Characterization of gene regulatory networks in cancers

Abstract

The initiation and progression of cancer is a complicated process, involving the abnormality of broad classes of genes, such as growth factors and its receptors, signal transducers, transcriptional factors, chromatin remodelers, DNA repair genes, regulators of cell cycle and apoptosis, and noncoding RNAs. These genes exert critical functions in the regulation of cell proliferation and differentiation, cell cycle, and apoptosis. It is critical to understand these genes’ deregulations at transcriptional level, such as DNA methylation of their promoters, and transcription activation by microRNA-argonaute complex and transcription factors associated with their promoters/enhancers. Network-based strategies provide practical approaches to understand the underpinnings mediating cancer development by analyzing protein- DNA and protein-protein interaction. In this thesis, I identified the key gene-regulatory network between miRs and SOXs in liver cancer, and also distinguished critical regulators for Ikaros expression that is an important tumor suppressor in leukemia and liver cancer. Cancer stem cell (CSC) causes tumor heterogeneity, relapse, and resistance to therapy. The underpinnings of CSC remain to be elucidated, especially the underlying gene regulatory circuitry. I here conducted integrated analyses and identified a miRNA- regulatory network defining a stemness subtype with poor-prognosis from TCGA hepatocellular carcinoma (HCC) cohort with independent validation. The poor- prognosis subtype was characterized by the signature expression pattern of CSCs, orchestrated by two miRNAs targeting two SOX transcriptional factors that together formed a core regulatory circuitry (CRC). Within the CRC, miR483-3p bound a complementary sequence on SOX9 promoter, facilitating the recruitment of RNA polymerase II and STAT3, which was essential for SOX9 transcription activation. SOX9 can further activate SOX4 expression with SOX9 binding site on SOX4 promoter. Both SOX4 and its associated activator lncSOX4 were the direct targets of miR204-5p that recognized the complementary sites in its target genes’ 3’-end. SOX4 and miR204- 5p formed double-negative feedback loop through mutual inhibition. The expression level of miR204-5p was tightly modulated by miR483-3p. The functional significance of the CRC for HCC metastasis and drug resistance was demonstrated with different in vitro and in vivo assays. Activation of the CRC caused by downregulation of miR204- 5p and upregulation of miR483-3p, SOX9, and SOX4, were confirmed with liver CSCs, and required for the self-renewal and maintenance of tumor spheroid. Ikaros plays a critical role in the hematopoietic system development. The development of lymphoid leukemia is closely associated with the dysfunction of Ikaros, a well-established tumor suppressor in human leukemia. In order to identify the regulatory network for Ikaros, I employed CRISPR/Cas9 and Cre/lox to figure out the cis-elements and trans-factors that are responsible for the transcription activation of Ikaros expression. Transcriptional factors Oct2 and Gata3 were identified to bind at Enhancer H of Ikzf1 (gene coding for Ikaros) to mediate the robust expression of Ikzf1 in mouse T-cell lymphoma. In conclusion, I identified the gene regulatory networks that are important for liver cancer development and Ikaros regulation.