A new common mechanism for increasing RhoA-GTP is identified in Tripathi et al. The increased RhoA-GTP results from signaling mechanisms that phosphorylate and attenuate the DLC1 tumor suppressor, which encodes RhoGAP. The potentially reversible nature of this attenuation may have therapeutic relevance in cancer.

Many human cancers have high AKT activity and constitutive up-regulation of Rho-GTP, which is a major factor in the neoplastic process, but AKT is not known to regulate Rho-GTP. In this study, we show that AKT positively regulates Rho-GTP by directly phosphorylating tumor suppressor DLC1 and attenuating the Rho-GAP activity of DLC1, which catalyzes the conversion of active Rho-GTP to inactive Rho-GDP. Interestingly, AKT was found to increase Rho-GTP and its downstream activities in DLC1-positive cancer lines whether the DLC1 was endogenous or transfected, but not in DLC1-negative lines. Similarly, when epithelial cell lines were stimulated with EGF, they activated AKT, which increased Rho-GTP in a DLC1-dependent manner. Three Serines (S298, S329, S567) in DLC1 have AKT consensus motifs and were phosphorylated by AKT in vitro and in vivo. Their phosphorylation attenuated the Rho-GAP and tumor suppressor activities of DLC1 - including decreased cell migration, focal adhesion turnover, anchorage-independent growth, and tumor growth in mice - as did the combined phosphomimetic mutant DLC1-3D. By contrast, the combined Serine to Alanine mutant DLC1-3A was even more active than wild type DLC1 (DLC1-WT). Remarkably, an AKT inhibitor stimulated the tumor suppressor and Rho-GAP activities of DLC1-WT, but did not influence DLC1-3A or DLC1-3D. The N-terminal half of DLC1 (amino acids 1-600) bound the DLC1 Rho-GAP domain (amino acids 609-878), as determined by in vivo complex formation and by microscale thermophoresis, which measures protein interactions in close-to-native conditions. There was increased binding and decreased Rho-GAP activity when the three Serines in the N-terminus were phosphorylated or carried the 3D mutations. Conversely, there was decreased binding and increased Rho-GAP activity when the Serines were not phosphorylated or carried the 3A mutations. In a xenograft mouse model, AKT inhibitor treatment of DLC1-positive palpable tumors reduced their size, their Rho-GTP level, and their Rho-dependent signaling, but did not affect these three parameters in isogenic DLC1-negative tumors. Similarly, in the MMTV-PyMT breast cancer model, which had high AKT activity, high Rho-GTP, and expressed DLC1, an AKT inhibitor reduced the size of palpable tumors, and reduced Rho-GTP and its downstream signaling. AKT inhibition reduced DLC1 phosphorylation in both tumor models. We conclude: AKT can increase Rho-GTP by phosphorylating three N-terminal Serines in DLC1, which attenuates its Rho-GAP and tumor suppressor functions; the N-terminus of DLC1 is an auto-inhibitory domain that reversibly binds the Rho-GAP domain; AKT attenuates DLC1 functions by phosphorylating the Serines in the N-terminus, which increases its binding the Rho-GAP domain; and AKT inhibition has greater anti-tumor activity in DLC1-positive tumors than in DLC1-negative tumors. Citation Format: Brajendra K. Tripathi, Tiera Grant, Philipp Mertins, Xiaolan Qian, Dunrui Wang, Alex G. Papageorge, Steven A. Carr, Douglas R. Lowy. AKT positively regulates Rho-GTP by attenuating the GAP activity of the DLC1 tumor suppressor: a mechanistic study with translational implications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4377.

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