Abstract 530: PDK1 regulates cell migration and 3D invasion of breast tumor cells by a kinase independent mechanism

Abstract

Abstract 3-Phosphoinositide Dependent Kinase 1 (PDK1) is the kinase that phosphorylates the activation loop of several protein kinases such as Akt, PKC, RSK or SGK. In melanoma, breast and prostate carcinomas PDK1 was found amplified and overexpressed and this event correlates with a more aggressive phenotype. The purpose of our study is to unveil the mechanism by which PDK1 regulates cell migration and invasion with the aim to rationally target it in order to prevent tumor cell dissemination. Combining PDK1 stable silencing and PDK1 stable overexpression, we studied PDK1 role in different assays of breast tumor cells migration and invasion. Our data showed that PDK1 silencing in T47D, MDAMB231, MCF10A and MCF10DCIS breast tumor cells determined a potent reduction of chemotactic ability towards different chemoattractants, including serum, Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF). Complementary, PDK1 exogenous overexpression, a condition that mimics PDK1 amplification and overexpression in tumors, significantly increased directional migration of MCF10A and MCF10DCIS cells. Surprisingly, the overexpression of a kinase dead mutant of PDK1 was equally able to increase cell migration, suggesting a kinase independent mechanism. We further confirmed this result by treating cells with an ATP competitive small molecule which inhibits PDK1 kinase activity but failed to impair cell migration. Moreover, we found that the kinase dead PDK1 overexpression was able to potently increase the ability of MCF10DCIS cells to invade through a basement membrane layer and to induce the formation of 3D invasive spheroids when cultured embedded in basement membrane matrix. We found that the molecular mechanism underlying the PDK1-mediated regulation of cell migration and invasion is the activation of Myotonic dystrophy kinase-related CDC42-binding kinase alpha (MRCKα). This kinase protein is an important regulator of myosin contraction and cytoskeletal dynamics. According to our data, PDK1 is able to increase the kinase activity of MRCKα. Moreover, we confirmed the MRCKα role by silencing it, resulting in a complete abolition of the effects of PDK1 overexpression. The widespread role of PDK1 in the regulation of tumor cells migration and invasion together with its overexpression status in different tumors, makes it an appealing target for precision medicine. However the simple inhibition of PDK1 kinase activity by ATP competitors couldn't be effective on invasive properties of tumor cells. Future strategies to target PDK1 should take in account these findings by developing allosteric PDK1 inhibitors able to block its kinase independent functions. Citation Format: Paolo Armando Gagliardi, Laura di Blasio, Desiana Somale, Alberto Puliafito, Giulia Chiaverina, Federico Bussolino, Luca Primo. PDK1 regulates cell migration and 3D invasion of breast tumor cells by a kinase independent mechanism. [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 530. doi:10.1158/1538-7445.AM2015-530