Abstract 2809: ROS signaling by NADPH oxidase 5 (Nox5) modulates proliferation of human melanoma UACC-257 cells and prolongs growth in the absence of serum or growth factors

Abstract

Abstract Reactive oxygen species (ROS) produced by the NADPH oxidase (Noxs) homologs participate in signaling cascades regulating proliferation. Recently, we have reported substantial overexpression of Nox5 in several human cancers including those of prostate, ovary and melanomas. Despite being upregulated in many human cancers and implicated in promoting cell proliferation, the molecular mechanisms modulated by Nox5 are poorly understood. In this study, the functional significance of Nox5 was assessed in human UACC-257 and WM-852 melanoma cells by generating Nox5-overexpressing and knockdown cells. Stable overexpression of Nox5 in UACC-257 cells resulted in enhanced cell growth, increased BrdU positive cells and decreased protein tyrosine phosphatase activity. Additionally, these cells had increased normoxic Hif-1α expression and decreased p27Kip1 expression. Conversely, knockdown of endogenous Nox5 in UACC-257 cells resulted in decreased cell growth, decreased BrdU positive cells, decreased Hif-1α expression and increased p27Kip1 expression. Likewise, in human WM-852 melanoma cells, transient overexpression of Nox5 decreased p27Kip1 expression and knockdown of endogenous Nox5 resulted in increased p27Kip1 and gamma-H2AX expression with decreased cell growth. Cadherin switch, loss of E-cadherin expression and upregulation of N-cadherin was observed in UACC-257 Nox5 overexpressing cells indicative of an invasive phenotype; conversely, an upregulation of E-cadherin expression in Nox5 knockdown cells was noted. Cell invasion assay through matrigel-coated membranes also demonstrated enhanced invasion by Nox5 overexpressing cells. Additionally, 3D cultures of Nox5 overexpressing UACC-257 cells exhibit an amoeboid morphology compared to that of the Nox5 knockdown cells that were mesenchymal, suggestive of an amoeboid - mesenchymal (AMT) transition. Strikingly, UACC-257 cells that overexpress Nox5 were able to proliferate in serum-free conditions for over a month. In summary, our findings suggest that ROS signaling by Nox5 in human melanoma could modulate multiple signaling networks that regulate Hif-1α and p27Kip1 expression, contributing in part, to cell proliferation and the ability to grow in the absence of serum or growth factors. Citation Format: Smitha Antony, Yongzhong Wu, Diana C. Haines, Guojian Jiang, Jennifer L. Meitzler, Agnes Juhasz, Jiamo Lu, Krishnendu K. Roy, James H. Doroshow. ROS signaling by NADPH oxidase 5 (Nox5) modulates proliferation of human melanoma UACC-257 cells and prolongs growth in the absence of serum or growth factors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2809.