Abstract 2586: Identification of cancer-associated RhoA mutants and analogous K-Ras mutants that induce gain-of function despite low GTP binding
Authors: Qian Xiaolan , Papageorge Alex , Wang Dunrui , Tripathi Brajendra , Gao Yucheng , Durkin Marian , Sanchez-Solana Beatriz , Esposito Dominic , Waybright Timothy , Stephen Andrew , Lowy Douglas R. 
Published: September 2020

While the Ras GTPases are frequently mutated, especially at codons 12, 13, or 61, the structurally related Rho GTPases, although they are implicated in tumor invasion and metastasis, are rarely mutated in solid tumors, except for diffuse gastric cancer. The vast majority of cancer-associated RHO mutations are not located in codons 14, 15, or 63, which correspond to the 3 canonical mutant Ras codons. Here we have evaluated the biological and biochemical phenotypes of several cancer-associated RhoA mutants, which indicated that some RhoA mutants have unusual phenotypes and therefore prompted us to generalize our findings to other small GTPases by constructing and analyzing the analogous mutants in K-Ras.

We constructed 8 cancer-associated RhoA mutants from the TCGA database, and found that each was gain-of-function biologically. Using the Rhotekin binding assay to infer RhoGTP levels, 5 mutants (Y42C, L57V, D59G, A61D, D76G), had higher binding than WT RhoA. However, 3 mutants (G62E. L69P and L69R) did not bind Rhotekin, but did bind strongly to Citron, a Rho-dependent kinase implicated in cytokinesis and cell cycle regulation. Because the three mutants were highly transforming, we directly determined GTP/GDP binding levels, the “gold-standard” assay, for all mutants, which indicated G62E had high GTP-binding and low GDP-binding, in contrast to its negative binding to Rhotekin, while L69P and L69R had high GDP-binding and low GTP-binding similar to WT. Purified GDP-bound L69P and L69R formed a complex with Citron more efficiently than did WT GTP-bound or GDP-bound RhoA. Endogenous Citron and CDK1 contribute to anchorage-independent growth, as siRNA knockdown of either gene in lines transformed by G62E, L69R or L69P inhibited this phenotype.

The cancer-associated K-Ras mutants G60E and M67L (which correspond to RhoA G62E and L69R) and also K-Ras M67R displayed gain-of-function biologically, and the GTP/GDP-binding phenotype of each was analogous to the respective RhoA mutant: high GTP-binding for G60E, and low Raf-RBD binding and high GDP-binding for M67L and M67R. The low GTP K-Ras mutants bound more strongly to the RBD domain of Ras effectors other than Raf, resulting in activation of RAP and AKT in transfected cells. Our microarray data revealed that these GDP-bound mutant K-Ras or RhoA can up-regulate cell-cycle progression genes and down-regulate tumor suppressor genes compared to WT, which may account for their transforming phenotype.

We conclude that some cancer-associated RhoA mutants are transforming when GDP-bound, a phenotype that has not been described for any small GTPase. The equivalent K-Ras mutants display a similar phenotype, which implies the findings should be extrapolatable to many small GTPases. Such mutants can be strongly transforming, although they only bind a subset of preferred downstream effectors and activate their respective pathways. In addition, the widely used Rhotekin binding assay to infer RhoGTP levels may give misleading results for some mutants.

Citation Format: Xiaolan Qian, Alex Papageorge, Dunrui Wang, Brajendra Tripathi, Yucheng Gao, Marian Durkin, Beatriz Sanchez-Solana, Dominic Esposito, Timothy Waybright, Andrew Stephen, Douglas R. Lowy. Identification of cancer-associated RhoA mutants and analogous K-Ras mutants that induce gain-of function despite low GTP binding [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2586.