Identification and characterization of two oncogenes LEF1 and GMNN in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC), a highly metastatic and fatal cancer, is one of the most common tumors worldwide. The carcinogenesis of HCC is believed to stem from multiple factors involving multiple genetic alterations and epigenetic changes. Compared with other cancers, HCC’s prognosis is extremely poor. The unsatisfactory clinical outcomes of HCC stem largely from its late diagnosis and propensity for recurrence and metastasis. However, the molecular mechanisms of the development and progression of HCC remain unclear. Discovery of HCC’s genetic alterations and potential mechanism is thus needed to develop novel approaches to early diagnosis and to attain better therapeutic effects. Activating dominant-acting oncogenes and disrupting the function of specific tumor suppressor genes in HCC progression could also prove clinically significant in HCC prevention and treatment. In previous studies, our team accomplished complete RNA-Seq analysis of three pairs of HCC tissues. Among the nearly 1000 RNA-Seq upregulated genes identified, two in particular aroused our interest, namely, lymphoid enhancer binding factor-1 (LEF1) and Geminin (GMNN). LEF1, an essential regulatory protein, plays a crucial role in the self-renewal and differentiation properties of several cancers, contributing to the degree of tumor malignancy and prognosis. In this study, I found that LEF is frequently overexpressed in HCC and significantly associated with HCC differentiation (P = 0.049) and recurrence (P = 0.048). In addition, a functional study involving both in vivo and in vitro experiments also showed that LEF1 can enhance cell proliferation, invasion, and migration and exhibit self-renewal abilities. I also demonstrated that LEF1 can induce Notch signaling via transcriptionally up-regulating Notch expression through direct DNA binding. The expression of several downstream members in the Notch signaling pathway, including Notch1 and Notch2, subsequently decreased. Moreover, LEF1 was found to regulate various differentiation markers and tumor stemness-related genes. Moreover, the other novel gene investigated, i.e., GMNN, was found to be frequently overexpressed in HCC in the mRNA level, as well as significantly related to a reduction in overall survival time (P = 0.001). A functional study also demonstrated GMNN to promote cell survival, clonogenicity, proliferation, and invasion and tumor metastasis both in vitro and in vivo. The mechanisms of GMNN amplification are beleived to be closely related to down-regulated miRNA449 in HCC. Further molecular study implied that cell motility and invasiveness can be enhanced by GMNN inducing epithelial-mesenchymal transition (EMT) and cytoskeleton remodeling. I also demonstrated GMNN to be significantly correlated with EZH2 expression, and to transcriptionally activate such expression, which plays a critical role in cell cycle regulation. The expression of several downstream members of the EZH2 signaling pathway was significantly associated with a change in GMNN expression. In this study, I identified and characterized the genetic alterations and potential mechanisms of two novel genes, (LEF1)-(NICD)-(HEY and HES family) and (GMNN)-(EZH2)), in the tumorigenesis and progression of HCC for the first time. LEF1 and GMNN may together constitute a dynamic tumor indexes for HCC prognosis evaluation and clinical treatments. The further delineation of their molecular mechanisms may illuminate the way for HCC gene diagnosis and therapy.