Abstract C49: Regulation of S100A8 and S100A9 expression by hypoxia and HIF-1 and their potential role as prognostic markers in prostate cancer

Abstract

Abstract S100A8 and S100A9, two heterodimer-forming members of the cytosolic S100 Ca2+ signaling protein family, are overexpressed in various cancer types, including prostate cancer. They act as pro-inflammatory danger signals when released to the extracellular space, and are thought to play an important role during tumorigenesis, affecting inflammatory processes, proliferation, invasion and metastasis of tumor cells. They are proposed as prognostic markers for several tumor types, including breast, bladder and non-small lung cancer. Despite their emerging role in tumorigenesis, little is known about tumor environmental factors influencing S100A8/A9 expression. Therefore, the aim of this study was to test the effect of hypoxia and its master transcriptional regulator HIF-1 on S100A8/A9 expression. Furthermore, the capacity of S100A8 and S100A9 as prognostic factors for prostate cancer recurrence after radical prostatectomy was investigated. The effect of hypoxia treatment (1 % O2) and overexpression of HIF-1α, the oxygen-sensitive subunit of HIF-1, on S100A8 and S100A9 expression was investigated by western blotting and qRT-PCR using prostate epithelial cell line BPH-1 and prostate cancer cell lines PC-3 and DU-145. S100A8/A9 promoters were analyzed for hypoxia responsive elements (HREs) by Transfaq database analysis and generation of promoter luciferase reporter constructs. Chromatin immunoprecipitation (ChIP) was performed to test interaction of HIF-1α with S100A8 and S100A9 promoters. Correlation of S100A8 and S100A9 expression with HIF-1α expression in prostate cancer tissue was tested by immunohistochemical staining using a tissue microarray (samples of 167 patients who underwent radical prostatectomy) and Pearson correlation analysis. Association of protein expression with time to tumor recurrence (re-appearance of prostate specific antigen in patients' serum) was tested by Cox proportional hazard modeling. Hypoxia treatment resulted in increased protein as well as mRNA expression of S100A8/A9 in BPH-1 cells, the latter was also confirmed in PC-3 and DU-145 cells. Furthermore, overexpression of HIF-1α led to up-regulation of S100A8/A9 mRNA and protein expression as well as secretion. S100A8 and S100A9 promoter analysis using promoter luciferase reporter constructs lead to identification of functional HREs mediating induction of promoter activity upon HIF-1α overexpression. Binding of HIF-1α to S100A8/A9 promoters was confirmed by ChIP. Immunohistochemical analysis of prostate cancer tissue microarray showed clear correlation of S100A8 and S100A9 with HIF-1α expression (Pearson r = 0.31 and r = 0.54, respectively, P < 0.0001). High S100A9 expression was significantly associated with shorter time to recurrence in univariate hazard analysis (HR = 2.16, 95% CI: 1.02 – 4.60, P = 0.045). Multivariate analysis, adjusted for known prognostic factors (Gleason score, tumor and lymph node stage), also showed relevant correlation of S100A9 expression with time to recurrence, albeit not significantly (HR = 1.78, 95% CI: 0.52 – 6.12). In conclusion, we identified hypoxia and HIF-1 as novel regulators of S100A8/A9 expression in prostate cancer. S100A9 could be useful as prognostic marker for prostate cancer recurrence after radical prostatectomy. Citation Format: Sina Grebhardt, Christian Veltkamp, Doris Mayer. Regulation of S100A8 and S100A9 expression by hypoxia and HIF-1 and their potential role as prognostic markers in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C49.