Sharon A. Glynn

The yin and yang of nitric oxide in cancer progression

Nitric oxide (NO) is a short-lived, pleiotropic molecule that affects numerous critical functions in the body. Presently, there are markedly conflicting findings in the literature regarding NO and its role in carcinogenesis and tumor progression. NO has been shown to have dichotomous effects on cellular proliferation, apoptosis, migration, invasion, angiogenesis and many other important processes in cancer biology. It has been shown to be both pro- and antitumorigenic, depending on the concentration and the tumor microenvironment in question. NO is generated by three isoforms of NO synthase (NOS) that are widely expressed and sometimes upregulated in human tumors. Due to its vast array of physiological functions, it presents a huge challenge to researchers to discover its true potential in cancer biology and consequently, its use in anticancer therapies. In this study, we review the current knowledge in this area, with an emphasis placed on NO modulation as an anticancer therapy, focusing on NO-donating drugs and NOS inhibitors.

Increased NOS2 predicts poor survival in estrogen receptor–negative breast cancer patients

Inducible nitric oxide synthase (NOS2) is involved in wound healing, angiogenesis, and carcinogenesis. NOS2 upregulation and increased nitric oxide (NO) production affect the redox state of cells and can induce protein, lipid, and DNA modifications. To investigate whether NOS2 levels influence survival of breast cancer patients, we examined NOS2 expression and its association with tumor markers and survival in 248 breast tumors. In multivariable survival analysis, increased NOS2 predicted inferior survival in women with estrogen receptor α-negative (ER-negative) tumors. Microdissected tumor epithelium from ER-negative tumors with high NOS2 had increased IL-8 and a gene expression signature characteristic of basal-like breast cancer with poor prognosis. In cell culture, NO only induced selected signature genes in ER-negative breast cancer cells. ER transgene expression in ER-negative cells inhibited NO-induced upregulation of the stem cell marker CD44 and other proteins encoded by signature genes, but not of IL-8. Exposure to NO also enhanced cell motility and invasion of ER-negative cells. Last, pathway analysis linked the tumor NOS2 gene signature to c-Myc activation. Thus, NOS2 is associated with a basal-like transcription pattern and poor survival of ER-negative patients.

Molecular Pathways: Toll-like Receptors in the Tumor Microenvironment—Poor Prognosis or New Therapeutic Opportunity

Numerous reports have described Toll-like receptor (TLR) expression in the tumor microenvironment as it relates to cancer progression, as well as their involvement in inflammation. While TLRs mediate immune surveillance, clinical studies have associated TLR expression in the tumor with poor patient survival, indicating that TLR expression may affect cancer treatment and survival. This review will examine mechanisms in which TLR activation upregulates protumorigenic pathways, including the induction of inducible nitric oxide synthase (iNOS2) and COX2, which in turn increase TLR expression and promote a feed-forward loop leading to tumor progression and the development of more aggressive tumor phenotypes. These propagating loops involve cancer cell, stroma, and/or immune cell TLR expression. Because of abundant TLR expression in many human tumors, several TLR agonists are now in clinical and preclinical trials and some have shown enhanced efficacy when used as adjuvant with radiation, chemotherapy, or cancer vaccines. These findings suggest that TLR expression influences cancer biology and therapeutic response, which may involve specific interactions within the tumor microenvironment, including mediators of inflammation such as nitric oxide and the arachidonic acid signaling pathways. Clin Cancer Res; 19(6); 1340–6. ©2012 AACR.

The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, simvastatin, lovastatin and mevastatin inhibit proliferation and invasion of melanoma cells

BackgroundA number of recent studies have suggested that cancer incidence rates may be lower in patients receiving statin treatment for hypercholesterolemia. We examined the effects of statin drugs on in vitro proliferation, migration and invasion of melanoma cells.MethodsThe ability of lovastatin, mevastatin and simvastatin to inhibit the melanoma cell proliferation was examined using cytotoxicity and apoptosis assays. Effects on cell migration and invasion were assessed using transwell invasion and migration chambers. Hypothesis testing was performed using 1-way ANOVA, and Students t-test.ResultsLovastatin, mevastatin and simvastatin inhibited the growth, cell migration and invasion of HT144, M14 and SK-MEL-28 melanoma cells. The concentrations required to inhibit proliferation of melanoma cells (0.8–2.1 μM) have previously been achieved in a phase I clinical trial of lovastatin in patients with solid tumours, (45 mg/kg/day resulted in peak plasma concentrations of approximately 3.9 μM).ConclusionOur results suggest that statin treatment is unlikely to prevent melanoma development at standard doses. However, higher doses of statins may have a role to play in adjuvant therapy by inhibiting growth and invasion of melanoma cells.

The specificity of nitroxyl chemistry is unique among nitrogen oxides in biological systems

The importance of nitric oxide in mammalian physiology has been known for nearly 30 years. Similar attention for other nitrogen oxides such as nitroxyl (HNO) has been more recent. While there has been speculation as to the biosynthesis of HNO, its pharmacological benefits have been demonstrated in several pathophysiological settings such as cardiovascular disorders, cancer, and alcoholism. The chemical biology of HNO has been identified as related to, but unique from, that of its redox congener nitric oxide. A summary of these findings as well as a discussion of possible endogenous sources of HNO is presented in this review.

Tumor microenvironment-based feed-forward regulation of NOS2 in breast cancer progression

Significance More than 92% of ER− breast cancer patients die with moderate to high NOS2. In this report, we show that tumor cell NOS2, through formation of a specific flux of NO, drives ER− disease to a more aggressive phenotype. Correlation between NOS2-related genes from patient cohorts, in vitro, and animal experiments show an inflammatory loop mediated by NOS2 that makes ER− breast cancer an aggressive disease. Inflammation is widely recognized as an inducer of cancer progression. The inflammation-associated enzyme, inducible nitric oxide synthase (NOS2), has emerged as a candidate oncogene in estrogen receptor (ER)-negative breast cancer, and its increased expression is associated with disease aggressiveness and poor survival. Although these observations implicate NOS2 as an attractive therapeutic target, the mechanisms of both NOS2 induction in tumors and nitric oxide (NO)-driven cancer progression are not fully understood. To enhance our mechanistic understanding of NOS2 induction in tumors and its role in tumor biology, we used stimulants of NOS2 expression in ER− and ER+ breast cancer cells and examined downstream NO-dependent effects. Herein, we show that up-regulation of NOS2 occurs in response to hypoxia, serum withdrawal, IFN-γ, and exogenous NO, consistent with a feed-forward regulation of NO production by the tumor microenvironment in breast cancer biology. Moreover, we found that key indicators of an aggressive cancer phenotype including increased S100 calcium binding protein A8, IL-6, IL-8, and tissue inhibitor matrix metalloproteinase-1 are up-regulated by these NOS2 stimulants, whereas inhibition of NOS2 in MDA-MB-231 breast cancer cells suppressed these markers. Moreover, NO altered cellular migration and chemoresistance of MDA-MB-231 cells to Taxol. Most notably, MDA-MB-231 tumor xenographs and cell metastases from the fat pad to the brain were significantly suppressed by NOS2 inhibition in nude mice. In summary, these results link elevated NOS2 to signals from the tumor microenvironment that arise with cancer progression and show that NO production regulates chemoresistance and metastasis of breast cancer cells.

Immunotherapeutic Potential of Anti-Human Endogenous Retrovirus-K Envelope Protein Antibodies in Targeting Breast Tumors

BACKGROUND The envelope (env) protein of the human endogenous retrovirus type K (HERV-K) family is commonly expressed on the surface of breast cancer cells. We assessed whether HERV-K env is a potential target for antibody-based immunotherapy of breast cancer. METHODS We examined the expression of HERV-K env protein in various malignant (MDA-MB-231, MCF-7, SKBR3, MDA-MB-453, T47D, and ZR-75-1) and nonmalignant (MCF-10A and MCF-10AT) human breast cell lines by immunoblot, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry. Anti-HERV-K env monoclonal antibodies (mAbs; 6H5, 4D1, 4E11, 6E11, and 4E6) were used to target expression of HERV-K, and antitumor effects were assessed by quantifying growth and apoptosis of breast cancer cells in vitro, and tumor growth in vivo in mice (n = 5 per group) bearing xenograft tumors. The mechanisms responsible for 6H5 mAb-mediated effects were investigated by microarray assays, flow cytometry, immunoblot, and immunofluorescence staining. The expression of HERV-K env protein was assessed in primary breast tumors (n = 223) by immunohistochemistry. All statistical tests were two-sided. RESULTS The expression of HERV-K env protein in malignant breast cancer cell lines was substantially higher than nonmalignant breast cells. Anti-HERV-K-specific mAbs inhibited growth and induced apoptosis of breast cancer cells in vitro. Mice treated with 6H5 mAb showed statistically significantly reduced growth of xenograft tumors compared with mice treated with control immunoglobulin (control [mIgG] vs 6H5 mAb, for tumors originating from MDA-MB-231 cells, mean size = 1448.33 vs 475.44 mm(3); difference = 972.89 mm(3), 95% CI = 470.17 to 1475.61 mm(3); P < .001). Several proteins involved in the apoptotic signaling pathways were overexpressed in vitro in 6H5 mAb-treated malignant breast cells compared with mIgG-treated control. HERV-K expression was detected in 148 (66%) of 223 primary breast tumors, and a higher rate of lymph node metastasis was associated with HERV-K-positive compared with HERV-K-negative tumors (43% vs 23%, P = .003). CONCLUSION Monoclonal antibodies against HERV-K env protein show potential as novel immunotherapeutic agents for breast cancer therapy.

S-Nitrosylation of EGFR and Src Activates an Oncogenic Signaling Network in Human Basal-Like Breast Cancer

Increased inducible nitric oxide synthase (NOS2) expression in breast tumors is associated with decreased survival of estrogen receptor negative (ER−) breast cancer patients. We recently communicated the preliminary observation that nitric oxide (NO) signaling results in epidermal growth factor receptor (EGFR) tyrosine phosphorylation. To further define the role of NO in the pathogenesis of ER− breast cancer, we examined the mechanism of NO-induced EGFR activation in human ER− breast cancer. NO was found to activate EGFR and Src by a mechanism that includes S-nitrosylation. NO, at physiologically relevant concentrations, induced an EGFR/Src-mediated activation of oncogenic signal transduction pathways (including c-Myc, Akt, and β-catenin) and the loss of PP2A tumor suppressor activity. In addition, NO signaling increased cellular EMT, expression and activity of COX-2, and chemoresistance to adriamycin and paclitaxel. When connected into a network, these concerted events link NO to the development of a stem cell–like phenotype, resulting in the upregulation of CD44 and STAT3 phosphorylation. Our observations are also consistent with the finding that NOS2 is associated with a basal-like transcription pattern in human breast tumors. These results indicate that the inhibition of NOS2 activity or NO signaling networks may have beneficial effects in treating basal-like breast cancer patients. Mol Cancer Res; 10(9); 1203–15. ©2012 AACR.

MicroRNA-106b-25 cluster expression is associated with early disease recurrence and targets caspase-7 and focal adhesion in human prostate cancer

The miR-106b-25 microRNA (miRNA) cluster is a candidate oncogene in human prostate cancer. Here, we report that miRNAs encoded by miR-106b-25 are upregulated in both primary tumors and distant metastasis. Moreover, increased tumor miR-106b expression was associated with disease recurrence and the combination of high miR-106b and low CASP7 (caspase-7) expressions in primary tumors was an independent predictor of early disease recurrence (adjusted hazard ratio=4.1; 95% confidence interval: 1.6–12.3). To identify yet unknown oncogenic functions of miR-106b, we overexpressed it in LNCaP human prostate cancer cells to examine miR-106b-induced global expression changes among protein-coding genes. The approach revealed that CASP7 is a direct target of miR-106b, which was confirmed by western blot analysis and a 3′-untranslated region reporter assay. Moreover, selected phenotypes induced by miR-106b knockdown in DU145 human prostate cancer cells did not develop when both miR-106b and CASP7 expression were inhibited. Further analyses showed that CASP7 is downregulated in primary prostate tumors and metastatic lesions across multiple data sets and is by itself associated with disease recurrence and disease-specific survival. Using bioinformatics, we also observed that miR-106b-25 may specifically influence focal adhesion-related pathways. This observation was experimentally examined using miR-106b-25-transduced 22Rv1 human prostate cancer cells. After infection with a miR-106b-25 lentiviral expression construct, 22Rv1 cells showed increased adhesion to basement membrane- and bone matrix-related filaments and enhanced soft agar growth. In summary, miR-106b-25 was found to be associated with prostate cancer progression and disease outcome and may do so by altering apoptosis- and focal adhesion-related pathways.

Lack of prognostic significance of survivin, survivin-ΔEx3, survivin-2B, galectin-3, bag-1, bax-α and MRP-1 mRNAs in breast cancer

Abstract The expression of transcripts for anti-apoptotic (survivin, survivin-ΔEx3, survivin-2B, galectin-3, bag-1 and bcl-2) and pro-apoptotic (bax-α) genes, and for multiple drug resistance related protein-1 (MRP-1) gene were investigated, using RT-PCR, in 106 breast tumour biopsies. Normal breast tissue was also analysed for comparative purposes. Overall, survivin, survivin-ΔEx3, survivin-2B, bcl-2, bag-1, galectin-3, bax-α and MRP-1 mRNAs were detected in 68, 54.7, 9.4, 78.4, 80.9, 98.9, 97.8 and 72.8%, respectively, of tumour specimens. Uniquely among the mRNAs analysed, the expression of bcl-2 correlated significantly with disease outcome, with bcl-2 expression indicative of favourable outcome in terms of both relapse-free survival and overall survival. This suggests that bcl-2 mRNA expression may be a key prognostic marker for breast cancer and that routine analysis of expression of this transcript should be considered. The results from this study suggest, however, that the expression of survivin, survivin-ΔEx3, survivin-2B, bag-1, galectin-3, bax-α and MRP-1 mRNAs cannot be considered as prognostic indicators of disease outcome for patients with breast cancer.