Approximately 30 to 50% of colorectal cancers contain KRas mutations, which confer resistance to standard therapy and have therefore been termed undruggable. Since no curative treatments for KRas driven colon cancer are available, there is a critical need to develop toxicologically innocuous KRas therapeutic approaches that are free of safety problems intrinsic to drugs administered over long periods of time. High fidelity signaling of Ras is dependent on its spatial organization into defined nanoclusters on the plasma membrane. This is noteworthy, because select nonsteroidal anti-inflammatory drugs, through direct modulation of the biophysical properties of the plasma membrane, alter oncogenic Ras nanoclustering and attenuate signal transduction. These findings suggest that Ras nanoclusters represent a novel target for future interventions. Consistent with this rationale, we hypothesize that select amphiphilic membrane targeted dietary bioactives (MTDBs), e.g., polyunsaturated fatty acids (n-3 PUFA) docosahexaenoic acid (DHA, 22:64,7,10,13,16,19) and eicosapentaenoic acid (EPA, 20:55,8,11,14,17),  modulate the rigidity of the plasma membrane,  modify Ras nanocluster formation,  disrupt oncogenic Ras driven signaling (pERK), and  suppress phenotype (hyper-proliferation) in vitro and in vivo.Our studies utilized a variety of complementary models including mouse colonic crypts, Drosophila midguts, and isogenic human and mouse colonic cell lines. Using a membrane order sensitive dye, Di-4-ANEPPDHQ, we initially characterized the rigidity of the plasma membrane in mouse colonic crypts and young adult mouse colonocytes (YAMC) cells. Quantification of Ras nanocluster formation was determined by fluorescence lifetime imaging microscopy (FLIM) combined with fluorescence resonance energy transfer (FRET) of fluorescently tagged Ras membrane targeting domains. In addition, oncogenic Ras driven signaling (pERK) and phenotype (hyper-proliferation) was determined by targeting expression of RasV12 or KRasG12D to Drosophila midgut stem cells and mouse colon, respectively. In vitro and in vivo incorporation of n-3 PUFA consistently reduced plasma membrane rigidity and tH-Ras clustering, while increasing tK-Ras clustering and the mixing of tH and tK proteins. These changes in membrane spatiotemporal organization were associated with a reduction in oncogenic RasV12 driven ERK phosphorylation and intestinal stem cell proliferation.These novel findings demonstrate a unique role for MTDBs in the modulation of Ras nanoscale spatial organization and signaling. Establishing a role for MTDBs in Ras-dependent oncogenesis would have a major translational impact because these bioactives are safe, well tolerated, relatively inexpensive, and provide additional health benefits, such as reduction in mortality. This work was support by NIH grant R35CA197707.
Citation Format: Natividad R. Fuentes, Rola Barhoumi, Mohamed Mlih, Jason Karpac, Paul Hardin, Trevor Steele, Spencer Behmer, Ian Prior, Robert S. Chapkin. Plasma membrane lipid therapy: disruption of oncogenic Ras spatiotemporal organization by membrane-targeted dietary bioactives (MTDB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 179. doi:10.1158/1538-7445.AM2017-179