RESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
Aberrant TGFbeta signaling is common in human cancers and contributes to tumor metastasis. Here, we demonstrate that Gr-1+CD11b+ myeloid cells are recruited into mammary carcinomas with type II TGF beta receptor gene (Tgfbr2) deletion and directly promote tumor metastasis. Gr-1+CD11b+ cells infiltrate into the invasive front of tumor tissues and facilitate tumor cell invasion and metastasis through a process involving metalloproteinase activity. This infiltration of Gr-1+CD11b+ cells also results in increased abundance of TGF beta 1 in tumors with Tgfbr2 deletion. The recruitment of Gr-1+CD11b+ cells into tumors with Tgfbr2 deletion involves two chemokine receptor axes, the SDF-1/CXCR4 and CXCL5/CXCR2 axes. Together, these data indicate that Gr-1+CD11b+ cells contribute to TGFbeta-mediated metastasis through enhancing tumor cell invasion and metastasis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Antígeno CD11b/metabolismo , Células Mieloides/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Femenino , Eliminación de Gen , Humanos , Metaloproteinasas de la Matriz/biosíntesis , Ratones , Modelos Biológicos , Células Mieloides/enzimología , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
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
Despite its clinical significance, joint morphogenesis is still an obscure process. In this study, we determine the role of transforming growth factor beta (TGF-beta) signaling in mice lacking the TGF-beta type II receptor gene (Tgfbr2) in their limbs (Tgfbr2(PRX-1KO)). In Tgfbr2(PRX-1KO) mice, the loss of TGF-beta responsiveness resulted in the absence of interphalangeal joints. The Tgfbr2(Prx1KO) joint phenotype is similar to that in patients with symphalangism (SYM1-OMIM185800). By generating a Tgfbr2-green fluorescent protein-beta-GEO-bacterial artificial chromosome beta-galactosidase reporter transgenic mouse and by in situ hybridization and immunofluorescence, we determined that Tgfbr2 is highly and specifically expressed in developing joints. We demonstrated that in Tgfbr2(PRX-1KO) mice, the failure of joint interzone development resulted from an aberrant persistence of differentiated chondrocytes and failure of Jagged-1 expression. We found that TGF-beta receptor II signaling regulates Noggin, Wnt9a, and growth and differentiation factor-5 joint morphogenic gene expressions. In Tgfbr2(PRX-1KO) growth plates adjacent to interphalangeal joints, Indian hedgehog expression is increased, whereas Collagen 10 expression decreased. We propose a model for joint development in which TGF-beta signaling represents a means of entry to initiate the process.
Asunto(s)
Articulaciones/crecimiento & desarrollo , Morfogénesis , Transducción de Señal , Factor de Crecimiento Transformador beta/fisiología , Animales , Embrión de Mamíferos , Extremidades , Articulaciones/química , Articulaciones/embriología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/análisis , Receptores de Factores de Crecimiento Transformadores beta/deficienciaRESUMEN
We investigated whether TGF-beta induced by anticancer therapies accelerates tumor progression. Using the MMTV/PyVmT transgenic model of metastatic breast cancer, we show that administration of ionizing radiation or doxorubicin caused increased circulating levels of TGF-beta1 as well as increased circulating tumor cells and lung metastases. These effects were abrogated by administration of a neutralizing pan-TGF-beta antibody. Circulating polyomavirus middle T antigen-expressing tumor cells did not grow ex vivo in the presence of the TGF-beta antibody, suggesting autocrine TGF-beta is a survival signal in these cells. Radiation failed to enhance lung metastases in mice bearing tumors that lack the type II TGF-beta receptor, suggesting that the increase in metastases was due, at least in part, to a direct effect of TGF-beta on the cancer cells. These data implicate TGF-beta induced by anticancer therapy as a pro-metastatic signal in tumor cells and provide a rationale for the simultaneous use of these therapies in combination with TGF-beta inhibitors.
Asunto(s)
Anticuerpos Bloqueadores/uso terapéutico , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/terapia , Neoplasias Inducidas por Radiación/prevención & control , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/inmunología , Animales , Antígenos Transformadores de Poliomavirus/fisiología , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Pulmonares/inmunología , Virus del Tumor Mamario del Ratón/fisiología , Ratones , Ratones Transgénicos , Neoplasias Inducidas por Radiación/inmunología , Neoplasias Inducidas por Radiación/patología , Células Neoplásicas Circulantes/inmunología , Células Neoplásicas Circulantes/patología , Infecciones por Retroviridae/patología , Infecciones por Retroviridae/prevención & control , Transducción de Señal/fisiología , Infecciones Tumorales por Virus/patología , Infecciones Tumorales por Virus/prevención & controlRESUMEN
A subset of neoplastic cells within human high-grade gliomas has features associated with stem cells. These cells may sustain glioma growth, and their stem-like properties may confer resistance to standard glioma treatments. Whether glioma stem cells derive from indigenous neural stem cells (NSC), or from tumor cells that have reacquired stem cell-like properties, is unknown. However, signaling pathways that are tightly regulated and central to NSC biology, including the Ras/Raf/Erk pathway, are hyperactive and pathogenic in gliomagenesis. Furthermore, data in animal models suggests that, in some cases, tumors are initiated in the subventricular zone (SVZ), a stem/progenitor cell niche in the mature brain. We activated oncogenic K-ras in mouse glioneuronal precursor cells and adult SVZ cells using GFAP-Cre. GFAP-Cre+/K-ras(G12D) mice showed a marked expansion of glial fibriallary acidic protein (GFAP)- and TUJ1-expressing cell populations in the SVZ. In addition, mice developed intermediate grade, infiltrating glioma with 100% penetrance. Tumors were consistently located in the amygdalohippocampal region and nearby cortex, often in association with the lateral ventricle and expanded SVZ. Tumor cells expressed markers associated with neural progenitor cells, including Olig2, Bmi-1, and PDGFR-alpha. These data suggest that infiltrating tumor cells may arise from NSC transformed by activation of oncogenic K-ras in vivo.
Asunto(s)
Ventrículos Cerebrales/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/metabolismo , Integrasas/metabolismo , Proteínas ras/metabolismo , Animales , Ventrículos Cerebrales/patología , Femenino , Proteína Ácida Fibrilar de la Glía/genética , Glioma/patología , Inmunohistoquímica , Integrasas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neuronas/metabolismo , Neuronas/patología , Proteínas ras/genéticaRESUMEN
Fibroblasts are major cellular components of the tumor microenvironment, regulating tumor cell behavior in part through secretion of extracellular matrix proteins, growth factors, and angiogenic factors. In previous studies, conditional deletion of the type II transforming growth factor-beta (TGF-beta) receptor in fibroblasts (Tgfbr2FspKO) was shown to promote mammary tumor metastasis in fibroblast-epithelial cell cotransplantation studies in mice, correlating with increased expression of hepatocyte growth factor (HGF). Here, we advance our findings to show that Tgfbr2(FspKO) fibroblasts enhance HGF/c-Met and HGF/Ron signaling to promote scattering and invasion of mammary carcinoma cells. Blockade of c-Met and Ron by small interfering RNA silencing and pharmacologic inhibitors significantly reduced mammary carcinoma cell scattering and invasion caused by Tgfbr2FspKO fibroblasts. Moreover, neutralizing antibodies to c-Met and Ron significantly inhibited HGF-induced cell scattering and invasion, correlating with reduced Stat3 and p42/44MAPK phosphorylation. Investigation of the signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways by pharmacologic inhibition and small interfering RNA silencing revealed a cooperative interaction between the two pathways to regulate HGF-induced invasion, scattering, and motility of mammary tumor cells. Furthermore, whereas c-Met was found to regulate both the Stat3 and MAPK signaling pathways, Ron was found to regulate Stat3 but not MAPK signaling in mammary carcinoma cells. These studies show a tumor-suppressive role for TGF-beta signaling in fibroblasts, in part by suppressing HGF signaling between mammary fibroblasts and epithelial cells. These studies characterize complex functional roles for HGF and TGF-beta signaling in mediating tumor-stromal interactions during mammary tumor cell scattering and invasion, with important implications in the metastatic process.
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Fibroblastos/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Medios de Cultivo Condicionados , Femenino , Factor de Crecimiento de Hepatocito/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Neoplasias Mamarias Experimentales/enzimología , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Modelos Biológicos , Invasividad Neoplásica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Transforming growth factor-beta (TGF-beta) plays complex dual roles as an inhibitor and promoter of tumor progression. Although the influence of the stromal microenvironment on tumor progression is well recognized, little is known about the functions of TGF-beta signaling in the stroma during tumor progression. Using cre-lox technology, expression of the type II TGF-beta receptor was selectively knocked out in fibroblasts (Tgfbr2(FspKO)). In a co-xenograft model, we show that Tgfbr2(FspKO) fibroblasts enhance mammary carcinoma growth and metastasis in mice while increasing hepatocyte growth factor (HGF) expression and c-Met signaling downstream pathways including signal transducers and activators of transcription 3 (Stat3) and p42/44 mitogen-activated protein kinase (MAPK). Treatment of tumor-bearing mice with a pharmacologic inhibitor (EXEL-7592) of c-Met blocks tumor progression and reduces levels of phospho-Stat3 and phospho-p42/44 MAPK. Similarly, small interfering RNA knockdown of c-Met expression in mammary tumor cells reduces metastasis and c-Met signaling caused by Tgfbr2(FspKO) fibroblasts. The results show that TGF-beta signaling in fibroblasts suppresses tumor metastasis by antagonizing HGF/c-Met signaling within tumor epithelial cells. Furthermore, this co-xenograft model represents a unique context to study stromal TGF-beta and HGF signaling in mammary tumorigenesis.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Interferente Pequeño/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/biosíntesis , Receptores de Factores de Crecimiento Transformadores beta/deficiencia , Receptores de Factores de Crecimiento Transformadores beta/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1-Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMTV) promoter results in mammary gland hyperplasia and fibrosis, and mammary tumors. Cell lines isolated from MMTV-cyclin D1-Cdk2 (MMTV-D1K2) tumors exhibit Rb and p130 hyperphosphorylation and up-regulation of the protein products of E2F-dependent genes. These results suggest that cyclin D1/Cdk2 complexes may mediate some of the transforming effects that result from cyclin D1 overexpression in human breast cancers. MMTV-D1K2 cancer cells express the hepatocyte growth factor (HGF) receptor, c-Met. MMTV-D1K2 cancer cells also secrete transforming growth factor beta (TGFbeta), but are relatively resistant to TGFbeta antiproliferative effects. Fibroblasts derived from MMTV-D1K2 tumors secrete factors that stimulate the proliferation of MMTV-D1K2 cancer cells, stimulate c-Met tyrosine phosphorylation, and stimulate the phosphorylation of the downstream signaling intermediates p70(s6k) and Akt on activating sites. Together, these results suggest that deregulation of the Cdk/Rb/E2F axis reprograms mammary epithelial cells to initiate a paracrine loop with tumor-associated fibroblasts involving TGFbeta and HGF, resulting in desmoplasia. The MMTV-D1K2 mice should provide a useful model system for the development of therapeutic approaches to block the stromal desmoplastic reaction that likely plays an important role in the progression of multiple types of human tumors.
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Ciclina D1/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Ciclina D1/genética , Quinasa 2 Dependiente de la Ciclina/genética , Progresión de la Enfermedad , Activación Enzimática , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Factor de Crecimiento de Hepatocito/biosíntesis , Hiperplasia , Glándulas Mamarias Animales/enzimología , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/virología , Neoplasias Mamarias Experimentales/virología , Virus del Tumor Mamario del Ratón/genética , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteína p130 Similar a la del Retinoblastoma/metabolismoRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.26567.].
RESUMEN
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.
RESUMEN
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
Transforming growth factor-beta 1 (TGF-beta1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. TGF-beta1 signals through the TGF-beta type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Since little is known about the selective activation of Smad2 versus Smad3, we set out to identify novel Smad2 and Smad3 interacting proteins in epithelial cells. A non-transformed human cell line was transduced with Myc-His(6)-Smad2 or Myc-His(6)-Smad3-expressing retrovirus and was treated with TGF-beta1. Myc-His(6)-Smad2 or Myc-His(6)-Smad3 was purified by tandem affinity purification, eluates were subject to SDS-PAGE and Colloidal Blue staining, and select protein bands were digested with trypsin. The resulting tryptic peptides were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) and the SEQUEST algorithm was employed to identify proteins in the bands. A number of proteins that are known to interact with Smad2 or Smad3 were detected in the eluates. In addition, a number of putative novel Smad2 and Smad3 associated proteins were identified that have functions in cell proliferation, apoptosis, actin cytoskeleton regulation, cell motility, transcription, and Ras or insulin signaling. Specifically, the interaction between Smad2/3 and the Cdc42 guanine nucleotide exchange factor, Zizimin1, was validated by co-immunoprecipitation. The discovery of these novel Smad2 and/or Smad3 associated proteins may reveal how Smad2 and Smad3 are regulated and/or uncover new functions of Smad2 and Smad3 in TGF-beta1 signaling.
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Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Far-Western Blotting , Línea Celular , Humanos , Unión Proteica , Proteínas/análisis , Espectrometría de Masas en Tándem , Transducción GenéticaRESUMEN
Rapamycin and its derivatives are promising anticancer agents, but the exact mechanisms by which these drugs induce cell cycle arrest and inhibit tumor growth are unknown. A biochemical analysis of human mammary tumor cell lines indicated that rapamycin-induced antiproliferative effects correlated with down-regulation of cellular p21 levels and the levels of p21 in cyclin-dependent kinase (Cdk) 2 and 4 complexes. Cyclin D1 overexpression reversed rapamycin action and this reversal correlated with increased levels of cellular p21, higher levels of p21 associated with Cdk2, and stabilization of cyclin D1/Cdk2/p21/proliferating cell nuclear antigen (PCNA) complexes. Experiments using a novel cyclin D1-Cdk2 fusion protein or a kinase-dead mutant of the fusion protein indicated that reversal of rapamycin action required not only the formation of complexes with p21 and PCNA but also complex-associated kinase activity. Similar results were observed in vivo. The rapamycin derivative RAD001 (everolimus) inhibited the growth of mouse mammary tumors, which correlated with the disruption of cyclin D1/Cdk2 complexes. The potential implications of these results with respect to the use of rapamycin derivatives in breast cancer therapy are discussed.
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Antibióticos Antineoplásicos/farmacología , Ciclina D1/fisiología , Sirolimus/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Ciclina D1/biosíntesis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Regulación hacia Abajo , Humanos , Neoplasias Mamarias Animales/tratamiento farmacológico , Ratones , Antígeno Nuclear de Célula en Proliferación/metabolismo , Regulación hacia ArribaRESUMEN
The development and progression of malignancies is a complex multistage process that involves the contribution of a number of genes giving growth advantage to cells when transformed. The role of transforming growth factor-beta (TGF-beta) in carcinogenesis is complex with tumor-suppressor or prooncogenic activities depending on the cell type and the stage of the disease. We have previously reported the identification of a novel WD-domain protein, STRAP, that associates with both TGF-beta receptors and that synergizes with the inhibitory Smad, Smad7, in the negative regulation of TGF-beta-induced transcription. Here, we show that STRAP is ubiquitously expressed and is localized in both cytoplasm and nucleus. STRAP is up-regulated in 60% colon and in 78% lung carcinomas. Stable expression of STRAP results in activation of mitogen-activated protein kinase/extracellular signal-regulated kinase pathway and in down-regulation of the cyclin-dependent kinase inhibitor p21(Cip1), which results in retinoblastoma protein hyperphosphorylation. In addition, we have observed that Smad2/3 phosphorylation, TGF-beta-mediated transcription, and growth inhibition are induced in STRAP-knockout mouse embryonic fibroblasts compared with wild-type cells. Ectopic expression of STRAP in A549 lung adenocarcinoma cell line inhibits TGF-beta-induced growth inhibition and enhances anchorage-independent growth of these cells. Moreover, overexpression of STRAP increases tumorigenicity in athymic nude mice. Knockdown of endogenous STRAP by small interfering RNA increases TGF-beta signaling, reduces ERK activity, increases p21(Cip1) expression, and decreases tumorigenicity. Taken together, these results suggest that up-regulation of STRAP in human cancers may provide growth advantage to tumor cells via TGF-beta-dependent and TGF-beta-independent mechanisms, thus demonstrating the oncogenic function of STRAP.
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Transformación Celular Neoplásica/genética , Proteínas de Neoplasias/genética , Oncogenes , Proteínas Adaptadoras Transductoras de Señales , Animales , Células COS , Chlorocebus aethiops , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Visón , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células 3T3 NIH , Proteínas de Neoplasias/metabolismo , Fosforilación , Proteínas/genética , Proteínas/metabolismo , Proteínas de Unión al ARN , Proteína de Retinoblastoma/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The transforming growth factor-beta (TGF-beta) signaling pathway is a tumor-suppressor pathway that is commonly inactivated in colon cancer. TGF-beta is a secreted ligand that mediates its effects through a transmembrane heteromeric receptor complex, which consists of type I (TGFBR1) and type II subunits (TGFBR2). Approximately 30% of colon cancers carry TGFBR2 mutations, demonstrating that it is a common target for mutational inactivation in this cancer. To assess the functional role of TGFBR2 inactivation in the multistep progression sequence of colon cancer, we generated a mouse model that recapitulates two common genetic events observed in human colon cancer by mating Apc(1638N/wt) mice with mice that are null for Tgfbr2 in the intestinal epithelium, Villin-Cre;Tgfbr2(E2flx/E2flx) mice. In this model, we observed a dramatic increase in the number of intestinal adenocarcinomas in the Apc(1638N/wt);Villin-Cre;Tgfbr2(E2flx/E2flx) mice (called Apc(1638N/wt);Tgfbr2(IEKO)) compared with those mice with intact Tgfbr2 (Apc(1638N/wt);Tgfbr2(E2flx/E2flx)). Additionally, in vitro analyses of epithelial tumor cells derived from the Apc(1638N/wt);Tgfbr2(IEKO) mice showed enhanced expression and activity of matrix metalloproteinase MMP-2 and MMP-9, as well as increased TGF-beta1 secretion in the conditioned medium. Similarly, primary tumor tissues from the Apc(1638N/wt);Tgfbr2(IEKO) mice also showed elevated amounts of TGF-beta1 as well as higher MMP-2 activity in comparison with Apc(1638N/wt);Tgfbr2(E2flx/E2flx)-derived tumors. Thus, loss of TGFBR2 in intestinal epithelial cells promotes the invasion and malignant transformation of tumors initiated by Apc mutation, providing evidence that Wnt signaling deregulation and TGF-beta signaling inactivation cooperate to drive the initiation and progression, respectively, of intestinal cancers in vivo.