An oxanthroquinone derivative that disrupts RAS plasma membrane localization inhibits cancer cell growth

Abstract

Oncogenic RAS proteins are commonly expressed in human cancer. To be functional, RAS proteins must undergo post-translational modification and localize to the plasma membrane (PM). Therefore, compounds that prevent RAS PM targeting have potential as putative RAS inhibitors. Here we examine the mechanism of action of oxanthroquinone G01 (G01), a recently described inhibitor of KRAS PM localization. We show that G01 mislocalizes HRAS and KRAS from the PM with similar potency and disrupts the spatial organization of RAS proteins remaining on the PM. G01 also inhibited recycling of epidermal growth factor receptor and transferrin receptor, but did not impair internalization of cholera toxin, indicating suppression of recycling endosome function. In searching for the mechanism of impaired endosomal recycling we observed that G01 also enhanced cellular sphingomyelin (SM) and ceramide levels and disrupted the localization of several lipid and cholesterol reporters, suggesting that the G01 molecular target may involve SM metabolism. Indeed, G01 exhibited potent synergy with other compounds that target SM metabolism in KRAS localization assays. Furthermore, G01 significantly abrogated RAS-RAF-MAPK signaling in Madin-Darby canine kidney (MDCK) cells expressing constitutively activated, oncogenic mutant RASG12V. G01 also inhibited the proliferation of RAS-less mouse embryo fibroblasts expressing oncogenic mutant KRASG12V or KRASG12D but not RAS-less mouse embryo fibroblasts expressing oncogenic mutant BRAFV600E. Consistent with these effects, G01 selectively inhibited the proliferation of KRAS-transformed pancreatic, colon, and endometrial cancer cells. Taken together, these results suggest that G01 should undergo further evaluation as a potential anti-RAS therapeutic.

Acylpeptide hydrolase is a novel regulator of KRAS plasma membrane localization and function

“…2D) reflects KRAS localization to endomembrane and not the cytosol, as occurs when posttranslational processing is abrogated (Hancock et al, 1990). G01 also causes the mislocalization other RAS isoforms (Tan et al, 2018). Consistent with this, we observed a significant mislocalization of HRASG12V and NRASG12V from the PM in APEH-knockdown cells (Fig.…”

“…Normal functioning of the RE is required to maintain KRAS on the PM (Misaki et al, 2010;Schmick et al, 2014). We reported previously that G01 treatment disrupts the endocytic recycling of the EGFR and transferrin receptor (TfR) (Ghosh and Maxfield, 1995;Rodman and Wandinger-Ness, 2000;Roepstorff et al, 2009;Tan et al, 2018). Therefore, we investigated whether knockdown of APEH had a similar effect.…”

“…5A). This is a useful cell system because CHO cells do not express any endogenous EGFR and were shown previously to be highly sensitive to G01 treatment (Tan et al, 2018). Cells were serum starved, incubated with EGF on ice, and then imaged in a confocal microscope at intervals following warming to 37°C.…”

“…Multiple classes of inhibitors of sphingomyelin (SM) metabolism reduce PM PtdSer content and therefore mislocalize KRAS from the PM (Cho et al, 2012(Cho et al, , 2016Maekawa et al, 2016;van der Hoeven et al, 2018). We recently reported that the oxanthroquinone G01, a representative derivative of a novel class of Streptomyces polyketide, oxanthroquinones, caused the mislocalization KRAS by a mechanism that involved perturbation of both RE function and SM metabolism (Tan et al, 2018). In this current study, we report the identification of a molecular target of G01 and reveal a novel biological function of this protein in regulating KRAS PM localization.…”

Novel Targets in Pancreatic Cancer Therapy - Current Status and Ongoing Translational Efforts

“…Therefore, pharmacologically inhibiting oncogenic KRAS signaling has been suggested for a long time and is still considered as the Holy Grail in the quest for targeted therapeutic approaches directed against pancreatic cancer by many authors [29]. Nevertheless, developing direct inhibitors of oncogenic KRAS signaling has been found to be extremely challenging and, as of to date, no suitable inhibitors are available for routine application outside of early clinical and preclinical studies [30,31,32,33,34,35,36]. Moreover, accumulating preclinical evidence already suggests that, although being a potentially powerful therapeutic approach, pharmacological inhibition of oncogenic KRAS signaling alone will likely not be sufficient to fully eradicate and cure metastatic pancreatic cancer [27,29].…”