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
A strong correlation between NOS2 and COX2 tumor expression and poor clinical outcomes in ER breast cancer has been established. However, the mechanisms of tumor induction of these enzymes are unclear. Analysis of The Cancer Genome Atlas (TCGA) revealed correlations between NOS2 and COX2 expression and Th1 cytokines. Herein, single-cell RNAseq analysis of TNBC cells shows potent NOS2 and COX2 induction by IFNγ combined with IL1ß or TNFα. Given that IFNγ is secreted by cytolytic lymphocytes, which improve clinical outcomes, this role of IFNγ presents a dichotomy. To explore this conundrum, tumor NOS2, COX2, and CD8+ T cells were spatially analyzed in aggressive ER-, TNBC, and HER2 + breast tumors. High expression and clustering of NOS2-expressing tumor cells occurred at the tumor/stroma interface in the presence of stroma-restricted CD8+ T cells. High expression and clustering of COX2-expressing tumor cells extended into immune desert regions in the tumor core where CD8+ T cell penetration was limited or absent. Moreover, high NOS2-expressing tumor cells were proximal to areas with increased satellitosis, suggestive of cell clusters with a higher metastatic potential. Further in vitro experiments revealed that IFNγ + IL1ß/TNFα increased the elongation and migration of treated tumor cells. This spatial analysis of the tumor microenvironment provides important insight into distinct neighborhoods where stroma-restricted CD8+ T cells exist proximal to NOS2-expressing tumor niches that could have increased metastatic potential.
Assuntos
Interferon gama , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Feminino , Humanos , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Antitumor immune polarization is a key predictor of clinical outcomes to cancer therapy. An emerging concept influencing clinical outcome involves the spatial location of CD8+ T cells, within the tumor. Our earlier work demonstrated immunosuppressive effects of NOS2 and COX2 tumor expression. Here, we show that NOS2/COX2 levels influence both the polarization and spatial location of lymphoid cells including CD8+ T cells. Importantly, elevated tumor NOS2/COX2 correlated with exclusion of CD8+ T cells from the tumor epithelium. In contrast, tumors expressing low NOS2/COX2 had increased CD8+ T cell penetration into the tumor epithelium. Consistent with a causative relationship between these observations, pharmacological inhibition of COX2 with indomethacin dramatically reduced tumor growth of the 4T1 model of TNBC in both WT and Nos2- mice. This regimen led to complete tumor regression in â¼20-25% of tumor-bearing Nos2- mice, and these animals were resistant to tumor rechallenge. Th1 cytokines were elevated in the blood of treated mice and intratumoral CD4+ and CD8+ T cells were higher in mice that received indomethacin when compared to control untreated mice. Multiplex immunofluorescence imaging confirmed our phenotyping results and demonstrated that targeted Nos2/Cox2 blockade improved CD8+ T cell penetration into the 4T1 tumor core. These findings are consistent with our observations in low NOS2/COX2 expressing breast tumors proving that COX2 activity is responsible for limiting the spatial distribution of effector T cells in TNBC. Together these results suggest that clinically available NSAID's may provide a cost-effective, novel immunotherapeutic approach for treatment of aggressive tumors including triple negative breast cancer.
Assuntos
Neoplasias de Mama Triplo Negativas , Humanos , Camundongos , Animais , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Orientação Espacial , Imunoterapia , Progressão da Doença , Linfócitos/metabolismo , Indometacina/farmacologia , Indometacina/metabolismo , Indometacina/uso terapêuticoRESUMO
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
The tumor microenvironment (TME) is multi-cellular, spatially heterogenous, and contains cell-generated gradients of soluble molecules. Current cell-based model systems lack this complexity or are difficult to interrogate microscopically. We present a 2D live-cell chamber that approximates the TME and demonstrate that breast cancer cells and macrophages generate hypoxic and nutrient gradients, self-organize, and have spatially varying phenotypes along the gradients, leading to new insights into tumorigenesis.
Assuntos
Neoplasias da Mama/fisiopatologia , Carcinogênese , Macrófagos/fisiologia , Células Tumorais Cultivadas/fisiologia , Microambiente Tumoral , Animais , Técnicas de Cultura de Células , CamundongosRESUMO
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
Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.
Assuntos
Neoplasias de Mama Triplo Negativas/patologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Neoplasias Pulmonares/secundário , Camundongos , Fenótipo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The role of nitric oxide (NO) in cancer progression has largely been studied in the context of tumor NOS2 expression. However, pro- versus anti-tumor signaling is also affected by tumor cell-macrophage interactions. While these cell-cell interactions are partly regulated by NO, the functional effects of NO flux on proinflammatory (M1) macrophages are unknown. Using a triple negative murine breast cancer model, we explored the potential role of macrophage Nos2 on 4T1 tumor progression. The effects of NO on macrophage phenotype were examined in bone marrow derived macrophages from wild type and Nos2-/- mice following in vitro stimulation with cytokine/LPS combinations to produce low, medium, and high NO flux. Remarkably, Nos2 induction was spatially distinct, where Nos2high cells expressed low cyclooxygenase-2 (Cox2) and vice versa. Importantly, in vitro M1 polarization with IFNγ+LPS induced high NO flux that was restricted to cells harboring depolarized mitochondria. This flux altered the magnitude and spatial extent of hypoxic gradients. Metabolic and single cell analyses demonstrated that single cell Nos2 induction limited the generation of hypoxic gradients in vitro, and Nos2-dependent and independent features may collaborate to regulate M1 functionality. It was found that Cox2 expression was important for Nos2high cells to maintain NO tolerance. Furthermore, Nos2 and Cox2 expression in 4T1 mouse tumors was spatially orthogonal forming distinct cellular neighborhoods. In summary, the location and type of Nos2high cells, NO flux, and the inflammatory status of other cells, such as Cox2high cells in the tumor niche contribute to Nos2 inflammatory mechanisms that promote disease progression of 4T1 tumors.
Assuntos
Citocinas/metabolismo , Lipopolissacarídeos/efeitos adversos , Óxido Nítrico Sintase Tipo II/genética , Neoplasias de Mama Triplo Negativas/patologia , Animais , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Transplante de Neoplasias , Óxido Nítrico/metabolismo , Análise de Célula Única , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Pancreatic cancer is a highly metastatic tumor with an extremely low 5-year survival rate. Lack of efficient diagnostics and dearth of effective therapeutics that can target the cancer as well as the microenvironment niche are the reasons for limited success in treatment and management of this disease. Cell invasion through extracellular matrix (ECM) involves the complex regulation of adhesion to and detachment from ECM and its understanding is critical to metastatic potential of pancreatic cancer. To understand the characteristics of these cancer cells and their ability to metastasize, we compared human pancreatic cancer cell line, PANC-1 and its invading phenotype (INV) collected from transwell inserts. The invasive cell type, INV, exhibited higher resistance to Carbon-ion radiation compared to whole cultured (normally dish-cultured) PANC-1 (WCC), and had more efficient in vitro spheroid formation capability. Invasiveness of INV was hampered by nitric oxide synthase (NOS) inhibitors, suggesting that nitric oxide (NO) plays a cardinal role in PANC-1 invasion. In addition, in vitro studies indicated that a MEK-ERK-dependent, JAK independent mechanism through which NOS/NO modulate PANC-1 invasiveness. Suspended INV showed enhanced NO production as well as induction of several pro-metastatic, and stemness-related genes. NOS inhibitor, l-NAME, reduced the expression of these pro-metastatic or stemness-related genes, and dampened spheroid formation ability, suggesting that NO can potentially influence pancreatic cancer aggressiveness. Furthermore, xenograft studies with INV and WCC in NSG mouse model revealed a greater ability of INV compared to WCC, to metastasize to the liver and l-NAME diminished the metastatic lesions in mice injected with INV. Overall, data suggest that NO is a key player associated with resistance to radiation and metastasis of pancreatic cancer; and inhibition of NOS demonstrates therapeutic potential as observed in the animal model by specifically targeting the metastatic cells that harbor stem-like features and are potentially responsible for relapse.
Assuntos
Óxido Nítrico/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fenótipo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunofluorescência , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Invasividade Neoplásica , Células-Tronco Neoplásicas/metabolismo , Óxido Nítrico Sintase/metabolismoRESUMO
SIGNIFICANCE: Cancer is a complex disease, which not only involves the tumor but its microenvironment comprising different immune cells as well. Nitric oxide (NO) plays specific roles within tumor cells and the microenvironment and determines the rate of cancer progression, therapy efficacy, and patient prognosis. Recent Advances: Key understanding of the processes leading to dysregulated NO flux within the tumor microenvironment over the past decade has provided better understanding of the dichotomous role of NO in cancer and its importance in shaping the immune landscape. It is becoming increasingly evident that nitric oxide synthase 2 (NOS2)-mediated NO/reactive nitrogen oxide species (RNS) are heavily involved in cancer progression and metastasis in different types of tumor. More recent studies have found that NO from NOS2+ macrophages is required for cancer immunotherapy to be effective. CRITICAL ISSUES: NO/RNS, unlike other molecules, are unique in their ability to target a plethora of oncogenic pathways during cancer progression. In this review, we subcategorize the different levels of NO produced by cells and shed light on the context-dependent temporal effects on cancer signaling and metabolic shift in the tumor microenvironment. FUTURE DIRECTIONS: Understanding the source of NO and its spaciotemporal profile within the tumor microenvironment could help improve efficacy of cancer immunotherapies by improving tumor infiltration of immune cells for better tumor clearance.
Assuntos
Neoplasias/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Óxido Nítrico Sintase Tipo II/metabolismo , Prognóstico , Transdução de Sinais , Microambiente TumoralRESUMO
Recent findings suggest that co-expression of NOS2 and COX2 is a strong prognostic indicator in triple-negative breast cancer patients. These two key inflammation-associated enzymes are responsible for the biosynthesis of NO and PGE2 , respectively, and can exert their effect in both an autocrine and paracrine manner. Impairment of their physiological regulation leads to critical changes in both intra-tumoural and intercellular communication with the immune system and their adaptation to the hypoxic tumour micro-environment. Recent studies have also established a key role of NOS2-COX2 in causing metabolic shift. This review provides an extensive overview of the role of NO and PGE2 in shaping communication between the tumour micro-environment composed of tumour and immune cells that in turn favours tumour progression and metastasis. LINKED ARTICLES: This article is part of a themed section on Nitric Oxide 20 Years from the 1998 Nobel Prize. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.2/issuetoc.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Humanos , Óxido Nítrico/metabolismo , Microambiente TumoralRESUMO
It is known that Cancer Associated Fibroblast (CAFs) from the primary tumor site can accompany cancer cells to a secondary site during the process of metastasis. We hypothesize that these CAFs could be transformed to an altered cell type, which can be called as Metastasis Associated Fibroblasts (MAF) in turn can support, and convoy cancer cells for metastasis. There are no published reports that have characterized and distinguished CAFs from MAF. It is well established that some of the cancer cells within the tumor mass accumulate novel mutations prior to metastasis. Hence, we speculated that mutations in the tumor suppressor gene, BRCA1, which is already reported to induce metastasis via abnormal expression of Ezrin, Radixin and Moesin (ERM), could generate MAF. In the present study, we demonstrate for the first time that CAFs isolated from primary breast cancer tissues when co-cultured with BRCA1 mutated HCC1937 cells transform CAFs to MAF in vitro. As expected, MAF augmented proliferation, migration and invasion along with over-expression of epithelial mesenchymal transition (EMT) markers, Ezrin and CCL5, thereby facilitating metastasis. Therefore, we inhibited Ezrin and CCL5 in vitro in MAF and observed that the migration and invasion abilities of these cells were attenuated. This highlights the intriguing possibilities of combination therapy using MAF inhibitors as anti-metastatic agents along with anticancer drugs, to control the metastatic spread from primary tumor site.
Assuntos
Neoplasias da Mama/patologia , Transformação Celular Neoplásica , Genes BRCA1 , Metástase Neoplásica , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Transição Epitelial-Mesenquimal , Feminino , Fibroblastos/patologia , HumanosRESUMO
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
SIGNIFICANCE: Breast cancer is the second leading cause of cancer-related deaths among women in the United States. Development and progression of malignancy are associated with diverse cell signaling pathways that control cell proliferation, survival, motility, invasion, and metastasis. Recent Advances: An increasing number of clinical studies have implicated a strong relationship between elevated tumor nitric oxide synthase-2 (NOS2) expression and poor patient survival. CRITICAL ISSUES: Herein, we review what we believe to be key mechanisms in the role(s) of NOS2-derived nitric oxide (NO) as a driver of breast cancer disease progression. High NO increases cyclooxygenase-2 activity, hypoxia inducible factor-1 alpha protein stabilization, and activation of important cell signaling pathways, including phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinase, epidermal growth factor receptor, and Ras, through post-translational protein modifications. Moreover, dysregulated NO flux within the tumor microenvironment has other important roles, including the promotion of angiogenesis and modulation of matrix metalloproteinase/tissue inhibitor matrix metalloproteinase associated with tumor progression. FUTURE DIRECTIONS: The elucidation of these and other NO-driven pathways implicates NOS2 as a key driver of breast cancer disease progression and provides a new perspective in the identification of novel targets that may be therapeutically beneficial in the treatment of estrogen receptor-negative disease. Antioxid. Redox Signal. 26, 1044-1058.
Assuntos
Neoplasias da Mama/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico/metabolismo , Animais , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Estabilidade Proteica , Transdução de Sinais , Microambiente TumoralRESUMO
SIGNIFICANCE: Gastrointestinal (GI) cancer taken together constitutes one of the most common cancers worldwide with a broad range of etiological mechanisms. In this review, we have examined the impact of nitric oxide (NO) on the etiology of colon, colorectal, gastric, esophageal, and liver cancers. Recent Advances: Despite differences in etiology, initiation, and progression, chronic inflammation has been shown to be a common element within these cancers showing interactions of numerous pathways. NO generated at the inflammatory site contributes to the initiation and progression of disease. The amount of NO generated, time, and site vary and are an important determinant of the biological effects initiated. Among the nitric oxide synthase enzymes, the inducible isoform has the most diverse range, participating in numerous carcinogenic processes. There is emerging evidence showing that inducible nitric oxide synthase (NOS2) plays a central role in the process of tumor initiation and/or development. CRITICAL ISSUES: Redox inflammation through NOS2 and cyclooxygenase-2 participates in driving the mechanisms of initiation and progression in GI cancers. FUTURE DIRECTIONS: Understanding the underlying mechanism involved in NOS2 activation can provide new insights into important prevention and treatment strategies. Antioxid. Redox Signal. 26, 1059-1077.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Neoplasias Gastrointestinais/enzimologia , Inflamação/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Carcinogênese/metabolismo , Progressão da Doença , Neoplasias Gastrointestinais/etiologia , Humanos , Inflamação/etiologia , Óxido Nítrico/imunologia , OxirreduçãoRESUMO
In tumor biology, nitric oxide (NO) is generally regarded as an immunosuppressive molecule that impedes T-cell functions and activation of endothelial cells. Contrasting with this view, we here describe a critical role for NO derived from inducible nitric oxide (iNOS)-expressing tumor macrophages in T-cell infiltration and tumor rejection as shown by iNOS gene deletion, inhibition of iNOS, or NO donors. Specifically, macrophage-derived NO was found to induce on tumor vessels adhesion molecules that were required for T-cell extravasation. Experiments with human endothelial cells revealed a bimodal dose-dependent effect of NO. High doses of NO donors were indeed suppressive but lower, more physiological concentrations, induced adhesion molecules in an NFkB-dependent pathway and preferentially activated transcription of genes involved in lymphocyte diapedesis. iNOS+ macrophages in tumors appear to generate precisely the amount of NO that promotes endothelial activation and T-cell infiltration. These results will be valuable for the development of strategies designed to overcome the paucity of T-cell infiltration into tumors that is a major obstacle in clinical cancer immunotherapy.
RESUMO
BACKGROUND: Studies over the past decade and half have identified cancer stem cells (CSCs) to be responsible for tumorigenesis, invasion, sustenance of metastatic disease, radio- and chemo-resistance and tumor relapse. Recent reports have described the plasticity of breast CSCs (BCSCs) to shift between the epithelial and mesenchymal phenotypes via Epithelial-Mesenchymal Transition (EMT) and Mesenchymal-Epithelial Transition (MET) states as the reason for their invasive capabilities. Additionally, BRCA1 has been found to be a mammary stem cell fate determinant. However, it is not clear what would be the best marker that can be used for identifying CSCs in BRCA1 mutated cancers. Also, anticancer agents that can reduce CSC population in a BRCA1 defective condition have not been addressed so far. METHODS: Putative BCSCs were identified based on Hoechst exclusion, CD44(+)/24(-/low) expression and Aldehyde Dehydrogenase 1 (ALDH1) positivity using flow cytometry. The 'stemness' of the isolated ALDH1+ cells were analysed by immunofluorescence, western blotting for stem cell and EMT markers as well as in vitro mammosphere assays. Induction of Reactive Oxygen Species (ROS) by Plumbagin (PB) in BCSCs was assayed by Dichloro-dihydro-fluorescein diacetate (DCF-DA) staining. Ovarian cancer xenografts treated with PB were subjected to immunohistochemical analysis to study the ability of PB to target CSCs. RESULTS: We have confirmed that ALDH1 positivity is the best marker for the identification of BCSCs in BRCA1-defective breast cancer cell lines when compared to the CD marker profile and Side Population (SP) analysis. BRCA1 status was observed to be a determinant of the abundance of epithelial-like (ALDH1+) or mesenchymal-like (CD44(+)/24(-/low)) BCSCs, and the reconstitution of a full length, wild type BRCA1 in HCC1937 breast cancer cells possessing a mutated BRCA1, transforms them from 'stem-like' to more 'mesenchymal'. For the first time we have shown that Plumbagin (PB), a naturally occurring naphthoquinone which is predominantly a ROS inducer, could reduce BCSCs specifically in BRCA1-defective, basal-like cancer cells. CONCLUSIONS: The best marker for identifying BCSCs in BRCA1 defective condition could be ALDH1 and that BRCA1 mutated BCSCs would be mostly 'stem like' than 'mesenchymal'. Also ROS inducers like PB could reduce BCSCs in BRCA1 defective cancers.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Proteína BRCA1/genética , Neoplasias da Mama/patologia , Naftoquinonas/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Proteína BRCA1/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Carboplatina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal , Feminino , Humanos , Concentração Inibidora 50 , Camundongos SCID , Naftoquinonas/uso terapêutico , Células-Tronco Neoplásicas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We have earlier shown that Plumbagin (PB) can induce selective cytotoxicity to BRCA1 defective ovarian cancer cells; however, the effect of this molecule in BRCA1 mutated breast cancers has not been analyzed yet. Here, we report that reactive oxygen species (ROS) induced by PB resulted in DNA DSB and activates downstream signaling by ATR/ATM kinases and subsequent apoptosis. PB reduces DNA- dependent protein kinase (DNA-PK) expression and inhibits NHEJ (Non Homologous End Joining) activity in BRCA1 defective breast cancer cells. Also, PB induces apoptosis in two different BRCA1 conditional knock out murine models: MMTV-Cre; BRCA1(Co/Co) and WAP-Cre; BRCA1(Co/Co), at 2 mg/kg body weight, but 32 mg/kg of carboplatin (CN) was needed to induce apoptosis in them. This is the first study where two different tissue specific promoter driven transgenic mice models with BRCA1 exon 11 deletions are used for preclinical drug testing. The apoptosis induced by PB in HR (Homologous Recombination) defective triple negative BRCA1 mutant cell lines and in mouse models occur by inducing ROS mediated DNA DSB. The toxicity profile as compared with CN in transgenic mice provides evidence for PB's safer disposition as a therapeutic lead in breast cancer drug development.
Assuntos
Apoptose/efeitos dos fármacos , Proteína BRCA1/deficiência , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Naftoquinonas/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteínas Supressoras de Tumor/deficiência , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The identification of various biomolecules in cancer progression and therapy has led to the exploration of the roles of two cardinal players, namely Nitric Oxide (NO) and Reactive Oxygen Species (ROS) in cancer. Both ROS and NO display bimodal fashions of functional activity in a concentration dependent manner, by inducing either pro- or anti- tumorigenic signals. Researchers have identified the potential capability of NO and ROS in therapies owing to their role in eliciting pro-apoptotic signals at higher concentrations and their ability to sensitize cancer cells to one another as well as to other therapeutics. We review the prospects of NO and ROS in cancer progression and therapy, and analyze the role of a combinatorial therapy wherein an NO donor (SNAP) is used to sensitize the oxidative damage repair defective, triple negative breast cancer cells (HCC 1937) to a potent ROS inducer. Preliminary findings support the potential to employ various combinatorial regimes for anti-cancer therapies with regard to exploiting the chemo-sensitization property of NO donors.
Assuntos
Neoplasias da Mama/metabolismo , Óxido Nítrico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Humanos , Células-Tronco Neoplásicas/metabolismo , OxirreduçãoRESUMO
Eventhough the role of BRCA1/2 in hereditary prostatic cancer is being unleashed at a rapid rate; their optimal clinical management remains undefined. Cancer stem cells are thought to be responsible for cancer chemoresistance and relapse, thus they represent a significant concern for cancer prognosis and therapy. In this study, we have analyzed the effect of Plumbagin (PB) and structurally related naphthaquinones on BRCA1/2 silenced prostate cancer cells and the ability of PB to target stem cells. Our cell proliferation studies showed that both PC-3 and DU145 cells were more sensitive to PB, though all the compounds induced mitochondrial potential loss, DNA fragmentation and morphological changes which are indicative of apoptosis. Both BRCA1/2 siRNA transfected PC-3 and DU145 cells exhibited increased sensitivity to PB. Gene expression profiling post PB treatment in BRCA1/2 silenced cells revealed that PB has a putative role in tumor suppression in BRCA defective cancers. Using flow cytometric analysis we have proved that PB has the putative ability to directly target CSCs. Overall studies suggest that PB's antitumour mechanisms holds promise for novel therapeutic approaches against BRCA mutated cancers as well as CSCs.