Hypoxia-Inducible Factor 1 a Determines Gastric Cancer Chemosensitivity via Modulation of p 53 and NFk B

Background: Reduced chemosensitivity of solid cancer cells represents a pivotal obstacle in clinical oncology. Hence, the molecular characterization of pathways regulating chemosensitivity is a central prerequisite to improve cancer therapy. The hypoxia-inducible factor HIF-1a has been linked to chemosensitivity while the underlying molecular mechanisms remain largely elusive. Therefore, we comprehensively analysed HIF-1a’s role in determining chemosensitivity focussing on responsible molecular pathways. Methodology and Principal Findings: RNA interference was applied to inactivate HIF-1a or p53 in the human gastric cancer cell lines AGS and MKN28. The chemotherapeutic agents 5-fluorouracil and cisplatin were used and chemosensitivity was assessed by cell proliferation assays as well as determination of cell cycle distribution and apoptosis. Expression of p53 and p53 target proteins was analyzed by western blot. NF-kB activity was characterized by means of electrophoretic mobility shift assay. Inactivation of HIF-1a in gastric cancer cells resulted in robust elevation of chemosensitivity. Accordingly, HIF-1acompetent cells displayed a significant reduction of chemotherapy-induced senescence and apoptosis. Remarkably, this phenotype was completely absent in p53 mutant cells while inactivation of p53 per se did not affect chemosensitivity. HIF1a markedly suppressed chemotherapy-induced activation of p53 and p21 as well as the retinoblastoma protein, eventually resulting in cell cycle arrest. Reduced formation of reactive oxygen species in HIF-1a-competent cells was identified as the molecular mechanism of HIF-1a-mediated inhibition of p53. Furthermore, loss of HIF-1a abrogated, in a p53-dependent manner, chemotherapy-induced DNA-binding of NF-kB and expression of anti-apoptotic NF-kB target genes. Accordingly, reconstitution of the NF-kB subunit p65 reversed the increased chemosensitivity of HIF-1a-deficient cells. Conclusion and Significance: In summary, we identified HIF-1a as a potent regulator of p53 and NF-kB activity under conditions of genotoxic stress. We conclude that p53 mutations in human tumors hold the potential to confound the efficacy of HIF-1-inhibitors in cancer therapy. Citation: Rohwer N, Dame C, Haugstetter A, Wiedenmann B, Detjen K, et al. (2010) Hypoxia-Inducible Factor 1a Determines Gastric Cancer Chemosensitivity via Modulation of p53 and NF-kB. PLoS ONE 5(8): e12038. doi:10.1371/journal.pone. 0012038 Editor: Deb Fox, The Research Institute for Children at Children’s Hospital New Orleans, United States of America Received April 28, 2010; Accepted July 19, 2010; Published August 10, 2010 Copyright: 2010 Rohwer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the Deutsche Forschungsgemeinschaft (http://www.dfg.de) to TC (CR 133/2-1, 133/2-2 and 133/2-3), and NR (Graduiertenkolleg 276/4 – ‘‘Signalerkennung und –umsetzung’’). TC was also supported by a grant from the Berliner Krebsgesellschaft e.V. (http://www.berlinerkrebsgesellschaft.de, CRFF200804). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: thorsten.cramer@charite.de