Role of RALA and its Ser194 phosphorylation in tumorigenesis in RAS-independent and RAS- dependent cancers

Abstract

RALA (RAS Like Proto-Oncogene A) is a RAS family small GTPase that regulates vital cellular processes like cell spreading, migration and proliferation. Downstream of RAS, activation of RALA is critical for cancer cell tumorigenesis, mediated through RALGEFs. In recent years, the phosphorylation of RALA at S194 has emerged as a novel regulatory player, though its role in RALA activation and tumor progression remains ambiguous. To better understand this, we made stable CRISPR-Cas9-based RALA knockout (RALA KO) in RAS-dependent (MiaPaCa2, T24, UMUC3) and RAS-independent (MCF7, SKVO3) cancer cells. Detection of RALA S194 phosphorylation in these RALA KO cells when probed using the commercial anti-phospho-RALA antibody in western blot lacks specificity in RAS-dependent cancers. Despite the loss of RALA, the pS194RALA band stays detectable in these cell lines, making it difficult to evaluate its regulation or role. AURKA inhibitor MLN8237 delivered in a nanovesicle (VMLN) developed earlier in the lab specifically inhibits AURKA but did not affect the pS194RALA band detected in RAS-dependent cancers. RALA KO MiaPaCa2 (Ras-dependent) and MCF7 (Ras-independent) cells were stably reconstituted with WT-RALA, S194A-RALA, and S194D-RALA mutants and were used to test the role of this phosphorylation. They showed no difference in RALA activation status, detected by Sec5 RBD pulldown. Tumor growth disrupted by the loss of RALA was restored partly by WT-RALA, but not by S194A-RALA or S194D-RALA. These phosphomutants significantly affected tumor formation in both RAS-independent and RAS-dependent cancer cell lines. This supports a role for RALA S194 phosphorylation, independent of its activation in regulating RALA-dependent tumorigenesis, regulated by its localization or effector interaction. Finally, our studies also suggest the possible role the AURKA-RALGEF-RALA (AURKA-RGL1-RALA in the case of WTMEFs) pathway could have in AURKA-mediated regulation of RALA activation, independent of its S194 phosphorylation.