Brandon M. Schickling

NOX4 mediates cytoprotective autophagy induced by the EGFR inhibitor erlotinib in head and neck cancer cells

Most head and neck squamous cell carcinomas (HNSCCs) overexpress epidermal growth factor receptor (EGFR) and EGFR inhibitors are routinely used in the treatment of HNSCC. However, many HNSCC tumors do not respond or become refractory to EGFR inhibitors. Autophagy, which is a stress-induced cellular self-degradation process, has been reported to reduce the efficacy of chemotherapy in various disease models. The purpose of this study is to determine if the efficacy of the EGFR inhibitor erlotinib is reduced by activation of autophagy via NOX4-mediated oxidative stress in HNSCC cells. Erlotinib induced the expression of the autophagy marker LC3B-II and autophagosome formation in FaDu and Cal-27 cells. Inhibition of autophagy by chloroquine and knockdown of autophagy pathway genes Beclin-1 and Atg5 sensitized both cell lines to erlotinib-induced cytotoxicity, suggesting that autophagy may serve as a protective mechanism. Treatment with catalase (CAT) and diphenylene iodonium (DPI) in the presence of erlotinib suppressed the increase in LC3B-II expression in FaDu and Cal-27 cells. Erlotinib increased NOX4 mRNA and protein expression by increasing its promoter activity and mRNA stability in FaDu cells. Knockdown of NOX4 using adenoviral siNOX4 partially suppressed erlotinib-induced LC3B-II expression, while overexpression of NOX4 increased expression of LC3B-II. These studies suggest that erlotinib may activate autophagy in HNSCC cells as a pro-survival mechanism, and NOX4 may play a role in mediating this effect.

Phosphorylation of Nox1 Regulates Association With NoxA1 Activation Domain

Rationale: Activation of Nox1 initiates redox-dependent signaling events crucial in the pathogenesis of vascular disease. Selective targeting of Nox1 is an attractive potential therapy, but requires a better understanding of the molecular modifications controlling its activation. Objective: To determine whether posttranslational modifications of Nox1 regulate its activity in vascular cells. Methods and Results: We first found evidence that Nox1 is phosphorylated in multiple models of vascular disease. Next, studies using mass spectroscopy and a pharmacological inhibitor demonstrated that protein kinase C-beta1 mediates phosphorylation of Nox1 in response to tumor necrosis factor-&agr;. siRNA-mediated silencing of protein kinase C-beta1 abolished tumor necrosis factor-&agr;–mediated reactive oxygen species production and vascular smooth muscle cell migration. Site-directed mutagenesis and isothermal titration calorimetry indicated that protein kinase C-beta1 phosphorylates Nox1 at threonine 429. Moreover, Nox1 threonine 429 phosphorylation facilitated the association of Nox1 with the NoxA1 activation domain and was necessary for NADPH oxidase complex assembly, reactive oxygen species production, and vascular smooth muscle cell migration. Conclusions: We conclude that protein kinase C-beta1 phosphorylation of threonine 429 regulates activation of Nox1 NADPH oxidase.

Nox1 NADPH oxidase is necessary for late but not early myocardial ischaemic preconditioning

AIMS Ischaemic preconditioning (IPC) is an adaptive mechanism that renders the myocardium resistant to injury from subsequent hypoxia. Although reactive oxygen species (ROS) contribute to both the early and late phases of IPC, their enzymatic source and associated signalling events have not yet been understood completely. Our objective was to investigate the role of the Nox1 NADPH oxidase in cardioprotection provided by IPC. METHODS AND RESULTS Wild-type (WT) and Nox1-deficient mice were treated with three cycles of brief coronary occlusion and reperfusion, followed by prolonged occlusion either immediately (early IPC) or after 24 h (late IPC). Nox1 deficiency had no impact on the cardioprotection afforded by early IPC. In contrast, deficiency of Nox1 during late IPC resulted in a larger infarct size, cardiac remodelling, and increased myocardial apoptosis compared with WT hearts. Furthermore, expression of Nox1 in WT hearts increased in response to late IPC. Deficiency of Nox1 abrogated late IPC-mediated activation of cardiac nuclear factor-κB (NF-κB) and induction of tumour necrosis factor-α (TNF-α) in the heart and circulation. Finally, knockdown of Nox1 in cultured cardiomyocytes prevented TNF-α induction of NF-κB and the protective effect of IPC on hypoxia-induced apoptosis. CONCLUSIONS Our data identify a critical role for Nox1 in late IPC and define a previously unrecognized link between TNF-α and NF-κB in mediating tolerance to myocardial injury. These findings have clinical significance considering the emergence of Nox1 inhibitors for the treatment of cardiovascular disease.

Cullin-5, a ubiquitin ligase scaffold protein, is significantly underexpressed in endometrial adenocarcinomas and is a target of miR-182

Altered expression of cullin-5 (CUL5), a member of the cullin-RING E3 ubiquitin ligase family, has been implicated in a number of types of cancers including breast, cervical and hepatocellular cancers. In the present study, we found that CUL5 expression was significantly decreased in both endometrioid and serous endometrial adenocarcinomas with the more aggressive serous type displaying a higher reduction (−4.3-fold) than the less aggressive endometrioid type (−2.9-fold). Overexpression of CUL5 mRNA and protein in Ishikawa H endometrial cancer cells resulted in decreased cell proliferation and in a reduction in CUL5-RING E3 ligase downstream clients JAK2 and FAS-L. Finally, we demonstrated for the first time that CUL5 is a direct target of miR-182 that we previously showed to be significantly overexpressed in endometrial adenocarcinomas and we provided evidence that increased miR-182 expression is, at least in part, a result of demethylation of its upstream promoter. These data suggest a cascade in which miR-182 expression is epigenetically increased leading to decreased CUL5 expression and increased cellular proliferation. The final step in the cascade may be operating through a decrease in ubiquitination of pro-growth CUL5 ubiquitin ligase clients. This cascade offers a series of potential interventional steps involving epigenetic modification, miRNA and/or gene targeting and ubiquitination.

Smooth Muscle Cell–targeted RNA Aptamer Inhibits Neointimal Formation

Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-β phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease.Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-β phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease.

microRNA expression patterns across seven cancers are highly correlated and dominated by evolutionarily ancient families

microRNAs (miRNAs) are involved in almost all normal and pathogenic eukaryotic cell processes. One area in which the influence of miRNAs is most prominent is cancer. Numerous expression surveys and more focused studies have revealed miRNA involvement in carcinogenesis, cellular pathology, cell behavior and prognosis. Large-scale comparisons of miRNA expression in varioius types of cancer have not been previously possible. However, The Cancer Genome Atlas (TCGA), an extensive multi-centered effort to characterize the genomes of hundreds of types of cancer, has enabled such comparisons. In the present study, the expression patterns of hundreds of miRNAs in thousands of tumors covering seven types of cancer: uterine corpus adenocarcinoma, ovarian serous adenocarcinoma, breast adenocarcinoma, prostate adenocarcinoma, pancreatic adenocarcinoma, colorectal adenocarcinoma, and lung adenocarcinoma were analyzed. The results showed that miRNA expression patterns among these cancer types are highly correlated (0.874>ρ>0.974) and that miRNA expression in all seven cancer types is dominated by miRNAs belonging to the most evolutionarily ancient miRNA families. This raises the possibility that more ancient miRNAs are involved in the fundamental cell processes that are central to tumor evolution.

An inhibitor of K+ channels modulates human endometrial tumor-initiating cells

BackgroundMany potassium ion (K+) channels function as oncogenes to sustain growth of solid tumors, but their role in cancer progression is not well understood. Emerging evidence suggests that the early progenitor cancer cell subpopulation, termed tumor initiating cells (TIC), are critical to cancer progression.ResultsA non-selective antagonist of multiple types of K+ channels, tetraethylammonium (TEA), was found to suppress colony formation in endometrial cancer cells via inhibition of putative TIC. The data also indicated that withdrawal of TEA results in a significant enhancement of tumorigenesis. When the TIC-enriched subpopulation was isolated from the endometrial cancer cells, TEA was also found to inhibit growth in vitro.ConclusionsThese studies suggest that the activity of potassium channels significantly contributes to the progression of endometrial tumors, and the antagonists of potassium channels are candidate anti-cancer drugs to specifically target tumor initiating cells in endometrial cancer therapy.

BKCa channel inhibitor modulates the tumorigenic ability of hormone-independent breast cancer cells via the Wnt pathway

In breast cancers, the large conductance Ca2+ and voltage sensitive K+ (BKCa) channels have been hypothesized to function as oncoproteins, yet it remains unclear how inhibition of channel activity impacts oncogenesis. We demonstrated herein that iberiotoxin (IbTX), an inhibitor of BKCa channels, differentially modulated the in vitro tumorigenic activities of hormone-independent breast cancer cells. Specifically, in HER-2/neu-overexpressing UACC893 cells and triple-negative MDA-MB-231 cells, IbTX selectively attenuated anchorage-independent growth with concomitant downregulation of β-catenin as well as total and phosphorylated Akt and HER-2/neu. By contrast, HER-2/neu-overexpressing SK-BR-3 cells were insensitive to IbTX. Molecular analyses showed an absence of β-catenin and a dose-dependent upregulation of total and phosphorylated Akt and HER-2/neu in these cells. Taken together, these studies identify β-catenin as a putative modulator of the inhibitory actions of IbTX in sensitive breast cancer cells.

Dysregulation of miR-181c expression influences recurrence of endometrial endometrioid adenocarcinoma by modulating NOTCH2 expression: An NRG Oncology/Gynecologic Oncology Group study

OBJECTIVE Endometrial cancer can be diagnosed early and cured, yet cases that recur portend a very poor prognosis with over 10,000 women succumbing to the disease every year. In this study we addressed the question of how to recognize cases likely to recur early in the course of therapy using dysregulation of tumor microRNAs (miRNAs) as predictors. METHODS Using the tissue collection from Gynecologic Oncology Group Study-210, we selected and analyzed expression of miRNAs in 54 recurrent and non-recurrent cases. The three most common histologic types, endometrioid adenocarcinoma (EEA), serous adenocarcinoma (ESA) and carcinosarcoma (UCS), were analyzed as three independent sets and their miRNA expression profiles compared. RESULTS Only one miRNA was statistically different between recurrent and non-recurrent cases, and in only one histologic type: significant down-regulation of miR-181c was observed in EEA recurrence. Using several well-known databases to assess miR-181c targets, one target of particular relevance to cancer, NOTCH2, was well supported. Using The Cancer Genome Atlas and our validation tumor panel from the GOG-210 cohort, we confirmed that NOTCH2 is significantly over-expressed in EEA. In the most relevant endometrial adenocarcinoma cell model, Ishikawa H, altering miR-181c expression produces significant changes in NOTCH2 expression, consistent with direct targeting. CONCLUSIONS Our findings suggest that increased NOTCH2 via loss of miR-181c is a significant component of EEA recurrence. This presents an opportunity to develop miR-181c and NOTCH2 as markers for early identification of high risk cases and the use of NOTCH inhibitors in the prevention or treatment of recurrent disease.

p53 mutation status is a primary determinant of placenta-specific protein 1 expression in serous ovarian cancers

Placenta-specific protein 1 (PLAC1) expression is co-opted in numerous human cancers. As a consequence of PLAC1 expression, tumor cells exhibit enhanced proliferation and invasiveness. This characteristic is associated with increased aggressiveness and worse patient outcomes. Recently, the presence of the tumor suppressor p53 was shown in vitro to inhibit PLAC1 transcription by compromising the P1, or distal/cancer, promoter. We sought to determine if this phenomenon occurs in primary patient tumors as well. Furthermore, we wanted to know if p53 mutation influenced PLAC1 expression as compared with wild-type. We chose to study serous ovarian tumors as they are well known to have a high rate of p53 mutation. We report herein that the phenomenon of PLAC1 transcription repression does occur in serous ovarian carcinomas but only when TP53 is wild-type. We find that mutant or absent p53 protein de-represses PLAC1 transcription. We further propose that the inability of mutant p53 to repress PLAC1 transcription is due to the fact that the altered TP53 protein is unable to occupy a putative p53 binding site in the PLAC1 P1 promoter thus allowing transcription to occur. Finally, we show that PLAC1 transcript number is significantly negatively correlated with patient survival in our samples. Thus, we suggest that characterizing tumors for TP53 mutation status, p53 protein status and PLAC1 transcription could be used to predict likely prognosis and inform treatment options in patients diagnosed with serous ovarian cancer.