Andrew Little

Paradoxical roles of dual oxidases in cancer biology

Dysregulated oxidative metabolism is a well-recognized aspect of cancer biology, and many therapeutic strategies are based on targeting cancers by altering cellular redox pathways. The NADPH oxidases (NOXes) present an important enzymatic source of biological oxidants, and the expression and activation of several NOX isoforms are frequently dysregulated in many cancers. Cell-based studies have demonstrated a role for several NOX isozymes in controlling cell proliferation and/or cell migration, further supporting a potential contributing role for NOX in promoting cancer. While various NOX isoforms are often upregulated in cancers, paradoxical recent findings indicate that dual oxidases (DUOXes), normally prominently expressed in epithelial lineages, are frequently suppressed in epithelial-derived cancers by epigenetic mechanisms, although the functional relevance of such DUOX silencing has remained unclear. This review will briefly summarize our current understanding regarding the importance of reactive oxygen species (ROS) and NOXes in cancer biology, and focus on recent observations indicating the unique and seemingly opposing roles of DUOX enzymes in cancer biology. We will discuss current knowledge regarding the functional properties of DUOX, and recent studies highlighting mechanistic consequences of DUOX1 loss in lung cancer, and its consequences for tumor invasiveness and current anticancer therapy. Finally, we will also discuss potentially unique roles for the DUOX maturation factors. Overall, a better understanding of mechanisms that regulate DUOX and the functional consequences of DUOX silencing in cancer may offer valuable new diagnostic insights and novel therapeutic opportunities.

DUOX1 silencing in lung cancer promotes EMT, cancer stem cell characteristics and invasive properties

Dual oxidase 1 (DUOX1) is an oxidant-generating enzyme within the airway epithelium that participates in innate airway host defense and epithelial homeostasis. Recent studies indicate that DUOX1 is suppressed in lung cancers by epigenetic silencing, although the importance of DUOX1 silencing in lung cancer development or progression is unknown. Here we show that loss of DUOX1 expression in a panel of lung cancer cell lines is strongly associated with loss of the epithelial marker E-cadherin. Moreover, RNAi-mediated DUOX1 silencing in lung epithelial cells and the cancer cell line NCI-H292 was found to result in loss of epithelial characteristics/molecular features (altered morphology, reduced barrier function and loss of E-cadherin) and increased mesenchymal features (increased migration, anchorage-independent growth and gain of vimentin/collagen), suggesting a direct contribution of DUOX1 silencing to epithelial-to-mesenchymal transition (EMT), an important feature of metastatic cancer. Conversely, overexpression of DUOX1 in A549 cells was capable of reversing EMT features. DUOX1 silencing in H292 cells also led to enhanced resistance to epidermal growth factor receptor tyrosine kinase inhibitors such as erlotinib, and enhanced levels of cancer stem cell (CSC) markers CD133 and ALDH1. Furthermore, acquired resistance of H292 cells to erlotinib resulted in enhanced EMT and CSC features, as well as loss of DUOX1. Finally, compared with control H292 cells, H292-shDUOX1 cells displayed enhanced invasive features in vitro and in vivo. Collectively, our findings indicate that DUOX1 silencing in lung epithelial cancer cells promotes features of EMT, and may be strongly associated with invasive and metastatic lung cancer.

Acrolein and thiol-reactive electrophiles suppress allergen-induced innate airway epithelial responses by inhibition of DUOX1 and EGFR

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca2+-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.

Direct cysteine sulfenylation drives activation of the Src kinase

The Src kinase controls aspects of cell biology and its activity is regulated by intramolecular structural changes induced by protein interactions and tyrosine phosphorylation. Recent studies indicate that Src is additionally regulated by redox-dependent mechanisms, involving oxidative modification(s) of cysteines within the Src protein, although the nature and molecular-level impact of Src cysteine oxidation are unknown. Using a combination of biochemical and cell-based studies, we establish the critical importance of two Src cysteine residues, Cys-185 and Cys-277, as targets for H2O2-mediated sulfenylation (Cys-SOH) in redox-dependent kinase activation in response to NADPH oxidase-dependent signaling. Molecular dynamics and metadynamics simulations reveal the structural impact of sulfenylation of these cysteines, indicating that Cys-277-SOH enables solvent exposure of Tyr-416 to promote its (auto)phosphorylation, and that Cys-185-SOH destabilizes pTyr-527 binding to the SH2 domain. These redox-dependent Src activation mechanisms offer opportunities for development of Src-selective inhibitors in treatment of diseases where Src is aberrantly activated.The activity of several protein kinases is increased upon cellular production of reactive oxygen species, which can cause cysteine oxidation. Here the authors show that sulfenylation of specific cysteine residues within Src induce local structural changes that directly impact its activation.

Abstract 1343: DUOX1 silencing in lung cancer is associated with enhanced nuclear EGFR localization

Non-small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer-related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H2O2 producing enzyme located in the airway epithelium with key roles in mucosal host defense and wound repair mechanisms. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is closely associated with epithelial-to-mesenchymal transition (EMT) and enhanced tumor invasiveness and metastasis. However, the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1-dependent epithelial host defense pathways are mediated by redox-dependent activation of epithelial signaling via the non-receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We therefore hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently overexpressed and activated in lung cancer and strongly contribute to tumor growth and survival. In fact, recent studies have indicated that nuclear localization of EGFR in cancer cells is associated with metastatic cell behavior and poor clinical outcome, and the nuclear EGFR localization depends on Src-dependent phosphorylation of EGFR at Y1101. We observed that overexpression of DUOX1 in alveolar lung cancer A549 cells, which possess EMT-like features and in which DUOX1 is normally silenced, results in redistribution Src to the plasma membrane and decreased nuclear accumulation. DUOX1 overexpression in A549 cells also suppressed EGF-stimulated nuclear translocation of EGFR, which was associated with reduced EGFR phosphorylation at Y1101. Conversely, RNAi-mediated silencing of DUOX1 in the epithelial cancer cell line H292, which normally expresses DUOX1 expression, was found to promote EGF-mediated EGFR nuclear translocation and Y1101 phosphorylation. Since nuclear EGFR is thought to enhance the transcription of target genes related to cell cycle progression and proliferation (e.g. CDK1, Myc, others), we evaluated gene expression of these target genes in our cell models. Indeed, in cells lacking DUOX1, EGF stimulation significantly enhanced mRNA levels of CDK1, Myc, and other target genes for nuclear EGFR, whereas no such induction was seen in cells that express DUOX1. Our findings indicate that DUOX1 silencing in lung cancer may be associated with worse prognosis, partly due to altered spatiotemporal regulation of EGFR and Src and increased nuclear targeting. Since both EGFR and Src are subject to redox regulation by cysteine oxidation, we are currently aiming to elucidate the molecular mechanisms by which these mechanism are affected by altered DUOX1 status. Citation Format: Andrew C. Little, Karamathullah Danyal, David Heppner, Milena Hristova, Albert van der Vliet. DUOX1 silencing in lung cancer is associated with enhanced nuclear EGFR localization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1343. doi:10.1158/1538-7445.AM2017-1343

Abstract 1681: DUOX1 expression in lung cancer disrupts pro-oncogenic activation mechanisms and localization of Src and EGFR

Non-small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer-related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H2O2 producing enzyme located in the airway epithelium with key roles in mucosal host defense and wound repair mechanisms. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is associated with epithelial-to-mesenchymal transition (EMT), a key feature of tumor invasiveness and metastasis, and that RNAi-mediated DUOX1 suppression can promote EMT, but the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1-dependent epithelial host defense pathways are mediated by redox-dependent activation of epithelial signaling via the non-receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We therefore hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently overexpressed and activated in lung cancer and strongly contribute to tumor growth and survival. Furthermore, nuclear Src/EGFR localization and phosphorylation of EGFR-Y1101 in lung cancers was recently associated with metastatic cell behavior and poor clinical outcome. Preliminary findings in alveolar lung cancer A549 cells, which possesses some EMT-like features, indicate that DUOX1 overexpression redistributes Src localization to the plasma membrane and decreases its nuclear accumulation. Moreover, DUOX1 overexpression in A549 cells also suppressed EGF-stimulated EGFR internalization and nuclear translocation, in association with reduced EGFR phosphorylation on its Src target, Y1101. Conversely, RNAi-mediated silencing of DUOX1 in the epithelial cancer cell line H292 (which has retained DUOX1 expression) promoted EGF-mediated EGFR nuclear translocation and Y1101 phosphorylation. Further mechanistic studies will be performed to elucidate the molecular mechanisms by which DUOX1 is able to alter these events. Collectively, our findings indicate that DUOX1 silencing in lung cancer may contribute to EMT and/or tumor invasiveness by altering Src/EGFR localization and activation mechanisms. Citation Format: Andrew C. Little, Karamatullah Danyal, Robert A. Bauer, David E. Heppner, Milena Hristova, Christopher Dustin, Aida Habibovic, Albert van der Vliet. DUOX1 expression in lung cancer disrupts pro-oncogenic activation mechanisms and localization of Src and EGFR. [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 1681.