Potential mediators of prostate carcinogenesis identified through quantitative analysis of the extracellular proteome associated with prostate fibroblast senescence Free

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Introduction: Aging is the greatest risk factor for prostate carcinoma, the leading non-cutaneous malignancy and second-leading cause of cancer death in men. Molecular changes in the neoplastic cells and/or the tumor microenvironment are hypothesized to contribute to aging-related prostate cancer progression. We have previously shown that senescent prostate stromal cells can stimulate the proliferation of benign and neoplastic prostate epithelial cells in vitro. To further investigate the molecular basis for these effects, we employed quantitative proteomic analysis to characterize changes in the secreted proteome that occur with prostate stromal senescence.

Methods: We induced senescence in two independent prostate stromal isolates by oxidative damage and replicative exhaustion. The complement of extracellular proteins from pre-senescent and senescent cells was characterized by isotope coded affinity tag labeling, followed by multidimensional liquid chromatography and tandem mass spectrometry. Protein identifications and relative quantitations were determined by mass spectra analysis by the PeptideProphet, ProteinProphet and Xpress algorithms. Protein changes were confirmed by Western blot.

Results: A total of 430 proteins were identified of which 401 could be quantitated. Of these, 55% of the proteins were annotated as localized to the extracellular space, of which 15% were plasma membrane-localized. Fewer than half of the proteins with the greatest senescent-dependent changes have previously been reported in association with prostate cancer, although the majority have been associated with at least one other malignancy. To further validate the mass spectrometry-based quantitation, Western blot analysis confirmed senescence-associated increases in secreted proteins including CXCL5, Cystatin B, Cystatin S, Stanniocalcin 1, and Prostate Differentiation Factor (PLAB). Some of the most abundant identified proteins are components of the Insulin-like Growth Factor pathway, including IGFBP4, IGFBP2 and IGF2. Western blots identified increases in IGFBP2, IGFBP4, IGFBP5, and IGFBP6, with decreased levels of IGFBP3. Of these proteins, IGFBP2 and IGFBP5 have been reported to be growth stimulatory for prostate epithelium, while IGFBP3 is growth inhibitory.

Conclusion. These results define alterations in extracellular proteins associated with the process of senescence in prostate fibroblasts. Several of these senescence-associated proteins have been shown to contribute to the stimulation of prostate epithelial growth, survival, or metastasis. Linking these paracrine-acting mediators of carcinogenesis derived from cells comprising the microenvironment with organismal senescence and aging in vivo could provide a mechanistic explanation for age-related increases in prostate cancer incidence.