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Bioactive molecule library screening may empirically identify effective combination therapies, but molecular mechanisms underlying favorable drug-drug interactions often remain unclear, precluding further rational design. In the absence of an accepted systems theory to interrogate synergistic responses, we introduce Omics-Based Interaction Framework (OBIF) to reveal molecular drivers of synergy through integration of statistical and biological interactions in synergistic biological responses. OBIF performs full factorial analysis of feature expression data from single versus dual exposures to identify molecular clusters that reveal synergy-mediating pathways, functions and regulators. As a practical demonstration, OBIF analyzed transcriptomic and proteomic data of a dyad of immunostimulatory molecules that induces synergistic protection against influenza A and revealed unanticipated NF-κB/AP-1 cooperation that is required for antiviral protection. To demonstrate generalizability, OBIF analyzed data from a diverse array of Omics platforms and experimental conditions, successfully identifying the molecular clusters driving their synergistic responses. Hence, unlike existing synergy quantification and prediction methods, OBIF is a phenotype-driven systems model that supports multiplatform interrogation of synergy mechanisms.
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BACKGROUND: Despite some successes with checkpoint inhibitors for treating cancer, most patients remain refractory to treatment, possibly due to the inhibitory nature of the tumor stroma that impedes the function and entry of effector cells. We devised a new technique of combining immunotherapy with radiotherapy (XRT), more specifically low-dose XRT, to overcome the stroma and maximize systemic outcomes. METHODS: We bilaterally established 344SQ lung adenocarcinoma tumors in 129Sv/Ev mice. Primary and secondary tumors were irradiated with either high-dose or low-dose of XRT with systemic anti-programmed cell death protein 1 and anti-cytotoxic T-lymphocyte associated protein 4 administration. Survival and tumor growth were monitored for the various groups, and secondary tumors were phenotyped by flow cytometry for immune populations. Tumor growth factor-beta (TGF-ß) cytokine levels were assessed locally after low-dose XRT, and specific immune-cell depletion experiments were conducted to identify the major contributors to the observed systemic antitumor effect. RESULTS: Through our preclinical and clinical studies, we observed that when tumor burden was high, there was a necessity of combining high-dose XRT to 'prime' T cells at the primary tumor site, with low-dose XRT directed to secondary (metastatic) tumors to 'modulate the stroma'. Low-dose XRT improved the antitumor outcomes of checkpoint inhibitors by favoring M1 macrophage polarization, enhancing natural killer (NK) cell infiltration, and reducing TGF-ß levels. Depletion of CD4+ T cells and NK cells abrogated the observed antitumor effect. CONCLUSION: Our data extend the benefits of low-dose XRT to reprogram the tumor environment and improve the infiltration and function of effector immune cells into secondary tumors.
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Imunidade/imunologia , Imunoterapia/métodos , Neoplasias/radioterapia , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Neoplasias/imunologia , Microambiente TumoralRESUMO
K-ras mutant lung adenocarcinoma (LUAD) is the most common type of lung cancer, displays abysmal prognosis and is tightly linked to tumor-promoting inflammation, which is increasingly recognized as a target for therapeutic intervention. We have recently shown a gender-specific role for epithelial Stat3 signaling in the pathogenesis of K-ras mutant LUAD. The absence of epithelial Stat3 in male K-ras mutant mice (LR/Stat3Δ/Δ mice) promoted tumorigenesis and induced a nuclear factor-kappaB (NF-κB)-driven pro-tumor immune response while reducing tumorigenesis and enhancing anti-tumor immunity in female counterparts. In the present study, we manipulated estrogen and NF-κB signaling to study the mechanisms underlying this intriguing gender-disparity. In LR/Stat3Δ/Δ females, estrogen deprivation by bilateral oophorectomy resulted in higher tumor burden, an induction of NF-κB-driven immunosuppressive response, and reduced anti-tumor cytotoxicity, whereas estrogen replacement reversed these changes. On the other hand, exogenous estrogen in males successfully inhibited tumorigenesis, attenuated NF-κB-driven immunosuppression and boosted anti-tumor immunity. Mechanistically, genetic targeting of epithelial NF-κB activity resulted in reduced tumorigenesis and enhanced the anti-tumor immune response in LR/Stat3Δ/Δ males, but not females. Our data suggest that estrogen exerts a context-specific anti-tumor effect through inhibiting NF-κB-driven tumor-promoting inflammation and provide insights into developing novel personalized therapeutic strategies for K-ras mutant LUAD.
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Adenocarcinoma de Pulmão/imunologia , Transformação Celular Neoplásica/imunologia , Estrogênios/metabolismo , Imunomodulação , Neoplasias Pulmonares/imunologia , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Humanos , Imunidade/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Mutação , NF-kappa B/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fator de Transcrição STAT3/genética , Células Tumorais CultivadasRESUMO
PURPOSE: Radiotherapy (RT) traditionally has been used for local tumor control in the treatment of cancer. The recent discovery that radiotherapy can have anticancer effects on the immune system has led to recognition of its ability to sensitize the tumor microenvironment to immunotherapy. However, radiation can also prompt adverse immunosuppressive effects that block aspects of systemic response at other tumor sites. Our hypothesis was that inhibition of the MER proto-oncogene tyrosine kinase (MerTK) in combination with anti-programmed cell death-1 (α-PD1) checkpoint blockade will enhance immune-mediated responses to radiotherapy. EXPERIMENTAL DESIGN: We tested the efficacy of this triple therapy (Radiation + α-PD1 + α-MerTK mAbs) in 129Sv/Ev mice with bilateral lung adenocarcinoma xenografts. Primary tumors were treated with stereotactic radiotherapy (36 Gy in 3 12-Gy fractions), and tumors were monitored for response. RESULTS: The triple therapy significantly delayed abscopal tumor growth, improved survival rates, and reduced numbers of lung metastases. We further found that the triple therapy increased the activated CD8+ and NK cells populations measured by granzyme B expression with upregulation of CD8+CD103+ tissue-resident memory cells (TRM) within the abscopal tumor microenvironment relative to radiation only. CONCLUSIONS: The addition of α-PD1 + α-MerTK mAbs to radiotherapy could alter the cell death to be more immunogenic and generate adaptive immune response via increasing the retention of TRM cells in the tumor islets of the abscopal tumors which was proven to play a major role in survival of non-small cell lung cancer patients.
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Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Neoplasias Pulmonares/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Radiocirurgia/métodos , c-Mer Tirosina Quinase/antagonistas & inibidores , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Camundongos , Microambiente Tumoral/efeitos dos fármacosRESUMO
PURPOSE: The limitation of hypofractionated radiation efficacy is due partly to the immunosuppressive tumor microenvironment. Indoleamine 2,3-dioxygenase 1 (IDO1) is an important regulator of tumor immune suppression. We evaluated the effects of IDO1 in hypofractionated radiation using a Lewis lung carcinoma (LLC) mouse model and tested whether IDO1 inhibition could sensitize those tumors to hypofractionated radiation. METHODS AND MATERIALS: Bilateral LLC tumors were established in C57BL/6 mice. Primary tumors were treated with 3 fractions of either 12 Gy or 6 Gy, and the IDO1 inhibitor INCB023843 was given starting on the first day of radiation. Plasma tryptophan and kynurenine levels were quantified by liquid chromatography and tandem mass spectrometry. Tumor-infiltrating immune cells were isolated from the tumors, stained, and quantified by flow cytometry. RESULTS: The combination of INCB023843 and three 12-Gy fractions led to better tumor control and survival than radiation alone; INCB023843 plus three 6-Gy fractions had no benefit. IDO1 expression by tumor-infiltrating immune cells was increased by three 12-Gy doses and inhibited by the addition of INCB023843. Nearly all IDO1+ immune cells were also F4/80+. Percentages of IDO1+F4/80+ immune cells were drastically increased by three 12-Gy fractions and by three 6-Gy fractions, but only INCB023843 combined with three 12-Gy fractions reduced those percentages. IDO1+F4/80+ immune cells were further found to be CD11b+, Gr1-intermediate-expressing, CD206-, and CD11c- (ie, myeloid-derived suppressor cells). Three 12-Gy fractions also increased the percentages of tumor-infiltrating T regulatory cells and CD8+ T cells, but adding INCB023843 did not affect those percentages. CONCLUSIONS: In addition to its immune activation effects, hypofractionated radiation induced "rebound immune suppression" in the tumor microenvironment by activating and recruiting IDO1-expressing myeloid-derived suppressor cells in a dose-dependent manner. Adding an IDO1 inhibitor to hypofractionated radiation reduced the percentages of these cells, overcame the immune suppression, and sensitized LLC tumors to hypofractionated radiation.
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Carcinoma Pulmonar de Lewis/radioterapia , Tolerância Imunológica , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Células Supressoras Mieloides/citologia , Oximas/farmacologia , Tolerância a Radiação/imunologia , Sulfonamidas/farmacologia , Microambiente Tumoral/imunologia , Animais , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides/metabolismo , Hipofracionamento da Dose de Radiação , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/efeitos da radiação , Regulação para Cima/efeitos da radiaçãoRESUMO
Many metabolic alterations, including the Warburg effect, occur in cancer cells that influence the tumor microenvironment, including switching to glycolysis from oxidative phosphorylation, using opportunistic modes of nutrient acquisition, and increasing lipid biosynthesis. The altered metabolic landscape of the tumor microenvironment can suppress the infiltration of immune cells and other functions of antitumor immunity through the production of immune-suppressive metabolites. Metabolic dysregulation in cancer cells further affects the expression of cell surface markers, which interferes with immune surveillance. Immune checkpoint therapies have revolutionized the standard of care for some patients with cancer, but disease in many others is resistant to immunotherapy. Specific metabolic pathways involved in immunotherapy resistance include PI3K-Akt-mTOR, hypoxia-inducible factor (HIF), adenosine, JAK/STAT, and Wnt/Beta-catenin. Depletion of essential amino acids such as glutamine and tryptophan and production of metabolites like kynurenine in the tumor microenvironment also blunt immune cell function. Targeted therapies against metabolic checkpoints could work in synergy with immune checkpoint therapy. This combined strategy could be refined by profiling patients' mutation status before treatment and identifying the optimal sequencing of therapies. This personalized combinatorial approach, which has yet to be explored, may well pave the way for overcoming resistance to immunotherapy.
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Resistencia a Medicamentos Antineoplásicos , Imunoterapia , Neoplasias/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Humanos , Neoplasias/terapiaRESUMO
Lung adenocarcinomas (LUADs) with mutations in the K-ras oncogene display dismal prognosis. Proinflammatory and immunomodulatory events that drive development of K-ras mutant LUAD are poorly understood. Here, we develop a lung epithelial specific K-ras mutant/Stat3 conditional knockout (LR/Stat3Δ/Δ) mouse model. Epithelial Stat3 deletion results in intriguing sex-associated discrepancies; K-ras mutant tumors are decreased in female LR/Stat3Δ/Δ mice whereas tumor burdens are increased in males. RNA-sequencing and tumor microenvironment (TME) analysis demonstrate increased anti-tumor immune responses following Stat3 deletion in females and, conversely, elevated pro-tumor immune pathways in males. While IL-6 blockade in male LR/Stat3Δ/Δ mice reduces lung tumorigenesis, inhibition of estrogen receptor signaling in female mice augments K-ras mutant oncogenesis and reprograms lung TME toward a pro-tumor phenotype. Our data underscore a critical sex-specific role for epithelial Stat3 signaling in K-ras mutant LUAD, thus paving the way for developing personalized (e.g. sex-based) immunotherapeutic strategies for this fatal disease.
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Células Epiteliais/metabolismo , Neoplasias Pulmonares/genética , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fator de Transcrição STAT3/metabolismo , Caracteres Sexuais , Transdução de Sinais , Animais , Feminino , Deleção de Genes , Interleucina-6/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Modelos Biológicos , Neutrófilos/metabolismo , Receptores de Estrogênio/metabolismo , Microambiente TumoralRESUMO
Chronic obstructive pulmonary disease (COPD), an inflammatory disease of the lung, is an independent risk factor for lung cancer. Lung tissues obtained from human smokers with COPD and lung cancer demonstrate hypoxia and up-regulated hypoxia inducible factor-1 (HIF-1). HIF-1 activation is the central mechanism for controlling the cellular response to hypoxia during inflammation and tumor development. These facts suggest a link between COPD-related airway inflammation, HIF-1, and lung cancer. We have previously established a mouse model of COPD-like airway inflammation that promotes lung cancer in a K-ras mutant mouse model (CC-LR). Here we show that tumors in the CC-LR model have significantly elevated levels of HIF-1α and HIF-1 activity. To determine the tumor-promoting functions of HIF-1 in CC-LR mice, the gene Hif1a which encodes HIF-1α and is required for HIF-1 activity, was disrupted in the lung epithelium of CC-LR animals. Airway epithelial specific HIF-1α deficient mice demonstrated significant reductions in lung surface tumor numbers, tumor angiogenesis, and tumor cell proliferation in the absence or presence of COPD-like airway inflammation. In addition, when CC-LR mice were bred with transgenic animals that overexpress a constitutively active mutant form of human HIF-1α in the airway epithelium, both COPD- and adenocarcinoma-like phenotypes were observed. HIF-1α overexpressing CC-LR mice had significant emphysema, and they also showed potentiated tumorigenesis, angiogenesis, and cell proliferation accompanied by an invasive metastatic phenotype. Our gain and loss of function studies support a key role for HIF-1α in the promotion of lung cancer by COPD-like inflammation.
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Given the unprecedentedly rapid progression of clinical immuno-oncology, evaluation of currently ongoing and upcoming clinical trials is essential to address potential deficiencies for future trial design. This observational study examined trends in the current landscape of clinical trials investigating immunotherapy with radiotherapy (iRT). The National Institute of Health's clinicaltrials.gov database was queried for trials including radiation and one of the six currently US FDA approved immunotherapy drugs: ipilimumab, nivolumab, pembrolizumab, durvalumab, atezolizumab and avelumab. This study highlights that the construction of prospective iRT trials is rapidly escalating. This study provides a framework to not only understand ongoing multidisciplinary trials, but also for prudent iRT trial design in the future.
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Antineoplásicos Imunológicos/uso terapêutico , Quimiorradioterapia/métodos , Ensaios Clínicos como Assunto , Imunoterapia/métodos , Radioterapia/métodos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Humanos , Imunoterapia/tendências , Ipilimumab/uso terapêutico , Neoplasias/terapia , Nivolumabe/uso terapêutico , Radioterapia/tendênciasRESUMO
With more than 150,000 deaths per year in the US alone, lung cancer has the highest number of deaths for any cancer. These poor outcomes reflect a lack of treatment for the most common form of lung cancer, non-small cell lung carcinoma (NSCLC). Lung adenocarcinoma (ADC) is the most prevalent subtype of NSCLC, with the main oncogenic drivers being KRAS and epidermal growth factor receptor (EGFR). Whereas EGFR blockade has led to some success in lung ADC, effective KRAS inhibition is lacking. KRAS-mutant ADCs are characterized by high levels of gel-forming mucin expression, with the highest mucin levels corresponding to worse prognoses. Despite these well-recognized associations, little is known about roles for individual gel-forming mucins in ADC development causatively. We hypothesized that MUC5AC/Muc5ac, a mucin gene known to be commonly expressed in NSCLC, is crucial in KRAS/Kras-driven lung ADC. We found that MUC5AC was a significant determinant of poor prognosis, especially in patients with KRAS-mutant tumors. In addition, by using mice with lung ADC induced chemically with urethane or transgenically by mutant-Kras expression, we observed significantly reduced tumor development in animals lacking Muc5ac compared with controls. Collectively, these results provide strong support for MUC5AC as a potential therapeutic target for lung ADC, a disease with few effective treatments.
Assuntos
Adenocarcinoma de Pulmão/patologia , Carcinogênese/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Mucina-5AC/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/mortalidade , Animais , Biomarcadores Tumorais , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Receptores ErbB/genética , Feminino , Humanos , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Masculino , Camundongos , Camundongos Transgênicos , Mucina-5AC/genética , Mutação , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)/genética , Análise de SobrevidaRESUMO
Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras-induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4+ T-helper cell response. IL22 is an effector molecule secreted by CD4+ and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on nonhematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS-mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of protumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8+ T-cell responses, while suppressing the protumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC+CCSP+ stem cells. Thus, we conclude that IL22 promotes Kras-mutant lung tumorigenesis by driving a protumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells. Cancer Immunol Res; 6(7); 788-97. ©2018 AACR.
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Interleucinas/metabolismo , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Mutação , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Animais Geneticamente Modificados , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Humanos , Imuno-Histoquímica , Interleucinas/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Microambiente Tumoral , Interleucina 22RESUMO
Indoleamine 2,3-dioxygenase 1 (IDO1), involved in the catabolism of tryptophan (Trp) to kynurenine (Kyn) is an important regulator of tumor-mediated immunosuppression implicated in resistance to anti-PD1 immunotherapy. We investigated the role of IDO1 in an anti-PD1-resistant lung cancer model (344SQ_R) compared to the parental 344SQ tumors (344SQ_P). IDO1 was overexpressed in tumor-infiltrating leukocytes, and plasma Kyn levels were increased, in 344SQ_R vs. 344SQ_P. The IDO1 inhibitor INCB023843 retarded tumor growth and reduced lung metastases in 344SQ_R. IDO1 was expressed at higher levels in F4/80+Gr1intCD11b+ myeloid-derived suppressor cells (MDSCs) that were prominent in 344SQ_R. The INCB023843 reduced IDO1 expression and percentages of these MDSCs while increasing CD8+ T cells infiltration, hence reactivating antitumor T-cell responses in 344SQ_R. Therefore, IDO1 inhibition holds promise for treating lung cancer that does not respond to anti-PD1 therapy.
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Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Células Supressoras Mieloides/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Terapia de Imunossupressão , Imunossupressores/farmacologia , Cinurenina/química , Camundongos , Transplante de Neoplasias , Triptofano/química , Microambiente TumoralRESUMO
Purpose: Radiation is used extensively to treat localized cancer, but improved understanding of its effects on the immune system has increased interest in its potential systemic (abscopal) effects, particularly in combination with checkpoint inhibitors such as anti-PD1. The majority of patients either do not respond or develop resistance to monotherapy over time. Here, we investigated the efficacy of OX40 (CD134) stimulation as an alternative immunotherapeutic approach in combination with radiotherapy (XRT) in a murine model of anti-PD1-resistant lung tumors.Experimental Design: We established a bilateral tumor model in 129Sv/Ev mice using an anti-PD1-resistant lung tumor cell line. Primary tumors were treated with intratumoral injection of an OX40 agonist antibody, given as adjuvant therapy after XRT (36 Gy in three 12-Gy fractions), whereas secondary tumors were left untreated to investigate abscopal outcomes.Results: The combination of XRT followed by OX40 stimulation effectively inhibited local and systemic antitumor growth, limited lung metastases, and improved survival rates. This treatment regimen augmented CD4+ and CD8+ T-cell expansion. XRT induced the expression of OX40 on T cells in tumors and spleens and increased the percentages of splenic CD103+ dendritic cells.Conclusions: Our data extend the benefits of radiation to systemic disease control, especially when combined with anti-OX40 agonist to promote immunologically mediated abscopal effects. Moreover, this study provides a rational treatment approach and sequence to overcome anti-PD1-resistant poorly immunogenic tumors. Clin Cancer Res; 24(22); 5735-43. ©2018 AACR.
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Antineoplásicos Imunológicos/farmacologia , Neoplasias Pulmonares/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores OX40/metabolismo , Animais , Linhagem Celular Tumoral , Quimiorradioterapia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Feminino , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/efeitos da radiação , Camundongos , Receptores OX40/antagonistas & inibidores , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Several promising chemopreventive agents have for lung cancer emerged in preclinical models and in retrospective trials. These agents have been shown to modulate pathways altered in carcinogenesis and reduce markers of carcinogenesis in animal and cell culture models. Cancer-prone transgenic mice with oncogenic Kras expressed in the airway epithelium (CcspCre/+ ; KrasLSL-G12D/+ ) were raised on diets compounded with myo-inositol. These animals form lung premalignant lesions in a stereotypical fashion over the ten weeks following weaning. Mice raised on myo-inositol containing diets showed potent reduction in the number, size, and stage of lesions as compared to those raised on control diets. myo-inositol has previously been reported to inhibit phosphoinositide 3-kinase (PI3K) signaling. However, in mice raised on myo-inositol, total PI3K signaling was largely unaffected. Proteomic and cytokine analyses revealed large reduction in IL-6 related pathways, including STAT3 phosphorylation. This effect was not due to direct inhibition of IL-6 production and autocrine signaling within the tumor cell, but rather through alteration in macrophage recruitment and in phenotype switching, with an increase in antitumoral M1 macrophages.
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Inositol/farmacologia , Interleucina-6/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Animais , Anticarcinógenos/farmacologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , FosforilaçãoRESUMO
Activating mutations of K-ras are the most common oncogenic alterations found in lung cancer. Unfortunately, attempts to target K-ras-mutant lung tumors have thus far failed, clearly indicating the need for new approaches in patients with this molecular profile. We have previously shown NF-κB activation, release of IL6, and activation of its responsive transcription factor STAT3 in K-ras-mutant lung tumors, which was further amplified by the tumor-enhancing effect of chronic obstructive pulmonary disease (COPD)-type airway inflammation. These findings suggest an essential role for this inflammatory pathway in K-ras-mutant lung tumorigenesis and its enhancement by COPD. Therefore, here we blocked IL6 using a monoclonal anti-IL6 antibody in a K-ras-mutant mouse model of lung cancer in the absence or presence of COPD-type airway inflammation. IL6 blockade significantly inhibited lung cancer promotion, tumor cell-intrinsic STAT3 activation, tumor cell proliferation, and angiogenesis markers. Moreover, IL6 inhibition reduced expression of protumor type 2 molecules (arginase 1, Fizz 1, Mgl, and IDO), number of M2-type macrophages and granulocytic myeloid-derived suppressor cells, and protumor T-regulatory/Th17 cell responses. This was accompanied by increased expression of antitumor type 1 molecule (Nos2), and antitumor Th1/CD8 T-cell responses. Our study demonstrates that IL6 blockade not only has direct intrinsic inhibitory effect on tumor cells, but also reeducates the lung microenvironment toward an antitumor phenotype by altering the relative proportion between protumor and antitumor immune cells. This information introduces IL6 as a potential druggable target for prevention and treatment of K-ras-mutant lung tumors. Cancer Res; 76(11); 3189-99. ©2016 AACR.
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Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Interleucina-6/antagonistas & inibidores , Neoplasias Pulmonares/patologia , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Microambiente Tumoral/efeitos dos fármacos , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Apoptose , Western Blotting , Carcinogênese , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Humanos , Técnicas Imunoenzimáticas , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Estadiamento de Neoplasias , Prognóstico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
The transcriptional regulator STAT3 curbs pro-inflammatory cytokine production mediated by NF-κB signalling in innate immune cells, yet the mechanism by which this occurs has been unclear. Here we identify STAT3 as a pivotal negative regulator of Ubc13, an E2 ubiquitin-conjugating enzyme that facilitates TRAF6 K63-linked ubiquitination and NF-κB activation. Ubc13 accumulates intracellularly in the absence of STAT3. Depletion of Ubc13 in Stat3-deficient macrophages subdues excessive RANKL- or LPS-dependent gene expression, indicating that Ubc13 overexpression mediates enhanced transcriptional responses in the absence of STAT3. In RANKL-activated macrophages, STAT3 is stimulated by autocrine IL-6 and inhibits accrual of Ets-1, Set1 methyltransferase and trimethylation of histone H3 lysine 4 (H3K4me3) at the Ube2n (Ubc13) promoter. These results delineate a mechanism by which STAT3 operates as a transcriptional repressor on Ube2n, thus modulating NF-κB activity by regulation of Ubc13 abundance. Our data suggest that this pathway plays important roles in bone homeostasis and restraint of inflammation.
Assuntos
Fibroblastos/metabolismo , Macrófagos/metabolismo , Ligante RANK/farmacologia , Fator de Transcrição STAT3/genética , Receptor 4 Toll-Like/genética , Enzimas de Conjugação de Ubiquitina/genética , Animais , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Cultura Primária de Células , Proteína Proto-Oncogênica c-ets-1 , Ligante RANK/genética , Ligante RANK/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/metabolismo , Transcrição Gênica , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Lung cancer development is associated with extensive pulmonary inflammation. In addition, the linkage between chronic obstructive pulmonary disease (COPD) and lung cancer has been demonstrated in population-based studies. IL-17-producing CD4 helper T cells (Th17 cells) play a critical role in promoting chronic tissue inflammation. Although Th17 cells are found in human COPD and lung cancer, their role is not understood. We have thus used a mouse model of lung cancer, in which an oncogenic form of K-ras (K-ras(G12D)), frequently found in human lung cancer, is restrictedly expressed in lung epithelial cells [via Clara cell secretory protein (CCSP(cre))]. In this model, Th17 and Treg but not Th1 cells were found enriched at the tumor tissues. When CCSP(cre)/K-ras(G12D) mice were weekly challenged with a lysate of nontypeable Haemophilus influenza (NTHi), which induces COPD-type inflammation and accelerates the tumor growth, they showed greatly enhanced Th17 cell infiltration in the lung tissues. Lack of IL-17, but not IL-17F, resulted in a significant reduction in lung tumor numbers in CCSP(cre)/K-ras(G12D) mice and also those treated with NTHi. Absence of IL-17 not only resulted in reduction of tumor cell proliferation and angiogenesis, but also decreased the expression of proinflammatory mediators and reduced recruitment of myeloid cells. Depletion of Gr-1(+)CD11b(+) myeloid cells in CCSP(cre)/K-ras(G12D) mice suppressed tumor growth in lung, indicating Gr-1(+)CD11b(+) myeloid cells recruited by IL-17 play a protumor role. Taken together, our data demonstrate a critical role for Th17 cell-mediated inflammation in lung tumorigenesis and suggest a novel way for prevention and treatment of this disease.
Assuntos
Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/fisiopatologia , Pneumonia/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Células Th17/imunologia , Animais , Primers do DNA/genética , Citometria de Fluxo , Genes ras/genética , Haemophilus influenzae/imunologia , Imuno-Histoquímica , Neoplasias Pulmonares/complicações , Camundongos , Camundongos Mutantes , Mutação/genética , Células Mieloides/imunologia , Células Mieloides/metabolismo , Pneumonia/complicações , Doença Pulmonar Obstrutiva Crônica/complicações , Reação em Cadeia da Polimerase em Tempo Real , Uteroglobina/metabolismoRESUMO
BACKGROUND: Tumor cells produce various cytokines and chemokines that attract leukocytes. Leukocytes can amplify parenchymal innate immune responses, and have been shown to contribute to tumor promotion. Neutrophils are among the first cells to arrive at sites of inflammation, and the increased number of tumor-associated neutrophils is linked to poorer outcome in patients with lung cancer. RESULTS: We have previously shown that COPD-like airway inflammation promotes lung cancer in a K-ras mutant mouse model of lung cancer (CC-LR). This was associated with severe lung neutrophilic influx due to the increased level of neutrophil chemoattractant, KC. To further study the role of neutrophils in lung tumorigenesis, we depleted neutrophils in CC-LR mice using an anti-neutrophil antibody. This resulted in a significant reduction in lung tumor number. We further selectively inhibited the main receptor for neutrophil chemo-attractant KC, CXCR2. Similarly, this resulted in suppression of neutrophil recruitment into the lung of CC-LR mice followed by significant tumor reduction. Neutrophil elastase (NE) is a potent elastolytic enzyme produced by neutrophils at the site of inflammation. We crossed the CC-LR mice with NE knock-out mice, and found that lack of NE significantly inhibits lung cancer development. These were associated with significant reduction in tumor cell proliferation and angiogenesis. CONCLUSION: We conclude that lung cancer promotion by inflammation is partly mediated by activation of the IL-8/CXCR2 pathway and subsequent recruitment of neutrophils and release of neutrophil elastase. This provides a baseline for future clinical trials using the IL-8/CXCR2 pathway or NE inhibitors in patients with lung cancer.
Assuntos
Carcinogênese/imunologia , Elastase de Leucócito/fisiologia , Neoplasias Pulmonares/imunologia , Neutrófilos/imunologia , Receptores de Interleucina-8B/fisiologia , Animais , Antineoplásicos/farmacologia , Líquido da Lavagem Broncoalveolar , Quimiocinas/metabolismo , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Knockout , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Neutrófilos/enzimologia , Receptores de Interleucina-8B/antagonistas & inibidoresRESUMO
BACKGROUND: PCA3 is a non-coding RNA (ncRNA) that is highly expressed in prostate cancer (PCa) cells, but its functional role is unknown. To investigate its putative function in PCa biology, we used gene expression knockdown by small interference RNA, and also analyzed its involvement in androgen receptor (AR) signaling. METHODS: LNCaP and PC3 cells were used as in vitro models for these functional assays, and three different siRNA sequences were specifically designed to target PCA3 exon 4. Transfected cells were analyzed by real-time qRT-PCR and cell growth, viability, and apoptosis assays. Associations between PCA3 and the androgen-receptor (AR) signaling pathway were investigated by treating LNCaP cells with 100 nM dihydrotestosterone (DHT) and with its antagonist (flutamide), and analyzing the expression of some AR-modulated genes (TMPRSS2, NDRG1, GREB1, PSA, AR, FGF8, CdK1, CdK2 and PMEPA1). PCA3 expression levels were investigated in different cell compartments by using differential centrifugation and qRT-PCR. RESULTS: LNCaP siPCA3-transfected cells significantly inhibited cell growth and viability, and increased the proportion of cells in the sub G0/G1 phase of the cell cycle and the percentage of pyknotic nuclei, compared to those transfected with scramble siRNA (siSCr)-transfected cells. DHT-treated LNCaP cells induced a significant upregulation of PCA3 expression, which was reversed by flutamide. In siPCA3/LNCaP-transfected cells, the expression of AR target genes was downregulated compared to siSCr-transfected cells. The siPCA3 transfection also counteracted DHT stimulatory effects on the AR signaling cascade, significantly downregulating expression of the AR target gene. Analysis of PCA3 expression in different cell compartments provided evidence that the main functional roles of PCA3 occur in the nuclei and microsomal cell fractions. CONCLUSIONS: Our findings suggest that the ncRNA PCA3 is involved in the control of PCa cell survival, in part through modulating AR signaling, which may raise new possibilities of using PCA3 knockdown as an additional therapeutic strategy for PCa control.
Assuntos
Antígenos de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , RNA não Traduzido/metabolismo , Receptores Androgênicos/metabolismo , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Humanos , Immunoblotting , Masculino , Neoplasias da Próstata/genética , RNA Interferente Pequeno , RNA não Traduzido/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , TransfecçãoRESUMO
BACKGROUND: Clinical studies have shown antineoplastic effectiveness of monoclonal antibodies (MAbs) against EGFR for different indications. Several MAbs directed to EGFR were developed recently, such as matuzumab, but there is still lack of information on preclinical data on its combination with chemo-radiation. Thus, the present study intended to examine the molecular pathways triggered by matuzumab alone or associated to chemo-radiotherapy in gynecological cell lines and its impact on cell growth and signaling. RESULTS: Combination of matuzumab with radiation and cisplatin did not enhance its cytostatic effects on A431, Caski and C33A cells (high, intermediate and low EGFR expression, respectively) in clonogenic assays, when compared to controls. The lack of effect was mediated by persistent signaling through EGFR due to its impaired degradation. In spite of the fact that matuzumab inhibited phosphorylation of EGFR, it had no effect upon cell viability. To analyze which downstream molecules would be involved in the EGFR signaling in the presence of matuzumab, we have tested it in combination with either PD98059 (MAPK inhibitor), or LY294002 (PI3K inhibitor). Matuzumab exhibited a synergic effect with LY294002, leading to a reduction of Akt phosphorylation that was followed by a decrease in A431 and Caski cells survival. The combination of PD98059 and matuzumab did not show the same effect suggesting that PI3K is an important effector of EGFR signaling in matuzumab-treated cells. Nonetheless, matuzumab induced ADCC in Caski cells, but not in the C33A cell line, suggesting that its potential therapeutic effects in vitro are indeed dependent on EGFR expression. CONCLUSIONS: Matuzumab combined with chemoradiation did not induce cytotoxic effects on gynecological cancer cell lines in vitro, most likely due to impaired EGFR degradation. However, a combination of matuzumab and PI3K inhibitor synergistically inhibited pAkt and cell survival, suggesting that the use of PI3K/Akt inhibitors could overcome intrinsic resistance to matuzumab in vitro. Altogether, data presented here can pave the way to a rational design of clinical strategies in patients with resistant profile to anti-EGFR inhibitors based on combination therapy.