Gαs (GNAS) suppression of the p53 genomic‐stability checkpoint unleashes RAS‐driven oncogenesis

Abstract

As one of the most frequently mutated oncogenes, mutant RAS has been well established to drive proliferative cell programs that are exploited by cancer. Interestingly, RAS, like many oncogenes, has been found in benign tumors that never progress to cancer. Although seemingly counterintuitive, RAS mutant tumors require an additional mutational insult to progress to malignancy. Using an unbiased, pan‐cancer analysis from patient sequencing data, we discovered that RAS mutations significantly co‐occur with mutations in GNAS, the gene encoding the Gαs subunit of G protein couple receptors (GPCRs). Given the established tractability of GPCRs as drug targets, we aimed to explore GNAS as a potential co‐driver in RAS mutant cancers. To first validate the ability of mutations in RAS and GNAS to cooperate to induce malignancy, we expressed the constitutive active mutations, KRASG12D and GNASR201C in the skin of adult mice. These double mutant mice rapidly developed skin papillomas, while mice with either mutation alone had no phenotype. Furthermore, when GNASR201C mice were treated with the chemical carcinogen DMBA, known to induce Hras mutations, mice rapidly developed squamous cell carcinoma in only 3 weeks. Analysis of the lesions revealed a surprising lack of mutations in the Trp53 tumor suppressor gene, which controls genomic stability. In vitro experiments revealed that activation of signaling through Gαs, or its downstream kinase PKA, was sufficient to block DNA damage‐induced p53 protein accumulation. Analysis of the underlying mechanisms demonstrated that MDM2, the cognate p53 E3 ubiquitin ligase, is a direct substrate of PKA, which phosphorylates MDM2 on S166 resulting in enhanced ligase activity. In vitro ubiquitination assays revealed that activation of Gαs or PKA increased MDM2‐mediated p53 ubiquitination and degradation. In patients, appendix cancer is the best example of the GNAS‐KRAS co‐driver state, with 70% of patients harboring this co‐mutation. Ongoing experiments are aimed at validating the mechanism of Gαs‐PKA regulation of MDM2‐p53 growth checkpoints in relevant murine and human intestinal organoid models. In summary, we have uncovered a significant co‐occurrence of RAS and GNAS mutations in cancer and demonstrated that these mutations cooperate to initiate carcinogenesis in vivo. Mechanistically, the cooperation of RAS and GNAS can be explained by the ability of Gαs to functionally repress p53, through PKA mediated phosphorylation of MDM2, releasing the normal brakes that check aberrant RAS‐driven growth.Support or Funding InformationDana Steffen was supported by the National Science Foundation Graduate Research Fellowship Program (NSF GRFP) and the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences (T32 GM007752). This study is supported in part by the San Diego NCI Cancer Centers Council (C3) Collaborative Translational Cancer Research Pilot Grant.