ACQUISITION OF METASTATIC PROPERTIES IN CARCINOMA CELLS: IDENTIFICATION OF A NOVEL PHOSPHORYLATION SITE IN CIP4 REGULATED BY THE PROTEIN COMPLEX AKAP350/PKA WITH A CENTRAL ROLE IN TUMOR INVASIVENESS

PARIANI, ALEJANDRO; FAVRE, CRISTIÁN; ALMADA, EVANGELINA; GIRARDINI, JAVIER; HIDALGO, FLORENCIA; BORINI CARLA; LAROCCA, M. CECILIA; TONUCCI, FACUNDO M.; FERRETTI, ANABELA; RICO, MARÍA JOSÉ; MENACHO MÁRQUEZ, MAURICIO

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Cancer malignancy is associated to cancer cells capacity to invade neighboring and distant tissues. Carcinomas are defined by their epithelial origin and constitute the most common type of cancer. Carcinoma metastasis requires epithelial derived tumor cells to acquire a mesenchymal phenotype (EMT). During EMT, cells lose contacts due to ablation of E-cadherin and acquire increased motility. CIP4 is a cdc42 effector essential for EMT, whose expression levels correlate with cell invasiveness in breast cancer. CIP4 structure and role in membrane plasticity has been characterized; however, how CIP4 function is regulated remains elusive. We have previously shown that CIP4 is a PKA substrate that interacts with AKAP350, and that this interaction is essential for CIP4 promotion of hepatocellular carcinoma (HCC) cell migration. The aim of this study was to evaluate if CIP4 pro-invasive ability is regulated via phosphorylation by PKA. Using in silico analysis and in vitro phosphorylation assays, we characterized that CIP4 has a unique PKA phosphorylation site (CIP4T225). Expression of its phosphomimetic mutant CIPT225E decreased E-cadherin levels (-51%*) and increased migratory efficiency (+44%*) in HCC cells. Conversely, expression of CIP4 not phosphorylatable mutant CIP4T225A inhibited HCC cell migration (-43%*). Pharmacological inhibition of PKA decreased HCC migratory efficiency in control (-40%*), but not in CIP4T225E or CIP4T225A cells. Transwell invasion assays showed that HCC invasiveness was increased in CIP4T225E (+800%*) and inhibited in CIP4T225A (-90%*) cells. In vivo studies confirmed the in vitro results, showing increased formation of metastatic nodules in lungs of athymic mice injected with CIP4T225E cells. These findings unveil a novel-signaling pathway involving CIP4T225 phosphorylation by PKA as a crucial event in the acquisition of metastatic properties in HCC cells. We are currently evaluating this regulatory pathway in breast cancer cells. *p