RESUMEN
BACKGROUND: Parathyroid hormone-related protein (PTHrP) is required for embryonic breast development and has important functions during lactation, when it is produced by alveolar epithelial cells and secreted into the maternal circulation to mobilize skeletal calcium used for milk production. PTHrP is also produced by breast cancers, and GWAS studies suggest that it influences breast cancer risk. However, the exact functions of PTHrP in breast cancer biology remain unsettled. METHODS: We developed a tetracycline-regulated, MMTV (mouse mammary tumor virus)-driven model of PTHrP overexpression in mammary epithelial cells (Tet-PTHrP mice) and bred these mice with the MMTV-PyMT (polyoma middle tumor-antigen) breast cancer model to analyze the impact of PTHrP overexpression on normal mammary gland biology and in breast cancer progression. RESULTS: Overexpression of PTHrP in luminal epithelial cells caused alveolar hyperplasia and secretory differentiation of the mammary epithelium with milk production. This was accompanied by activation of Stat5 and increased expression of E74-like factor-5 (Elf5) as well as a delay in post-lactation involution. In MMTV-PyMT mice, overexpression of PTHrP (Tet-PTHrP;PyMT mice) shortened tumor latency and accelerated tumor growth, ultimately reducing overall survival. Tumors overproducing PTHrP also displayed increased expression of nuclear pSTAT5 and Elf5, increased expression of markers of secretory differentiation and milk constituents, and histologically resembled secretory carcinomas of the breast. Overexpression of PTHrP within cells isolated from tumors, but not PTHrP exogenously added to cell culture media, led to activation of STAT5 and milk protein gene expression. In addition, neither ablating the Type 1 PTH/PTHrP receptor (PTH1R) in epithelial cells nor treating Tet-PTHrP;PyMT mice with an anti-PTH1R antibody prevented secretory differentiation or altered tumor latency. These data suggest that PTHrP acts in a cell-autonomous, intracrine manner. Finally, expression of PTHrP in human breast cancers is associated with expression of genes involved in milk production and STAT5 signaling. CONCLUSIONS: Our study suggests that PTHrP promotes pathways leading to secretory differentiation and proliferation in both normal mammary epithelial cells and in breast tumor cells.
Asunto(s)
Neoplasias de la Mama , Neoplasias Mamarias Animales , Proteína Relacionada con la Hormona Paratiroidea , Factor de Transcripción STAT5 , Animales , Neoplasias de la Mama/patología , Femenino , Humanos , Lactancia/genética , Glándulas Mamarias Animales , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Ratones , Proteína Relacionada con la Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismoRESUMEN
Cells that form bone (osteoblasts) express both ephrinB2 and EphB4, and previous work has shown that pharmacological inhibition of the ephrinB2/EphB4 interaction impairs osteoblast differentiation in vitro and in vivo. The purpose of this study was to determine the role of ephrinB2 signaling in the osteoblast lineage in the process of bone formation. Cultured osteoblasts from mice with osteoblast-specific ablation of ephrinB2 showed delayed expression of osteoblast differentiation markers, a finding that was reproduced by ephrinB2, but not EphB4, RNA interference. Microcomputed tomography, histomorphometry, and mechanical testing of the mice lacking ephrinB2 in osteoblasts revealed a 2-fold delay in bone mineralization, a significant reduction in bone stiffness, and a 50% reduction in osteoblast differentiation induced by anabolic parathyroid hormone (PTH) treatment, compared to littermate sex- and age-matched controls. These defects were associated with significantly lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis, indicated by TUNEL staining and transmission electron microscopy of bone samples, and a 2-fold increase in annexin V staining and 7-fold increase in caspase 8 activation in cultured ephrinB2 deficient osteoblasts. We conclude that osteoblast differentiation and bone strength are maintained by antiapoptotic actions of ephrinB2 signaling within the osteoblast lineage.
Asunto(s)
Apoptosis , Calcificación Fisiológica , Osteoblastos/metabolismo , Osteogénesis , Receptor EphB2/metabolismo , Animales , Anexina A5/genética , Anexina A5/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoblastos/citología , Receptor EphB2/genética , Receptor EphB4/genética , Receptor EphB4/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Statins reduce cardiovascular events and may improve bone mineral density. METHODS: We conducted a sub-analysis of a randomized clinical trial that investigated the differential effect of moderate vs intensive low-density lipoprotein cholesterol (LDL-C) lowering therapies on coronary artery calcium (CAC) scores, and used the acquired images to assess the change in radiological attenuation of selected thoracic vertebrae. Baseline and 12-month unenhanced chest CT scans were performed in 420 hyperlipidemic, postmenopausal women randomized to atorvastatin (ATV) 80 mg/day or pravastatin (PRV) 40 mg/day in the Beyond Endorsed Lipid Lowering with Electron Beam Tomography Scanning (BELLES) trial. Bone attenuation was measured in three contiguous thoracic vertebrae at baseline and 12 months. RESULTS: There were no differences in baseline demographic and clinical characteristics between treatment arms. The median percent lowering (interquartile range) in LDL-C was significantly greater with ATV than PRV [-53 (-69 to 20)% vs -28 (-55 to 74)%, p < 0.001], although the CAC score change was similar [12 (-63 to 208)% vs 13 (-75 to 358)%; p = 0.44]. At follow-up, the median bone attenuation loss was significantly greater with PRV than with ATV [-2.6 (-27 to 11)% vs 0 (-11 to 25)%; p < 0.001]. The attenuation loss in the PRV group was comparable to that of a historical untreated general population sample. In the entire cohort, the changes in LDL-C and total cholesterol were inversely correlated with bone attenuation change (p < 0.01). In adjusted multivariable linear regression analyses, race and percent change in LDL-C were independent predictors of bone attenuation change. Age, body mass index, history of smoking, diabetes mellitus, hypertension, peripheral vascular disease, or hormone replacement therapy did not affect percent change in BMD. CONCLUSIONS: These findings support the hypothesis that there is an interaction between bone and cardiometabolic health and that intensive lipid lowering has a beneficial effect on bone health.
Asunto(s)
Anticolesterolemiantes , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Hiperlipidemias , Humanos , Femenino , Atorvastatina/uso terapéutico , Pravastatina/uso terapéutico , LDL-Colesterol , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Hiperlipidemias/tratamiento farmacológico , Pirroles/uso terapéutico , Anticolesterolemiantes/uso terapéuticoRESUMEN
Parathyroid hormone-related protein (PTHrP) contributes to the development and metastatic progression of breast cancer by promoting hypercalcemia, tumor growth, and osteolytic bone metastases, but it is not known how PTHrP is upregulated in breast tumors. Here we report a central role in this process for the calcium-sensing receptor, CaSR, which enables cellular responses to changes in extracellular calcium, through studies of CaSR-PTHrP interactions in the MMTV-PymT transgenic mouse model of breast cancer and in human breast cancer cells. CaSR activation stimulated PTHrP production by breast cancer cells in vitro and in vivo Tissue-specific disruption of the casr gene in mammary epithelial cells in MMTV-PymT mice reduced tumor PTHrP expression and inhibited tumor cell proliferation and tumor outgrowth. CaSR signaling promoted the proliferation of human breast cancer cell lines and tumor cells cultured from MMTV-PyMT mice. Further, CaSR activation inhibited cell death triggered by high extracellular concentrations of calcium. The actions of the CaSR appeared to be mediated by nuclear actions of PTHrP that decreased p27(kip1) levels and prevented nuclear accumulation of the proapoptotic factor apoptosis inducing factor. Taken together, our findings suggest that CaSR-PTHrP interactions might be a promising target for the development of therapeutic agents to limit tumor cell growth in bone metastases and in other microenvironments in which elevated calcium and/or PTHrP levels contribute to breast cancer progression. Cancer Res; 76(18); 5348-60. ©2016 AACR.
Asunto(s)
Neoplasias de la Mama/patología , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Receptores Sensibles al Calcio/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Ratones , Ratones Noqueados , Análisis de Matrices TisularesRESUMEN
Previous reports indicate that ephrinB2 expression by osteoblasts is stimulated by parathyroid hormone (PTH) and its related protein (PTHrP) and that ephrinB2/EphB4 signaling between osteoblasts and osteoclasts stimulates osteoblast differentiation while inhibiting osteoclast differentiation. To determine the role of the ephrinB2/EphB4 interaction in the skeleton, we used a specific inhibitor, soluble EphB4 (sEphB4), in vitro and in vivo. sEphB4 treatment of cultured osteoblasts specifically inhibited EphB4 and ephrinB2 phosphorylation and reduced mRNA levels of late markers of osteoblast/osteocyte differentiation (osteocalcin, dentin matrix protein-1 [DMP-1], sclerostin, matrix-extracellular phosphoglycoprotein [MEPE]), while substantially increasing RANKL. sEphB4 treatment in vivo in the presence and absence of PTH increased osteoblast formation and mRNA levels of early osteoblast markers (Runx2, alkaline phosphatase, Collagen 1α1, and PTH receptor [PTHR1]), but despite a substantial increase in osteoblast numbers, there was no significant change in bone formation rate or in late markers of osteoblast/osteocyte differentiation. Rather, in the presence of PTH, sEphB4 treatment significantly increased osteoclast formation, an effect that prevented the anabolic effect of PTH, causing instead a decrease in trabecular number. This enhancement of osteoclastogenesis by sEphB4 was reproduced in vitro but only in the presence of osteoblasts. These data indicate that ephrinB2/EphB4 signaling within the osteoblast lineage is required for late stages of osteoblast differentiation and, further, restricts the ability of osteoblasts to support osteoclast formation, at least in part by limiting RANKL production. This indicates a key role for the ephrinB2/EphB4 interaction within the osteoblast lineage in osteoblast differentiation and support of osteoclastogenesis.