Virus-induced hepatocarcinogenesis involves a series of histological developmental processes with the stepwise acquisition of several genetic changes that are necessary for the malignant transformation of hepatocytes. Although genetic alterations are known to be involved in the pathogenesis of hepatocellular carcinoma (HCC), little is known about the contributions of specific genes to this process. To gain insight into the genetic alterations involved in the neoplastic evolution from chronic hepatitis B virus infection to dysplastic nodules (DN) to HCC, we captured and sequenced the exomes of four DNA samples: one DN sample, two HCC samples, and one control peripheral blood sample from a single HCC patient. Mutations in the UBE3C gene (encoding ubiquitin ligase E3C) were observed in both tumor tissues. Then we resequenced the UBE3C gene in a cohort of 105 HCC patients and identified mutations in 17 out of a total of 106 (16.0%) HCC patients. The subsequent experiments showed that UBE3C promoted HCC progression by regulating HCC cells epithelial-mesenchymal transition. Clinically, a tissue microarray study of a cohort containing 323 HCC patients revealed that the overexpression of UBE3C in primary HCC tissues correlated with decreased survival (hazard ratio [HR] = 1.657, 95% confidence interval [CI] = 1.220-2.251, P = 0.001) and early tumor recurrence (HR = 1.653, 95% CI = 1.227-2.228, P = 0.001) in postoperative HCC patients. Conclusion: Our findings indicate that UBE3C is a candidate oncogene involved in tumor development and progression and therefore a potential therapeutic target in applicable HCC patients. (Hepatology 2014;59:2216–2227)
Hepatocellular carcinoma (HCC) remains the second leading cause of cancer-related deaths worldwide, with ∼700,000 cancer deaths and 750,000 new cases reported each year. While the incidence rates of most cancers have declined in recent decades, the HCC incidence has continued to increase among both men and women. Although recent progress in early diagnosis, surveillance programs in high-risk patients, and surgical interventions (liver resection and transplantation) have significantly improved the survival of patients with HCC, the overall survival (OS) of HCC patients remains poor. The mechanism of hepatocarcinogenesis remains unclear, and our understanding of HCC-associated molecular changes is still lacking. Thus, the discovery of novel HCC susceptibility genes will provide new targets for drug development and gene therapy.
Among the various etiologies related to HCC development, chronic hepatitis B viral (HBV) infection remains the most prominent. Virus-induced hepatocarcinogenesis involves an array of histological developmental processes, and the stepwise acquisition of genetic and genomic changes is necessary for the malignant transformation of hepatocytes, which involves hepatocyte damage, regeneration, and the development of regenerative nodules and dysplastic nodules (DN).[5-7] Hepatocytes with an increasing number of genetic hits gradually develop into precancerous lesions (such as DN) and eventually evolve into neoplasms. A DN is a premalignant lesion that displays a large number of genetic variations and abnormal cytological features, and it is known to invade surrounding tissues and occasionally demonstrate metastatic potential.[8, 9]
Although sequencing studies have made progress in the search for molecular changes in HCC,[10-12] no study has sequenced each histological lesion that is formed during the process of hepatocarcinogenesis. To gain insight into the genetic alterations involved in the neoplastic evolution from chronic HBV infection to DN to HCC, we captured and sequenced the exomes from four DNA samples: one DN sample, two HCC samples, and one control peripheral blood sample from the same HCC patient. The candidate mutated gene(s) identified in these samples were further validated in an additional 105 HCC patients. The functional relevance of these candidates was investigated in HCC cell lines, and clinical relevance was demonstrated in a tissue microarray study of a cohort comprising 323 HCC patients. Our goal was to identify frequent somatic alterations that might describe the biological characteristics of HCC and furnish new approaches for diagnosis and treatment.
Tumorigenesis is a complex biological process driven by genetic alterations that activate oncogenes or inactivate tumor suppressor genes. Similar to other solid tumors, HCC accumulates a large number of genetic variations during the development of hepatocarcinogenesis. As a normal liver develops an increasing number of genetic variants, it gradually develops precancerous lesions that ultimately evolve into a neoplasm. Hepatocarcinogenesis is known to be a histological developmental process that is induced by multiple factors and steps. Most HCCs evolve from cirrhosis-inducing conditions, with neoplasms induced by chronic HBV or HCV infection evolving from liver cirrhosis to DN and eventually HCC. Taguchi et al. and Kobayashi et al. reported that DNs arising from livers with chronic viral hepatitis infection or cirrhosis were predisposed to neoplasm formation and should be considered precancerous lesions. In our study, we identified five unique nonsynonymous variants in a DN within the CADPS, CAPG, FOXD4L1, EXOSC7 and FAM209A genes; however, these mutations were not identified in either tumor from this patient. In contrast, Miller et al. reported that CADPS was lost in 27.6% of primary central nervous system primitive neuroectodermal tumors and that the loss of this gene was associated with poor prognosis. Moreover, the expression of CAPG, which encodes a member of the gelsolin family of actin regulatory proteins, is elevated in some human cancers and is involved in tumor development and prognosis.[19, 20] The fact that these tumor-related genes were also mutated in the liver DN prompted us to speculate that the DN had already accumulated a number of genetic changes that endowed it with the capacity for malignant transformation prior to the appearance of changes in its cytomorphological features. This finding provides a better understanding of the mechanism of hepatocarcinogenesis.
The recent exome analysis of human HCC have revealed numerous novel mutations of cancer-related genes. According to previous studies, TP53 and CTNNB1 were thought to be the most recurrently mutated tumor suppressor and oncogene for HCC, respectively. Besides, a number of novel somatic alterations for HCC have been identified, such as the ARID2, ARID1A, and LEPR genes.[11, 12, 22, 23] Interestingly, various risk factors for HCC, such as HBV and HCV infections, showed various mutation profiles of the corresponding tumor genome. In this study we identified frequent mutations of the UBE3C gene in human HCCs. Further targeted resequencing of this gene showed that the UBE3C gene was mainly mutated in HBV-related HCCs, rarely in DNs or HCV- and nonviral-related HCCs, and not in the nontumoral tissues. These findings imply that etiology, such as HBV and HCV, may affect the UBE3C mutation profile.
The UBE3C protein contains two characteristic protein domains: an IQ motif and a HECT domain. The IQ motif in the N-terminal region mediates substrate targeting and may serve as a binding and regulatory site, whereas the HECT domain is a highly conserved C-terminal catalytic domain that binds to ubiquitin-conjugating enzymes (E2) and accepts and transfers ubiquitin to the target substrate. Thus, the HECT domain provides the E3 catalytic activity. Furthermore, we identified nine UBE3C mutations in HBV-related HCCs, three of which (Asn929Ile, Glu959Lys, and Thr1004Ala) occurred in the highly conserved HECT domain region of UBE3C. The available crystal structure of the HECT domain of E6AP (also known as UBE3A) reveals a bilobal protein conformation, with a broad catalytic cleft at the junction of the two lobes, which plays a critical role in transferring ubiquitin from the E2 to the E3. In this study, structure prediction analysis revealed that three UBE3C molecules were in close proximity to each other, forming a “center” in the trimer interface of the HECT domain. In light of the catalytic activity of HECT domain, we speculate that this “center” is the enzymatic activity center of UBE3C that binds to substrates and catalyzes reactions. Because Glu959 is situated on an α-helix of the HECT domain surface that may participate in protein-protein interactions, the Glu959Lys mutation in UBE3C may affect its substrate binding capacity, resulting in the excessive degradation of tumor suppressor proteins or a reduced degradation of oncoproteins involved in hepatocarcinogenesis.
To date, no association between UBE3C and tumorigenesis has been established. Previous studies reported that mutations in the HECT domain of E3s often lead to pathophysiological states, including neurological disorders and human cancers.[26-28] Thus, the high frequency of UBE3C mutations in HCCs provides a preliminary connection between UBE3C and human cancer. Furthermore, UBE3C was overexpressed in HCC tissues and promoted HCC progression in vivo and in vitro, extending its role in cancer development. The loss of contact inhibition of cell proliferation, which is a hallmark of cancer, is thought to be associated with cadherin-mediated cell-cell attachments. E-cadherin is expressed in epithelial cells and plays a vital role in cell adhesion and movement. The loss of E-cadherin function or expression is thought to promote tumor progression by increasing proliferation, invasion, and metastasis. Here we showed that UBE3C down-regulated the expression of E-cadherin and induced cancer cells to undergo EMT, which is known to promote carcinoma progression and metastasis in a variety of human cancers, including HCC. Thus, we speculate that UBE3C promotes HCC development and progression by targeting E-cadherin for ubiquitin-mediated proteolysis and inducing EMT in cancer cells.
The ubiquitin-proteasome system regulates crucial cellular functions, such as the cell cycle, DNA repair, cell signaling, and responses to hypoxia, and E3 enzymes play a key role in this process by way of selectively binding to their protein substrates. HECT E3s possess intrinsic catalytic activity and directly catalyze the ubiquitination of substrate proteins, thereby performing pivotal roles in maintaining cellular homeostasis and biological signaling. The dysfunction of HECT E3s frequently contributes to pathological disorders, including various tumors. E6AP is the first identified member of the HECT E3 family. E6AP targets the tumor suppressor protein p53 for ubiquitin-mediated proteolysis, which promotes p53 degradation and contributes to the development of a majority of human cervical cancer cases. Nedd4 was recently reported to promote the proteasomal degradation of PTEN, a tumor suppressor that negatively regulates the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and Nedd4 expression has also been associated with colorectal cancer, bladder, and prostate carcinomas.[34-36] An increasing number of tumor suppressor molecules have been identified as substrates of HECT E3s, and genetic aberrations in and altered expression of the HECT E3s are often observed in various human tumors.[37, 38] Although the present study found no correlation of UBE3C status and the expression of TP53 and PTEN, we confirmed that UBE3C expression was markedly increased in HCC tissues compared with peritumoral liver tissues. Additionally, we present clinically relevant data showing a correlation between high UBE3C expression and poor prognosis in HCC patients. This finding may be biased by the fact that aggressive clinicopathological features, such as multiple tumors, large tumor size, high TNM and BCLC staging of HCC, were more frequently observed in patients with high UBE3C expression than in those with low expression. Nevertheless, these data indicate that UBE3C protein expression may be a promising prognostic biomarker for HCC.
The ubiquitin-proteasome system yields a plentiful source of molecular targets for anticancer therapy. Proteasome inhibitors such as bortezomib (Velcade, Millennium Pharmaceuticals) are established cancer therapeutics and have been approved by the U.S. Food and Drug Administration for the treatment of multiple myeloma and mantle cell lymphoma. Given the clinical toxicity arising from the lack of specificity of proteasome inhibitors, targeting highly selective E3s will limit the adverse effects and broaden and diversify the scope of anticancer therapies, making them more specific and efficient. Furthermore, our findings indicate that UBE3C may represent a selective target for HCC patients. However, the roles of UBE3C in other types of tumors remain undetermined, and the specific substrate of UBE3C has not yet been identified. Thus, further study is needed to address these issues.
In summary, our findings identify a novel HCC susceptibility gene, UBE3C, as a candidate oncogene with a role in tumor development and progression that may serve as a potential therapeutic target in a subset of HCC patients.
We thank Jie Zong and Dai Chen (NovelBioinformatics Ltd., Co.) for technical support in the bioinformatics analysis process.