Transcriptional regulation of NOX genes expression in human breast adenocarcinoma MCF-7 cells is modulated by adaptor protein Ruk/CIN85

Abstract

NADPH oxidases are key components of redox-dependent signaling networks involved in the control of cancer cell proliferation, survival and invasion. The data have been accumulated that demonstrate specific expression patterns and levels of NADPH oxidase homologues (NOXs) and accessory genes in human cancer cell lines and primary tumors as well as modulation of these parameters by extracellular cues. Our previous studies revealed that ROS production by human colorectal adenocarcinoma HT-29 cells is positively correlated with adaptor protein Ruk/CIN85 expression while increased levels of Ruk/CIN85 in weakly invasive human breast adenocarcinoma MC F-7 cells contribute to their malignant phenotype through the constitutive activation of Src/Akt pathway. In this study, to investigate whether overexpression of Ruk/CIN85 in MC F-7 cells can influence transcriptional regulation of NOXs genes, the subclones of MCF-7 cells with different levels of Ruk/CIN85 were screened for NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2 as well as for regulatory subunit p22Phox mRNA contents by quantitative RT-PCR (qPCR). Systemic multidirectional changes in mRNA levels for NOX1, NOX2, NOX5, DUOX2 and p22Phox were revealed in Ruk/CIN85 overexpressing cells in comparison to control WT cells. Knocking down of Ruk/CIN85 using technology of RNA-interference resulted in the reversion of these changes. Further studies are necessary to elucidate, by which molecular mechanisms Ruk/CIN85 could affect transcriptional regulation of NOXs genes.

NOX5: Molecular biology and pathophysiology

“…Increased Nox activity and dysregulated production of ROS cause tissue injury, DNA damage and uncontrolled cell proliferation that are already evident in pre‐malignant conditions, especially Barrett's oesophagitis (Kalatskaya, ). Pathways implicated in these NOX5–ROS‐dependent processes include signalling molecules (MAP kinases, PI3K, PKC and p27Kip1), transcription factors (APE1/Ref‐1, hypoxia‐inducible factor‐1α, AP‐1, Nrf2, nuclear factor‐κB, p53, FOXO, STAT5A and β‐catenin) (Antony et al., ; Dho et al., ; Roy et al., ) and adaptor proteins (Ruk/CIN 85) (Bazalii, Horak, Pasi chn yk, Komisarenko, & Drobot, ).…”

“…-dependent processes include signalling molecules (MAP kinases, PI3K, PKC and p27Kip1), transcription factors (APE1/Ref-1, hypoxiainducible factor-1 , AP-1, Nrf2, nuclear factor-B, p53, FOXO, STAT5Aand -catenin)(Antony et al, 2017;Dho et al, 2017;Roy et al, 2015) and adaptor proteins (Ruk/CIN 85)(Bazalii, Horak, Pasi chn yk, Komisarenko, & Drobot, 2016).…”

Apocynin attenuates motility and induces transition from sustained to transient EGF-dependent Akt activation in MCF-7 cells that overexpress adaptor protein Ruk/CIN85

“…Taking into account our data regarding the direct physical interaction of Ruk/CIN85 with the organizer subunit Tks4 of NOX1-containing NADPH oxidase complex [10,11], a positive correlation between the level of Ruk/CIN85 expression and the ROS production in tumor cells [14, and our unpublished data], co-localization of adaptor protein and H 2 O 2 generation at the edges of certain vesicular structures in MCF-7 cells transiently transfected with the plasmid encoding fluorescent sensor of hydrogen peroxide Hyper fused with Ruk/CIN85 [17], the potential of Ruk/CIN85 to differentially influence NOX genes expression in MCF-7 cells stably overexpressing different levels of Ruk/CIN85 [15], it was the aim of this study to investigate a possible involvement of NADPH oxidases in the control of cell motility and Akt signaling depending on the Ruk/CIN85 expression levels in MCF-7 cells. For this purpose we used apocynin, which is considered to be one of the most specific inhibitor of NADPH oxidases, which blocks the assembly of the active enzyme complex [21].…”

“…Additionaly, it has been shown by us that the ROS production by human colorectal adenocarcinoma HT-29 cells is positively correlated with the Ruk/CIN85 expression [14]. Recently, we have demonstrated systemic multidirectional changes in mRNA levels for NOX1, NOX2, NOX5, DUOX2 and p22 Phox in Ruk/CIN85-overexpressing human breast adenocarcinoma MCF-7 cells [15] characterized by increased malignant phenotype [16]. Using the expression vector encoding fluorescent sensor of hydrogen peroxide HyPer fused with…”

Scientific and practical activity of the Laboratory of Cell Signaling Mechanisms of the Palladin Institute of Biochemistry of NAS of Ukraine

“…Також продемонстровано формування ендо-генного комплексу Ruk/CIN85 із протеїном Tks4 та виявлено потенційне біологічне зна-чення цієї взаємодії. Одержані дані є важливи-ми для пошуку і ідентифікації нових редокс-залежних мішеней для розробки відповідних фармакологічних препаратів скерованої дії [9,[13][14][15].…”