RESUMO
Nitric oxide (NO) and reactive nitrogen species (RNS) exert profound biological impacts dictated by their chemistry. Understanding their spatial distribution is essential for deciphering their roles in diverse biological processes. This review establishes a framework for the chemical biology of NO and RNS, exploring their dynamic reactions within the context of cancer. Concentration-dependent signaling reveals distinctive processes in cancer, with three levels of NO influencing oncogenic properties. In this context, NO plays a crucial role in cancer cell proliferation, metastasis, chemotherapy resistance, and immune suppression. Increased NOS2 expression correlates with poor survival across different tumors, including breast cancer. Additionally, NOS2 can crosstalk with the proinflammatory enzyme cyclooxygenase-2 (COX-2) to promote cancer progression. NOS2 and COX-2 co-expression establishes a positive feed-forward loop, driving immunosuppression and metastasis in estrogen receptor-negative (ER-) breast cancer. Spatial evaluation of NOS2 and COX-2 reveals orthogonal expression, suggesting the unique roles of these niches in the tumor microenvironment (TME). NOS2 and COX2 niche formation requires IFN-γ and cytokine-releasing cells. These niches contribute to poor clinical outcomes, emphasizing their role in cancer progression. Strategies to target these markers include direct inhibition, involving pan-inhibitors and selective inhibitors, as well as indirect approaches targeting their induction or downstream effectors. Compounds from cruciferous vegetables are potential candidates for NOS2 and COX-2 inhibition offering therapeutic applications. Thus, understanding the chemical biology of NO and RNS, their spatial distribution, and their implications in cancer progression provides valuable insights for developing targeted therapies and preventive strategies.
Assuntos
Neoplasias da Mama , Ciclo-Oxigenase 2 , Progressão da Doença , Óxido Nítrico Sintase Tipo II , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Ciclo-Oxigenase 2/metabolismo , Feminino , Óxido Nítrico Sintase Tipo II/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Animais , Óxido Nítrico/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismoRESUMO
BACKGROUND: There is a need to improve the treatment of prostate cancer (PCa) and reduce treatment side effects. Vascular-targeted photodynamic therapy (VTP) is a focal therapy for low-risk low-volume localised PCa, which rapidly disrupts targeted tumour vessels. There is interest in expanding the use of VTP to higher-risk disease. Tumour vasculature is characterised by vessel immaturity, increased permeability, aberrant branching and inefficient flow. FRT alters the tumour microenvironment and promotes transient 'vascular normalisation'. We hypothesised that multimodality therapy combining fractionated radiotherapy (FRT) and VTP could improve PCa tumour control compared against monotherapy with FRT or VTP. METHODS: We investigated whether sequential delivery of FRT followed by VTP 7 days later improves flank TRAMP-C1 PCa tumour allograft control compared to monotherapy with FRT or VTP. RESULTS: FRT induced 'vascular normalisation' changes in PCa flank tumour allografts, improving vascular function as demonstrated using dynamic contrast-enhanced magnetic resonance imaging. FRT followed by VTP significantly delayed tumour growth in flank PCa allograft pre-clinical models, compared with monotherapy with FRT or VTP, and improved overall survival. CONCLUSION: Combining FRT and VTP may be a promising multimodal approach in PCa therapy. This provides proof-of-concept for this multimodality treatment to inform early phase clinical trials.
Assuntos
Neovascularização Patológica/terapia , Fotoquimioterapia/métodos , Neoplasias da Próstata/terapia , Animais , Linhagem Celular Tumoral , Terapia Combinada , Fracionamento da Dose de Radiação , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Neoplasias da Próstata/irrigação sanguínea , Análise de Sobrevida , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mechanistic roles of nitric oxide (NO) during cancer progression have been important considerations since its discovery as an endogenously generated free radical. Nonetheless, the impacts of this signaling molecule can be seemingly contradictory, being both pro-and antitumorigenic, which complicates the development of cancer treatments based on the modulation of NO fluxes in tumors. At a fundamental level, low levels of NO drive oncogenic pathways, immunosuppression, metastasis, and angiogenesis, while higher levels lead to apoptosis and reduced hypoxia and also sensitize tumors to conventional therapies. However, clinical outcome depends on the type and stage of the tumor as well as the tumor microenvironment. In this Viewpoint, the current understanding of the concentration, spatial, and temporal dependence of responses to NO is correlated with potential treatment and prevention technologies and strategies.
Assuntos
Óxido NítricoRESUMO
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.
Assuntos
Neoplasias/imunologia , Óxido Nítrico/imunologia , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Animais , Humanos , Neoplasias/patologiaRESUMO
BACKGROUND: Radiotherapy enhances innate and adaptive anti-tumour immunity. It is unclear whether this effect may be harnessed by combining immunotherapy with radiotherapy fractions used to treat prostate cancer. We investigated tumour immune microenvironment responses of pre-clinical prostate cancer models to radiotherapy. Having defined this landscape, we tested whether radiotherapy-induced tumour growth delay could be enhanced with anti-PD-L1. METHODS: Hypofractionated radiotherapy was delivered to TRAMP-C1 and MyC-CaP flank allografts. Tumour growth delay, tumour immune microenvironment flow-cytometry, and immune gene expression were analysed. TRAMP-C1 allografts were then treated with 3 × 5 Gy ± anti-PD-L1. RESULTS: 3 × 5 Gy caused tumour growth delay in TRAMP-C1 and MyC-CaP. Tumour immune microenvironment changes in TRAMP-C1 at 7 days post-radiotherapy included increased tumour-associated macrophages and dendritic cells and upregulation of PD-1/PD-L1, CD8+ T-cell, dendritic cell, and regulatory T-cell genes. At tumour regrowth post-3 × 5 Gy the tumour immune microenvironment flow-cytometry was similar to control tumours, however CD8+, natural killer and dendritic cell gene transcripts were reduced. PD-L1 inhibition plus 3 × 5 Gy in TRAMP-C1 did not enhance tumour growth delay versus monotherapy. CONCLUSION: 3 × 5 Gy hypofractionated radiotherapy can result in tumour growth delay and immune cell changes in allograft prostate cancer models. Adjuncts beyond immunomodulation may be necessary to improve the radiotherapy-induced anti-tumour response.
Assuntos
Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias da Próstata/terapia , Hipofracionamento da Dose de Radiação , Microambiente Tumoral , Animais , Antígeno B7-H1/análise , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologiaRESUMO
BACKGROUND: Metabolic reprogramming is being recognized as a fundamental hallmark of cancer, and efforts to identify drugs that can target cancer metabolism are underway. In this study, we used human breast cancer (BC) cell lines and established their invading phenotype (INV) collected from transwell inserts to compare metabolome differences and evaluate prognostic significance of the metabolome in aggressive BC invasiveness. METHODS: The invasiveness of seven human BC cell lines were compared using the transwell invasion assay. Among these, INV was collected from SUM149, which exhibited the highest invasiveness. Levels of metabolites in INV were compared with those of whole cultured SUM149 cells (WCC) using CE-TOFMS. The impact of glycolysis in INV was determined by glucose uptake assay using fluorescent derivative of glucose (2-NBDG), and significance of glycolysis, or tricarboxylic acid cycle (TCA) and electron transport chain (ETC) in the invasive process were further determined in aggressive BC cell lines, SUM149, MDA-MB-231, HCC1937, using invasion assays in the presence or absence of inhibitors of glycolysis, TCA cycle or ETC. RESULTS: SUM149 INV sub-population exhibited a persistent hyperinvasive phenotype. INV were hyper-glycolytic with increased glucose (2-NBDG) uptake; diminished glucose-6-phosphate (G6P) levels but elevated pyruvate and lactate, along with higher expression of phosphorylated-pyruvate dehydrogenase (pPDH) compared to WCC. Notably, inhibiting of glycolysis with lower doses of 2-DG (1 mM), non-cytotoxic to MDA-MB-231 and HCC1937, was effective in diminishing invasiveness of aggressive BC cell lines. In contrast, 3-Nitropropionic acid (3-NA), an inhibitor of succinate dehydrogenase, the enzyme that oxidizes succinate to fumarate in TCA cycle, and functions as complex II of ETC, had no significant effect on their invasiveness, although levels of TCA metabolites or detection of mitochondrial membrane potential with JC-1 staining, indicated that INV cells originally had functional TCA cycles and membrane potential. CONCLUSIONS: Hyper-glycolytic phenotype of invading cells caters to rapid energy production required for invasion while TCA cycle/ETC cater to cellular energy needs for sustenance in aggressive BC. Lower, non-cytotoxic doses of 2-DG can hamper invasion and can potentially be used as an adjuvant with other anti-cancer therapies without the usual side-effects associated with cytotoxic doses.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Neoplasias da Mama/tratamento farmacológico , Reprogramação Celular/efeitos dos fármacos , Desoxiglucose/análogos & derivados , Invasividade Neoplásica/genética , 4-Cloro-7-nitrobenzofurazano/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/genética , Ciclo do Ácido Cítrico/efeitos dos fármacos , Desoxiglucose/farmacologia , Feminino , Glucose/metabolismo , Glucose/farmacologia , Glicólise/efeitos dos fármacos , Humanos , Metaboloma/genética , Invasividade Neoplásica/patologiaRESUMO
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.
Assuntos
Neoplasias da Mama/genética , Ciclo-Oxigenase 2/genética , Regulação Neoplásica da Expressão Gênica , Óxido Nítrico Sintase Tipo II/genética , Receptores de Estrogênio/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Aspirina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retroalimentação Fisiológica , Feminino , Guanidinas/farmacologia , Humanos , Indometacina/farmacologia , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , Receptores de Estrogênio/deficiência , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Nitric oxide (NO) is implicated in several biological processes, including cancer progression. At low concentrations, it promotes cell survival and tumor progression, and at high concentrations it causes apoptosis and cell death. Until now, the impact of NO donors has not been investigated on human endometrial tumors. Four cancer cell lines were exposed to different concentrations of DETA/NO for 24 to 120 h. The effects of DETA/NO on cell proliferation and invasion were determined utilizing MTS and Boyden chamber assays, respectively. The DETA/NO induced a dose and time-dependent reduction in cell viability by the activation of caspase-3 and cell cycle arrest at the G0/G1 phase that was associated with the attenuated expression of cyclin-D1 and D3. Furthermore, the reduction in the amount of CD133-expressing cancer stem-like cell subpopulation was observed following DETA/NO treatment of cells, which was associated with a decreased expression of stem cell markers and attenuation of cell invasiveness. To understand the mechanisms by which DETA/NO elicits anti-cancer effects, RNA sequencing (RNA-seq) was used to ascertain alterations in the transcriptomes of human endometrial cancer cells. RNA-seq analysis revealed that 14 of the top 21 differentially expressed genes were upregulated and seven were downregulated in endometrial cancer cells with DETA/NO. The genes that were upregulated in all four cell lines with DETA/NO were the tumor suppressors Ras association domain family 1 isoform A (RASSF1) and Cyclin-dependent kinase inhibitor 1A (CDKN1A). The expression patterns of these genes were confirmed by Western blotting. Taken together, the results provide the first evidence in support of the anti-cancer effects of DETA/NO in endometrial cancer.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Neoplasias do Endométrio/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Proteínas Supressoras de Tumor/genética , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Óxido Nítrico/metabolismo , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Progressão da Doença , Retroalimentação Fisiológica , Óxido Nítrico Sintase Tipo II/metabolismo , Microambiente Tumoral , Animais , Biomarcadores Tumorais/metabolismo , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura Livres de Soro , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Feminino , Guanidinas , Humanos , Interferon gama/farmacologia , Camundongos , Modelos Biológicos , Metástase Neoplásica , Óxido Nítrico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Modulating tissue responses to stress is an important therapeutic objective. Oxidative and genotoxic stresses caused by ionizing radiation are detrimental to healthy tissues but beneficial for treatment of cancer. CD47 is a signaling receptor for thrombospondin-1 and an attractive therapeutic target because blocking CD47 signaling protects normal tissues while sensitizing tumors to ionizing radiation. Here we utilized a metabolomic approach to define molecular mechanisms underlying this radioprotective activity. CD47-deficient cells and cd47-null mice exhibited global advantages in preserving metabolite levels after irradiation. Metabolic pathways required for controlling oxidative stress and mediating DNA repair were enhanced. Some cellular energetics pathways differed basally in CD47-deficient cells, and the global declines in the glycolytic and tricarboxylic acid cycle metabolites characteristic of normal cell and tissue responses to irradiation were prevented in the absence of CD47. Thus, CD47 mediates signaling from the extracellular matrix that coordinately regulates basal metabolism and cytoprotective responses to radiation injury.
Assuntos
Antígeno CD47/metabolismo , Redes e Vias Metabólicas/efeitos da radiação , Tolerância a Radiação , Animais , Antígeno CD47/genética , Ciclo do Ácido Cítrico/efeitos da radiação , Metabolismo Energético/efeitos da radiação , Deleção de Genes , Homeostase/efeitos da radiação , Humanos , Células Jurkat , Metabolômica , Camundongos , Nucleotídeos/biossíntese , Estresse Oxidativo/efeitos da radiação , Via de Pentose Fosfato/efeitos da radiaçãoRESUMO
Small redox active molecules such as reactive nitrogen and oxygen species and hydrogen sulfide have emerged as important biological mediators that are involved in various physiological and pathophysiological processes. Advancement in understanding of cellular mechanisms that tightly regulate both generation and reactivity of these molecules is central to improved management of various disease states including cancer and cardiovascular dysfunction. Imbalance in the production of redox active molecules can lead to damage of critical cellular components such as cell membranes, proteins and DNA and thus may trigger the onset of disease. These small inorganic molecules react independently as well as in a concerted manner to mediate physiological responses. This review provides a general overview of the redox biology of these key molecules, their diverse chemistry relevant to physiological processes and their interrelated nature in cellular signaling.
RESUMO
Nitric oxide (NO) synthase 2 (NOS2), a major inflammatory protein, modulates disease progression via NO in a number of pathologies, including cancer. The role of NOS2-derived NO is not only flux-dependent, which is higher in mouse vs human cells, but also varies based on spatial and temporal distribution both within tumor cells and in the tumor microenvironment. NO donors have been utilized to mimic NO flux conditions and to investigate the effects of varied NO concentrations. As a wide range of effects mediated by NO and other nitrogen oxides such as nitroxyl (HNO) have been elucidated, multiple NO- and HNO-releasing compounds have been developed as potential therapeutics, including as tumor modulators. One of the challenges is to determine differences in biomarker expression from extracellular vs intracellular generation of NO or HNO. Taking advantage of new NO and HNO releasing agents, we have characterized the gene expression profile of estrogen receptor-negative human breast cancer (MDA-MB-231) cells following exposure to aspirin, the NO donor DEA/NO, the HNO donor IPA/NO andtheir intracellularly-activated prodrug conjugates DEA/NO-aspirin and IPA/NO-aspirin. Comparison of the gene expression profiles demonstrated that several genes were uniquely expressed with respect to NO or HNO, such as miR-21, HSP70, cystathionine γ-lyase and IL24. These findings provide insight into targets and pathways that could be therapeutically exploited by the redox related species NO and HNO.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/farmacologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Óxido Nítrico Sintase/metabolismo , Transdução de SinaisRESUMO
RATIONALE: In the myocardium, redox/cysteine modification of proteins regulating Ca(2+) cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery. OBJECTIVE: To determine the effects of HNO modification in cardiac myofilament proteins. METHODS AND RESULTS: The HNO-donor, 1-nitrosocyclohexyl acetate, was found to act directly on the myofilament proteins, increasing maximum force (F(max)) and reducing the concentration of Ca(2+) for 50% activation (Ca(50)) in intact and skinned cardiac muscles. The effects of 1-nitrosocyclohexyl acetate are reversible by reducing agents and distinct from those of another HNO donor, Angeli salt, which was previously reported to increase F(max) without affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide-linked actin-tropomyosin and myosin heavy chain-myosin light chain 1. Comparison of the 1-nitrosocyclohexyl acetate and Angeli salt effects with the modifications induced by each donor indicated the actin-tropomyosin and myosin heavy chain-myosin light chain 1 interactions independently correlated with increased Ca(2+) sensitivity and force generation, respectively. CONCLUSIONS: HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca(2+) responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus, which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.
Assuntos
Dissulfetos/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/fisiologia , Miofibrilas/fisiologia , Óxidos de Nitrogênio/metabolismo , Acetatos/metabolismo , Acetatos/farmacologia , Actinas/química , Actinas/metabolismo , Animais , Cálcio/metabolismo , Cisteína/química , Cisteína/metabolismo , Dimerização , Dissulfetos/química , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Técnicas In Vitro , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miofibrilas/efeitos dos fármacos , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/química , Compostos Nitrosos/metabolismo , Compostos Nitrosos/farmacologia , Oxirredução , RatosRESUMO
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic that induces apoptosis in cancer cells while sparing the non-malignant cells in preclinical models. However, its efficacy in clinical trials has been limited, suggesting unknown modulatory mechanisms responsible for the lack of TRAIL activity in patients. Here, we hypothesized that TRAIL treatment elicits transcriptional changes in triple negative breast cancer (TNBC) cells that alter the immune milieu. To test this, we performed an RNAseq analysis of MDA-MB-231 cells treated with TRAIL, followed by validation in additional TNBC cell lines. TRAIL significantly induces expression of multiple cytokines such as CXCLs 1, 2, 3, 8,11 and IL-6, which are known to modify neutrophil function. Mechanistically, the induction of these cytokines was predominantly mediated by death receptor 5, caspase 8 (but not caspase 8 enzymatic activity), and the non-canonical NFKB2 pathway. The cytokines produced by the TRAIL-treated TNBC cells enhanced chemotaxis of healthy human donor isolated neutrophils. In vivo , TRAIL treated TNBC murine xenograft tumors showed activation of the NFKB2 pathway, elevated production of CXCLs and IL-6, and increased neutrophil recruitment into the tumors. Moreover, donor isolated neutrophils preincubated in supernatants from TRAIL-treated TNBC cells exhibited impaired cytotoxic effect against TNBC cells. Transcriptomic analysis of neutrophils incubated with either TRAIL alone or supernatant of TRAIL-treated TNBC cells revealed increased expression of inflammatory cytokines, immune modulatory genes, immune checkpoint genes, and genes implicated in delayed neutrophil apoptosis. Functional studies with these neutrophils confirmed their suppressive effect on T cell proliferation and an increase in Treg suppressive phenotype. Collectively, our study demonstrates a novel role of TRAIL-induced NFKB2-dependent cytokine production that promotes neutrophil chemotaxis and immune suppression.
RESUMO
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.
Assuntos
Proteínas de Membrana , Transdução de Sinais , Linfócitos T Citotóxicos , Animais , Proteínas de Membrana/metabolismo , Camundongos , Feminino , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/radioterapia , Indometacina/farmacologia , Indometacina/uso terapêutico , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Nucleotidiltransferases/metabolismo , Interferon Tipo I/metabolismo , Ciclo-Oxigenase 2/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Neoplasias da Mama , Linfócitos T CD8-Positivos , Ciclo-Oxigenase 2 , Células-Tronco Neoplásicas , Óxido Nítrico Sintase Tipo II , Linfócitos T CD8-Positivos/imunologia , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/imunologia , Feminino , Ciclo-Oxigenase 2/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/imunologia , Óxido Nítrico Sintase Tipo II/metabolismo , Nicho de Células-Tronco , Animais , Camundongos , Receptores de Estrogênio/metabolismo , Metástase Neoplásica , Microambiente Tumoral/imunologiaRESUMO
Nitric oxide (NO) and the enzyme that synthesizes it, nitric oxide synthase 2 (NOS2), have emerged as key players in inflammation and cancer. Expression of NOS2 in tumors has been correlated both with positive outcomes and with poor prognoses. The chemistry of NO is the major determinate to the biological outcome and the concentration of NO, which can range over five orders of magnitude, is critical in determining which pathways are activated. It is the activation of specific oncogenic and immunological mechanisms that shape the outcome. The kinetics of specific reactions determine the mechanisms of action. In this review, the relevant reactions of NO and related species are discussed with respect to these oncogenic and immunological signals.
Assuntos
Neoplasias , Óxido Nítrico Sintase Tipo II , Óxido Nítrico , Humanos , Neoplasias/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Transdução de SinaisRESUMO
Utilizing targeted therapies capable of reducing cancer metastasis, targeting chemoresistant and self-renewing cancer stem cells, and augmenting the efficacy of systemic chemo/radiotherapies is vital to minimize cancer-associated mortality. Targeting nitric oxide synthase (NOS), a protein within the tumor microenvironment, has gained interest as a promising therapeutic strategy to reduce metastatic capacity and augment the efficacy of chemo/radiotherapies in various solid malignancies. Our review highlights the influence of nitric oxide (NO) in tumor progression and cancer metastasis, as well as promising preclinical studies that evaluated NOS inhibitors as anticancer therapies. Lastly, we highlight the prospects and outstanding challenges of using NOS inhibitors in the clinical setting.
Assuntos
Neoplasias , Óxido Nítrico , Humanos , Óxido Nítrico/metabolismo , Neoplasias/tratamento farmacológico , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Microambiente TumoralRESUMO
The small endogenous signaling molecule nitric oxide (NO) has been linked with chronic inflammation and cancer. The effects of NO are both concentration and temporally dependent; under some conditions, NO protects against damage caused by reactive oxygen species and activates P53 signaling. During chronic inflammation, NO causes DNA damage and inhibits repair proteins. To extend our understanding of the roles of NO during carcinogenesis, we investigated the possible effects of chronic NO exposure on MCF10A breast epithelial cells, as defined by changes in cellular morphology, chromosome/genomic stability, RNA, and protein expression, and altered cell phenotypes. Human MCF10A cells were maintained in varying doses of the NO donor DETANO for three weeks. Distinct patterns of genomic modifications in TP53 and KRAS target genes were detected in NO-treated cells when compared to background mutations. In addition, quantitative real-time PCR demonstrated an increase in the expression of cancer stem cell (CSC) marker CD44 after prolonged exposure to 300 µM DETANO. While similar changes in cell morphology were found in cells exposed to 300-500 µM DETANO, cells cultured in 100 µM DETANO exhibited enhanced motility. In addition, 100 µM NO-treated cells proliferated in serum-free media and selected clonal populations and pooled cells formed colonies in soft agar that were clustered and disorganized. These findings show that chronic exposure to NO generates altered breast epithelial cell phenotypes with malignant characteristics.
Assuntos
Neoplasias da Mama , Óxido Nítrico , Humanos , Feminino , Óxido Nítrico/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células Epiteliais/metabolismo , Mutação , Inflamação/metabolismo , Neoplasias da Mama/metabolismoRESUMO
M1 macrophages enter a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase 2 and pyruvate dehydrogenase (PDH) activity. Here, we provide evidence that NO targets the PDH complex by using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 enzyme, also known as dihydrolipoamide dehydrogenase (DLD), is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated production of HNO, which interacts with thiols forming irreversible modifications including sulfinamide. In addition, we reveal a macrophage signature of proteins with reduction-resistant modifications, including in DLD, and identify potential HNO targets. Consistently, DLD enzyme is modified in an HNO-dependent manner at Cys477 and Cys484, and molecular modeling and mutagenesis show these modifications impair the formation of DLD homodimers. In conclusion, our work demonstrates that HNO is produced physiologically. Moreover, the production of HNO is dependent on the lipoate-rich PDH complex facilitating irreversible modifications that are critical to NO-dependent metabolic rewiring.