RESUMEN
BACKGROUND: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. METHODS: The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. RESULTS: Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. CONCLUSIONS: Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.
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Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Proteínas de Unión al GTP ral/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Ratones , Metástasis de la Neoplasia , Paclitaxel/uso terapéutico , Pronóstico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Proteínas de Unión al GTP ral/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is associated with an incredibly dense stroma, which contributes to its recalcitrance to therapy. Cancer-associated fibroblasts (CAFs) are one of the most abundant cell types within the PDAC stroma and have context-dependent regulation of tumor progression in the tumor microenvironment (TME). Therefore, understanding tumor-promoting pathways in CAFs is essential for developing better stromal targeting therapies. Here, we show that disruption of the STAT3 signaling axis via genetic ablation of Stat3 in stromal fibroblasts in a Kras G12D PDAC mouse model not only slows tumor progression and increases survival, but re-shapes the characteristic immune-suppressive TME by decreasing M2 macrophages (F480+CD206+) and increasing CD8+ T cells. Mechanistically, we show that loss of the tumor suppressor PTEN in pancreatic CAFs leads to an increase in STAT3 phosphorylation. In addition, increased STAT3 phosphorylation in pancreatic CAFs promotes secretion of CXCL1. Inhibition of CXCL1 signaling inhibits M2 polarization in vitro. The results provide a potential mechanism by which CAFs promote an immune-suppressive TME and promote tumor progression in a spontaneous model of PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Linfocitos T CD8-positivos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Fibroblastos/metabolismo , Ratones , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Platelet-derived growth factor receptor-beta (PDGFRß) is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand PDGFB is released by neighboring epithelial and endothelial cells. While expression of both PDGFRß and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRß tumor-stroma signaling mediates breast cancer initiation, progression, and metastasis remains unclear. Here we demonstrate this paracrine signaling pathway that mediates both primary tumor growth and metastasis, specifically, metastasis to the brain. Elevated levels of PDGFB accelerated orthotopic tumor growth and intracranial growth of mammary tumor cells, while mesenchymal-specific expression of an activating mutant PDGFRß (PDGFRßD849V) exerted proproliferative signals on adjacent mammary tumor cells. Stromal expression of PDGFRßD849V also promoted brain metastases of mammary tumor cells expressing high PDGFB when injected intravenously. In the brain, expression of PDGFRßD849V was observed within a subset of astrocytes, and aged mice expressing PDGFRßD849V exhibited reactive gliosis. Importantly, the PDGFR-specific inhibitor crenolanib significantly reduced intracranial growth of mammary tumor cells. In a tissue microarray comprised of 363 primary human breast tumors, high PDGFB protein expression was prognostic for brain metastases, but not metastases to other sites. Our results advocate the use of mice expressing PDGFRßD849V in their stromal cells as a preclinical model of breast cancer-associated brain metastases and support continued investigation into the clinical prognostic and therapeutic use of PDGFB-to-PDGFRß signaling in women with breast cancer. SIGNIFICANCE: These studies reveal a previously unknown role for PDGFB-to-PDGFRß paracrine signaling in the promotion of breast cancer brain metastases and support the prognostic and therapeutic clinical utility of this pathway for patients.See related article by Wyss and colleagues, p. 594.
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Neoplasias de la Mama , MicroARNs , Animales , Encéfalo/metabolismo , Neoplasias de la Mama/genética , Células Endoteliales/metabolismo , Humanos , Ratones , Receptor beta de Factor de Crecimiento Derivado de PlaquetasRESUMEN
Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.
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Neoplasias Pulmonares/patología , Patología/métodos , Cola (estructura animal) , Animales , Recuento de Células , Línea Celular Tumoral , Transformación Celular Neoplásica , Humanos , Procesamiento de Imagen Asistido por Computador , Inyecciones Intravenosas , Ratones , Metástasis de la NeoplasiaRESUMEN
Although tremendous progress has been made in understanding the functions of Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in tumor cells, only recently have tumor cell-non-autonomous PTEN actions within the tumor microenvironment (TME) been appreciated. While it is accepted that the TME actively communicates with cancer cells to influence disease progression, our understanding of the genes and pathways responsible is still evolving. Given that inactivation of PTEN in the stroma is correlated with worse outcomes in human cancers, determining the unique functions and mechanisms of PTEN regulation in various TME cell compartments is essential. In this review, the evidence for PTEN function in different TME cell compartments, the mechanisms governing PTEN inactivation, and the downstream pathways regulated by PTEN that are critical for intracellular communication, are covered. The potential clinical implications of these findings as well as the future directions for the study of stromal PTEN are discussed.
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Regulación Neoplásica de la Expresión Génica , Fosfohidrolasa PTEN/metabolismo , Células del Estroma/metabolismo , Microambiente Tumoral/genética , Humanos , Fosfohidrolasa PTEN/genética , Transducción de SeñalRESUMEN
Bone-resorbing osteoclasts (OCs) are derived from myeloid precursors (MPs). Several transcription factors are implicated in OC differentiation and function; however, their hierarchical architecture and interplay are not well known. Analysis for enriched motifs in PU.1 and MITF chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) data from differentiating OCs identified eomesodermin (EOMES) as a potential novel binding partner of PU.1 and MITF at genes critical for OC differentiation and function. We were able to demonstrate using co-immunoprecipitation and sequential ChIP analysis that PU.1, MITF, and EOMES are in the same complex and present as a complex at OC genomic loci. Furthermore, EOMES knockdown in MPs led to osteopetrosis associated with decreased OC differentiation and function both in vitro and in vivo. Although EOMES is associated with embryonic development and other hematopoietic lineages, this is the first study demonstrating the requirement of EOMES in the myeloid compartment.
RESUMEN
The importance of the tumor-associated stroma in cancer progression is clear. However, it remains uncertain whether early events in the stroma are capable of initiating breast tumorigenesis. Here, we show that in the mammary glands of non-tumor bearing mice, stromal-specific phosphatase and tensin homolog (Pten) deletion invokes radiation-induced genomic instability in neighboring epithelium. In these animals, a single dose of whole-body radiation causes focal mammary lobuloalveolar hyperplasia through paracrine epidermal growth factor receptor (EGFR) activation, and EGFR inhibition abrogates these cellular changes. By analyzing human tissue, we discover that stromal PTEN is lost in a subset of normal breast samples obtained from reduction mammoplasty, and is predictive of recurrence in breast cancer patients. Combined, these data indicate that diagnostic or therapeutic chest radiation may predispose patients with decreased stromal PTEN expression to secondary breast cancer, and that prophylactic EGFR inhibition may reduce this risk.
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Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/genética , Fosfohidrolasa PTEN/genética , Tolerancia a Radiación/genética , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Transformación Celular Neoplásica , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Rayos gamma/efectos adversos , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/efectos de la radiación , Humanos , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/efectos de la radiación , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/metabolismo , Glándulas Mamarias Humanas/efectos de la radiación , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/radioterapia , Ratones , Fosfohidrolasa PTEN/deficiencia , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/efectos de la radiaciónRESUMEN
The contribution of the tumor microenvironment to pancreatic ductal adenocarcinoma (PDAC) development is currently unclear. We therefore examined the consequences of disrupting paracrine Hedgehog (HH) signaling in PDAC stroma. Herein, we show that ablation of the key HH signaling gene Smoothened (Smo) in stromal fibroblasts led to increased proliferation of pancreatic tumor cells. Furthermore, Smo deletion resulted in proteasomal degradation of the tumor suppressor PTEN and activation of oncogenic protein kinase B (AKT) in fibroblasts. An unbiased proteomic screen identified RNF5 as a novel E3 ubiquitin ligase responsible for degradation of phosphatase and tensin homolog (PTEN) in Smo-null fibroblasts. Ring Finger Protein 5 (Rnf5) knockdown or pharmacological inhibition of glycogen synthase kinase 3ß (GSKß), the kinase that marks PTEN for ubiquitination, rescued PTEN levels and reversed the oncogenic phenotype, identifying a new node of PTEN regulation. In PDAC patients, low stromal PTEN correlated with reduced overall survival. Mechanistically, PTEN loss decreased hydraulic permeability of the extracellular matrix, which was reversed by hyaluronidase treatment. These results define non-cell autonomous tumor-promoting mechanisms activated by disruption of the HH/PTEN axis and identifies new targets for restoring stromal tumor-suppressive functions.
RESUMEN
The contribution of the tumor microenvironment to the development of pancreatic adenocarcinoma (PDAC) is unclear. The LSL-KrasG12D/+;LSL-p53R172H/+;Pdx-1-Cre (KPC) tumor model, which is widely utilized to faithfully recapitulate human pancreatic cancer, depends on Cre-mediated recombination in the epithelial lineage to drive tumorigenesis. Therefore, specific Cre-loxP recombination in stromal cells cannot be applied in this model, limiting the in vivo investigation of stromal genetics in tumor initiation and progression. To address this issue, we generated a new Pdx1FlpO knock-in mouse line, which represents the first mouse model to physiologically express FlpO recombinase in pancreatic epithelial cells. This mouse specifically recombines Frt loci in pancreatic epithelial cells, including acinar, ductal, and islet cells. When combined with the Frt-STOP-Frt KrasG12D and p53Frt mouse lines, simultaneous Pdx1FlpO activation of mutant Kras and deletion of p53 results in the spectrum of pathologic changes seen in PDAC, including PanIN lesions and ductal carcinoma. Combination of this KPF mouse model with any stroma-specific Cre can be used to conditionally modify target genes of interest. This will provide an excellent in vivo tool to study the roles of genes in different cell types and multiple cell compartments within the pancreatic tumor microenvironment.
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Transformación Celular Neoplásica/patología , ADN Nucleotidiltransferasas/metabolismo , Modelos Animales de Enfermedad , Proteínas de Homeodominio/fisiología , Neoplasias Pancreáticas/patología , Transactivadores/fisiología , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , ADN Nucleotidiltransferasas/genética , Progresión de la Enfermedad , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Microambiente Tumoral , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The extracellular matrix (ECM) is critical for mammary ductal development and differentiation, but how mammary fibroblasts regulate ECM remodeling remains to be elucidated. Herein, we used a mouse genetic model to activate platelet derived growth factor receptor-alpha (PDGFRα) specifically in the stroma. Hyperactivation of PDGFRα in the mammary stroma severely hindered pubertal mammary ductal morphogenesis, but did not interrupt the lobuloalveolar differentiation program. Increased stromal PDGFRα signaling induced mammary fat pad fibrosis with a corresponding increase in interstitial hyaluronic acid (HA) and collagen deposition. Mammary fibroblasts with PDGFRα hyperactivation also decreased hydraulic permeability of a collagen substrate in an in vitro microfluidic device assay, which was mitigated by inhibition of either PDGFRα or HA. Fibrosis seen in this model significantly increased the overall stiffness of the mammary gland as measured by atomic force microscopy. Further, mammary tumor cells injected orthotopically in the fat pads of mice with stromal activation of PDGFRα grew larger tumors compared to controls. Taken together, our data establish that aberrant stromal PDGFRα signaling disrupts ECM homeostasis during mammary gland development, resulting in increased mammary stiffness and increased potential for tumor growth.