Oncogenicitiy Comparison of Human Papillomavirus Type 52 E6 Variants.

Human papillomavirus (HPV) infection contributes to virtually all cases of cervical cancer, the fourth most common cancer affecting women worldwide. The oncogenicity of HPV is mainly attributable to the E6 and E7 oncoproteins. HPV-52 is the seventh most common HPV type globally, but it has a remarkably high prevalence in East Asia. In previous studies it has been speculated that the oncogenicity might vary among different HPV-52 variants. In the present study, we compared the oncogenicity of E6 derived from the HPV-52 prototype and three commonly found variants, V1 (K93R), V2 (E14D/V92L) and V3 (K93R/N122K), through molecular and phenotypic approaches. We demonstrated that cells containing V1 achieved higher colony formation and showed greater cell migration ability when compared to other variants, but no difference in cell immortalization ability was observed. At the molecular level, the three variants formed complexes with E6-associated protein (E6AP) and p53 as efficiently as the prototype. They degraded p53 and PSD95/Dlg/ZO-1(PDZ) proteins, including MAGI-1c and Dlg, to a similar extent. They also exhibited a similar subcellular localization, and shared a half-life of approximately 45 min. Our findings provide a clearer picture of HPV-52 E6 variant oncogenicity, which is important for further studies aiming to understand the unusually high prevalence of HPV-52 among cervical cancers in East Asia.

Oncogenic comparison of human papillomavirus type 58 E7 variants.

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony-forming ability to primary murine epithelial cells than prototype (P < 0.05) and other variants, implicating its higher immortalising potential. Further experiments showed that both V1 and prototype enhanced the anchorage-independent growth of NIH/3T3 cells (P < 0.001), implicating their stronger transforming power than the two other variants. Moreover, they possessed an increased ability to degrade pRb (P < 0.001), which is a major effector pathway of E7-driven oncogenesis. Our work represents the first study to compare the oncogenicities of HPV58 E7 prototype and variants. These findings deepened our understanding of HPV58 and might inform clinical screening and follow-up strategy.

Deep sequencing of small RNA transcriptome reveals novel non-coding RNAs in hepatocellular carcinoma.

BACKGROUND & AIMS: Small non-coding RNAs (ncRNA) are increasingly recognized to play important roles in tumorigenesis. With the advent of deep sequencing, efforts have been put forth to profile the miRNome in a number of human malignancies. However, information on ncRNA in hepatocellular carcinoma (HCC), especially the non-microRNA transcripts, is still lacking. METHODS: Small RNA transcriptomes of two HCC cell lines (HKCI-4 and HKCI-8) and an immortalized hepatocyte line (MIHA) were examined using Illumina massively parallel sequencing. Dysregulated ncRNAs were verified in paired HCC tumors and non-tumoral livers (n=73) by quantitative reverse transcription-polymerase chain reaction. Clinicopathologic correlations and in vitro functional investigations were further carried out. RESULTS: The combined bioinformatic and biological analyses showed the presence of ncRNAs and the involvement of a new PIWI-interacting RNA (piRNA), piR-Hep1, in liver tumorigenesis. piR-Hep1 was found to be upregulated in 46.6% of HCC tumors compared to the corresponding adjacent non-tumoral liver. Silencing of piR-Hep1 inhibited cell viability, motility, and invasiveness, with a concomitant reduction in the level of active AKT phosphorylation. In the analysis of miRNA, we showed for the first time, the abundant expression of miR-1323 in HCC and its distinct association in tumors arising from a cirrhotic background. Furthermore, miR-1323 overexpression in cirrhotic HCC correlated with poorer disease-free and overall survivals of patients (p<0.009). CONCLUSIONS: Our study demonstrated the value of next-generation sequencing in dissecting the ncRNome in cancer. The comprehensive definition of transcriptome unveils virtually all types of ncRNAs and provides new insight into liver carcinogenetic events.

MiR-145 modulates multiple components of the insulin-like growth factor pathway in hepatocellular carcinoma.

Profiling of microRNA expression in human cancers has highlighted downregulation of miR-145 as a common event in epithelial malignancies. Here, we describe recurrent underexpression of miR-145 in hepatocellular carcinoma (HCC) and the identification of a biological pathway by which miR-145 exerts its functional effects in liver tumorigenesis. In a cohort of 80 HCC patients, quantitative reverse transcription polymerase chain reaction corroborated reduced miR-145 expression in 50% of tumors, which also correlated with a shorter disease-free survival of patients. One HCC tumor analyzed with low endogenous miR-145 was propagated as cell line. This in vitro model HKCI-C2 maintained low miR-145 level and upon restoration of miR-145 expression, a consistent inhibitory effect on cell viability and proliferation was readily found. Flow cytometric analysis indicated that miR-145 re-expression could induce G(2)-M cell cycle arrest and apoptosis. Multiple in silico algorithms predicted that miR-145 could target a number of genes along the insulin-like growth factor (IGF) signaling, including insulin receptor substrate (IRS1)-1, IRS2 and insulin-like growth factor 1 receptor. We found protein expression of these putative targets was concordantly downregulated in the presence of miR-145. Luciferase reporter assay further verified direct target association of miR-145 to specific sites of the IRS1 and IRS2 3'-untranslated regions. Subsequent analysis also affirmed miR-145 modulation on the IGF signaling cascade by reducing its downstream mediator, namely the active ß-catenin level. Taken together, our study shows for the first time the pleiotropic effect of miR-145 in targeting multiple components of the oncogenic IGF signaling pathway in HCC.

Emerging roles of microRNA in the intracellular signaling networks of hepatocellular carcinoma.

MicroRNAs (miRNAs) are small non-coding RNAs of 19-23 nucleotides that negatively regulate gene expression through binding to the 3'-untranslated regions of target messenger RNAs (mRNAs). Although the miRNA family constitutes only a minor fraction of the human genome, they hold fundamental importance in diverse physiological and developmental processes due to their pleiotropic effects on the post-transcriptional regulation of many vital genes. This class of regulatory RNAs has also emerged as important players in carcinogenesis; most, if not all, cancer types have abnormal miRNA expression patterns. In hepatocellular carcinoma (HCC), miRNA dysregulation plays a key role in mediating the pathogenicity of several etiologic risk factors and, more importantly, they promote a number of cancer-inducing signaling pathways. Recent studies have also demonstrated their potential values in the clinical management of HCC patients as some miRNAs may be used as prognostic or diagnostic markers. The significance of miRNAs in liver carcinogenesis emphasizes their values as therapeutic targets, while technological advances in the delivery of miRNA has shed new possibilities for their use as novel therapeutic agents against HCC.

Re-expression of transcription factor ATF5 in hepatocellular carcinoma induces G2-M arrest.

Transcription factors represent an important class of genes that play key roles in controlling cellular proliferation, cell cycle modulation, and attractive targets for cancer therapy. Here, we report on the novel finding of common ATF5 down-regulations in hepatocellular carcinoma (HCC), a highly malignant tumor with a dismal clinical course. Array-based mapping in HCC highlighted a high and consistent incidence of transcription factor ATF5 repressions on regional chr.19q13. By quantitative reverse transcription-PCR, profound down-regulations of ATF5 were further suggested in 78% of HCC tumors (60 of 77 cases) compared to their adjacent nontumoral liver (P = 0.0004). Restoration of ATF5 expression in 3 nonexpressing HCC cell lines demonstrated a consistent growth inhibitory effect (P < 0.029) but minimal induction on cellular apoptosis. Subsequent flow cytometric investigations revealed a G(2)-M cell cycle arrest in HCC cells that were ectopically transfected with ATF5 (P < 0.002). The differential expressed genes from the functional effects of ATF5 were examined by array profiling. Over a hundred genes were identified, among which ID1 contains the ATF/CREB target binding sequences within its promoter region. An inverse relationship between ATF5 expressions with ID1 transcriptions was verified in HCC (P = 0.019), and a direct interaction of ATF5 on the promoter of ID1 was further demonstrated from electromobility shift assay. Examination of causal events underlying the silencing of ATF5 in HCC suggested copy number losses, promoter hypermethylation, histone deacetylation, and DNA mutations to be the likely inactivating mechanisms. In conclusion, our finding supports a tumor suppressive role for ATF5 in HCC, and highlighted ID1 as a potential downstream target.

MicroRNA-223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1.

BACKGROUND & AIMS: Recent studies have emphasized causative links between microRNA (miRNA) deregulations and cancer development. In hepatocellular carcinoma (HCC), information on differentially expressed miRNA remained largely undefined. METHODS: Array-based miRNA profiling was performed on HCC cells that were derived from chronic carriers of hepatitis B virus (HBV) and hepatitis C virus (HCV), and nonviral-associated patients. Specific microRNA (miR)-223 and miR-222 deregulations were verified in an independent series of tumors. The functional effect of miR-223 was examined further. An integrative analysis of messenger RNA (mRNA) array with in silico predictions defined potential downstream targets of miR-223. A luciferase reporter assay was conducted to confirm target association. RESULTS: Distinct up-regulations of miR-222, miR-221, and miR-31, and down-regulations of miR-223, miR-126, and miR-122a were identified. Further investigations suggested the highly deregulated miR-223 and miR-222 could unequivocally distinguish HCC from adjacent nontumoral liver, irrespective of viral associations (P