Growth inhibition of KRAS mutant cell lines via cleavage of RAS by RAS/RAP1 specific protease

Stubbs, Caleb Kawun

doi:https://doi.org/10.21985/n2-a95a-3589

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Growth inhibition of KRAS mutant cell lines via cleavage of RAS by RAS/RAP1 specific protease

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RAS is one of the most frequently mutated oncogenes in cancer with ~30% of all human tumors harboring a mutation in either HRAS, NRAS, or KRAS isoforms. Despite countless efforts for the development of small molecule inhibitors for RAS, it remains an elusive target in the clinic. Here I demonstrate that the pan-RAS biological inhibitor RAS/RAP1-specific endopeptidase (RRSP) has proteolytic activity in Ras-dependent mouse embryonic fibroblasts expressing human RAS isoforms (H/N/KRAS) or major oncogenic KRAS mutants (G12C, G12V, G12D, G13D, and Q61R). The cleavage of RAS inhibited phosphorylation of ERK and cell proliferation. To investigate how RAS processing might lead to varying outcomes in cell fate within cancer cells, I tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo apoptosis, RRSP treatment of GP5d and SW620 cells induces G1 cell cycle arrest. In some cell lines, growth effects were dictated by expression of the tumor suppressor protein p27 (Kip1). To further understand the growth inhibitory effects of RRSP outside of the RAS pathway, I investigated RRSP activity in RAS-independent BRAF mutant cell lines. These studies demonstrated that RRSP was still capable of growth inhibition in BRAF mutant cells, however growth was inhibited temporarily. These results were followed by studies that demonstrate previously unknown roles of ERK regulation of RAP1a gene expression in mouse embryonic fibroblasts (MEFs). I next examined the effect of RRSP in vivo mouse models for pancreatic cancer using the KRAS, p53, Cre (KPC) syngeneic mouse models. Despite RRSP effects in this model being minimized for several possible reasons, it has introduced several questions that need to be addressed for future application of RRSP as a cancer therapeutic. Together, this work demonstrated that complete ablation of RAS in cells induces growth inhibition, but the mechanism of inhibition can vary in different tumor cell lines. This ability of RAS processing to halt cell proliferation by multiple strategies highlights RRSP both as a potential anti-tumor therapy and as a tool for studying RAS signaling across tumor types.

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