Up-regulation of Skp2 after Prostate Cancer Cell Adhesion to Basement Membranes Results in BRCA2 Degradation and Cell Proliferation

Arnaldo A. Arbini,Margherita Greco,Loredana Moro,Ersilia Marra

doi:10.1074/jbc.m604636200

Abstract

Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.

Highlights

With epithelial and endothelial cells, muscle fibers, and nerves
We provided evidence for a novel pathological mechanism whereby prostate carcinoma cell adhesion to collagen type I (COL1), a major extracellular matrix (ECM) protein at osseous metastatic sites, promotes cancer cell proliferation through depletion of BRCA2 protein, the product of a tumor suppressor gene whose inactivation accounts for an increased risk in cancer development [21,22,23]
Prostate Normal and Carcinoma Cells Adhere Differently to basement membranes (BM)—We investigated the adhesive properties of PNT1A and PC-3 cells to LN, PLN, and COL4, major components of BM, and to FN, a widely expressed extracellular matrix protein [25] enriched at the prostatic stroma but a minor component in BM

Summary

Introduction

With epithelial and endothelial cells, muscle fibers, and nerves. They consist mostly of collagen type IV (COL4) admixed with laminins (LN), nidogens, and the heparan sulfate proteoglycan perlecan (PLN) and may contain small amounts of fibronectin (FN) [1, 2]. BRCA2 Protein Depletion after Cancer Cell Adhesion to BM Increases DNA Synthesis—To investigate whether the signaling cascade initiated by BM proteins had any effect on cell proliferation, we measured DNA incorporation of [3H]thymidine in PNT1A and PC-3 cells onto various substrates (Fig. 3A).

Results

Conclusion