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.

TONUCCI, FACUNDO M.; ALMADA E; PARIANI, ALEJANDRO; FERRETI AC; HIDALGO F; FAVRE C; RICO MJ; BORINI ETICHETTI C; GIRARDINI J; MENACHO MARQUEZ M; LAROCCA MC

Buenos Aires

Congreso; Reunion cientifica anual conjunta de Sociedades de Biociencias; 2017

Sociedades de biociencias

Cancer malignancy is associated to cancer cells capacityto invade neighboring and distant tissues. Carcinomasare defined by their epithelial origin and constitute themost common type of cancer. Carcinoma metastasis requiresepithelial derived tumor cells to acquire a mesenchymalphenotype (EMT). During EMT, cells losecontacts due to ablation of E-cadherin and acquire increasedmotility. CIP4 is a cdc42 effector essential forEMT, whose expression levels correlate with cell invasivenessin breast cancer. CIP4 structure and role inmembrane plasticity has been characterized; however,how CIP4 function is regulated remains elusive. We havepreviously shown that CIP4 is a PKA substrate that interactswith AKAP350, and that this interaction is essentialfor CIP4 promotion of hepatocellular carcinoma (HCC) cell migration. The aim of this study was to evaluate ifCIP4 pro-invasive ability is regulated via phosphorylationby PKA. Using in silico analysis and in vitro phosphorylationassays, we characterized that CIP4 has aunique PKA phosphorylation site (CIP4T225). Expressionof its phosphomimetic mutant CIPT225E decreasedE-cadherin levels (-51%*) and increased migratory efficiency(+44%*) in HCC cells. Conversely, expression ofCIP4 not phosphorylatable mutant CIP4T225A inhibitedHCC cell migration (-43%*). Pharmacological inhibitionof PKA decreased HCC migratory efficiency in control(-40%*), but not in CIP4T225E or CIP4T225A cells. Transwellinvasion assays showed that HCC invasivenesswas increased in CIP4T225E (+800%*) and inhibitedin CIP4T225A (-90%*) cells. In vivo studies confirmedthe in vitro results, showing increased formation of metastaticnodules in lungs of athymic mice injected withCIP4T225E cells. These findings unveil a novel-signalingpathway involving CIP4T225 phosphorylation by PKA asa crucial event in the acquisition of metastatic propertiesin HCC cells. We are currently evaluating this regulatorypathway in breast cancer cells. *p