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

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 ␤ 1 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 downregulation 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. Basement membranes (BM)2 are thin layers of specialized extracellular matrix (ECM) that surround and closely associate 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). Although the BM structural role in defining tissue architecture and compartmentalization has long been recognized, its dynamic role in the modulation of cell behavior has only recently been documented (1).Aberrant cancer cell interactions with BM proteins play a crucial role in the biology of metastasis (3). Cancer cells must be able to coordinately decrease cell-cell interactions and increase cell adhesion to an adjacent BM in order to become motile, which along with the capacity of degrading/remodeling a BM directly relates to their metastatic potential (4). This cell behavior is accompanied by changes in the expression and/or usage of various adhesion receptors, including integrins (5). Integrins are transmembrane adhesion receptors for ECM proteins that not only provide physical anchoring cell support but also play a pivotal role in triggering intracellular signaling in response to environmental changes through interactions with molecules ...

Mitochondrial DNA depletion in prostate epithelial cells promotes anoikis resistance and invasion through activation of PI3K/Akt2

“…We chose the immortalized human prostate epithelial cell line PNT1A as a model to explore the role of mtDNA mutations in prostate malignant progression. PNT1A are non-tumorigenic cells 12 that exhibit molecular and biochemical properties close to normal prostate epithelium, [13][14][15][16] albeit they may be considered preneoplastic as they have suffered an initial genetic hit (expression of SV40T for immortalization) that renders them susceptible to progression towards malignant transformation. Long-term cell exposure to low concentration of EtBr selectively reduces the mtDNA content in numerous cell types, and this effect is reversible after EtBr removal from the culture medium.…”

“…Antibodies to phospho-Akt Ser 473, murine double minute 2 (Mdm2), matrix metalloproteinase-9 (MMP-9) and b-tubulin have been described earlier. 15,16,30 Polyclonal antibody to p85a and monoclonal antibody to c-Myc (9E10) were from Sigma. Monoclonal antibody to p53 (Ab-6) was purchased from Calbiochem (San Diego, CA, USA).…”

“…Total protein extracts were analyzed by immunoblotting as described previously. 15,16 Monoclonal antibody to Cox I was purchased from Molecular Probes (Carlsbad, CA, USA). Antibodies to phospho-Akt Ser 473, murine double minute 2 (Mdm2), matrix metalloproteinase-9 (MMP-9) and b-tubulin have been described earlier.…”

“…Transient transfections with DNp85, a dominant negative form of PI3K, 15,16 or empty vector (pcDNA3; Invitrogen) were performed as described earlier. 15 Akt1, Akt2 and Akt3 siRNAs were purchased from Santa Cruz Biotechnologies and used for transient transfections according to the manufacturer's instructions.…”

Skp2 Overexpression Is Associated with Loss of BRCA2 Protein in Human Prostate Cancer

“…6 -8 In addition to cancer predisposition, recent clinical evidence suggests that patients with PCa who carry germline BRCA2 mutations display a more aggressive phenotype with worse survival rates than noncarrier patients 9 -14 ; we have provided mechanistic evidence demonstrating that loss of BRCA2 pro-motes PCa cell proliferation and invasion in experimental cell-line models. [15][16][17][18] These latter findings are important despite the fact that mutations in BRCA2 are rare in sporadic PCas 2,9,19,20 ; in sporadic prostate tumors, nonmutational functional inactivation of BRCA2 may occur through different mechanisms, including down-regulation of its expression. [15][16][17][18]21 Indeed, there is preliminary evidence indicating that BRCA2 protein is significantly reduced in most sporadic PCas.…”