Targeting plasma membrane phosphatidylserine content to inhibit oncogenic KRAS function.

John F Hancock,Wei Chen,Ransome Van Der Hoeven,Walaa E Kattan,Tien Hung Lan,Xiaoping Ma,Dharini Van Der Hoeven

doi:10.26508/lsa.201900431

Abstract

The small GTPase KRAS, which is frequently mutated in human cancers, must be localized to the plasma membrane (PM) for biological activity. We recently showed that the KRAS C-terminal membrane anchor exhibits exquisite lipid-binding specificity for select species of phosphatidylserine (PtdSer). We, therefore, investigated whether reducing PM PtdSer content is sufficient to abrogate KRAS oncogenesis. Oxysterol-related binding proteins ORP5 and ORP8 exchange PtdSer synthesized in the ER for phosphatidyl-4-phosphate synthesized in the PM. We show that depletion of ORP5 or ORP8 reduced PM PtdSer levels, resulting in extensive mislocalization of KRAS from the PM. Concordantly, ORP5 or ORP8 depletion significantly reduced proliferation and anchorage-independent growth of multiple KRAS-dependent cancer cell lines, and attenuated KRAS signaling in vivo. Similarly, functionally inhibiting ORP5 and ORP8 by inhibiting PI4KIIIα-mediated synthesis of phosphatidyl-4-phosphate at the PM selectively inhibited the growth of KRAS-dependent cancer cell lines over normal cells. Inhibiting KRAS function through regulating PM lipid PtdSer content may represent a viable strategy for KRAS-driven cancers.

Highlights

RAS proteins are membrane-localized GTPases that regulate cell proliferation, differentiation, and apoptosis
We have shown previously that KRAS plasma membrane (PM) localization and nanoclustering are critically dependent on the PtdSer content of the inner PM leaflet (Zhou et al, 2014)
ORP5 and ORP8 are lipid exchangers involved in the transport of PtdSer from the ER to the PM (Fig 1A); we hypothesized that inhibition of either protein will deplete the PM of PtdSer and inhibit KRAS signaling through mislocalization of KRAS from the PM

Summary

Introduction

RAS proteins are membrane-localized GTPases that regulate cell proliferation, differentiation, and apoptosis. Two classes of proteins regulate RAS.GTP levels: guanine nucleotide exchange factors activate RAS by promoting exchange of GDP for GTP, and GTPase-activating proteins stimulate RAS GTPase activity to return RAS.GTP to the inactive ground state. This regulatory circuit is subverted in 15–20% of all human tumors that express oncogenic RAS with mutations at residues 12, 13, or 61 (Cox et al, 2014). HRAS, NRAS, KRAS4A, and KRAS4B (hereafter referred to as KRAS) are ubiquitously expressed in mammalian cells These RAS isoforms have a near identical G-domain that binds guanine nucleotides and interacts with effector proteins, GTPase activating proteins, and guanine nucleotide exchange factors but have different C termini and membrane anchors. The major clinical problem is KRAS, which is mutated in >90% of pancreatic cancers, ~50% of colon cancers, and ~25% of non–small cell lung cancer (Prior et al, 2012)

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