic development, is induced by transforming growth factor (TGF)-␤ in mouse mammary NMuMG epithelial cells. Id proteins have previously been reported to inhibit major features of TGF-␤-induced EMT.In this study, we show that expression of the ␦EF1 family proteins, ␦EF1 (ZEB1) and SIP1, is gradually increased by TGF-␤ with expression profiles reciprocal to that of E-cadherin. SIP1 and ␦EF1 each dramatically down-regulated the transcription of E-cadherin in NMuMG cells through direct binding to the E-cadherin promoter. Silencing of the expression of both SIP1 and ␦EF1, but not either alone, completely abolished TGF-␤-induced E-cadherin repression. However, expression of mesenchymal markers, including fibronectin, N-cadherin, and vimentin, was not affected by knockdown of SIP1 and ␦EF1. TGF-␤-induced the expression of Ets1, which in turn activated ␦EF1 promoter activity. Moreover, up-regulation of SIP1 and ␦EF1 expression by TGF-␤ was suppressed by knockdown of Ets1 expression. In addition, Id2 suppressed the TGF-␤-and Ets1-induced up-regulation of ␦EF1. Taken together, these findings suggest that the ␦EF1 family proteins, SIP1 and ␦EF1, are necessary, but not sufficient, for TGF-␤-induced EMT and that Ets1 induced by TGF-␤ may function as an upstream transcriptional regulator of SIP1 and ␦EF1. INTRODUCTIONTransforming growth factor (TGF)-␤, a prototypical member of the TGF-␤ family, regulates a broad range of cellular responses, including cell proliferation, differentiation, adhesion, migration, and apoptosis (Bierie and Moses, 2006). TGF-␤ and related factors exhibit their pleiotropic effects through binding to transmembrane serine-threonine kinase receptors type I (T␤R-I) and type II (T␤R-II). On ligand-induced heteromeric complex formation between T␤R-I and T␤R-II, T␤R-I is phosphorylated and activated by T␤R-II kinase and mediates specific intracellular signaling through phosphorylation of receptor-regulated Smads (R-Smads). Phosphorylated R-Smads interact with coSmad (Smad4) and translocate into the nucleus, where they regulate transcription of target genes in cooperation with various transcription factors and transcriptional coactivators or corepressors (Miyazawa et al., 2002;Miyazono et al., 2003;Shi and Massague, 2003).TGF-␤ has potent antiproliferative effects on a wide variety of cells, including epithelial cells, endothelial cells, and hematopoietic cells, although under certain conditions it promotes the proliferation of mesenchymal cells, including fibroblasts, chondrocytes, and osteoblasts. TGF-␤ also induces the deposition of extracellular matrix proteins. In early stages of tumorigenesis, TGF-␤ inhibits the growth of epithelial cells, and insensitivity to this growth-inhibitory effect is associated with progression of tumors Derynck et al., 2001). Transgenic mice expressing a dominant-negative T␤R-II in epidermis exhibit malignant conversion of epithelial cells and promotion of tumor formation (Gorska et al., 20...

TGF-β regulates isoform switching of FGF receptors and epithelial–mesenchymal transitionBoth TGF-β and FGF signalling regulate the epithelial–mesenchymal transition. Here, TGF-β is found to promote myofibroblast differentiation, while concomitant FGF pathway activation instead drives cells towards an invasive mesenchymal fate.

Hepatocellular carcinoma, one of the most common virus-associated cancers, is the third most frequent cause of cancer-related death worldwide. By massively parallel sequencing of a primary hepatitis C virus-positive hepatocellular carcinoma (36× coverage) and matched lymphocytes (>28× coverage) from the same individual, we identified more than 11,000 somatic substitutions of the tumor genome that showed predominance of T>C/A>G transition and a decrease of the T>C substitution on the transcribed strand, suggesting preferential DNA repair. Gene annotation enrichment analysis of 63 validated non-synonymous substitutions revealed enrichment of phosphoproteins. We further validated 22 chromosomal rearrangements, generating four fusion transcripts that had altered transcriptional regulation (BCORL1-ELF4) or promoter activity. Whole-exome sequencing at a higher sequence depth (>76× coverage) revealed a TSC1 nonsense substitution in a subpopulation of the tumor cells. This first high-resolution characterization of a virus-associated cancer genome identified previously uncharacterized mutation patterns, intra-chromosomal rearrangements and fusion genes, as well as genetic heterogeneity within the tumor.

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Epithelial-mesenchymal transdifferentiation (EMT) is a critical morphogenic event that occurs during embryonic development and during the progression of various epithelial tumors. EMT can be induced by transforming growth factor (TGF)-b in mouse NMuMG mammary epithelial cells. Here, we demonstrate a central role of helix-loop-helix factors, E2A and inhibitor of differentiation (Id) proteins, in TGF-b-induced EMT. Epithelial cells ectopically expressing E2A adopt a fibroblastic phenotype and acquire migratory/invasive properties, concomitant with the suppression of E-cadherin expression. Id proteins interacted with E2A proteins and antagonized E2A-dependent suppression of the E-cadherin promoter. Levels of Id proteins were dramatically decreased by TGF-b. Moreover, NMuMG cells overexpressed Id2 showed partial resistance to TGF-b-induced EMT. Id proteins thus inhibit the action of E2A proteins on the expression of E-cadherin, but after TGF-b stimulation, E2A proteins are present in molar excess of the Id proteins, thus over-riding their inhibitory function and leading to EMT.

Genetic alterations and deregulation of the miRNA biogenesis pathway components have been reported in human tumors. Tissue‐specific deletion of the Dicer gene, which encodes an essential miRNA processing enzyme, promotes carcinogenesis in animal models. These features indicate that aberrant miRNA biogenesis components are directly associated with cancer. For the present study, we conducted quantitative RT‐PCR of 14 genes that are related to the miRNA biogenesis pathway in 47 paired samples of primary hepatocellular carcinoma (HCC) and matched non‐cancerous liver. Expression of seven genes (Dgcr8, p68, p72, Dicer, Ago3, Ago4 and Piwil4) was significantly decreased in primary HCC, especially in non‐viral HCC subtypes, compared to the non‐cancerous liver. Combinations of decreased expression of the miRNA biogenesis components in non‐cancerous liver were related to cigarette smoking, alcohol intake and diabetes, which are known to be risk factors for HCC, and were also associated with the occurrence of multicentric tumors. Reduction of two of these genes (Dicer and p68) in HCC was associated with poor prognosis. Trimethylation of histone H3 lysine 27 in the promoters is implicated in the deregulation of these miRNA‐biogenesis‐related genes in non‐HBV genome integrated HCC cell lines. In conclusion, deregulation of the miRNA biogenesis pathway components is frequently observed in non‐viral‐associated HCC and is linked to etiological risk factors and poor prognosis. Our study further showed that epigenetic regulation could be implicated in the deregulation of these genes during hepatocarcinogenesis.

Epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Transforming growth factor (TGF)-b induces EMT in mouse epithelial cells. During prolonged treatment, TGF-b successively induces myofibroblastic differentiation with increased expression of myofibroblast marker proteins, including smooth muscle a actin and calponin. We recently showed that fibroblast growth factor-2 prevented myofibroblastic differentiation induced by TGF-b, and transdifferentiated the cells to those with much more aggressive characteristics (enhanced EMT). To identify the molecular markers specifically expressed in cells undergoing enhanced EMT induced by the combination of TGF-b and fibroblast growth factor-2, we carried out a microarray-based analysis and found that integrin a3 (ITGA3) and Ret were upregulated. Intriguingly, ITGA3 was also overexpressed in breast cancer cells with aggressive phenotypes and its expression was correlated with that of dEF-1, a key regulator of EMT. Moreover, the expression of both genes was downregulated by U0126, a MEK 1 ⁄ 2 inhibitor. Therefore, ITGA3 is a potential marker protein for cells undergoing enhanced EMT and for cancer cells with aggressive phenotypes, which is positively regulated by dEF-1 and the MEK-ERK pathway. (Cancer Sci 2013; 104: 1189-1197 E pithelial-mesenchymal transition (EMT) serves as a switch directing polarized epithelial cells to transdifferentiate into mesenchymal cells. During the processes of embryonic development, wound healing, and reorganization of adult tissues, epithelial cells have been shown to lose their epithelial polarity and acquire mesenchymal phenotypes.(1) Furthermore, EMT is involved in the process of tumor-cell invasion, which also includes the loss of cell-cell interaction. Thus far, in most cases, EMT appears to be regulated by ECM components and soluble growth factors or cytokines. Of these, transforming growth factor-b (TGF-b) is considered to be the key inducer of EMT during physiological processes.(2) TGF-b is frequently and abundantly expressed in various tumors, and also induces EMT in cancer cells during cancer progression. Several extracellular signaling molecules, including Wnt, epidermal growth factor, fibroblast growth factor (FGF)-2, and tumor necrosis factor-a, cooperate with TGF-b to promote tumor invasion and metastasis as well as EMT. In addition, constitutively active Ras dramatically enhances TGF-binduced expression of Snail, a key mediator of EMT, whereas representative target genes of TGF-b are either unaffected or slightly inhibited by Ras signaling, leading to selective synergism between TGF-b and Ras as well as soluble factors in cancer progression. TGF-b has been found to induce EMT in normal mouse mammary epithelial NMuMG cells, and we recently showed that prolonged treatment of NMuMG cells with TGF-b induces the epithelial-myofibroblastic transition (EMyoT) with the expression of myofibroblast markers, smooth muscle a actin (a-SMA), and calponin.(4) During T...