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

Lingxiao Tan,Kwang-Jin Cho,Pratik Neupane,Robert J Capon,John F Hancock

doi:10.1074/jbc.ra118.003907

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.

Highlights

Oncogenic RAS proteins are commonly expressed in human cancer
We reported previously the synthesis of a series of oxanthroquinone derivatives based on the original microbial polyketide structures identified in a high content screen for inhibitors of KRAS plasma membrane (PM) binding [25]
The results show that G01 mislocalizes oncogenic KRAS, HRAS, and KRAS4A from the PM with approximately equal potencies, the dose-response curves yielding IC50 values of ϳ1 ␮M (Fig. 1, B and C)

Summary

Results

We reported previously the synthesis of a series of oxanthroquinone derivatives based on the original microbial polyketide structures identified in a high content screen for inhibitors of KRAS PM binding [25]. In control cells Tf-555 was rapidly internalized and substantially returned to the PM by 60 min; as shown in the images in Fig. 6B that were quantified using Manders coefficients to evaluate the extent of co-localization between mGFP-LactC2 and Tf-555. Given that G01 synergizes with compounds that perturb SM metabolism for KRASG12V mislocalization, we tested RAS-RAFMAPK signaling in MDCK cells expressing KRASG12V treated with synergistic drug combinations. 5 ␮M G01 treatment for 72 h significantly inhibited proliferation of KRASG12V and KRASG12D RAS-less MEFs, but not BRAFV600E RAS-less MEFs (Fig. 9A) This result suggests that G01 inhibits cell growth at the level of KRAS but not RAF, consistent with an effect on KRAS PM binding. G01 inhibited the proliferation lung tumor cell lines, but without evidence of increased potency in lines expressing oncogenic mutant KRAS rather than WT KRAS (Fig. 9B)

Discussion

HRASG12V ppERK

Experimental procedures