Abstract 4036: Palmitoylated Ras-driven MAPK signaling, transformation and tumorigenesis, but not tumor progression, are spatially regulated by plasma membrane microdomains

Abstract

Abstract In this study, we assessed the contributions of plasma membrane (PM) microdomain targeting to the functions of H-Ras and paralogue R-Ras. Activated H-Ras is a mitogen which can drive tumorigenesis through Raf activation, while R-Ras is weakly mitogenic and does not induce the MAPK pathway in cells, despite binding Raf in vitro. These paralogues have identical effector-binding regions, but variant C-terminal targeting domains (tDs). Ras tDs are responsible for lateral distribution within PM microdomains as a result of distinct lipid modifications: activated H-Ras targets to lipid ordered/disordered (Lo/Ld) domain borders, whereas active R-Ras is sequestered in Lo domains (rafts). We hypothesize that PM segregation regulates effector interactions and downstream signaling. We used tD swap mutants, and assessed effects on MAPK pathway activation, cell proliferation, transformation, and tumorigenesis. Activated R-Ras harboring the H-Ras tD (R-Ras-tH), stably expressed in NIH3T3 fibroblasts, interacted with Raf and induced MEK and ERK phosphorylation. Furthermore, R-Ras-tH stimulated cell growth, and supported anchorage-independent growth in soft agar, indicating induction of a transformed phenotype in vitro. Conversely, stable transfectants of H-Ras harboring the R-Ras tD (H-Ras-tR) had reduced MEK and ERK phosphorylation, cell growth, and colony formation in soft agar compared with H-Ras cells. Thus, Ras access to Raf at the PM is a critical step for Ras mitogenic signaling. However, tumor progression driven by these cells in xenografts in mice did not match in vitro phenotypes. R-Ras-expressing cells produced few solid tumors, while R-Ras-tH cells produced relatively larger, but size-limited, avascular tumors with a higher frequency per xenograft. Surprisingly, both H-Ras and H-Ras-tR xenografts produced large and heavily vascularized tumors, implicating an important role of Ras in modulating tumor angiogenesis in this model. Indeed, the conditioned media (secretome) of R-Ras-expressing cells did not support endothelial cell cord formation in growth factor-reduced Matrigel, while the secretome from H-Ras cells robustly induced cord formation; these effects were independent of Ras microdomain localization. Thus, targeting Ras to the Lo/Ld border is sufficient to initiate tumor formation, however, a tD-independent angiogenic effect is required for progression of Ras-induced tumors. Citation Format: James Michael, Jeremy G.T. Wurtzel, Lawrence E. Goldfinger. Palmitoylated Ras-driven MAPK signaling, transformation and tumorigenesis, but not tumor progression, are spatially regulated by plasma membrane microdomains. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4036. doi:10.1158/1538-7445.AM2015-4036