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Proteomic analysis of zoledronic-acid resistant prostate cancer cells unveils novel pathways characterizing an invasive phenotype.

ABSTRACT: Proteomic analysis identified differentially expressed proteins between zoledronic acid-resistant and aggressive DU145R80 prostate cancer (PCa) cells and their parental DU145 cells. Ingenuity Pathway Analysis (IPA) showed a strong relationship between the identified proteins within a network associated with cancer and with homogeneous cellular functions prevalently related with regulation of cell organization, movement and consistent with the smaller and reduced cell-cell contact morphology of DU145R80 cells. The identified proteins correlated in publically available human PCa genomic data with increased tumor expression and aggressiveness. DU145R80 exhibit also a clear increase of alpha-v-(?v) integrin, and of urokinase receptor (uPAR), both included within the same network of the identified proteins. Interestingly, the actin-rich structures localized at the cell periphery of DU145R80 cells are rich of Filamin A, one of the identified proteins and uPAR which, in turn, co-localizes with ?v-integrin, in podosomes and/or invadopodia. Notably, the invasive feature of DU145R80 may be prevented by blocking anti-?v antibody. Overall, we unveil a signaling network that physically links the interior of the nucleus via the cytoskeleton to the extracellular matrix and that could dictate PCa aggressiveness suggesting novel potential prognostic markers and therapeutic targets for PCa patients.

SUBMITTER: Milone MR

PROVIDER: S-EPMC4467152 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Proteomic analysis of zoledronic-acid resistant prostate cancer cells unveils novel pathways characterizing an invasive phenotype.

Milone Maria Rita MR Pucci Biagio B Bifulco Katia K Iannelli Federica F Lombardi Rita R Ciardiello Chiara C Bruzzese Francesca F Carriero Maria Vincenza MV Budillon Alfredo A

Oncotarget 20150301 7

Proteomic analysis identified differentially expressed proteins between zoledronic acid-resistant and aggressive DU145R80 prostate cancer (PCa) cells and their parental DU145 cells. Ingenuity Pathway Analysis (IPA) showed a strong relationship between the identified proteins within a network associated with cancer and with homogeneous cellular functions prevalently related with regulation of cell organization, movement and consistent with the smaller and reduced cell-cell contact morphology of D ...[more]

PMID: 25481874

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Project description:BackgroundBone metastases are a major burden in men with advanced prostate cancer. We compared denosumab, a human monoclonal antibody against RANKL, with zoledronic acid for prevention of skeletal-related events in men with bone metastases from castration-resistant prostate cancer.MethodsIn this phase 3 study, men with castration-resistant prostate cancer and no previous exposure to intravenous bisphosphonate were enrolled from 342 centres in 39 countries. An interactive voice response system was used to assign patients (1:1 ratio), according to a computer-generated randomisation sequence, to receive 120 mg subcutaneous denosumab plus intravenous placebo, or 4 mg intravenous zoledronic acid plus subcutaneous placebo, every 4 weeks until the primary analysis cutoff date. Randomisation was stratified by previous skeletal-related event, prostate-specific antigen concentration, and chemotherapy for prostate cancer within 6 weeks before randomisation. Supplemental calcium and vitamin D were strongly recommended. Patients, study staff, and investigators were masked to treatment assignment. The primary endpoint was time to first on-study skeletal-related event (pathological fracture, radiation therapy, surgery to bone, or spinal cord compression), and was assessed for non-inferiority. The same outcome was further assessed for superiority as a secondary endpoint. Efficacy analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00321620, and has been completed.Findings1904 patients were randomised, of whom 950 assigned to denosumab and 951 assigned to receive zoledronic acid were eligible for the efficacy analysis. Median duration on study at primary analysis cutoff date was 12·2 months (IQR 5·9-18·5) for patients on denosumab and 11·2 months (IQR 5·6-17·4) for those on zoledronic acid. Median time to first on-study skeletal-related event was 20·7 months (95% CI 18·8-24·9) with denosumab compared with 17·1 months (15·0-19·4) with zoledronic acid (hazard ratio 0·82, 95% CI 0·71-0·95; p = 0·0002 for non-inferiority; p = 0·008 for superiority). Adverse events were recorded in 916 patients (97%) on denosumab and 918 patients (97%) on zoledronic acid, and serious adverse events were recorded in 594 patients (63%) on denosumab and 568 patients (60%) on zoledronic acid. More events of hypocalcaemia occurred in the denosumab group (121 [13%]) than in the zoledronic acid group (55 [6%]; p<0·0001). Osteonecrosis of the jaw occurred infrequently (22 [2%] vs 12 [1%]; p = 0·09).InterpretationDenosumab was better than zoledronic acid for prevention of skeletal-related events, and potentially represents a novel treatment option in men with bone metastases from castration-resistant prostate cancer.FundingAmgen.

Project description:The nitrogen-containing bisphosphonates (N-BP) zoledronic acid (ZOL) inhibits osteoclast-mediated bone resorption, and it is used to prevent skeletal complications from bone metastases. ZOL has also demonstrated anticancer activities in preclinical models and, recently, in cancer patients, highlighting the interest in determining eventual mechanisms of resistance against this agent. In our study, we selected and characterised a resistant subline of prostate cancer (PCa) cells to better understand the mechanisms, by which tumour cells can escape the antitumour effect of ZOL. DU145R80-resistant cells were selected in about 5 months using stepwise increasing concentrations of ZOL from DU145 parental cells. DU145R80 cells showed a resistance index value of 5.5 and cross-resistance to another N-BP, pamidronate, but not to the non-nitrogen containing BP clodronate. Notably, compared with DU145 parental cells, DU145R80 developed resistance to apoptosis and anoikis, as well as overexpressed the anti-apoptotic protein Bcl-2 and oncoprotein c-Myc. Moreover, DU145R80 cells underwent epithelial to mesenchymal transition (EMT) and showed increased expression of the metalloproteases MMP-2/9, as well as increased invading capability. Interestingly, compared with DU145, DU145R80 cells also increased the gene expression and protein secretion of VEGF and the cytokines Eotaxin-1 and IL-12. At the molecular level, DU145R80 cells showed strong activation of the p38-MAPK-dependent survival pathway compared with parental sensitive cells. Moreover, using the p38-inhibitor SB203580, we completely reversed the resistance to ZOL, as well as EMT marker expression and invasion. Furthermore, SB203580 treatment reduced the expression of VEGF, Eotaxin-1, IL-12, MMP-9, Bcl-2 and c-Myc. Thus, for the first time, we demonstrate that the p38-MAPK pathway can be activated under continuous extensive exposure to ZOL in PCa cells and that the p38-MAPK pathway has a critical role in the induction of resistance, as well as in the acquisition of a more aggressive and invasive phenotype.

Project description:BackgroundBisphosphonates (BPs) were designed for the prevention of skeletal-related events secondary to bone metastases. The purpose of this study was to show that zoledronic acid (ZA) directly eradicates highly tumorigenic and potentially metastatic cancer cells.Materials and methodsHuman prostate and breast highly tumorigenic (PC3, MCF 7) and low- or non-tumorigenic (LNCaP, MCF 10a) cell lines, respectively, were exposed to different concentrations of ZA (0-10 μM). Reverse transcriptase double quantitative polymerase chain reaction was used for quantitative gene expression analysis. Apoptosis and cell proliferation were determined using microscopic observation and MTS assays. Western blot was used to confirm the translational effects of apoptotic genes on protein expression.ResultsHuman prostate and breast highly tumorigenic (PC3, MCF 7) and low- or non-tumorigenic (LNCaP, MCF 10a) cell lines, respectively, showed multiple genes demonstrating differential expressions, including TRAF, TRADD, BCL2, CASPASES and IAP families. Increasing ZA concentrations showed a greater concentration-time response on cell proliferation and apoptosis in the highly tumorigenic cells. These results were confirmed by both reversing and enhancing the effect of ZA on cell proliferation with caspase 3, 7 or survivin siRNA, respectively. Pro-apoptotic proteins bax and caspase 2, 3, 7 and 9 were up-regulated, while the anti-apoptotic proteins bcl2, birc3 and survivin were down-regulated only in the highly tumorigenic cells.ConclusionsThis explains the ability of ZA to inhibit bony metastasis in highly tumorigenic cells compared with the low- or non-tumorigenic cells through a significant decrease in cell proliferation and increase in apoptosis through gene-regulated and translational-mediated down-regulation of survivin coupled with the inhibition of caspase 3 or 7. This has significant implications toward understanding the pharmacophysiology of BPs in metastasis and supports the clinically observed effect of BPs when administered adjunctively with anticancer drugs such as cyclophosphamide/methotrexate/5-fluorouracil, epirubicin in combination with cyclophosphamide or docetaxel, and doxorubicin.

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Proteomic analysis of zoledronic-acid resistant prostate cancer cells unveils novel pathways characterizing an invasive phenotype.

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Proteomic analysis of zoledronic-acid resistant prostate cancer cells unveils novel pathways characterizing an invasive phenotype.

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