Abstract 2155: Inactivation of the tumor suppressor DLC1 by the oncogenes SRC and ERK1 in lung adenocarcinoma

The oncogenes SRC and ERK1 are frequently activated in a wide variety of human cancer, while the tumor suppressor DLC1, which encodes a Rho-GAP (GTPase activating protein) essential for its tumor suppressor functions, is frequently down-regulated. However, no prior research has mechanistically linked SRC and ERK1 to DLC1. In this study, we report that SRC and ERK1 cooperate to attenuate the Rho-GAP and tumor suppressor functions of DLC1 by a previously undescribed mechanism. We determined the direct phosphorylation of Y701 of DLC1, which lies in the Rho-GAP domain, by the SRC kinase reduces the binding of Rho-GTP (active Rho) to the Rho-GAP domain of DLC1 and abolishes its Rho-GAP activity. In untransformed and lung adenocarcinoma cell lines, endogenous active SRC and DLC1 co-localized at focal adhesions and formed a protein complex in vivo, implying this interaction is physiologically relevant. The phosphorylation of S129 of DLC1, which lies N-terminal to the Rho-GAP domain, by ERK1 enhanced the binding of the SRC SH3 domain to this region of DLC1 and increased the phosphorylation of Y701 by SRC. These changes reduced the Rho-GAP activity of DLC1, increased Rho-GTP in the cell, and attenuated the DLC1 tumor suppressor functions, as measured by cell migration rate, anchorage-independent growth, and tumor formation in nude mice. Consistent with these observations, mutation of Y701 to F701 increased the Rho-GAP and tumor suppressor activities of DLC1 and decreased Rho-GTP and Rho/ROCK/MRLC signaling. Conversely, mutation of Y701 to the phosphomimetic D701 produced a mutant DLC1 with the opposite phenotype, similar to a ‘GAP-dead’ DLC1 mutant. The Rho-GAP domain of DLC1 was necessary and sufficient for the attenuated Rho-GAP activity attributable to Y701 phosphorylation, as the isolated Rho-GAP domain (residues 609-878) with the Y701F and Y701D mutants displayed, respectively, high and low Rho-GAP activities, as in full-length DLC1. In considering the potential relevance of these findings to human tumors, it is important to recognize that while the Rho-GAP activity of DLC1 is necessary for its full tumor suppressor activity, it is not sufficient, as DLC1 binds several ligands that contribute to this function without attenuating its Rho-GAP activity. Thus, it would be predicted that there would be selective pressure for down-regulation of DLC1 expression even in the presence of high SRC activity. Consistent with this hypothesis, the combination high SRC expression and low DLC1 expression was associated with a poor prognosis in lung adenocarcinomas (p = 0.005) in the TCGA cohort. In summary, the cooperation between the SRC and ERK1 pathways contribute to phosphorylation of Y701, which directly inactivates the Rho-GAP function of DLC1 and attenuates its tumor suppressor activity. These findings are relevant to normal physiology and human cancer. Citation Format: Brajendra K. Tripathi, Xiaolan Qian, Tiera Grant, Philipp Mertins, Dunrui Wang, Alex G. Papageorge, Steven A. Carr, Douglas R. Lowy. Inactivation of the tumor suppressor DLC1 by the oncogenes SRC and ERK1 in lung adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2155. doi:10.1158/1538-7445.AM2015-2155