Date of Award
Ph.D. in Pharmaceutical Sciences
Kristine L Willett
Robert J. Doerksen
DNA-binding proteins play a pivotal role in cell biology. The major class of DNA-binding proteins are transcription factors (TFs). TFs are central to almost every fundamental cellular process such as cell development, differentiation, cell growth, and gene expression. They account for 10% of the genes in eukaryotes. In mammals, more than 700 TFs are identified to be DNA-binding TFs. They bind to the TF binding sites (TFBSs) in the genome and regulate the expression of their target genes. kRAS is a proto-oncogene with intrinsic GTPase activity, that contributes to cell proliferation, division, and apoptosis. kRAS mutations are observed in >95% of pancreatic adenocarcinoma and in 30 % of all human tumors. Pancreatic cancer is the fourth most deadly cancer, with 5 year survival rate of ~6%. When kRAS is mutated it leads to constitutive activity and uncontrolled proliferation, which results in increased tumorigenicity and poor prognosis. Other than mutation, kRAS gene amplification, overexpression, or increased upstream activation is also observed. Downregulating kRAS expression has shown to halt proliferation and lead to cellular death in pancreatic cancer models, but to date no small molecule capable of silencing expression has been described. Moreover, the kRAS promoter region is G-rich and is a hot spot for binding of TFs. TF binding and function in respect to kRAS transcription, is not yet mapped, leading to a gap in understanding of kRAS transcriptional regulation. In the current study, our purpose was to: a) identify and evaluate the function and binding interactions of TF’s on the regulation of kRAS, with a particular focus on two putative G-quadruplex (G4)-forming regions (herein termed near and mid) and the core region from 0 - +50, respective to the transcriptional start site and (b) to evaluate the effect of novel G4 stabilizing compounds on the kRAS expression. This study evaluated biological effects in both an isolated system with plasmids in HEK-293 cells by luciferase assay, and in complex in vitro milieus within pancreatic cancer cell lines by RT-qPCR. Protein changes were evaluated by western blotting. TF binding to the kRAS promoter was predicted based on consensus binding sites by online tools, and by direct binding was probed by Qiagen, and by us using a promoter binding array kit, and DNA pulldown followed by LC-MS/MS. EMSA was utilized for binding studies and effects on G4 formation and stability profile was probed by ECD. For the identification of kRAS-G4 interactive molecules we used FRET, ECD, luciferase assay and RT-qPCR. This mapping of TF binding to the kRAS promoter, and the demonstration of their function as their transcriptional silencers and activators and identification of G4-interactive molecules is important piece of the puzzle associated with the kRAS regulation.
Batra, Harshul, "Regulation of the kRAS Promoter in Pancreatic Cancer by Proteins and Small Molecules" (2017). Electronic Theses and Dissertations. 1481.