RESUMEN
Parathyroid hormone-related protein (PTHrP) plays a major role in skeletal metastasis but its action mechanism has not been fully defined. We previously demonstrated the crucial importance of PTHrP in promoting mammary tumor initiation, growth, and metastasis in a mouse model with a mammary epithelium-targeted Pthlh gene ablation. We demonstrate here a novel mechanism for bone invasion involving PTHrP induction of epithelial to mesenchymal transition (EMT) and cancer stem cells (CSCs) regulation. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated Pthlh gene ablation was used to study EMT markers, phenotype, and invasiveness in two triple-negative breast cancer (TNBC) cell types (established MDA-MB-231 and patient-derived PT-TNBC cells). In vitro, Pthlh ablation in TNBC cells reduced EMT markers, mammosphere-forming ability, and CD44high/CD24low cells ratio. In vivo, cells were injected intratibially into athymic nude mice, and therapeutic treatment with our anti-PTHrP blocking antibody was started 2 weeks after skeletal tumors were established. In vivo, compared to control, lytic bone lesion from Pthlh -ablated cells decreased significantly over 2 weeks by 27% for MDA-MB-231 and by 75% for PT-TNBC-injected mice (p < 0.001). Micro-CT (µCT) analyses also showed that antibody therapy reduced bone lytic volume loss by 52% and 48% for non-ablated MDA-MB-231 and PT-TNBC, respectively (p < 0.05). Antibody therapy reduced skeletal tumor burden by 45% and 87% for non-ablated MDA-MB-231 and PT-TNBC, respectively (p < 0.002) and caused a significant decrease of CSC/EMT markers ALDH1, vimentin, and Slug, and an increase in E-cadherin in bone lesions. We conclude that PTHrP is a targetable EMT molecular driver and suggest that its pharmacological blockade can provide a potential therapeutic approach against established TNBC-derived skeletal lesions. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
RESUMEN
Pancreatic cancer metastasis is a leading cause of cancer-related deaths, yet very little is understood regarding the underlying biology. As a result, targeted therapies to inhibit metastasis are lacking. Here, we report that the parathyroid hormone-related protein (PTHrP encoded by PTHLH) is frequently amplified as part of the KRAS amplicon in patients with pancreatic cancer. PTHrP upregulation drives the growth of both primary and metastatic tumors in mice and is highly enriched in pancreatic ductal adenocarcinoma metastases. Loss of PTHrP-either genetically or pharmacologically-dramatically reduces tumor burden, eliminates metastasis, and enhances overall survival. These effects are mediated in part through a reduction in epithelial-to-mesenchymal transition, which reduces the ability of tumor cells to initiate metastatic cascade. Spp1, which encodes osteopontin, is revealed to be a downstream effector of PTHrP. Our results establish a new paradigm in pancreatic cancer whereby PTHrP is a driver of disease progression and emerges as a novel therapeutic vulnerability. SIGNIFICANCE: Pancreatic cancer often presents with metastases, yet no strategies exist to pharmacologically inhibit this process. Herein, we establish the oncogenic and prometastatic roles of PTHLH, a novel amplified gene in pancreatic ductal adenocarcinoma. We demonstrate that blocking PTHrP activity reduces primary tumor growth, prevents metastasis, and prolongs survival in mice.This article is highlighted in the In This Issue feature, p. 1601.
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Neoplasias Pancreáticas/metabolismo , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Animales , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Metástasis de la Neoplasia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteína Relacionada con la Hormona Paratiroidea/antagonistas & inhibidores , Proteína Relacionada con la Hormona Paratiroidea/genéticaRESUMEN
UNLABELLED: While in vitro studies have shown that nanoscale surface modifications influence cell fate and activity, there is little information on how they modulate healing at the bone-implant interface. AIM: This study aims to investigate the effect of nanotopography at early time intervals when critical events for implant integration occur. MATERIALS & METHODS: Untreated and sulfuric acid/hydrogen peroxide-treated machined-surface titanium alloy implants were placed in rat tibiae. Samples were processed for DNA microarray analysis and histomorphometry. RESULTS: At both 3 and 5 days, the gene expression profile of the healing tissue around nanotextured implants differed from that around machined-surface implants or control empty holes, and were accompanied by an increase in bone-implant contact on day 5. While some standard pathways such as the immune response predominated, a number of unclassified genes were also implicated. CONCLUSION: Nanotexture elicits an initial gene response that is more complex than suspected so far and favors healing at the bone-implant interface.
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Perfilación de la Expresión Génica , Nanoestructuras/uso terapéutico , Osteogénesis/genética , Prótesis e Implantes , Animales , Desarrollo Óseo/genética , Humanos , Nanoestructuras/efectos adversos , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Tibia/cirugía , Titanio/uso terapéutico , Cicatrización de Heridas/genéticaRESUMEN
Ameloblastin (AMBN) is an enamel matrix protein produced by ameloblasts. It has been suggested that AMBN might also be implicated in craniofacial bone formation. Our objective was to determine whether AMBN has an effect on osteogenic mineralisation and influences bone remodelling and repair. MC3T3-E1 cells were screened for endogenous expression of enamel proteins using real time PCR. Various osteogenic cells were infected with lentivirus encoding for AMBN and protein expression was verified using immunochemistry. Cultures were stained with alizarin red and mineralisation was quantified. Healing bone was probed for expression of AMBN by DNA microarray analysis. Tooth extraction, experimental tooth movement (ETM), and creation of a non-critical size bone defect in the tibia (BDT) were carried out in wild type and AMBN(Δ5-6) mutant mice. Tissues were processed for immunolabelling of AMBN and Bril, an osteoblast specific protein associated with active bone formation. MC3T3-E1 cells and healing bone showed no significant expression of AMBN. Overexpression of AMBN in osteogenic cultures induced no noticeable changes in mineralisation. In wild type mice, AMBN was immunodetected in ameloblasts and enamel, but not in normal bone, and at sites where bone remodelling and repair were induced. Bone remodelling during ETM and BDT repair in AMBN(Δ5-6) mice were not significantly different from that in wild type animals. Our results suggest that AMBN does not influence osteogenic activity in vitro under the conditions used, and does not participate in craniofacial bone remodelling under mechanical stress and in repair of non-critical size bone defects.
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Regeneración Ósea , Remodelación Ósea , Proteínas del Esmalte Dental/fisiología , Cicatrización de Heridas , Ameloblastos/citología , Ameloblastos/metabolismo , Animales , Calcificación Fisiológica , Ratones , Ratones Mutantes , Tibia/lesionesRESUMEN
In the course of attempting to define the bone "secretome" using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 134 amino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus-mediated Bril overexpression and lentivirus-mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose-dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.
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Huesos/metabolismo , Calcificación Fisiológica , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Adenoviridae , Secuencia de Aminoácidos , Animales , Biomarcadores/metabolismo , Western Blotting , Línea Celular , Clonación Molecular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Hibridación in Situ , Lentivirus , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Osteoblastos/citología , Osteoblastos/metabolismo , Proteínas/química , Proteínas/genética , Ratas , Alineación de SecuenciaRESUMEN
Osteocrin (Ostn) is a recently discovered secreted protein produced by cells of the osteoblast lineage that shows a well conserved homology with members of the natriuretic peptide (NP) family. We hypothesized that Ostn could interact with the NP receptors, thereby modulating NP actions on the skeleton. Ostn binds specifically and saturably to the NP peptide receptor-C (NPR-C) receptor with a Kd of approximately 5 nM with no binding to the GC-A or GC-B receptors. Deletion of several of the residues deemed important for NP binding to NPR-C led to abolition of Ostn binding, confirming the presence of a "natriuretic motif." Functionally, Ostn was able to augment C-type natriuretic peptide-stimulated cGMP production in both pre-chondrocytic (ATDC5) and osteoblastic (UMR106) cells, suggesting increased NP levels due to attenuation of NPR-C associated NP clearance. Ostn-transgenic mice displayed elongated bones and a marked kyphosis associated with elevated bone cGMP levels, suggesting that elevated natriuretic peptide activity contributed to the increased bone length possibly through an increase in growth plate chondrocyte proliferation. Thus, we have demonstrated that Ostn is a naturally occurring ligand of the NPR-C clearance receptor and may act to locally modulate the actions of the natriuretic system in bone by blocking the clearance action of NPR-C, thus locally elevating levels of C-type natriuretic peptide.