Investigating Cancer Molecular Genetics using Genome-wide RNA Interference Screens: A Dissertation

Abstract

The development of RNAi based technologies has given researchers the tools to interrogate processes as diverse as cancer biology, metabolism and organ development. Here I employ genome-wide shRNA screens to discover the genes involved in two different processes in carcinogenesis, oncogene-induced senescence [OIS] and epigenetic silencing of tumor suppressor genes [TSGs]. OIS is a poorly studied yet significant tumor suppressing mechanism in normal cells where they enter cell cycle arrest [senescence] or programmed cell death [apoptosis] in the presence of an activated oncogene. Here I employ a genomewide shRNA screen and identify a secreted protein, IGFBP7, that induces senescence and apoptosis in melanocytes upon introduction of the oncogene BRAF V600E . Expression of BRAF V600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 has potent pro-apoptotic and anti-tumor activity in mouse xenograft models using BRAF V600E -postive melanoma cell lines. Finally, IGFBP7 is epigenetically silenced in human melanoma samples suggesting IGFBP7 expression is a key barrier to melanoma formation. Next I investigated the factors involved in epigenetic silencing in cancer. The TSG p14 ARF is inactivated in a wide range of cancers by promoter hypermethylation through unknown mechanisms. To discover p14 ARF epigenetic silencing factors, I performed a genome-wide shRNA screen and identified ZNF304, a zinc finger transcription factor that contains a Kruppel-associated box [KRAB] repressor domain. I show that ZNF304 binds to the p14 AR F promoter and recruits a KRAB co-repressor complex containing KAP1, SETDB1 and DNMT1 for silencing. We find oncogenic RAS signaling to promote the silencing of p14ARF by USP28-mediated stabilization of ZNF304. In addition I find ZNF304 to be overexpressed in human colorectal cancers and responsible for hypermethylation of over 50 TSGs known as Group 2 CIMP marker genes. My findings establish ZNF304 as a novel oncogene that directs epigenetic silencing and facilitates tumorigenicity in colorectal cancer.