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Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.
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Neoplasias/metabolismo , Neoplasias/patología , Nutrientes/metabolismo , Microambiente Tumoral , Animales , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Femenino , Glucosa/metabolismo , Glutamina/metabolismo , Humanos , Metabolismo de los Lípidos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Células Mieloides/inmunología , Células Mieloides/metabolismo , Neoplasias/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.26567.].
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Upregulation of the PI3K pathway has been implicated in the initiation and progression of several types of cancer, including renal cell carcinoma (RCC). Although several targeted therapies have been developed for RCC, durable and complete responses are exceptional. Thus, advanced RCC remains a lethal disease, underscoring the need of robust biomarker-based strategies to treat RCC. We report a synthetic lethal interaction between inhibition of phosphatidylinositol 3-kinase beta (PI3Kß) and loss of SETD2 methyltransferase. Clear cell RCC (ccRCC)-derived SETD2 knockout 786-0 and SETD2 mutant A498 cells treated with TGX221 (PI3Kß-specific) and AZD8186 (PI3Kß- and δ-specific) inhibitors displayed decreased cell viability, cell growth, and migration compared to SETD2 proficient 786-0 cells. Inhibition of the p110 δ and α isoforms alone had modest (δ) and no (α) effect on ccRCC cell viability, growth, and migration. In vivo, treatment of SETD2 mutant A498 cells, but not SETD2 proficient 786-0 cells, with AZD8186 significantly decreased tumor growth. Interestingly, inhibition of the downstream effector AKT (MK2206) recapitulated the effects observed in AZD8186-treated SETD2 deficient cells. Our data show that specific inhibition of PI3Kß causes synthetic lethality with SETD2 loss and suggest targeting of the AKT downstream effector pathway offers a rationale for further translational and clinical investigation of PI3Kß-specific inhibitors in ccRCC.
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BACKGROUND: Functional and molecular changes often precede gross anatomical changes, so early assessment of a tumor's functional and molecular response to therapy can help reduce a patient's exposure to the side effects of ineffective chemotherapeutics or other treatment strategies. OBJECTIVE: Our intent was to test the hypothesis that an ultrasound microvascular imaging approach might provide indications of response to therapy prior to assessment of tumor size. METHODS: Mice bearing clear-cell renal cell carcinoma xenograft tumors were treated with antiangiogenic and Notch inhibition therapies. An ultrasound measurement of microvascular density was used to serially track the tumor response to therapy. RESULTS: Data indicated that ultrasound-derived microvascular density can indicate response to therapy a week prior to changes in tumor volume and is strongly correlated with physiological characteristics of the tumors as measured by histology ([Formula: see text]). Furthermore, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups with high sensitivity and specificity. CONCLUSION/SIGNIFICANCE: Results suggests that future applications utilizing ultrasound imaging to monitor tumor response to therapy may be able to provide earlier insight into tumor behavior from metrics of microvascular density rather than anatomical tumor size measurements.
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Inhibidores de la Angiogénesis/farmacología , Carcinoma de Células Renales , Neoplasias Renales , Microvasos , Ultrasonografía/métodos , Angiografía/métodos , Animales , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/diagnóstico por imagen , Monitoreo de Drogas , Femenino , Xenoinjertos , Riñón/irrigación sanguínea , Riñón/diagnóstico por imagen , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/diagnóstico por imagen , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microvasos/diagnóstico por imagen , Microvasos/efectos de los fármacos , Microvasos/patologíaRESUMEN
Metastatic clear-cell renal cell carcinoma (ccRCC) affects thousands of patients worldwide each year. Antiangiogenic therapy has been shown to have beneficial effects initially, but resistance is eventually developed. Therefore, it is important to accurately track the response of cancer to different therapeutics in order to appropriately adjust the therapy to maximize efficacy. Change in tumor volume is the current gold standard for determining efficacy of treatment. However, functional variations can occur much earlier than measurable volume changes. Contrast-enhanced ultrasound (CEUS) is an important tool for assessing tumor progression and response to therapy, since it can monitor functional changes in the physiology. In this study, we demonstrate how ultrasound molecular imaging (USMI) can accurately track the evolution of the disease and molecular response to treatment. Methods A cohort of NSG (NOD/scid/gamma) mice was injected with ccRCC cells and treated with either the VEGF inhibitor SU (Sunitinib malate, Selleckchem, TX, USA) or the Notch pathway inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY, USA), or started on SU and later switched to GSI (Switch group). The therapies used in the study focus on disrupting angiogenesis and proper vessel development. SU inhibits signaling of vascular endothelial growth factor (VEGF), which is responsible for the sprouting of new vasculature, and GSI inhibits the Notch pathway, which is a key factor in the correct maturation of newly formed vasculature. Microbubble contrast agents targeted to VEGFR-2 (VEGF Receptor) were delivered as a bolus, and the bound agents were imaged in 3D after the free-flowing contrast was cleared from the body. Additionally, the tumors were harvested at the end of the study and stained for CD31. Results The results show that MI can detect changes in VEGFR-2 expression in the group treated with SU within a week of the start of treatment, while differences in volume only become apparent after the mice have been treated for three weeks. Furthermore, USMI can detect response to therapy in 92% of cases after 1 week of treatment, while the detection rate is only 40% for volume measurements. The amount of targeting for the GSI and Control groups was high throughout the duration of the study, while that of the SU and Switch groups remained low. However, the amount of targeting in the Switch group increased to levels similar to those of the Control group after the treatment was switched to GSI. CD31 staining indicates significantly lower levels of patent vasculature for the SU group compared to the Control and GSI groups. Therefore, the results parallel the expected physiological changes in the tumor, since GSI promotes angiogenesis through the VEGF pathway, while SU inhibits it. Conclusion This study demonstrates that MI can track disease progression and assess functional changes in tumors before changes in volume are apparent, and thus, CEUS can be a valuable tool for assessing response to therapy in disease. Future work is required to determine whether levels of VEGFR-2 targeting correlate with eventual survival outcomes.
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Carcinoma de Células Renales/diagnóstico por imagen , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Imagen Molecular/métodos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de la Angiogénesis , Animales , Carcinoma de Células Renales/genética , Medios de Contraste , Femenino , Inmunohistoquímica , Neoplasias Renales/diagnóstico por imagen , Neoplasias Renales/genética , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48TGFßRIIKO), we discovered an effect of TGFß signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G+ cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b+ cells. Deletion of csf3 in PDAC cells or use of a G-CSF-blocking antibody decreased tumor growth. Anti-G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G+ cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. Cancer Immunol Res; 5(9); 718-29. ©2017 AACR.
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Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Factor Estimulante de Colonias de Granulocitos/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Factor de Crecimiento Transformador beta/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Proliferación Celular/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos/antagonistas & inhibidores , Factor Estimulante de Colonias de Granulocitos/inmunología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Humanos , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Ratones , Ratones Noqueados , Transducción de Señal/genética , Linfocitos T/inmunologíaRESUMEN
The TGF-ß pathway plays a major role in tumor progression through regulation of epithelial and stromal cell signaling. Dysfunction of the pathway can lead to carcinoma progression and metastasis. To gain insight into the stromal role of the TGF-ß pathway in breast cancer, we performed laser capture microdissection (LCM) from breast cancer patients and reduction mammoplasty patients. Microdissected tumor stroma and normal breast stroma were examined for gene expression. Expression of the TGF-ß type III receptor (TGFBR3) was greatly decreased in the tumor stroma compared to control healthy breast tissue. These results demonstrated a 44-fold decrease in TGFBR3 mRNA in tumor stroma in comparison to control tissue. We investigated publicly available databases, and have identified that TGFBR3 mRNA levels are decreased in tumor stroma. We next investigated fibroblast cell lines derived from cancerous and normal breast tissue and found that in addition to mRNA levels, TßRIII protein levels were significantly reduced. Having previously identified that cancer-associated fibroblasts secrete greater levels of tumor promoting cytokines, we investigated the consequences of soluble-TßRIII (sTßRIII) on fibroblasts. Fibroblast conditioned medium was analyzed for 102 human secreted cytokines and distinct changes in response to sTßRIII were observed. Next, we used the fibroblast-conditioned medium to stimulate human monocyte cell line THP-1. These results indicate a distinct transcriptional response depending on sTßRIII treatment and whether it was derived from normal or cancerous breast tissue. We conclude that the effect of TßRIII has distinct roles not only in cancer-associated fibroblasts but that sTßRIII has distinct paracrine functions in the tumor microenvironment.
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Bone Morphogenetic Proteins (BMPs) are secreted cytokines/growth factors belonging to the Transforming Growth Factor ß (TGFß) family. BMP ligands have been shown to be overexpressed in human breast cancers. Normal and cancerous breast tissue display active BMP signaling as indicated by phosphorylated Smads 1, 5 and 9. We combined mice expressing the MMTV.PyMT oncogene with mice having conditional knockout (cKO) of BMP receptor type 1a (BMPR1a) using whey acidic protein (WAP)-Cre and found this deletion resulted in delayed tumor onset and significantly extended survival. Immunofluorescence staining revealed that cKO tumors co-expressed Keratin 5 and mesenchymal cell markers such as Vimentin. This indicates that epithelial-to-mesenchymal (EMT)-like transitions occurred in cKO tumors. We performed microarray analysis on these tumors and found changes that support EMT-like changes. We established primary tumor cell lines and found that BMPR1a cKO had slower growth in vitro and in vivo upon implantation. cKO tumor cells had reduced migration in vitro. We analyzed human databases from TCGA and survival data from microarrays to confirm BMPR1a tumor promoting functions, and found that high BMPR1a gene expression correlates with decreased survival regardless of molecular breast cancer subtype. In conclusion, the data indicate that BMP signaling through BMPR1a functions as a tumor promoter.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/deficiencia , Neoplasias de la Mama/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Metástasis de la Neoplasia , Transducción de SeñalRESUMEN
Postnatal mammary gland development and differentiation occur during puberty and pregnancy. To explore the role of DNA methylation in these processes, we determined the genome-wide DNA methylation and gene expression profiles of CD24(+)CD61(+)CD29(hi), CD24(+)CD61(+)CD29(lo), and CD24(+)CD61(-)CD29(lo) cell populations that were previously associated with distinct biological properties at different ages and reproductive stages. We found that pregnancy had the most significant effects on CD24(+)CD61(+)CD29(hi) and CD24(+)CD61(+)CD29(lo) cells, inducing distinct epigenetic states that were maintained through life. Integrated analysis of gene expression, DNA methylation, and histone modification profiles revealed cell-type- and reproductive-stage-specific changes. We identified p27 and TGFß signaling as key regulators of CD24(+)CD61(+)CD29(lo) cell proliferation, based on their expression patterns and results from mammary gland explant cultures. Our results suggest that relatively minor changes in DNA methylation occur during luminal differentiation compared with the effects of pregnancy on CD24(+)CD61(+)CD29(hi) and CD24(+)CD61(+)CD29(lo) cells.
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Metilación de ADN , Células Epiteliales/citología , Células Epiteliales/metabolismo , Glándulas Mamarias Animales/citología , Factores de Edad , Animales , Antígenos de Superficie/metabolismo , Diferenciación Celular/genética , Análisis por Conglomerados , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Elementos de Facilitación Genéticos , Activación Enzimática , Epigénesis Genética , Células Epiteliales/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Histonas/metabolismo , Inmunofenotipificación , Ratones , Ratones Noqueados , Especificidad de Órganos/genética , Fenotipo , Embarazo , Regiones Promotoras Genéticas , Maduración Sexual/genética , Transducción de SeñalRESUMEN
Metastasis is the most devastating aspect of cancer, however we know very little about the mechanisms of local invasion, the earliest step of metastasis. During tumor growth CD11b+ Gr1+ cells, known also as MDSCs, have been shown to promote tumor progression by a wide spectrum of effects that suppress the anti-tumor immune response. In addition to immunosuppression, CD11b+ Gr1+ cells promote metastasis by mechanisms that are currently unknown. CD11b+ Gr1+ cells localize near fibroblasts, which remodel the ECM and leave tracks for collective cell migration of carcinoma cells. In this study we discovered that CD11b+ Gr1+ cells promote invasion of mammary carcinoma cells by increasing fibroblast migration. This effect was directed by secreted factors derived from CD11b+ Gr1+ cells. We have identified several CD11b+ Gr1+ cell secreted proteins that activate fibroblast migration, including CXCL11, CXCL15, FGF2, IGF-I, IL1Ra, Resistin, and Shh. The combination of CXCL11 and FGF2 had the strongest effect on fibroblast migration that is associated with Akt1 and ERK1/2 phosphorylation. Analysis of subsets of CD11b+ Gr1+ cells identified that CD11b+ Ly6Chigh Ly6Glow cells increase fibroblast migration more than other myeloid cell populations. Additionally, tumor-derived CD11b+ Gr1+ cells promote fibroblast migration more than splenic CD11b+ Gr1+ cells of tumor-bearing mice. While TGFß signaling in fibroblasts does not regulate their migration toward CD11b+ Gr1+ cells, however deletion of TGFß receptor II on CD11b+ Gr1+ cells downregulates CXCL11, Shh, IGF1 and FGF2 resulting in reduced fibroblast migration. These studies show that TGFß signaling in CD11b+ Gr1+ cells promotes fibroblast directed carcinoma invasion and suggests that perivascular CD11b+ Ly6Chigh Ly6Glow cells may be the stimulus for localized invasion leading to metastasis.
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Fibroblastos/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Células Mieloides/metabolismo , Invasividad Neoplásica/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/fisiología , Femenino , Fibroblastos/patología , Neoplasias Mamarias Experimentales/patología , Ratones , Células Mieloides/patología , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal/fisiologíaRESUMEN
Bone Morphogenetic Protein (BMP) receptors mediate a diverse range of signals to regulate both development and disease. BMP activity has been linked to both tumor promoting and suppressive functions in both tumor cells and their surrounding microenvironment. We sought to investigate the requirement for BMPR2 in stromal fibroblasts during mammary tumor formation and metastasis. We utilized FSP1 (Fibroblast Specific Protein-1) promoter driven Cre to genetically delete BMPR2 in mice expressing the MMTV.PyVmT mammary carcinoma oncogene. We found that abrogation of stromal BMPR2 expression via FSP1 driven Cre resulted in increased tumor metastasis. Additionally, similar to epithelial BMPR2 abrogation, stromal loss of BMPR2 results in increased inflammatory cell infiltration. We proceeded to isolate and establish fibroblast cell lines without BMPR2 and found a cell autonomous increase in inflammatory cytokine secretion. Fibroblasts were co-implanted with syngeneic tumor cells and resulted in accelerated tumor growth and increased metastasis when fibroblasts lacked BMPR2. We observed that the loss of BMPR2 results in increased chemokine expression, which facilitates inflammation by a sustained increase in myeloid cells. The chemokines increased in BMPR2 deleted cells correlated with poor outcome in human breast cancer patients. We conclude that BMPR2 has tumor suppressive functions in the stroma by regulating inflammation.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Fibroblastos/metabolismo , Eliminación de Gen , Neoplasias Mamarias Experimentales/metabolismo , Proteínas Supresoras de Tumor/genética , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Fibroblastos/patología , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Metástasis de la Neoplasia , Proteínas Supresoras de Tumor/metabolismoRESUMEN
INTRODUCTION: Transforming growth factor beta (TGFß) plays a major role in the regulation of tumor initiation, progression, and metastasis. It is depended on the type II TGFß receptor (TßRII) for signaling. Previously, we have shown that deletion of TßRII in mammary epithelial of MMTV-PyMT mice results in shortened tumor latency and increased lung metastases. However, active TGFß signaling increased the number of circulating tumor cells and metastases in MMTV-Neu mice. In the current study, we describe a newly discovered connection between attenuated TGFß signaling and human epidermal growth factor receptor 2 (HER2) signaling in mammary tumor progression. METHODS: All studies were performed on MMTV-Neu mice with and without dominant-negative TßRII (DNIIR) in mammary epithelium. Mammary tumors were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence staining. The levels of secreted proteins were measured by enzyme-linked immunosorbent assay. Whole-lung mount staining was used to quantitate lung metastasis. The Cancer Genome Atlas (TCGA) datasets were used to determine the relevance of our findings to human breast cancer. RESULTS: Attenuated TGFß signaling led to a delay tumor onset, but increased the number of metastases in MMTVNeu/DNIIR mice. The DNIIR tumors were characterized by increased vasculogenesis, vessel leakage, and increased expression of vascular endothelial growth factor (VEGF). During DNIIR tumor progression, both the levels of CXCL1/5 and the number of CD11b+Gr1+ cells and T cells decreased. Analysis of TCGA datasets demonstrated a significant negative correlation between TGFBR2 and VEGF genes expression. Higher VEGFA expression correlated with shorter distant metastasis-free survival only in HER2+ patients with no differences in HER2-, estrogen receptor +/- or progesterone receptor +/- breast cancer patients. CONCLUSION: Our studies provide insights into a novel mechanism by which epithelial TGFß signaling modulates the tumor microenvironment, and by which it is involved in lung metastasis in HER2+ breast cancer patients. The effects of pharmacological targeting of the TGFß pathway in vivo during tumor progression remain controversial. The targeting of TGFß signaling should be a viable option, but because VEGF has a protumorigenic effect on HER2+ tumors, the targeting of this protein could be considered when it is associated with attenuated TGFß signaling.
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Neoplasias Pulmonares/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinogénesis/metabolismo , Quimiocinas/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/patología , Ratones Transgénicos , Neovascularización Patológica/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Linfocitos T/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and proangiogenic properties of myeloid cells. We have previously shown that the deletion of the TGF-ß type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-ß signaling. In this study, we tested the hypothesis that TGF-ß drives differentiation of myeloid-derived suppressor cells into protumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell-surface CD39/CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in the tumor microenvironment. In tumors isolated from mice with spontaneous tumor formation of mammary gland and conditional deletion of the type II TGF-ß receptor in mammary epithelium, an increased level of TGF-ß protein was associated with further increase in number of CD39(+)CD73(+) TDMMCs compared with MMTV-PyMT/TGFßRII(WT) control tumors with intact TGF-ß signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-ß signaling mediates maturation of myeloid-derived suppressor cells into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGF-ß signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and CD73, and was accompanied by increased infiltration of T lymphocytes, reduced density of blood vessels, and diminished progression of both Lewis lung carcinoma and spontaneous mammary carcinomas. We propose that TGF-ß signaling can directly induce the generation of CD39(+)CD73(+) TDMMCs, thus contributing to the immunosuppressive, proangiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment.
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5'-Nucleotidasa/biosíntesis , Antígenos CD/biosíntesis , Apirasa/biosíntesis , Células Mieloides/inmunología , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Crecimiento Transformador beta/inmunología , Animales , Células de la Médula Ósea/inmunología , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular/inmunología , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/inmunología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/biosíntesis , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Transducción de Señal/inmunología , Linfocitos T/inmunología , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
INTRODUCTION: There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a mesenchymal-stem like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-ß) pathway-associated genes relative to other subtypes, including the TGF-ß receptor type III (TßRIII). We hypothesize that TßRIII is tumor promoter in mesenchymal-stem like TNBC cells. METHODS: Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of TßRIII in the MSL subtype. We stably expressed short hairpin RNAs specific to TßRIII (TßRIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine TßRIII expression patterns across all TNBC subtypes. RESULTS: TßRIII was the most differentially expressed TGF-ß signaling gene in the MSL subtype. Silencing TßRIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when TßRIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed TßRIII-dependent phenotypes, we discovered that integrin-α2 was expressed at higher level in MSL cells after TßRIII-KD. Stable knockdown of integrin-α2 in TßRIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells. CONCLUSIONS: We have found that TßRIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that TßRIII-KD elevates expression of integrin-α2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both TßRIII and integrin-α2. Overall, our results indicate a potential mechanism in which TßRIII modulates integrin-α2 expression to effect MSL cell migration, invasion, and tumorigenicity.
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Proteoglicanos/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Análisis por Conglomerados , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Integrina alfa2/genética , Células Madre Mesenquimatosas/patología , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Esferoides Celulares , Carga Tumoral , Células Tumorales CultivadasRESUMEN
The tumor stromal environment can dictate many aspects of tumor progression. A complete understanding of factors driving stromal activation and their role in tumor metastasis is critical to furthering research with the goal of therapeutic intervention. Polyoma middle T-induced mammary carcinomas lacking the type II TGF-ß receptor (PyMT(mgko)) are highly metastatic compared with control PyMT-induced carcinomas (PyMT(fl/fl)). We hypothesized that the PyMT(mgko)-activated stroma interacts with carcinoma cells to promote invasion and metastasis. We show that the extracellular matrix associated with PyMT(mgko) tumors is stiffer and has more fibrillar collagen and increased expression of the collagen crosslinking enzyme lysyl oxidase (LOX) compared with PyMT(fl/fl) mammary carcinomas. Inhibition of LOX activity in PyMT(mgko) mice had no effect on tumor latency and size, but significantly decreased tumor metastasis through inhibition of tumor cell intravasation. This phenotype was associated with a decrease in keratin 14-positive myoepithelial cells in PyMT(mgko) tumors following LOX inhibition as well as a decrease in focal adhesion formation. Interestingly, the primary source of LOX was found to be activated fibroblasts. LOX expression in these fibroblasts can be driven by myeloid cell-derived TGF-ß, which is significantly linked to human breast cancer. Overall, stromal expansion in PyMT(mgko) tumors is likely caused through the modulation of immune cell infiltrates to promote fibroblast activation. This feeds back to the epithelium to promote metastasis by modulating phenotypic characteristics of basal cells. Our data indicate that epithelial induction of microenvironmental changes can play a significant role in tumorigenesis and attenuating these changes can inhibit metastasis. Cancer Res; 73(17); 5336-46. ©2013 AACR.
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Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Proteínas Serina-Treonina Quinasas/fisiología , Proteína-Lisina 6-Oxidasa/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Células del Estroma/enzimología , Factor de Crecimiento Transformador beta/fisiología , Animales , Carcinogénesis , Colágeno/metabolismo , Inhibidores Enzimáticos/farmacología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Hibridación in Situ , Queratina-14/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Transgénicos , Microscopía de Fuerza Atómica , Células Mieloides/metabolismo , Células Mieloides/patología , Fosforilación , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Receptor Tipo II de Factor de Crecimiento Transformador beta , Transducción de Señal , Células del Estroma/patologíaRESUMEN
INTRODUCTION: Transforming growth factor beta (TGF-ß) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-ß signaling regulates fibroblast recruitment and activation. Concurrently, TGF-ß signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-ß signaling to tumorigenesis, yet the role of TGF-ß in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-ß is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion. METHODS: Fluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TßRII KO) or TßRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-ß signaling loss. RESULTS: Intravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-ß signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TßRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TßRII KO carcinoma cells was identified by E-cadherin+/p120+/ß-catenin+ clusters. TßRII KO tumors also exhibited a twofold greater metastasis than TßRIIfl/fl tumors, attributed to enhanced extravasation ability. In TßRII KO tumor epithelium compared with TßRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-ß-insensitive mammary cancer. CONCLUSION: Fibroblast-stimulated carcinoma cells utilize TGF-ß signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-ß signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-ß signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.
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Comunicación Celular , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal/genética , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Uniones Intercelulares/metabolismo , Ratones , Neoplasias/genética , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/genética , beta Catenina/metabolismoRESUMEN
By crossing LysM-Cre and TGF-ß type II receptor (Tgfbr2) floxed mice we achieved specific deletion of Tgfbr2 in myeloid cells (Tgfbr2(MyeKO) mice). S.c.-injected (LLC, EL4-OVA) and implanted (MMTV-PyMT) carcinoma cells grow slower in Tgfbr2(MyeKO) mice. The number of CD45(+) cells in the tumor tissue was the same in both genotypes of mice, but upon analysis, the percentage of T cells (CD45(+)CD3(+)) in the KO mice was increased. By flow cytometry analysis, we did not detect any differences in the number and phenotype of TAMs, CD11b(+)Gr1(+), and DCs in Tgfbr2(MyeKO) compared with Tgfbr2(MyeWT) mice. ELISA and qRT-PCR data showed differences in myeloid cell functions. In Tgfbr2(MyeKO) TAMs, TNF-α secretion was increased, basal IL-6 secretion was down-regulated, TGF-ß did not induce any VEGF response, and there was decreased MMP9 and increased MMP2 and iNOS expression. TGF-ß did not have any effect on CD11b(+)Gr1(+) cells isolated from Tgfbr2(MyeKO) mice in the regulation of Arg, iNOS, VEGF, and CXCR4, and moreover, these cells have decreased suppressive activity relative to T cell proliferation. Also, we found that DCs from tumor tissue of Tgfbr2(MyeKO) mice have increased antigen-presented properties and an enhanced ability to stimulate antigen-specific T cell proliferation. We conclude that Tgfbr2 in myeloid cells has a negative role in the regulation of anti-tumorigenic functions of these cells, and deletion of this receptor decreases the suppressive function of CD11b(+)Gr1(+) cells and increases antigen-presenting properties of DCs and anti-tumorigenic properties of TAMs.
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Células Mieloides/inmunología , Neoplasias Experimentales/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Transducción de Señal/inmunología , Animales , Presentación de Antígeno/inmunología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Neoplasias Experimentales/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Bone morphogenetic proteins (BMPs) are members of the TGF-ß superfamily of signaling molecules. BMPs can elicit a wide range of effects in many cell types and have previously been shown to induce growth inhibition in carcinoma cells as well as normal epithelia. Recently, it has been demonstrated that BMP4 and BMP7 are overexpressed in human breast cancers and may have tumor suppressive and promoting effects. We sought to determine whether disruption of the BMP receptor 2 (BMPR2) would alter mammary tumor progression in mice that express the Polyoma middle T antigen. Mice expressing Polyoma middle T antigen under the mouse mammary tumor virus promoter were combined with mice that have doxycycline-inducible expression of a dominant-negative (DN) BMPR2. We did not observe any differences in tumor latency. However, mice expressing the BMPR2-DN had a fivefold increase in lung metastases. We characterized several cell autonomous changes and found that BMPR2-DN-expressing tumor cells had higher rates of proliferation. We also identified unique changes in inflammatory cells and secreted chemokines/cytokines that accompanied BMPR2-DN-expressing tumors. By immunohistochemistry, it was found that BMPR2-DN primary tumors and metastases had an altered reactive stroma, indicating specific changes in the tumor microenvironment. Among the changes we discovered were increased myeloid derived suppressor cells and the chemokine CCL9. BMP was shown to directly regulate CCL9 expression. We conclude that BMPR2 has tumor-suppressive function in mammary epithelia and microenvironment and that disruption can accelerate mammary carcinoma metastases.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Comunicación Paracrina , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Movimiento Celular , Proliferación Celular , Quimiocinas/metabolismo , Progresión de la Enfermedad , Femenino , Humanos , Inflamación/patología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/irrigación sanguínea , Virus del Tumor Mamario del Ratón/metabolismo , Ratones , Células Mieloides/patología , Invasividad Neoplásica , Metástasis de la Neoplasia , Neovascularización Patológica , Transducción de Señal , Microambiente TumoralRESUMEN
Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal neoplasms, is characterized by an expanded stroma with marked fibrosis (desmoplasia). We previously generated pancreas epithelium-specific TGF-ß receptor type II (Tgfbr2) knockout mice in the context of Kras activation (mice referred to herein as Kras+Tgfbr2KO mice) and found that they developed aggressive PDAC that recapitulated the histological manifestations of the human disease. The mouse PDAC tissue showed strong expression of connective tissue growth factor (Ctgf), a profibrotic and tumor-promoting factor, especially in the tumor-stromal border area, suggesting an active tumor-stromal interaction. Here we show that the PDAC cells in Kras+Tgfbr2KO mice secreted much higher levels of several Cxc chemokines compared with mouse pancreatic intraepithelial neoplasia cells, which are preinvasive. The Cxc chemokines induced Ctgf expression in the pancreatic stromal fibroblasts, not in the PDAC cells themselves. Subcutaneous grafting studies revealed that the fibroblasts enhanced growth of PDAC cell allografts, which was attenuated by Cxcr2 inhibition. Moreover, treating the Kras+Tgfbr2KO mice with the CXCR2 inhibitor reduced tumor progression. The decreased tumor progression correlated with reduced Ctgf expression and angiogenesis and increased overall survival. Taken together, our data indicate that tumor-stromal interactions via a Cxcr2-dependent chemokine and Ctgf axis can regulate PDAC progression. Further, our results suggest that inhibiting tumor-stromal interactions might be a promising therapeutic strategy for PDAC.