Publication Date

2022

Document Type

Thesis

Committee Members

Kwang-Jin Cho, Ph.D. (Advisor); Michael Leffak, Ph.D. (Committee Member); Hongmei Ren, Ph.D. (Committee Member)

Degree Name

Master of Science (MS)

Abstract

Constitutively active oncogenic mutant K-Ras is one the principal contributors to human cancers including 90% of pancreatic, 50% of colorectal and 32% of non-small cell lung cancers. However, except for K-Ras G12C oncogenic mutant, which only presents in about 13% of non-small cell lung cancer patients, there is no anti-K-Ras therapy for a considerable subset of K-Ras mutations in human tumors, reflecting challenges for targeting oncogenic K-Ras activity. K-Ras is a membrane-bound small GTPase; when active, it triggers multiple signaling pathways regulating a variety of key cellular functions such as cell growth, proliferation and survival. To initiate these signaling cascades, K-Ras must localize to the plasma membrane (PM), where gets activated by guanine-nucleotide exchange factors and interact with its downstream effectors. Based on this dependency of K-Ras localization to the PM for its biological activity, removing K-Ras from the PM has been suggested as an anti-K-Ras approach. The exact mechanism of K-Ras interaction with the PM is not fully elucidated. Extensive studies have shown a specific preference of K-Ras for interacting with phosphatidylserine (PS), an acidic phospholipid in the inner PM leaflet, and decrease in the PS contents dissociates K-Ras from the PM and blocks K-Ras signaling. Towards our goal of targeting the PM/K-Ras interaction, our recent study has indicated that phosphatidylinositol 4-kinase IIIβ (PI4KB), which converts PI to PI 4-phosphate at the Golgi complex, is involved in the PM enrichment of PS and K-Ras. We found that upon PI4KB inhibition, K-Ras and PS redistribute from the PM to mitochondria and other endomembranes, respectively. The aims of this dissertation are to 1) characterize PI4KB as an anti-K-Ras target in human pancreatic cancer cell lines expressing oncogenic mutant K-Ras, and 2) assess the mechanism of K-Ras translocation to mitochondria upon PI4KB inhibition. Our proliferation assay data demonstrate that chemical inhibitors for PI4KB reduced the growth of human pancreatic cancer cells harboring oncogenic mutant K-Ras, but not wild-type K-Ras. These data suggest that PI4KB could be a target for treating human pancreatic cancers. In the second aim of study, we sought to elucidate whether the polybasic domain (PBD) of K-Ras is involved in mitochondrial translocation of K-Ras in PI4KB inhibited cells. K-Ras is distinct from the other Ras isoforms, H-Ras and N-Ras, for its PM targeting signal. While H- and N-Ras bind the PM via the C-terminal prenyl and palmitoyl lipid moieties, K-Ras binds the PM through the C-terminal prenyl lipid moiety and PBD made of hexa-Lys residues. Also, the PBD electrostatically interacts with the PM PS, and depleting PM PS content dissociates K-Ras from the PM. Our data show that PI4KB inhibition translocates K-Ras, but not H -Ras, to mitochondria. Thus, we propose that K-Ras PBD is involved in translocation to mitochondria upon PI4KB inhibition. To test this, we examined the mitochondrial localization of four small GTPases containing PBD in PI4KB-inhibited cells: Rac1, RalA, Arl4a and Arl4c. Our data demonstrate that PI4KB inhibition promotes mitochondrial translocation of RalA, which has a closer sequence and structure to K-Ras compared to the other three small GTPase, even though K-Ras and RalA use different prenyl groups for the PM anchoring. Taken together, here we identified PI4KB as a possible therapeutic candidate for treating human pancreatic cancers harboring oncogenic mutant K-Ras. Moreover, different observations made for the effect of PI4KB inhibition on the localization of four small GTPases containing PBD indicate that PBD-mediated PM anchoring is not a deceptively simple electrostatic interaction, which only is based on positive charges provided by PBD. Although further studies are needed to explore the exact mechanism of this interaction, we propose that in addition to positive charges, primary sequences of PBD, and intramolecular interactions (non-covalent bonds) also play roles. Moreover, it is feasible to consider the possibility that the membrane localization of small GTPase is also regulated by their effector proteins, where they recruit to and/or stabilize the membrane localization of small GTPases.

Page Count

76

Department or Program

Department of Biochemistry and Molecular Biology

Year Degree Awarded

2022

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