Timp1 Promotes Cell Survival by Activating the PDK1 Signaling Pathway in Melanoma.

Mariana Toricelli,Aline Hunger,Miriam Jasiulionis,Fabiana Melo,Daniela Zanatta,Bryan Strauss

doi:10.3390/cancers9040037

Abstract

High TIMP1 expression is associated with poor prognosis in melanoma, where it can bind to CD63 and β1 integrin, inducing PI3-kinase pathway and cell survival. Phosphatidylinositol (3,4,5)-trisphosphate (PIP3), generated under phosphatidylinositol-3-kinase (PI3K) activation, enables the recruitment and activation of protein kinase B (PKB/AKT) and phosphoinositide-dependent kinase 1 (PDK1) at the membrane, resulting in the phosphorylation of a host of other proteins. Using a melanoma progression model, we evaluated the impact of Timp1 and AKT silencing, as well as PI3K, PDK1, and protein kinase C (PKC) inhibitors on aggressiveness characteristics. Timp1 downregulation resulted in decreased anoikis resistance, clonogenicity, dacarbazine resistance, and in vivo tumor growth and lung colonization. In metastatic cells, pAKTThr308 is highly expressed, contributing to anoikis resistance. We showed that PDK1Ser241 and PKCβIISer660 are activated by Timp1 in different stages of melanoma progression, contributing to colony formation and anoikis resistance. Moreover, simultaneous inhibition of Timp1 and AKT in metastatic cells resulted in more effective anoikis inhibition. Our findings demonstrate that Timp1 promotes cell survival with the participation of PDK1 and PKC in melanoma. In addition, Timp1 and AKT act synergistically to confer anoikis resistance in advanced tumor stages. This study brings new insights about the mechanisms by which Timp1 promotes cell survival in melanoma, and points to novel perspectives for therapeutic approaches.

Highlights

Cutaneous melanoma is a melanocytic tumor whose incidence and mortality are on the rise worldwide
We demonstrated a progressive increase in Timp1 expression along the melanoma progression [9]
Previous data from our laboratory showed increased Timp1 expression along melanoma progression and more important the assembly of a supramolecular complex containing Timp1, CD63, and β1-integrins associated with a more aggressive phenotype [9,11]

Summary

Introduction

Cutaneous melanoma is a melanocytic tumor whose incidence and mortality are on the rise worldwide. NRAS, BRAF, MEK1/2, and ERK1/2, is one of most-studied signaling pathways in melanoma This path is considered the main route changed in melanoma and is involved in cell survival, proliferation, and migration, and is related to both development and melanoma progression [2]. Du and coworkers revealed that mammary-specific ablation of PDK1 could delay tumor initiation, progression, and metastasis in a spontaneous mouse tumor model [7] They demonstrated that inducible deletion of PDK1 could noticeably shrink the growing breast tumors. We reported, for the first time, the assembly of a supramolecular complex containing Timp, CD63, and β1-integrins at the cell surface in melanoma cells, and its involvement in the acquisition of an anoikis-resistant phenotype through the PI3K signaling pathway [11]. We observed that the simultaneous deletion of Timp and AKT prevents more efficiently cell survival, providing a possible new therapeutic strategy for metastatic melanoma

Results

Timp1 Modulates the PKC Activation via PDK1 in the Early Stages of Melanoma

Materials and Methods