Chronic nitric oxide exposure induces prostate cell carcinogenesis, involving genetic instability and a pro-tumorigenic secretory phenotype.

Prostate cancer is a leading cause of cancer death in men. Inflammation and overexpression of inducible nitric oxide synthase (NOS2) have been implicated in prostate carcinogenesis. We aimed to explore the hypothesis that nitric oxide NO exerts pro-tumorigenic effects on prostate cells at physiologically relevant levels contributing to carcinogenesis. We investigated the impact of acute exposure of normal immortalised prostate cells (RWPE-1) to NO on cell proliferation and activation of DNA damage repair pathways. Furthermore we investigated the long term effects of chronic NO exposure on RWPE-1 cell migration and invasion potential and hallmarks of transformation. Our results demonstrate that NO induces DNA damage as indicated by γH2AX foci and p53 activation resulting in a G1/S phase block and activation of 53BP1 DNA damage repair protein. Long term adaption to NO results in increased migration and invasion potential, acquisition of anchorage independent growth and increased resistance to chemotherapy. This was recapitulated in PC3 and DU145 prostate cancer cells which upon chronic exposure to NO displayed increased cell migration, colony formation and increased resistance to chemotherapeutics. These findings indicate that NO may play a key role in the development of prostate cancer and the acquisition of an aggressive metastatic phenotype.

Nitric Oxide Modulates Metabolic Processes in the Tumor Immune Microenvironment.

The metabolic requirements and functions of cancer and normal tissues are vastly different. Due to the rapid growth of cancer cells in the tumor microenvironment, distorted vasculature is commonly observed, which creates harsh environments that require rigorous and constantly evolving cellular adaption. A common hallmark of aggressive and therapeutically resistant tumors is hypoxia and hypoxia-induced stress markers. However, recent studies have identified alterations in a wide spectrum of metabolic pathways that dictate tumor behavior and response to therapy. Accordingly, it is becoming clear that metabolic processes are not uniform throughout the tumor microenvironment. Metabolic processes differ and are cell type specific where various factors promote metabolic heterogeneity within the tumor microenvironment. Furthermore, within the tumor, these metabolically distinct cell types can organize to form cellular neighborhoods that serve to establish a pro-tumor milieu in which distant and spatially distinct cellular neighborhoods can communicate via signaling metabolites from stroma, immune and tumor cells. In this review, we will discuss how biochemical interactions of various metabolic pathways influence cancer and immune microenvironments, as well as associated mechanisms that lead to good or poor clinical outcomes.

The role of extracellular matrix in tumour angiogenesis: the throne has NOx servants.

The extracellular matrix (ECM) dynamics in tumour tissue are deregulated compared to the ECM in healthy tissue along with disorganized architecture and irregular behaviour of the residing cells. Nitric oxide (NO) as a pleiotropic molecule exerts different effects on the components of the ECM driving or inhibiting augmented angiogenesis and tumour progression and tumour cell proliferation and metastasis. These effects rely on the concentration of NO within the tumour tissue, the nature of the surrounding microenvironment and the sensitivity of resident cells to NO. In this review article, we summarize the recent findings on the correlation between the levels of NO and the ECM components towards the modulation of tumour angiogenesis in different types of cancers. These are discussed principally in the context of how NO modulates the expression of ECM proteins resulting in either the promotion or inhibition of tumour growth via tumour angiogenesis. Furthermore, the regulatory effects of individual ECM components on the expression of the NO synthase enzymes and NO production were reviewed. These findings support the current efforts for developing effective therapeutics for cancers.

Coexpression of NOS2 and COX2 accelerates tumor growth and reduces survival in estrogen receptor-negative breast cancer.

Proinflammatory signaling pathways are commonly up-regulated in breast cancer. In estrogen receptor-negative (ER-) and triple-negative breast cancer (TNBC), nitric oxide synthase-2 (NOS2) and cyclooxygenase-2 (COX2) have been described as independent predictors of disease outcome. We further explore these findings by investigating the impact of their coexpression on breast cancer survival. Elevated coexpression of NOS2/COX2 proteins is a strong predictor of poor survival among ER- patients (hazard ratio: 21). Furthermore, we found that the key products of NOS2 and COX2, NO and prostaglandin E2 (PGE2), respectively, promote feed-forward NOS2/COX2 crosstalk in both MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal-like) TNBC cell lines in which NO induced COX2 and PGE2 induced NOS2 proteins. COX2 induction by NO involved TRAF2 activation that occurred in a TNFα-dependent manner in MDA-MB-468 cells. In contrast, NO-mediated TRAF2 activation in the more aggressive MDA-MB-231 cells was TNFα independent but involved the endoplasmic reticulum stress response. Inhibition of NOS2 and COX2 using amino-guanidine and aspirin/indomethacin yielded an additive reduction in the growth of MDA-MB-231 tumor xenografts. These findings support a role of NOS2/COX2 crosstalk during disease progression of aggressive cancer phenotypes and offer insight into therapeutic applications for better survival of patients with ER- and TNBC disease.

The Role of Nitric Oxide in Cancer: Master Regulator or NOt?

Nitric oxide (NO) is a key player in both the development and suppression of tumourigenesis depending on the source and concentration of NO. In this review, we discuss the mechanisms by which NO induces DNA damage, influences the DNA damage repair response, and subsequently modulates cell cycle arrest. In some circumstances, NO induces cell cycle arrest and apoptosis protecting against tumourigenesis. NO in other scenarios can cause a delay in cell cycle progression, allowing for aberrant DNA repair that promotes the accumulation of mutations and tumour heterogeneity. Within the tumour microenvironment, low to moderate levels of NO derived from tumour and endothelial cells can activate angiogenesis and epithelial-to-mesenchymal transition, promoting an aggressive phenotype. In contrast, high levels of NO derived from inducible nitric oxide synthase (iNOS) expressing M1 and Th1 polarised macrophages and lymphocytes may exert an anti-tumour effect protecting against cancer. It is important to note that the existing evidence on immunomodulation is mainly based on murine iNOS studies which produce higher fluxes of NO than human iNOS. Finally, we discuss different strategies to target NO related pathways therapeutically. Collectively, we present a picture of NO as a master regulator of cancer development and progression.

Outcome for triple negative breast cancer in a retrospective cohort with an emphasis on response to platinum-based neoadjuvant therapy.

PURPOSE: The rate of pathological complete response (pCR) for patients with triple negative breast cancer (TNBC) is increased when carboplatin is added to neo-adjuvant chemotherapy (NACT). However, while phase III trial data showing a survival benefit are awaited, carboplatin is not yet standard-of-care for TNBC. The aim of this study was to examine the rate of pCR and the outcome for those treated with carboplatin and to examine the predictors of response to therapy. METHODS: The retrospective series comprised 333 consecutive patients with TNBC (median follow-up time, 43 months). Adjuvant chemotherapy was given to 51% (n = 168) of patients and 29% (n = 97) received anthracycline-taxane NACT with carboplatin given to 9% (n = 31) of patients. RESULTS: Overall, 25% (n = 78) of patients experienced a breast cancer recurrence and 22% (n = 68) died from disease. A pCR breast and pCR breast/axilla was more common in those who received carboplatin (n = 18, 58% and n = 17, 55%, respectively) compared those who did not (n = 23, 36% and n = 18, 28%, respectively) (p = 0.041 and p = 0.011, respectively). By multivariable analysis, carboplatin and high tumor grade were independent predictors of pCR breast/axilla (ORnon-pCR = 0.17; 95% CI 0.06-0.54; p = 0.002; and ORnon-pCR = 0.05, 95% CI 0.01-0.27; p < 0.001, respectively). pCR breast/axilla was an independent predictor of DFS (HRnon-pCR=6.23; 95% CI 1.36-28.50; p = 0.018), metastasis-free survival (HRnon-pCR = 5.08; 95% CI 1.09-23.65; p = 0.038) and BCSS (HRnon-pCR = 8.52; 95% CI 1.09-66.64; p = 0.041). CONCLUSION: Carboplatin therapy and high tumor grade are associated with a significant increase in the rate of pCR, which is an independent predictor of outcome. These data support the use of carboplatin in NACT for TNBC, while results from phase III studies are awaited.

Secreted factors from metastatic prostate cancer cells stimulate mesenchymal stem cell transition to a pro-tumourigenic 'activated' state that enhances prostate cancer cell migration.

Mesenchymal stem cells (MSCs) are a heterogeneous population of multipotent cells that are capable of differentiating into osteocytes, chondrocytes and adipocytes. Recently, MSCs have been found to home to the tumour site and engraft in the tumour stroma. However, it is not yet known whether they have a tumour promoting or suppressive function. We investigated the interaction between prostate cancer cell lines 22Rv1, DU145 and PC3, and bone marrow-derived MSCs. MSCs were 'educated' for extended periods in prostate cancer cell conditioned media and PC3-educated MSCs were found to be the most responsive with a secretory profile rich in pro-inflammatory cytokines. PC3-educated MSCs secreted increased osteopontin (OPN), interleukin-8 (IL-8) and fibroblast growth factor-2 (FGF-2) and decreased soluble fms-like tyrosine kinase-1 (sFlt-1) compared to untreated MSCs. PC3-educated MSCs showed a reduced migration and proliferation capacity that was dependent on exposure to PC3-conditioned medium. Vimentin and α-smooth muscle actin (αSMA) expression was decreased in PC3-educated MSCs compared to untreated MSCs. PC3 and DU145 education of healthy donor and prostate cancer patient-derived MSCs led to a reduced proportion of FAP+ αSMA+ cells contrary to characteristics commonly associated with cancer associated fibroblasts (CAFs). The migration of PC3 cells was increased toward both PC3-educated and DU145-educated MSCs compared to untreated MSCs, while DU145 migration was only enhanced toward patient-derived MSCs. In summary, MSCs developed an altered phenotype in response to prostate cancer conditioned medium which resulted in increased secretion of pro-inflammatory cytokines, modified functional activity and the chemoattraction of prostate cancer cells.

Reproducibility and predictive value of scoring stromal tumour infiltrating lymphocytes in triple-negative breast cancer: a multi-institutional study.

BACKGROUND: Several studies have demonstrated a prognostic role for stromal tumour infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC). The reproducibility of scoring sTILs is variable with potentially excellent concordance being achievable using a software tool. We examined agreement between breast pathologists across Europe scoring sTILs on H&E-stained sections without software, an approach that is easily applied in clinical practice. The association between sTILs and response to anthracycline-taxane NACT was also examined. METHODOLOGY: Pathologists from the European Working Group for Breast Screening Pathology scored sTILs in 84 slides from 75 TNBCs using the immune-oncology biomarker working group guidance in two circulations. There were 16 participants in the first and 19 in the second circulation. RESULTS: Moderate agreement was achieved for absolute sTILs scores (intraclass correlation coefficient (ICC) = 0.683, 95% CI 0.601-0.767, p-value < 0.001). Agreement was less when a 25% threshold was used (ICC 0.509, 95% CI 0.416-0.614, p-value < 0.001) and for lymphocyte predominant breast cancer (LPBC) (ICC 0.504, 95% CI 0.412-0.610, p-value < 0.001). Intra-observer agreement was strong for absolute sTIL values (Spearman ρ = 0.727); fair for sTILs ≥ 25% (κ = 0.53) and for LPBC (κ = 0.49), but poor for sTILs as 10% increments (κ = 0.24). Increasing sTILs was significantly associated with an increased likelihood of a pathological complete response (pCR) on multivariable analysis. CONCLUSION: Increasing sTILs in TNBCs improves the likelihood of a pCR. However, inter-observer agreement is such that H&E-based assessment is not sufficiently reproducible for clinical application. Other methodologies should be explored, but may be at the cost of ease of application.

Mesenchymal stem cells: key players in cancer progression.

Tumour progression is dependent on the interaction between tumour cells and cells of the surrounding microenvironment. The tumour is a dynamic milieu consisting of various cell types such as endothelial cells, fibroblasts, cells of the immune system and mesenchymal stem cells (MSCs). MSCs are multipotent stromal cells that are known to reside in various areas such as the bone marrow, fat and dental pulp. MSCs have been found to migrate towards inflammatory sites and studies have shown that they also migrate towards and incorporate into the tumour. The key question is how they interact there. MSCs may interact with tumour cells through paracrine signalling. On the other hand, MSCs have the capacity to differentiate to various cell types such as osteocytes, chondrocytes and adipocytes and it is possible that MSCs differentiate at the site of the tumour. More recently it has been shown that cross-talk between tumour cells and MSCs has been shown to increase metastatic potential and promote epithelial-to-mesenchymal transition. This review will focus on the role of MSCs in tumour development at various stages of progression from growth of the primary tumour to the establishment of distant metastasis.

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

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.

Human endogenous retrovirus K and cancer: Innocent bystander or tumorigenic accomplice?

Harbored as relics of ancient germline infections, human endogenous retroviruses (HERVs) now constitute up to 8% of our genome. A proportion of this sequence has been co-opted for molecular and cellular processes, beneficial to human physiology, such as the fusogenic activity of the envelope protein, a vital component of placentogenesis. However, the discovery of high levels of HERV-K mRNA and protein and even virions in a wide array of cancers has revealed that HERV-K may be playing a more sinister role-a role as an etiological agent in cancer itself. Whether the presence of this retroviral material is simply an epiphenomenon, or an actual causative factor, is a hotly debated topic. This review will summarize the current state of knowledge regarding HERV-K and cancer and attempt to outline the potential mechanisms by which HERV-K could be involved in the onset and promotion of carcinogenesis.

A close connection between the PERK and IRE arms of the UPR and the transcriptional regulation of autophagy.

Endoplasmic reticulum (ER) stress is known to lead to activation of both the unfolded protein response (UPR) and autophagy. Although regulatory connections have been identified between the UPR and autophagy, it is still unclear to what extent the UPR regulates the genes involved at the different stages of the autophagy pathway. Here, we carried out a microarray analysis of HCT116 cells subjected to ER stress and observed the transcriptional upregulation of a large cohort of autophagy-related genes. Of particular interest, we identified the transcriptional upregulation of the autophagy receptor genes SQSTM1/p62, NBR1 and BNIP3L/NIX in response to ER stress and show that the inhibition of the UPR transmembrane receptors, PERK and IRE1, abrogates this upregulation.

Elevated HERV-K mRNA expression in PBMC is associated with a prostate cancer diagnosis particularly in older men and smokers.

Aberrant expression of subgroup k human endogenous retroviruses (HERV-K) has been observed in prostate cancer. This subgroup is unique because it encodes sequences in the human genome containing open reading frames for near intact retroviruses. We hypothesized that HERV-K reactivation could serve as a non-invasive early disease detection marker for prostate cancer. We evaluated HERV-K gag messenger RNA (mRNA) expression in blood samples of African-American and European-American men using a case-control design via quantitative real-time PCR. Additionally, we examined HERV-K envelope protein expression in prostate tumors by immunohistochemistry. HERV-K envelope protein was commonly upregulated in prostate tumors, but more so in tumors of African-American than European-American patients (61% versus 40%, P < 0.01). Examining HERV-K gag expression in peripheral blood mononuclear cells (PBMC) from 294 cases and 135 healthy men, we found that the abundance of HERV-K gag message was significantly higher in cases than controls and was associated with increased plasma interferon-γ. Men with gag expression in the highest quartile had >12-fold increased odds {odds ratio = 12.87 [95% confidence interval 6.3-26.25]} of being diagnosed with prostate cancer than those in the lowest quartile. Moreover, our results showed that HERV-K expression may perform better as a disease biomarker in older than younger men (whereas the sensitivity of prostate-specific antigen (PSA) testing decreases with age) and in men with a smoking history compared with never smokers. Combining non-invasive HERV-K testing with PSA testing may improve the efficacy of prostate cancer detection specifically among older men and smokers who tend to develop a more aggressive disease.

The impact of nitric oxide on HER family post-translational modification and downstream signaling in cancer.

The human epidermal growth factor receptor (HER) family consists of four members, activated by two families of ligands. They are known for mediating cell-cell interactions in organogenesis, and their deregulation has been associated with various cancers, including breast and esophageal cancers. In particular, aberrant epidermal growth factor receptor (EGFR) and HER2 signaling drive disease progression and result in poorer patient outcomes. Nitric oxide (NO) has been proposed as an alternative activator of the HER family and may play a role in this aberrant activation due to its ability to induce s-nitrosation and phosphorylation of the EGFR. This review discusses the potential impact of NO on HER family activation and downstream signaling, along with its role in the efficacy of therapeutics targeting the family.

Adjuvant COX inhibition augments STING signaling and cytolytic T cell infiltration in irradiated 4T1 tumors.

Immune therapy is the new frontier of cancer treatment. Therapeutic radiation is a known inducer of immune response and can be limited by immunosuppressive mediators including cyclooxygenase-2 (COX2) that is highly expressed in aggressive triple negative breast cancer (TNBC). A clinical cohort of TNBC tumors revealed poor radiation therapeutic efficacy in tumors expressing high COX2. Herein, we show that radiation combined with adjuvant NSAID (indomethacin) treatment provides a powerful combination to reduce both primary tumor growth and lung metastasis in aggressive 4T1 TNBC tumors, which occurs in part through increased antitumor immune response. Spatial immunological changes including augmented lymphoid infiltration into the tumor epithelium and locally increased cGAS/STING1 and type I IFN gene expression were observed in radiation-indomethacin-treated 4T1 tumors. Thus, radiation and adjuvant NSAID treatment shifts "immune desert phenotypes" toward antitumor M1/TH1 immune mediators in these immunologically challenging tumors. Importantly, radiation-indomethacin combination treatment improved local control of the primary lesion, reduced metastatic burden, and increased median survival when compared with radiation treatment alone. These results show that clinically available NSAIDs can improve radiation therapeutic efficacy through increased antitumor immune response and augmented local generation of cGAS/STING1 and type I IFNs.

Tumor NOS2 and COX2 Spatial Juxtaposition with CD8+ T Cells Promote Metastatic and Cancer Stem Cell Niches that Lead to Poor Outcome in ER- Breast Cancer.

SIGNIFICANCE: This work identifies CD8-NOS2+COX2+ and CD8-NOS2-COX2+ unique cellular neighborhoods that drive the tumor immune spatial architecture of CD8+ T cells predictive of clinical outcome and can be targeted with clinically available NOS inhibitors and NSAIDs.

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.

Nitric Oxide-Scavenging, Anti-Migration Effects, and Glycosylation Changes after Hemin Treatment of Human Triple-Negative Breast Cancer Cells: A Mechanistic Study.

The enhanced expression of nitric oxide (•NO) synthase predicts triple-negative breast cancer outcome and its resistance to different therapeutics. Our earlier work demonstrated the efficiency of hemin to scavenge the intra- and extracellular •NO, proposing its potency as a therapeutic agent for inhibiting cancer cell migration. In continuation, the present work evaluates the effects of •NO on the migration of MDA-MB-231 cells and how hemin modulates the accompanied cellular behavior, focusing on the corresponding expression of cellular glycoproteins, migration-associated markers, and mitochondrial functions. We demonstrated for the first time that while •NO induced cell migration, hemin contradicted that by •NO-scavenging. This was in combination with modulation of the •NO-enhanced glycosylation patterns of cellular proteins with inhibition of the expression of specific proteins involved in the epithelial-mesenchymal transition. These effects were in conjunction with changes in the mitochondrial functions related to both •NO, hemin, and its nitrosylated product. Together, these results suggest that hemin can be employed as a potential anti-migrating agent targeting •NO-scavenging and regulating the expression of migration-associated proteins.