Abstract 2648: Hypoxia-induced NADPH oxidase 5 (NOX5) promotes tumor growth of head and neck squamous cell carcinoma through HIF-2á signaling

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Abstract Background: Head and neck squamous cell carcinoma (HNSCC) has an increased content of reactive oxygen species (ROS). However, clinical trials of antioxidant resulted in no benefit towards HNSCC anti-cancer treatment. NADPH oxidase (NOX) family is found to be the major generator of ROS which might contribute to the failure of antioxidant therapy. For the past 10 years, NOX family have been found with a range of functions in different cancers. Of which, NOX5 is the least well-understood one with limited details of signaling roles. Purpose of the study: To investigate the role of NOX5 in HNSCC. Experimental procedures: Online databases were used to analyze the expression of NOX5 in HNSCC patients. Immunohistochemistry was performed to detect the NOX5 expression level and its co-expression with hypoxia-inducible factors (HIFs) in paraffin-embedded tumor tissues surgically resected from HNSCC patients in Hong Kong. Subcutaneous xenograft model was established using NOX5-silenced HNSCC cells to evaluate the tumor growth potential in vivo. Sulforhodamine B assay and colony formation assay were used to measure the proliferation potential of NOX5-silenced HNSCC cells in vitro. Hypoxia chamber was applied to mimic the hypoxic condition of tumor tissue in vitro. Flow cytometry with Dihydroethidium staining was performed to measure the production of superoxide when silencing or overexpressing NOX5. Superoxide scavenger TEMPOL was applied to test the signaling role of superoxide generated by NOX5. Quantitative PCR and western blot were used to measure the mRNA and protein expression level of NOX5 and HIFs in HNSCC cell lines. Results: Relative mRNA expression of NOX5 was found significantly increased in HNSCC tumor tissues comparing to normal tissues both in Oncomine and TCGA datasets. Interestingly, NOX5 expression was found higher in central areas of tumor nests with the co-expression of HIF-1α and HIF-2α in our HNSCC patients’ samples. Further, NOX5 was upregulated in HNSCC cell lines under hypoxic condition (1% oxygen). Knocking-down of NOX5 impaired HNSCC cell growth both in vivo and in vitro, and this reduction was more significant under hypoxic condition in vitro. Loss of NOX5 resulted in superoxide reduction and HIF-2α impairment but not HIF-1α. Overexpressed NOX5 induced superoxide production and HIF-2α expression, and this induction was attenuated by TEMPOL. Conclusions: Hypoxia-induced NOX5 promotes HNSCC tumor growth by regulating HIF-2α via generating superoxide. NOX5 might serve as a novel therapeutic target for treatment of HNSCC. Citation Format: Siqi Chen, Wei Gao, Jimmy Yu-Wai Chan, Thian-Sze Wong. Hypoxia-induced NADPH oxidase 5 (NOX5) promotes tumor growth of head and neck squamous cell carcinoma through HIF-2á signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2648.