RESUMEN
BACKGROUND: Previous results from our lab indicate a tumor suppressor role for the transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) in prostate cancer (PCa). Here, we further characterize this role and uncover new functions for TMEFF2 in cancer and adult prostate regeneration. METHODS: The role of TMEFF2 was examined in PCa cells using Matrigel(TM) cultures and allograft models of PCa cells. In addition, we developed a transgenic mouse model that expresses TMEFF2 from a prostate specific promoter. Anatomical, histological, and metabolic characterizations of the transgenic mouse prostate were conducted. The effect of TMEFF2 in prostate regeneration was studied by analyzing branching morphogenesis in the TMEFF2-expressing mouse lobes and alterations in branching morphogenesis were correlated with the metabolomic profiles of the mouse lobes. The role of TMEFF2 in prostate tumorigenesis in whole animals was investigated by crossing the TMEFF2 transgenic mice with the TRAMP mouse model of PCa and analyzing the histopathological changes in the progeny. RESULTS: Ectopic expression of TMEFF2 impairs growth of PCa cells in Matrigel or allograft models. Surprisingly, while TMEFF2 expression in the TRAMP mouse did not have a significant effect on the glandular prostate epithelial lesions, the double TRAMP/TMEFF2 transgenic mice displayed an increased incidence of neuroendocrine type tumors. In addition, TMEFF2 promoted increased branching specifically in the dorsal lobe of the prostate suggesting a potential role in developmental processes. These results correlated with data indicating an alteration in the metabolic profile of the dorsal lobe of the transgenic TMEFF2 mice. CONCLUSIONS: Collectively, our results confirm the tumor suppressor role of TMEFF2 and suggest that ectopic expression of TMEFF2 in mouse prostate leads to additional lobe-specific effects in prostate regeneration and tumorigenesis. This points to a complex and multifunctional role for TMEFF2 during PCa progression.
Asunto(s)
Adenocarcinoma , Carcinogénesis/metabolismo , Proteínas de la Membrana/metabolismo , Tumores Neuroendocrinos , Próstata , Neoplasias de la Próstata , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Trasplante de Neoplasias/patología , Trasplante de Neoplasias/fisiología , Tumores Neuroendocrinos/metabolismo , Tumores Neuroendocrinos/patología , Próstata/patología , Próstata/fisiología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Regeneración , Células Tumorales CultivadasRESUMEN
Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFP(Low)) and reserve/facultative ISCs (Sox9-EGFP(High)) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFP(Low) ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFP(High) ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFP(High) facultative ISCs but not Sox9-EGFP(Low) actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.
Asunto(s)
Células Madre Adultas/clasificación , Factor I del Crecimiento Similar a la Insulina/fisiología , Intestino Delgado/citología , Células Madre Adultas/efectos de los fármacos , Células Madre Adultas/fisiología , Animales , Ciclo Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/fisiología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/fisiología , Ratones , Ratones Transgénicos , Células Madre Multipotentes/clasificación , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Traumatismos Experimentales por Radiación/patología , Traumatismos Experimentales por Radiación/fisiopatología , Receptor IGF Tipo 1/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Regeneración/efectos de los fármacos , Regeneración/fisiología , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismoRESUMEN
INTRODUCTION: Basal-like and luminal breast cancers have distinct stromal-epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal-epithelial interactions evolve in benign and pre-invasive lesions. METHODS: To study epithelial-stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis. RESULTS: Our results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basallike subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival. CONCLUSIONS: Coordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that targeting stroma-derived HGF signaling in early carcinogenesis may block progression of basal-like precursor lesions.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/metabolismo , Carcinoma Intraductal no Infiltrante/patología , Transformación Celular Neoplásica/metabolismo , Células Epiteliales/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Células del Estroma/metabolismo , Neoplasias de la Mama/genética , Carcinoma Intraductal no Infiltrante/genética , Comunicación Celular , Línea Celular Tumoral , Análisis por Conglomerados , Técnicas de Cocultivo , Citocinas/biosíntesis , Citocinas/metabolismo , Femenino , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Factor de Crecimiento de Hepatocito/genética , Humanos , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Esferoides Celulares , Células Tumorales Cultivadas , Microambiente Tumoral/genéticaRESUMEN
Obesity is associated with basal-like breast cancer (BBC), an aggressive breast cancer subtype. The objective of this study was to determine whether obesity promotes BBC onset in adulthood and to evaluate the role of stromal-epithelial interactions in obesity-associated tumorigenesis. We hypothesized that hepatocyte growth factor (HGF) plays a promoting role in BBC, which express the HGF receptor, c-Met. In C3(1)-T(Ag) mice, a murine model of BBC, we demonstrated that obesity leads to a significant increase in HGF secretion and an associated decrease in tumor latency. By immunohistochemical analysis, normal mammary gland exhibited obesity-induced HGF, c-Met and phospho-c-Met, indicating that the activation of the cascade was obesity-driven. HGF secretion was also increased from primary mammary fibroblasts isolated from normal mammary glands and tumors of obese mice compared to lean. These results demonstrate that obesity-induced elevation of HGF expression is a stable phenotype, maintained after several passages, and after removal of dietary stimulation. Conditioned media from primary tumor fibroblasts from obese mice drove tumor cell proliferation. In co-culture, neutralization of secreted HGF blunted tumor cell migration, further linking obesity-mediated HGF-dependent effects to in vitro measures of tumor aggressiveness. In sum, these results demonstrate that HGF/c-Met plays an important role in obesity-associated carcinogenesis. Understanding the effects of obesity on risk and progression is important given that epidemiologic studies imply a portion of BBC could be eliminated by reducing obesity.
Asunto(s)
Neoplasias de la Mama/etiología , Transformación Celular Neoplásica/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Animales , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma Basocelular/etiología , Carcinoma Basocelular/mortalidad , Carcinoma Basocelular/patología , Movimiento Celular , Proliferación Celular , Citocinas/sangre , Citocinas/metabolismo , Dieta , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Humanos , Glándulas Mamarias Animales/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de SeñalRESUMEN
Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such, they have significant therapeutic potential. However, whether ISCs can survive tissue storage is unknown. We hypothesize that, although the majority of epithelial cells might die, ISCs would remain viable for at least 24 h at 4 °C. To explore this hypothesis, jejuna of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate-buffered saline at 4 °C. Delayed isolation of epithelium was performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact for 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retained high integrity for 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Cultured isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80 %. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated in human tissue, storage at 4 °C might offer a valuable temporal window for the harvesting of crypts or ISCs for therapeutic application.
Asunto(s)
Yeyuno/citología , Células Madre/citología , Conservación de Tejido/métodos , Animales , Apoptosis , Técnicas de Cultivo de Célula , Proliferación Celular , Separación Celular , Supervivencia Celular , Células Cultivadas , Humanos , Mucosa Intestinal/citología , Yeyuno/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24(+) fraction that contained the majority of Lgr5-EGFP(+) putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24(+)EpCAM(+) fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent putative CESCs. Furthermore, the presence of CD24 expression in normal colonic epithelium may have important implications for the use of anti-CD24-based colorectal cancer therapies.
Asunto(s)
Antígeno CD24/metabolismo , Separación Celular/métodos , Colon/inmunología , Células Epiteliales/inmunología , Citometría de Flujo , Mucosa Intestinal/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/inmunología , Animales , Antígenos de Neoplasias/metabolismo , Biomarcadores/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proliferación Celular , Colon/citología , Molécula de Adhesión Celular Epitelial , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Inmunohistoquímica , Mucosa Intestinal/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
Recent identification of intestinal epithelial stem cell (ISC) markers and development of ISC reporter mice permit visualization and isolation of regenerating ISCs after radiation to define their functional and molecular phenotypes. Previous studies in uninjured intestine of Sox9-EGFP reporter mice demonstrate that ISCs express low levels of Sox9-EGFP (Sox9-EGFP Low), whereas enteroendocrine cells (EEC) express high levels of Sox9-EGFP (Sox9-EGFP High). We hypothesized that Sox9-EGFP Low ISCs would expand after radiation, exhibit enhanced proliferative capacities, and adopt a distinct gene expression profile associated with rapid proliferation. Sox9-EGFP mice were given 14 Gy abdominal radiation and studied between days 3 and 9 postradiation. Radiation-induced changes in number, growth, and transcriptome of the different Sox9-EGFP cell populations were determined by histology, flow cytometry, in vitro culture assays, and microarray. Microarray confirmed that nonirradiated Sox9-EGFP Low cells are enriched for Lgr5 mRNA and mRNAs enriched in Lgr5-ISCs and identified additional putative ISC markers. Sox9-EGFP High cells were enriched for EEC markers, as well as Bmi1 and Hopx, which are putative markers of quiescent ISCs. Irradiation caused complete crypt loss, followed by expansion and hyperproliferation of Sox9-EGFP Low cells. From nonirradiated intestine, only Sox9-EGFP Low cells exhibited ISC characteristics of forming organoids in culture, whereas during regeneration both Sox9-EGFP Low and High cells formed organoids. Microarray demonstrated that regenerating Sox9-EGFP High cells exhibited transcriptomic changes linked to p53-signaling and ISC-like functions including DNA repair and reduced oxidative metabolism. These findings support a model in which Sox9-EGFP Low cells represent active ISCs, Sox9-EGFP High cells contain radiation-activatable cells with ISC characteristics, and both participate in crypt regeneration.
Asunto(s)
Proteínas Fluorescentes Verdes/biosíntesis , Yeyuno/efectos de la radiación , Factor de Transcripción SOX9/biosíntesis , Células Madre/efectos de la radiación , Animales , Proliferación Celular/efectos de la radiación , Células Cultivadas , Expresión Génica/efectos de la radiación , Proteínas de Homeodominio/biosíntesis , Yeyuno/metabolismo , Ratones , Ratones Transgénicos , Proteínas Nucleares/biosíntesis , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/biosíntesis , Receptores Acoplados a Proteínas G/biosíntesis , Regeneración/efectos de la radiación , Proteínas Represoras/biosíntesisRESUMEN
Epigenetic in vitro and in vivo studies suggest that suppressor of cytokine signaling-2 (SOCS2) may normally limit tumorigenesis in the intestine; however, this theory has not been directly tested. We hypothesized that SOCS2 deficiency promotes spontaneous intestinal tumorigenesis in Apc(Min/+) mice. Therefore, we quantified tumor number, size, and load in the small intestine and colon using SOCS2(+/+)/Apc(Min/+), SOCS2(+/-)/Apc(Min/+), and SOCS2(-/-)/Apc(Min/+) mice and assayed hematocrit as an indirect marker of disease severity. Biochemical and histological assays were used to assess mechanisms. Heterozygous and homozygous disruption of SOCS2 alleles promoted 166 and 441% increases in tumor load in the small intestine, respectively, accelerated development of colon tumors, and caused severe anemia. SOCS2 deletion promoted significant increases in intestinal insulin-like growth factor-I mRNA but did not affect plasma insulin-like growth factor-I. Western blots and immunohistochemical analysis demonstrated that tumor and nontumor intestinal tissue of SOCS2(-/-)/Apc(Min/+) mice had increased serine 727 phosphorylation of signal transducer and activator of transcription 3 compared with SOCS2(+/+)/Apc(Min/+) mice. Moreover, electromobility shift assays showed that SOCS2 deletion did not alter signal transducer and activator of transcription 3 DNA binding. However, tumors and small intestine from SOCS2(-/-)/Apc(Min/+) showed dramatic increases in activator protein-1 (AP-1) DNA binding, and SOCS2 overexpression in vitro reduced levels of AP-1. These studies indicate that SOCS2 deletion promotes the spontaneous development of intestinal tumors driven by mutations in the adenomatous polyposis coli/beta-catenin pathway and activates AP-1. Therefore, reduced expression or epigenetic silencing of SOCS2 may serve as a useful biomarker for colorectal cancer risk.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes APC , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Factor de Transcripción AP-1/metabolismo , Alelos , Animales , Biomarcadores de Tumor , Epigénesis Genética , Eliminación de Gen , Silenciador del Gen , Homocigoto , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
In breast tumors, it is well established that intratumoral angiogenesis is crucial for malignant progression, but little is known about the vascular characteristics of extratumoral, cancer-adjacent breast. Genome-wide transcriptional data suggest that extratumoral microenvironments may influence breast cancer phenotypes; thus, histologic features of cancer-adjacent tissue may also have clinical implications. To this end, we developed a digital algorithm to quantitate vascular density in approximately 300 histologically benign tissue specimens from breast cancer patients enrolled in the UNC Normal Breast Study (NBS). Specimens were stained for CD31, and vascular content was compared to demographic variables, tissue composition metrics, and tumor molecular features. We observed that the vascular density of cancer-adjacent breast was significantly higher in older and obese women, and was strongly associated with breast adipose tissue content. Consistent with observations that older and heavier women experience higher frequencies of ER+ disease, higher extratumoral vessel density was also significantly associated with positive prognostic tumor features such as lower stage, negative nodal status, and smaller size (<2 cm). These results reveal biological relationships between extratumoral vascular content and body size, breast tissue composition, and tumor characteristics, and suggest biological plausibility for the relationship between weight gain (and corresponding breast tissue changes) and breast cancer progression.
Asunto(s)
Neoplasias de la Mama/patología , Mama/irrigación sanguínea , Mama/patología , Neovascularización Patológica/patología , Adulto , Factores de Edad , Anciano , Neoplasias de la Mama/irrigación sanguínea , Femenino , Humanos , Persona de Mediana Edad , Obesidad/patologíaRESUMEN
Reduced apoptosis of crypt stem/progenitor cells and elevated insulin and IGFs are linked to colon cancer risk. Insulin receptor substrate-1 (IRS-1) mediates the actions of insulin, IGF-I, and IGF-II, but the role of endogenous IRS-1 in crypt apoptosis and cancer is undefined. Using IRS-1(-/-), IRS-1(+/-), and IRS-1(+/+) mice, we tested the hypothesis that reduced IRS-1 expression increases apoptosis of intestinal crypt cells and protects against Apc(min/+) (Min)/beta-catenin-driven intestinal tumors. Expression of Sox9, a transcriptional target of Tcf/beta-catenin and putative biomarker of crypt stem cells, was assessed in intestine of different IRS-1 genotypes and cell lines. Irradiation-induced apoptosis was significantly increased in the crypts and crypt stem cell region of IRS-1-deficient mice. Tumor load was significantly reduced by 31.2 +/- 14.6% in IRS-1(+/-)/Min and by 64.1 +/- 7.6% in IRS-1(-/-)/Min mice, with more prominent reductions in tumor number than size. Compared with IRS-1(+/+)/Min, IRS-1(-/-)/Min mice had fewer Sox9-positive cells in intestinal crypts and reduced Sox9 mRNA in intestine. IRS-1 overexpression increased Sox9 expression in an intestinal epithelial cell line. We conclude that even small reductions in endogenous IRS-1 increase apoptosis of crypt stem or progenitor cells, protect against beta-catenin-driven intestinal tumors, and reduce Sox9, a Tcf/beta-catenin target and putative stem/progenitor cell biomarker.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Adenoma/genética , Apoptosis/genética , Genes APC , Proteínas del Grupo de Alta Movilidad/genética , Mucosa Intestinal/fisiología , Neoplasias Intestinales/genética , Células Madre/fisiología , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenoma/patología , Animales , Apoptosis/efectos de la radiación , Progresión de la Enfermedad , Femenino , Dosificación de Gen , Regulación de la Expresión Génica , Heterocigoto , Proteínas Sustrato del Receptor de Insulina , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de la radiación , Mucosa Intestinal/ultraestructura , Neoplasias Intestinales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvellosidades , Mutación , Factor de Transcripción SOX9 , Células Madre/metabolismo , Células Madre/efectos de la radiaciónRESUMEN
GH may improve intestinal growth or function in patients with short bowel syndrome. Excessive trophic effects of GH or IGF-I may contribute to neoplastic growth or increased colorectal cancer risk in acromegaly. Identification of mechanisms that limit the tumorigenic potential of GH and IGF-I is desirable. Suppressor of cytokine signaling-2 (SOCS2) limits GH action on body and organ growth, but its role in GH action on intestine is unknown. We tested the hypothesis that SOCS2 limits GH-induced intestinal growth or neoplasia in vivo. GH-transgenic (GH-TG) mice were crossed with SOCS2 null mice to generate wild-type (WT) or transgenic (TG) mice with zero (HO-WT; HO-TG), one (HT-WT; HT-TG), or two (WT-WT; WT-TG) functional SOCS2 genes. No HO-TG mice were derived from crossbreeding. WT-WT, HT-WT, WT-TG, and HT-TG were compared. Body weight, small intestine and colon growth, and levels of jejunal IGF-I and sucrase-isomaltase mRNAs were assessed. Colon was analyzed for abnormal lesions. HT-WT did not differ from WT-WT. Compared with WT-TG, HT-TG had significantly increased body weight, small intestine growth, and local IGF-I expression and decreased sucrase-isomaltase expression. HT-TG colon spontaneously developed multiple hyperplastic and lymphoid polyps. GH-induced activation of STAT5 DNA binding activity was enhanced in intestine of SOCS2 null mice compared with WT control. Haplotype insufficiency for SOCS2 promotes trophic actions of GH in small intestine and promotes preneoplastic growth in colon during excess GH. Small variations in SOCS2 expression levels may significantly influence the outcome of therapeutic GH or acromegaly in intestine.
Asunto(s)
Hormona del Crecimiento/fisiología , Haplotipos , Mucosa Intestinal/patología , Pólipos Intestinales/etiología , Proteínas Supresoras de la Señalización de Citocinas/genética , Animales , Proliferación Celular , Colon/patología , Femenino , Factor I del Crecimiento Similar a la Insulina/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/análisis , Factor de Transcripción STAT5/metabolismo , Complejo Sacarasa-Isomaltasa/genética , Proteínas Supresoras de la Señalización de Citocinas/deficiencia , Proteínas Supresoras de la Señalización de Citocinas/fisiologíaRESUMEN
UNLABELLED: Epidemiologic studies have associated obesity with increased risk of the aggressive basal-like breast cancer (BBC) subtype. Hepatocyte growth factor (HGF) signaling through its receptor, cMET, is elevated in obesity and is a pro-tumorigenic pathway strongly associated with BBC. We previously reported that high fat diet (HFD) elevated HGF, cMET, and phospho-cMET in normal mammary gland, with accelerated tumor development, compared to low fat diet (LFD)-fed lean controls in a murine model of BBC. We also showed that weight loss resulted in a significant reversal of HFD-induced effects on latency and elevation of HGF/cMET signaling in normal mammary and cMET in normal mammary and tumors. Here, we sought to inhibit BBC tumor progression in LFD- and HFD-fed C3(1)-Tag BBC mice using a small molecule cMET inhibitor, and began crizotinib treatment (50 mg/kg body weight by oral gavage) upon identification of the first palpable tumor. We next investigated if administering crizotinib in a window prior to tumor development would inhibit or delay BBC tumorigenesis. TREATMENT: Crizotinib significantly reduced mean tumor burden by 27.96 and 37.29 %, and mean tumor vascularity by 35.04 and 33.52 %, in our LFD- and HFD-fed C3(1)-Tag BBC mice, respectively. PREVENTION: Crizotinib significantly accelerated primary tumor progression in both diet groups but had no effect on total tumor progression or total tumor burden. In sum, cMET inhibition by crizotinib limited tumor development and microvascular density in basal-like tumor-bearing mice but did not appear to be an effective preventive agent for BBC.
RESUMEN
INTRODUCTION: Overall survival of early-stage breast cancer patients is similar for those who undergo breast-conserving therapy (BCT) and mastectomy; however, 10% to 15% of women undergoing BCT suffer ipsilateral breast tumor recurrence. The risk of recurrence may vary with breast cancer subtype. Understanding the gene expression of the cancer-adjacent tissue and the stromal response to specific tumor subtypes is important for developing clinical strategies to reduce recurrence risk. METHODS: We utilized two independent datasets to study gene expression data in cancer-adjacent tissue from invasive breast cancer patients. Complementary in vitro cocultures were used to study cell-cell communication between fibroblasts and specific breast cancer subtypes. RESULTS: Our results suggest that intrinsic tumor subtypes are reflected in histologically normal cancer-adjacent tissue. Gene expression of cancer-adjacent tissues shows that triple-negative (Claudin-low or basal-like) tumors exhibit increased expression of genes involved in inflammation and immune response. Although such changes could reflect distinct immune populations present in the microenvironment, altered immune response gene expression was also observed in cocultures in the absence of immune cell infiltrates, emphasizing that these inflammatory mediators are secreted by breast-specific cells. In addition, although triple-negative breast cancers are associated with upregulated immune response genes, luminal breast cancers are more commonly associated with estrogen-response pathways in adjacent tissues. CONCLUSIONS: Specific characteristics of breast cancers are reflected in the surrounding histologically normal tissue. This commonality between tumor and cancer-adjacent tissue may underlie second primaries and local recurrences. IMPACT: Biomarkers derived from cancer-adjacent tissue may be helpful in defining personalized surgical strategies or in predicting recurrence risk.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Neoplasias/genética , Microambiente Tumoral/genética , Adulto , Anciano , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Genes MHC Clase II/genética , Humanos , Persona de Mediana Edad , Invasividad Neoplásica , Recurrencia Local de Neoplasia , Análisis de Matrices Tisulares , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Adulto JovenRESUMEN
The ventral or inner region of spinal substantia gelatinosa (SG; lamina II(i)) is a heterogeneous sublamina important for the generation and maintenance of hyperalgesia and neuropathic pain. To test whether II(i) neurons can be hyperpolarized by the mu-opioid agonist [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO; 500 nM) and to address possible downstream consequences of mu-opioid-evoked inhibition of II(i) neurons, we combined in vitro whole-cell, tight-seal recording methods with fluorescent labeling of the intracellular tracer biocytin and confocal microscopy. Twenty-one of 23 neurons studied had identifiable axons. Nine possessed axons that projected ventrally into laminae III-V; six of these were hyperpolarized by DAMGO. Three of four neurons with identifiable axons that projected to lamina I were hyperpolarized by DAMGO. Most neurons could be classified as either islet cells or stalked cells. Five of nine labeled islet cells and only two of seven stalked cells were hyperpolarized by DAMGO. Three were stellate cells: one resembled a spiny cell and three could not be classified. DAMGO hyperpolarized each of the stellate cells, the spiny cell, and 1 of the unclassified cells. Our data support the hypothesis that part of the action of mu-opioid agonists involves the inhibition of interneurons that are part of a polysynaptic excitatory pathway from primary afferents to neurons in the deep and/or superficial dorsal horn.
Asunto(s)
Analgésicos Opioides/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Inhibición Neural/fisiología , Dolor/metabolismo , Células del Asta Posterior/citología , Terminales Presinápticos/ultraestructura , Receptores Opioides mu/metabolismo , Vías Aferentes/citología , Vías Aferentes/efectos de los fármacos , Vías Aferentes/metabolismo , Animales , Dendritas/efectos de los fármacos , Dendritas/metabolismo , Dendritas/ultraestructura , Femenino , Interneuronas/citología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microscopía Confocal , Inhibición Neural/efectos de los fármacos , Dolor/fisiopatología , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Ratas Long-Evans , Receptores Opioides delta/efectos de los fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/efectos de los fármacos , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/agonistas , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
It is widely thought that pregnancy reduces breast cancer risk, but this lacks consideration of breast cancer subtypes. While a full term pregnancy reduces risk for estrogen receptor positive (ER+) and luminal breast cancers, parity is associated with increased risk of basal-like breast cancer (BBC) subtype. Basal-like subtypes represent less than 10% of breast cancers and are highly aggressive, affecting primarily young, African American women. Our previous work demonstrated that high fat diet-induced obesity in nulliparous mice significantly blunted latency in C3(1)-TAg mice, a model of BBC, potentially through the hepatocyte growth factor (HGF)/c-Met oncogenic pathway. Experimental studies have examined parity and obesity individually, but to date, the joint effects of parity and obesity have not been studied. We investigated the role of obesity in parous mice on BBC. Parity alone dramatically blunted tumor latency compared to nulliparous controls with no effects on tumor number or growth, while obesity had only a minor role in further reducing latency. Obesity-associated metabolic mediators and hormones such as insulin, estrogen, and progesterone were not significantly regulated by obesity. Plasma IL-6 was also significantly elevated by obesity in parous mice. We have previously reported a potential role for stromal-derived hepatocyte growth factor (HGF) via its cognate receptor c-Met in the etiology of obesity-induced BBC tumor onset and in both human and murine primary coculture models of BBC-aggressiveness. Obesity-associated c-Met concentrations were 2.5-fold greater in normal mammary glands of parous mice. Taken together, our studies demonstrate that, parity in C3(1)-TAg mice dramatically reduced BBC latency compared to nulliparous mice. In parous mice, c-Met is regulated by obesity in unaffected mammary gland and is associated with tumor onset. C3(1)-TAg mice recapitulate epidemiologic findings such that parity drives increased BBC risk and potential microenvironmental alterations in c-Met signaling may play a role in etiology.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Mamarias Experimentales/patología , Obesidad/metabolismo , Paridad , Animales , Femenino , Factor de Crecimiento de Hepatocito/genética , Interleucina-6/sangre , Neoplasias Mamarias Experimentales/complicaciones , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Obesidad/complicaciones , Factores de TiempoRESUMEN
Epidemiologic studies demonstrate that obesity is associated with an aggressive subtype of breast cancer called basal-like breast cancer (BBC). Using the C3(1)-TAg murine model of BBC, we previously demonstrated that mice displayed an early onset of tumors when fed obesogenic diets in the adult window of susceptibility. Obesity was also shown to elevate mammary gland expression and activation of hepatocyte growth factor (HGF)/c-Met compared to lean controls, a pro-tumorigenic pathway associated with BBC in patients. Epidemiologic studies estimate that weight loss could prevent a large proportion of BBC. We sought to investigate whether weight loss in adulthood prior to tumor onset would protect mice from accelerated tumorigenesis observed in obese mice. Using a life-long model of obesity, C3(1)-TAg mice were weaned onto and maintained on an obesogenic high-fat diet. Obese mice displayed significant elevations in tumor progression, but not latency or burden. Tumor progression was significantly reversed when obese mice were induced to lose weight by switching to a control low-fat diet prior to tumor onset compared to mice maintained on obesogenic diet. We investigated the HGF/c-Met pathway known to regulate tumorigenesis. Importantly, HGF/c-Met expression in normal mammary glands and c-Met in tumors was elevated with obesity and was significantly reversed with weight loss. Changes in tumor growth could not be explained by measures of HGF action including phospho-AKT or phospho-S6. Other mediators associated with oncogenesis such as hyperinsulinemia and a high leptin:adiponectin ratio were elevated by obesity and reduced with weight loss. In sum, weight loss significantly blunted the obesity-responsive pro-tumorigenic HGF/c-Met pathway and improved several metabolic risk factors associated with BBC, which together may have contributed to the dramatic reversal of obesity-driven tumor progression. Future research aims to evaluate the role of obesity and the HGF/c-Met pathway in basal-like breast cancer progression.
RESUMEN
BACKGROUND: Injury and intestinal inflammation trigger wound healing responses that can restore mucosal architecture but if chronic, can promote intestinal fibrosis. Intestinal fibrosis is a major complication of Crohn's disease. The cellular and molecular basis of mucosal healing and intestinal fibrosis are not well defined and better understanding requires well characterized mouse models. METHODS: FVB-N wild type mice and C57BL6 procollagen α1(I)-GFP reporter mice were given one (DSS1) or two (DSS2) cycles of 3% DSS (5 days/cycle) followed by 7 days recovery. Histological scoring of inflammation and fibrosis were performed at DSS1, DSS1+3, DSS1+7, DSS2, DSS2+3, and DSS2+7. Procollagen α1(I)-GFP activation was assessed in DSS and also TNBS models by whole colon GFP imaging and fluorescence microscopy. Colocalization of GFP with α-smooth muscle actin (α-SMA) or vimentin was examined. GFP mRNA levels were tested for correlation with endogenous collagen α1(I) mRNA. RESULTS: Males were more susceptible to DSS-induced disease and mortality than females. In FVB-N mice one DSS cycle induced transient mucosal inflammation and fibrosis that resolved by 7 days of recovery. Two DSS cycles induced transmural inflammation and fibrosis in a subset of FVB-N mice but overall, did not yield more consistent, severe or sustained fibrosis. In C57BL6 mice, procollagen α1(I)-GFP reporter was activated at the end of DSS1 and through DSS+7 with more dramatic and transmural activation at DSS2 through DSS2+7, and in TNBS treated mice. In DSS and TNBS models GFP reporter expression localized to vimentin(+) cells and much fewer α-SMA(+) cells. GFP mRNA strongly correlated with collagen α1(I) mRNA. CONCLUSIONS: One DSS cycle in FVB-N mice provides a model to study mucosal injury and subsequent mucosal healing. The procollagen α1(I)-GFP transgenic provides a useful model to study activation of a gene encoding a major extracellular matrix protein during acute or chronic experimental intestinal inflammation and fibrosis.
Asunto(s)
Colágeno Tipo I/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Inflamación/patología , Mucosa Intestinal/patología , Regiones Promotoras Genéticas/genética , Cicatrización de Heridas , Enfermedad Aguda , Animales , Antígenos/metabolismo , Enfermedad Crónica , Cadena alfa 1 del Colágeno Tipo I , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Femenino , Fibrosis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Análisis de Supervivencia , Ácido Trinitrobencenosulfónico , Vimentina/metabolismo , Pérdida de PesoRESUMEN
Suppressors of cytokine signaling (SOCS) typically limit cytokine receptor signaling via the JAK-STAT pathway. Considerable evidence demonstrates that SOCS2 limits growth hormone (GH) action on body and organ growth. Biochemical evidence that SOCS2 binds to the IGF-I receptor (IGF-IR) supports the novel possibility that SOCS2 limits IGF-I action. The current study tested the hypothesis that SOCS2 normally limits basal or IGF-I-induced intestinal growth and limits IGF-IR signaling in intestinal epithelial cells. Intestinal growth was assessed in mice homozygous for SOCS2 gene deletion (SOCS2 null) and wild-type (WT) littermates at different ages and in response to infused IGF-I or vehicle or EGF and vehicle. The effects of SOCS2 on IGF-IR signaling were examined in ex vivo cultures of SOCS2 null and WT intestine and Caco-2 cells. Compared with WT, SOCS2 null mice showed significantly enhanced small intestine and colon growth, mucosal mass, and crypt cell proliferation and decreases in radiation-induced crypt apoptosis in jejunum. SOCS2 null mice showed significantly greater growth responses to IGF-I in small intestine and colon. IGF-I-stimulated activation of IGF-IR and downstream signaling intermediates were enhanced in the intestine of SOCS2 null mice and were decreased by SOCS2 overexpression in Caco-2 cells. SOCS2 bound directly to the endogenous IGF-IR in Caco-2 cells. The intestine of SOCS2 null mice also showed enhanced growth responses to infused EGF. We conclude that SOCS2 normally limits basal and IGF-I- and EGF-induced intestinal growth in vivo and has novel inhibitory effects on the IGF-IR tyrosine kinase pathway in intestinal epithelial cells.