Constitutive activation of MAPK/ERK inhibits prostate cancer cell proliferation through upregulation of BRCA2

Abstract

Abstract. BRCA2 is central to an utterly diverse biological behavior elicited after integrin-mediated normal and prostate cancer cell adhesion to basement membrane (BM) and extracellular matrix (ECM) proteins. Unlike normal cells, adhesive stimuli in cancer cells activate PI 3-kinase/AKT signaling resulting in BRCA2 degradation and unchecked cancer cell proliferation and metastasis. However, the precise mechanisms involved in normal BRCA2 homeostasis are unknown. We investigated ERK and AKT phosphorylation in normal (PNT1A) and cancer (PC-3) prostate cells after adhesion to ECM and the effects upon BRCA2 and cell proliferation. PNT1A cell adhesion to ECM triggered MAPK/ERK signaling resulting in upregulation of BRCA2 mRNA and protein, with negligible effects upon cell proliferation. Disruption of MAPK/ERK with PD98059 prevented any BRCA2 upregulation inhibiting DNA synthesis below basal levels. PC-3 cells exhibited a defective MAPK/ERK pathway that was unresponsive to adhesion to the ECM, which instead triggered PI 3-kinase/AKT signaling leading to BRCA2 protein depletion and cell proliferation. Reconstitution of MAPK/ERK by recombinant expression of a constitutively active form of MAPK kinase 1 (MEK1) effectively reversed the neoplastic phenotype by increasing BRCA2 expression and preventing any aberrant cell proliferation at rest and upon interaction with ECM proteins. Our results suggest that aberrant loss of MAPK/ERK activity in prostate cancer may play a pivotal role in the malignant phenotype, and provide evidence that interventions aimed at bypassing the signaling block are able to effectively reverse neoplastic unchecked cell proliferation. IntroductionIn adult human prostate, the relationship between cell proliferation and cell death is a precise balancing act that prevents organ involution or overgrowth. Upon neoplastic transformation this equilibrium is ultimately deranged leading to abnormal organ growth and tumor formation (1). Both processes are exquisitely regulated by a continuous cross-talk between cells and the surrounding microenvironment. Chemical stimuli such as growth factors or physical interaction with BM or ECM proteins activate diverse cell signaling pathways that exert control over cell proliferation and survival (2-5). The MAPK/ERK (mitogen-activated protein kinase/ extracellularregulated kinase) and PI 3-kinase (phosphatidyl inositol 3-kinase)/AKT signaling pathways are two major transducers of extracellular stimuli through modulation of the expression of genes exerting stringent control upon cell survival, motility, and cell proliferation (6,7). ERK accumulation in the nucleus regulates transcription factors involved in DNA synthesis (8), cell differentiation (9,10), and promotion of acinar formation in the normal prostate (11). Similarly, the PI 3-kinase/AKT signaling pathway regulates many cellular processes that are critically involved in cell proliferation and survival (12). The role of these pathways in oncogenesis has been extensively investigated and altered expression/activi...

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

“…By using PCa cell lines, a recent study 16 demonstrated that the Skp1-cullin-F-box ubiquitin ligase Skp2 interacts with and promotes ubiquitination of BRCA2, targeting it for degradation in the proteasome; in addition, the loss of BRCA2 promotes PCa cell proliferation and invasion. [15][16][17][18] However, although experimental models based on cultured cell lines are amenable for the assessment of signaling cascades and molecules that are selectively deregulated in cancer, in vivo validation remains a stepping stone in translating relevant scientific findings to the patients' bedside. In the current study, we investigated whether the expression of Skp2 correlates with BRCA2 protein levels in prostate epithelial neoplasms in vivo; we also assessed the functional role of Skp2 overexpression and loss of BRCA2 protein in the early stages of prostate neoplastic transformation.…”

“…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.…”

“…[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. 18 Researchers have previously reported that BRCA2 protein levels are regulated by ubiquitin-mediated proteolysis 16,22 in a process that requires its association with the E3 ubiquitin ligase Skp2, 16 an oncogenic protein that is upregulated in prostatic intraepithelial neoplasia (PIN) and PCa.…”

“…Antibodies to Skp2, poly(ADP-ribose) polymerase (PARP), and ␤-tubulin were previously described. 15,17,29 Antibody to p85 was from Sigma (St Louis, MO).…”

Important Clues of Current Phosphoproteomic Approaches for Clinical Research

“…Proteins in the MAPK signalling pathways are activated by both RTKs and TNFα, which allows cells to integrate multiple signals. [8][9][10][11][12][13][14][15][16][17][18][19][20]. ) by converting acidic peptides to methyl esters but increasing the spectra complexity and requiring lyophilization of the sample, which causes adsorptive losses of especially phosphopeptides [28].…”