RESUMEN
RATIONALE: Wnt signaling regulates key aspects of diabetic vascular disease. OBJECTIVE: We generated SM22-Cre;LRP6(fl/fl);LDLR(-/-) mice to determine contributions of Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6) in the vascular smooth muscle lineage of male low-density lipoprotein receptor-null mice, a background susceptible to diet (high-fat diet)-induced diabetic arteriosclerosis. METHODS AND RESULTS: As compared with LRP6(fl/fl);LDLR(-/-) controls, SM22-Cre;LRP6(fl/fl);LDLR(-/-) (LRP6-VKO) siblings exhibited increased aortic calcification on high-fat diet without changes in fasting glucose, lipids, or body composition. Pulse wave velocity (index of arterial stiffness) was also increased. Vascular calcification paralleled enhanced aortic osteochondrogenic programs and circulating osteopontin (OPN), a matricellular regulator of arteriosclerosis. Survey of ligands and Frizzled (Fzd) receptor profiles in LRP6-VKO revealed upregulation of canonical and noncanonical Wnts alongside Fzd10. Fzd10 stimulated noncanonical signaling and OPN promoter activity via an upstream stimulatory factor (USF)-activated cognate inhibited by LRP6. RNA interference revealed that USF1 but not USF2 supports OPN expression in LRP6-VKO vascular smooth muscle lineage, and immunoprecipitation confirmed increased USF1 association with OPN chromatin. ML141, an antagonist of cdc42/Rac1 noncanonical signaling, inhibited USF1 activation, osteochondrogenic programs, alkaline phosphatase, and vascular smooth muscle lineage calcification. Mass spectrometry identified LRP6 binding to protein arginine methyltransferase (PRMT)-1, and nuclear asymmetrical dimethylarginine modification was increased with LRP6-VKO. RNA interference demonstrated that PRMT1 inhibits OPN and TNAP, whereas PRMT4 supports expression. USF1 complexes containing the histone H3 asymmetrically dimethylated on Arg-17 signature of PRMT4 are increased with LRP6-VKO. Jmjd6, a demethylase downregulated with LRP6 deficiency, inhibits OPN and TNAP expression, USF1: histone H3 asymmetrically dimethylated on Arg-17 complex formation, and transactivation. CONCLUSIONS: LRP6 restrains vascular smooth muscle lineage noncanonical signals that promote osteochondrogenic differentiation, mediated in part via USF1- and arginine methylation-dependent relays.
Asunto(s)
Arteriosclerosis/prevención & control , Calcinosis/prevención & control , Diabetes Mellitus Experimental/complicaciones , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/fisiología , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/metabolismo , Receptores de LDL/deficiencia , Vía de Señalización Wnt , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Arteriosclerosis/etiología , Arteriosclerosis/metabolismo , Calcinosis/etiología , Calcinosis/metabolismo , Diabetes Mellitus Experimental/patología , Grasas de la Dieta/efectos adversos , Receptores Frizzled/fisiología , Regulación de la Expresión Génica/fisiología , Histonas/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/deficiencia , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/patología , Osteopontina/biosíntesis , Osteopontina/genética , Comunicación Paracrina , Mapeo de Interacción de Proteínas , Proteína-Arginina N-Metiltransferasas/metabolismo , Receptores de Superficie Celular , Receptores de LDL/genética , Factores Estimuladores hacia 5'/fisiología , Rigidez Vascular/fisiologíaRESUMEN
BACKGROUND: Despite advances in early diagnosis and treatment of cancer patients, metastasis remains the major cause of mortality. TP53 is one of the most frequently mutated genes in human cancer, and these alterations can occur during the early stages of oncogenesis or as later events as tumors progress to more aggressive forms. Previous studies have suggested that p53 plays a role in cellular pathways that govern metastasis. To investigate how p53 deficiency contributes to late-stage tumor growth and metastasis, we developed paired isogenic patient-derived xenograft (PDX) models of triple-negative breast cancer (TNBC) differing only in p53 status for longitudinal analysis. METHODS: Patient-derived isogenic human tumor lines differing only in p53 status were implanted into mouse mammary glands. Tumor growth and metastasis were monitored with bioluminescence imaging, and circulating tumor cells (CTCs) were quantified by flow cytometry. RNA-Seq was performed on p53-deficient and p53 wild-type tumors, and functional validation of a lead candidate gene was performed in vivo. RESULTS: Isogenic p53 wild-type and p53-deficient tumors metastasized out of mammary glands and colonized distant sites with similar frequency. However, p53-deficient tumors metastasized earlier than p53 wild-type tumors and grew faster in both primary and metastatic sites as a result of increased proliferation and decreased apoptosis. In addition, greater numbers of CTCs were detected in the blood of mice engrafted with p53-deficient tumors. However, when normalized to tumor mass, the number of CTCs isolated from mice bearing parental and p53-deficient tumors was not significantly different. Gene expression profiling followed by functional validation identified B cell translocation gene 2 (BTG2), a downstream effector of p53, as a negative regulator of tumor growth both at primary and metastatic sites. BTG2 expression status correlated with survival of TNBC patients. CONCLUSIONS: Using paired isogenic PDX-derived metastatic TNBC cells, loss of p53 promoted tumor growth and consequently increased tumor cell shedding into the blood, thus enhancing metastasis. Loss of BTG2 expression in p53-deficient tumors contributed to this metastatic potential by enhancing tumor growth in primary and metastatic sites. Furthermore, clinical data support conclusions generated from PDX models and indicate that BTG2 expression is a candidate prognostic biomarker for TNBC.
Asunto(s)
Proliferación Celular/genética , Proteínas Inmediatas-Precoces/biosíntesis , Neoplasias de la Mama Triple Negativas/genética , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/biosíntesis , Animales , Apoptosis/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Proteínas Inmediatas-Precoces/genética , Ratones , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/patología , Neoplasias de la Mama Triple Negativas/patología , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: Endothelial cells (ECs) can undergo an endothelial-mesenchymal transition with tissue fibrosis. Wnt- and Msx2-regulated signals participate in arteriosclerotic fibrosis and calcification. We studied the impact of Wnt7, Msx2, and Dkk1, a Wnt7 antagonist, on endothelial-mesenchymal transition in primary aortic ECs. APPROACH AND RESULTS: Transduction of aortic ECs with vectors expressing Dkk1 suppressed EC differentiation and induced a mineralizing myofibroblast phenotype. Dkk1 suppressed claudin 5, PECAM, cadherin 5 (Cdh5), Tie1, and Tie2. Dkk1 converted the cuboidal cell monolayer into a spindle-shaped multilayer and inhibited EC cord formation. Myofibroblast and osteogenic markers, SM22, type I collagen, Osx, Runx2, and alkaline phosphatase, were upregulated by Dkk1 via activin-like kinase/Smad pathways. Dkk1 increased fibrotic mineralization of aortic ECs cultured under osteogenic conditions--the opposite of mesenchymal cell responses. Msx2 and Wnt7b maintained morphology and upregulated markers of differentiated ECs. Deleting EC Wnt7b with the Cdh5-Cre transgene in Wnt7b(fl/fl);LDLR(-/-) mice upregulated aortic osteogenic genes (Osx, Sox9, Runx2, and Msx2) and nuclear phospho-Smad1/5, and increased collagen and calcium accumulation. CONCLUSIONS: Dkk1 enhances endothelial-mesenchymal transition in aortic ECs, whereas Wnt7b and Msx2 signals preserve EC phenotype. EC responses to Dkk1, Wnt7b, and Msx2 are the opposite of mesenchymal responses, coupling EC phenotypic stability with osteofibrogenic predilection during arteriosclerosis.
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Aorta/metabolismo , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal , Proteínas de Homeodominio/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Miofibroblastos/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Arteriosclerosis/genética , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Biomarcadores/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Bovinos , Diferenciación Celular , Forma de la Célula , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Fibrosis , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Ratones Noqueados , Miofibroblastos/patología , Neovascularización Fisiológica , Osificación Heterotópica/metabolismo , Fenotipo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transducción Genética , Transfección , Proteínas Wnt/deficiencia , Proteínas Wnt/genéticaRESUMEN
Triple negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases in the United States. Systemic neoadjuvant chemotherapy (NACT), with or without immunotherapy, is the current standard of care for patients with early-stage TNBC. However, up to 70% of TNBC patients have significant residual disease once NACT is completed, which is associated with a high risk of developing recurrence within two to three years of surgical resection. To identify targetable vulnerabilities in chemoresistant TNBC, we generated longitudinal patient-derived xenograft (PDX) models from TNBC tumors before and after patients received NACT. We then compiled transcriptomes and drug response profiles for all models. Transcriptomic analysis identified the enrichment of aberrant protein homeostasis pathways in models from post-NACT tumors relative to pre-NACT tumors. This observation correlated with increased sensitivity in vitro to inhibitors targeting the proteasome, heat shock proteins, and neddylation pathways. Pevonedistat, a drug annotated as a NEDD8-activating enzyme (NAE) inhibitor, was prioritized for validation in vivo and demonstrated efficacy as a single agent in multiple PDX models of TNBC. Pharmacotranscriptomic analysis identified a pathway-level correlation between pevonedistat activity and post-translational modification (PTM) machinery, particularly involving neddylation and sumoylation targets. Elevated levels of both NEDD8 and SUMO1 were observed in models exhibiting a favorable response to pevonedistat compared to those with a less favorable response in vivo. Moreover, a correlation emerged between the expression of neddylation-regulated pathways and tumor response to pevonedistat, indicating that targeting these PTM pathways may prove effective in combating chemoresistant TNBC.
RESUMEN
Patient-derived xenograft (PDX) models of breast cancer are an effective discovery platform and tool for preclinical pharmacologic testing and biomarker identification. We established orthotopic PDX models of triple negative breast cancer (TNBC) from the primary breast tumors of patients prior to and following neoadjuvant chemotherapy (NACT) while they were enrolled in the ARTEMIS trial (NCT02276443). Serial biopsies were obtained from patients prior to treatment (pre-NACT), from poorly responsive disease after four cycles of Adriamycin and cyclophosphamide (AC, mid-NACT), and in cases of AC-resistance, after a 3-month course of different experimental therapies and/or additional chemotherapy (post-NACT). Our study cohort includes a total of 269 fine needle aspirates (FNAs) from 217 women, generating a total of 62 PDX models (overall success-rate = 23%). Success of PDX engraftment was generally higher from those cancers that proved to be treatment-resistant, whether poorly responsive to AC as determined by ultrasound measurements mid-NACT (p = 0.063), RCB II/III status after NACT (p = 0.046), or metastatic relapse within 2 years of surgery (p = 0.008). TNBC molecular subtype determined from gene expression microarrays of pre-NACT tumors revealed no significant association with PDX engraftment rate (p = 0.877). Finally, we developed a statistical model predictive of PDX engraftment using percent Ki67 positive cells in the patient's diagnostic biopsy, positive lymph node status at diagnosis, and low volumetric reduction of the patient's tumor following AC treatment. This novel bank of 62 PDX models of TNBC provides a valuable resource for biomarker discovery and preclinical therapeutic trials aimed at improving neoadjuvant response rates for patients with TNBC.
RESUMEN
RATIONALE: Vascular fibrosis and calcification contribute to diabetic arteriosclerosis, impairing Windkessel physiology necessary for distal tissue perfusion. Wnt family members, upregulated in arteries by the low-grade inflammation of "diabesity," stimulate type I collagen expression and osteogenic mineralization of mesenchymal progenitors via beta-catenin. Conversely, parathyroid hormone (PTH) inhibits aortic calcification in low-density lipoprotein receptor (LDLR)-deficient mice fed high fat diabetogenic diets (HFD). OBJECTIVE: We sought to determine the impact of vascular PTH receptor (PTH1R) activity on arteriosclerotic Wnt/beta-catenin signaling in vitro and in vivo. We generated SM-caPTH1R transgenic mice, a model in which the constitutively active PTH1R variant H223R (caPTH1R) is expressed only in the vasculature. METHODS AND RESULTS: The caPTH1R inhibited Wnt/beta-catenin signaling, collagen production, and vascular smooth muscle cell proliferation and calcification in vitro. Transgenic SM-caPTH1R;LDLR(+/-) mice fed HFD develop diabesity, with no improvements in fasting serum glucose, cholesterol, weight, body composition, or bone mass versus LDLR(+/-) siblings. SM-caPTH1R downregulated aortic Col1A1, Runx2, and Nox1 expression without altering TNF, Msx2, Wnt7a/b, or Nox4. The SM-caPTH1R transgene decreased aortic beta-catenin protein accumulation and signaling in diabetic LDLR(+/-) mice. Levels of aortic superoxide (a precursor of peroxide that activates pro-matrix metalloproteinase 9 and osteogenic signaling in vascular smooth muscle cells) were suppressed by the SM-caPTH1R transgene. Aortic calcification, collagen accumulation, and wall thickness were concomitantly reduced, enhancing vessel distensibility. CONCLUSIONS: Cell-autonomous vascular smooth muscle cell PTH1R activity inhibits arteriosclerotic Wnt/beta-catenin signaling and reduces vascular oxidative stress, thus limiting aortic type I collagen and calcium accrual in diabetic LDLR-deficient mice.
Asunto(s)
Arteriosclerosis/metabolismo , Diabetes Mellitus/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor de Hormona Paratiroídea Tipo 1/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Arteriosclerosis/genética , Arteriosclerosis/patología , Calcinosis/metabolismo , Calcinosis/patología , Proliferación Celular , Células Cultivadas , Colágeno/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Modelos Animales de Enfermedad , Fibrosis , Humanos , Masculino , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/patología , Músculo Liso Vascular/patología , Mutación , Miocitos del Músculo Liso/patología , Estrés Oxidativo , Ratas , Receptor de Hormona Paratiroídea Tipo 1/genética , Receptores de LDL/deficiencia , Receptores de LDL/genética , Superóxidos/metabolismo , Transcripción Genética , Transducción GenéticaRESUMEN
OBJECTIVE: Calcification and fibrosis reduce vascular compliance in arteriosclerosis. To better understand the role of osteopontin (OPN), a multifunctional protein upregulated in diabetic arteries, we evaluated contributions of OPN in male low-density lipoprotein receptor (LDLR)-/- mice fed a high-fat diet. METHODS AND RESULTS: OPN had no impact on high-fat diet-induced hyperglycemia, dyslipidemia, or body composition. However, OPN-/-;LDLR-/- mice exhibited an altered time-course of aortic calcium accrual-reduced during initiation but increased with progression-versus OPN+/+;LDLR-/- controls. Collagen accumulation, chondroid metaplasia, and mural thickness were increased in aortas of OPN-/-;LDLR-/- mice. Aortic compliance was concomitantly reduced. Vascular reexpression of OPN (SM22-OPN transgene) reduced aortic Col2A1 and medial chondroid metaplasia but did not affect atherosclerotic calcification, Col1A1 expression, collagen accumulation, or arterial stiffness. Dosing with the proinflammatory OPN fragment SVVYGLR upregulated aortic Wnt and osteogenic gene expression, increased aortic ß-catenin, and restored early-phase aortic calcification in OPN-/-;LDLR-/- mice. CONCLUSIONS: OPN exerts stage-specific roles in arteriosclerosis in LDLR-/- mice. Actions phenocopied by the OPN metabolite SVVYGLR promote osteogenic calcification processes with disease initiation. OPN limits vascular chondroid metaplasia, endochondral mineralization, and collagen accumulation with progression. Complete deficiency yields a net increase in arteriosclerotic disease, reducing aortic compliance and conduit vessel function in LDLR-/- mice.
Asunto(s)
Aorta/patología , Aorta/fisiopatología , Arteriosclerosis/patología , Arteriosclerosis/fisiopatología , Angiopatías Diabéticas/patología , Angiopatías Diabéticas/fisiopatología , Osteopontina/fisiología , Secuencia de Aminoácidos , Animales , Aorta/efectos de los fármacos , Arteriosclerosis/etiología , Calcinosis/etiología , Calcinosis/patología , Calcinosis/fisiopatología , Calcio , Colágeno/metabolismo , Angiopatías Diabéticas/etiología , Elastina/metabolismo , Fibrosis , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Osteopontina/deficiencia , Osteopontina/genética , Osteopontina/farmacología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Regiones Promotoras Genéticas , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de LDL/fisiología , Transducción de Señal , Resistencia Vascular , beta Catenina/metabolismoRESUMEN
There is an unmet clinical need for stratification of breast lesions as indolent or aggressive to tailor treatment. Here, single-cell transcriptomics and multiparametric imaging applied to a mouse model of breast cancer reveals that the aggressive tumor niche is characterized by an expanded basal-like population, specialization of tumor subpopulations, and mixed-lineage tumor cells potentially serving as a transition state between luminal and basal phenotypes. Despite vast tumor cell-intrinsic differences, aggressive and indolent tumor cells are functionally indistinguishable once isolated from their local niche, suggesting a role for non-tumor collaborators in determining aggressiveness. Aggressive lesions harbor fewer total but more suppressed-like T cells, and elevated tumor-promoting neutrophils and IL-17 signaling, disruption of which increase tumor latency and reduce the number of aggressive lesions. Our study provides insight into tumor-immune features distinguishing indolent from aggressive lesions, identifies heterogeneous populations comprising these lesions, and supports a role for IL-17 signaling in aggressive progression.
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Neoplasias de la Mama/inmunología , Mama/patología , Escape del Tumor , Animales , Mama/inmunología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Femenino , Humanos , Interleucina-17/genética , Interleucina-17/inmunología , Ratones , Neutrófilos/inmunología , Análisis de la Célula IndividualRESUMEN
Eradicating triple-negative breast cancer (TNBC) resistant to neoadjuvant chemotherapy (NACT) is a critical unmet clinical need. In this study, patient-derived xenograft (PDX) models of treatment-naïve TNBC and serial biopsies from TNBC patients undergoing NACT were used to elucidate mechanisms of chemoresistance in the neoadjuvant setting. Barcode-mediated clonal tracking and genomic sequencing of PDX tumors revealed that residual tumors remaining after treatment with standard frontline chemotherapies, doxorubicin (Adriamycin) combined with cyclophosphamide (AC), maintained the subclonal architecture of untreated tumors, yet their transcriptomes, proteomes, and histologic features were distinct from those of untreated tumors. Once treatment was halted, residual tumors gave rise to AC-sensitive tumors with similar transcriptomes, proteomes, and histological features to those of untreated tumors. Together, these results demonstrated that tumors can adopt a reversible drug-tolerant state that does not involve clonal selection as an AC resistance mechanism. Serial biopsies obtained from patients with TNBC undergoing NACT revealed similar histologic changes and maintenance of stable subclonal architecture, demonstrating that AC-treated PDXs capture molecular features characteristic of human TNBC chemoresistance. Last, pharmacologic inhibition of oxidative phosphorylation using an inhibitor currently in phase 1 clinical development delayed residual tumor regrowth. Thus, AC resistance in treatment-naïve TNBC can be mediated by nonselective mechanisms that confer a reversible chemotherapy-tolerant state with targetable vulnerabilities.
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Doxorrubicina/uso terapéutico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Ciclofosfamida/uso terapéutico , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Ratones SCID , Terapia Neoadyuvante , Transcriptoma/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In diabetic LDLR-/- mice, an ectopic BMP2-Msx2 gene regulatory program is upregulated in association with vascular calcification. We verified the procalcific actions of aortic Msx2 expression in vivo. CMV-Msx2 transgenic (CMV-Msx2Tg(+)) mice expressed 3-fold higher levels of aortic Msx2 than nontransgenic littermates. On high-fat diets, CMV-Msx2Tg(+) mice exhibited marked cardiovascular calcification involving aortic and coronary tunica media. This corresponded to regions of Msx2 immunoreactivity in adjacent adventitial myofibroblasts, suggesting a potential paracrine osteogenic signal. To better understand Msx2-regulated calcification, we studied actions in 10T1/2 cells. We found that conditioned media from Msx2-transduced 10T1/2 cells (Msx2-CM) is both pro-osteogenic and adipostatic; these features are characteristic of Wnt signaling. Msx2-CM stimulated Wnt-dependent TCF/LEF transcription, and Msx2-transduced cells exhibited increased nuclear beta-catenin localization with concomitant alkaline phosphatase induction. Msx2 upregulated Wnt3a and Wnt7a but downregulated expression of the canonical inhibitor Dkk1. Dkk1 treatment reversed osteogenic and adipostatic actions of Msx2. Teriparatide, a PTH1R agonist that inhibits murine vascular calcification, suppressed vascular BMP2-Msx2-Wnt signaling. Analyses of CMV-Msx2Tg(+) mice confirmed that Msx2 suppresses aortic Dkk1 and upregulates vascular Wnts; moreover, TOPGAL(+) (Wnt reporter); CMV-Msx2Tg(+) mice exhibited augmented aortic LacZ expression. Thus, Msx2-expressing cells elaborated an osteogenic milieu that promotes vascular calcification in part via paracrine Wnt signals.
Asunto(s)
Calcinosis/metabolismo , Sistema Cardiovascular/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Comunicación Paracrina/fisiología , Fosfatasa Alcalina/metabolismo , Animales , Medios de Cultivo Condicionados , Citomegalovirus , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Vectores Genéticos , Proteínas de Homeodominio , Metabolismo de los Lípidos , Ratones , Ratones Transgénicos , Transactivadores/metabolismo , Proteínas Wnt , beta CateninaRESUMEN
Osteopontin (OPN) is a cytokine upregulated in diabetic vascular disease. To better understand its role in vascular remodeling, we assessed how OPN controls metalloproteinase (MMP) activation in aortic adventitial myofibroblasts (AMFs) and A7r5 vascular smooth muscle cells (VSMCs). By zymography, OPN and tumor necrosis factor (TNF)-alpha preferentially upregulate pro-matrix metalloproteinase 9 (pro-MMP9) activity. TNF-alpha upregulated pro-MMP9 in AMFs isolated from wild-type (OPN(+/+)) mice, but pro-MMP9 induction was abrogated in AMFs from OPN(-/-) mice. OPN treatment of VSMCs enhanced pro-MMP9 activity, and TNF-alpha induction of pro-MMP9 was inhibited by anti-OPN antibody and apocynin. Superoxide and the oxylipid product 8-isoprostaglandin F(2) alpha-isoprostane (8-IsoP) were increased by OPN treatment, and anti-OPN antibody suppressed 8-IsoP production. Like OPN and TNF-alpha, 8-IsoP preferentially activated pro-MMP9. Superoxide, 8-IsoP, and NADPH oxidase 2 (Nox2) subunits were reduced in OPN(-/-) AMFs. Treatment of A7r5 VSMCs with OPN upregulated NADPH oxidase subunit accumulation. OPN structure/function studies mapped these activities to the SVVYGLR heptapeptide motif in the thrombin-liberated human OPN N-terminal domain (SLAYGLR in mouse OPN). Treatment of aortic VSMCs with SVVYGLR upregulated pro-MMP9 activity and restored TNF-alpha activation of pro-MMP9 in OPN(-/-) AMFs. Injection of OPN-deficient OPN(+/-) mice with SVVYGLR peptide upregulated pro-MMP9 activity, 8-IsoP levels, and Nox2 protein levels in aorta and increased panmural superoxide production (dihydroethidium staining). At equivalent hyperglycemia and dyslipidemia, 8-IsoP levels and aortic pro-MMP9 were reduced with complete OPN deficiency in a model of diet-induced diabetes, achieved by comparing OPN(-/-)/LDLR(-/-) versus OPN(+/-)/LDLR(-/-) siblings. Thus, OPN provides a paracrine signal that augments vascular pro-MMP9 activity, mediated in part via superoxide generation and oxylipid formation.
Asunto(s)
Aorta/metabolismo , Colagenasas/metabolismo , Precursores Enzimáticos/metabolismo , Fibroblastos/metabolismo , Miocitos del Músculo Liso/metabolismo , NADPH Oxidasas/metabolismo , Sialoglicoproteínas/metabolismo , Transducción de Señal/fisiología , Acetilcisteína/farmacología , Animales , Aorta/citología , Bovinos , Células Cultivadas , Grasas de la Dieta/administración & dosificación , Dinoprost/análogos & derivados , Dinoprost/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Precursores Enzimáticos/antagonistas & inhibidores , Glucosa/farmacología , Humanos , Isoenzimas/metabolismo , Metaloproteinasa 9 de la Matriz , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Noqueados , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Osteopontina , Fragmentos de Péptidos/farmacología , Receptores de LDL/deficiencia , Sialoglicoproteínas/química , Sialoglicoproteínas/deficiencia , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacología , Superóxidos/antagonistas & inhibidores , Superóxidos/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVE: Aortic calcification is prevalent in type II diabetes (T2DM), enhancing morbidity and tracking metabolic syndrome parameters. Ldlr(-/-) mice fed high-fat "Westernized" diets (HFD) accumulate aortic calcium primarily in the tunica media, mediated via osteogenic morphogens and transcriptional programs that induce aortic alkaline phosphatase (ALP). Because elevated TNF-alpha is characteristic of obesity with T2DM, we examined contributions of this inflammatory cytokine. METHODS AND RESULTS: HFD promoted obesity, hyperglycemia, and hyperlipidemia, and upregulated serum TNF-alpha in Ldlr(-/-) mice. Serum haptoglobin (inflammatory marker) was increased along with aortic expression of BMP2, Msx2, Wnt3a, and Wnt7a. Dosing with the TNF-alpha neutralizing antibody infliximab did not reduce obesity, hypercholesterolemia, or hyperglycemia; however, haptoglobin, aortic BMP2, Msx2, Wnt3a, and Wnt7a and aortic calcium accumulation were downregulated by infliximab. Mice with vascular TNF-alpha augmented by a transgene (SM22-TNFalphaTg) driven from the SM22 promoter upregulated aortic Msx2, Wnt3a, and Wnt7a. Furthermore, SM22-TNFalphaTg;TOPGAL mice exhibited greater aortic beta-galactosidase reporter staining versus TOPGAL sibs, indicating enhanced mural Wnt signaling. In aortic myofibroblast cultures, TNF-alpha upregulated Msx2, Wnt3a, Wnt7a, and ALP. ALP induction was inhibited by Dkk1, an antagonist of paracrine Wnt actions. CONCLUSIONS: TNF-alpha promote aortic Msx2-Wnt programs that contribute to aortic calcium accumulation in T2DM.
Asunto(s)
Enfermedades de la Aorta/metabolismo , Calcinosis/metabolismo , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Homeodominio/metabolismo , Receptores de LDL/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Wnt/metabolismo , Fosfatasa Alcalina , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Aorta/enzimología , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Calcinosis/etiología , Calcinosis/patología , Calcinosis/prevención & control , Células Cultivadas , Proteínas de Unión al ADN/genética , Diabetes Mellitus Tipo 2/inducido químicamente , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/patología , Grasas de la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Fibroblastos/enzimología , Fibroblastos/metabolismo , Haptoglobinas/metabolismo , Proteínas de Homeodominio/genética , Inflamación/metabolismo , Inflamación/patología , Inflamación/prevención & control , Infliximab , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas Musculares/genética , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/genética , Proteínas Wnt/genéticaRESUMEN
Tumor cells disseminate early in tumor development making metastasis-prevention strategies difficult. Identifying proteins that promote the outgrowth of disseminated tumor cells may provide opportunities for novel therapeutic strategies. Despite multiple studies demonstrating that the mesenchymal-to-epithelial transition (MET) is critical for metastatic colonization, key regulators that initiate this transition remain unknown. We serially passaged lung metastases from a primary triple negative breast cancer xenograft to the mammary fat pads of recipient mice to enrich for gene expression changes that drive metastasis. An unbiased transcriptomic signature of potential metastatic drivers was generated, and a high throughput gain-of-function screen was performed in vivo to validate candidates. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver. CEACAM5 overproduction enriched for an epithelial gene expression pattern and facilitated tumor outgrowth at metastatic sites. Tissues from patients with metastatic breast cancer confirmed elevated levels of CEACAM5 in lung metastases relative to breast tumors, and an inverse correlation between CEACAM5 and the mesenchymal marker vimentin was demonstrated. Thus, CEACAM5 facilitates tumor outgrowth at metastatic sites by promoting MET, warranting its investigation as a therapeutic target and biomarker of aggressiveness in breast cancer.
RESUMEN
Most triple negative breast cancers (TNBCs) are aggressively metastatic with a high degree of intra-tumoral heterogeneity (ITH), but how ITH contributes to metastasis is unclear. Here, clonal dynamics during metastasis were studied in vivo using two patient-derived xenograft (PDX) models established from the treatment-naive primary breast tumors of TNBC patients diagnosed with synchronous metastasis. Genomic sequencing and high-complexity barcode-mediated clonal tracking reveal robust alterations in clonal architecture between primary tumors and corresponding metastases. Polyclonal seeding and maintenance of heterogeneous populations of low-abundance subclones is observed in each metastasis. However, lung, liver, and brain metastases are enriched for an identical population of high-abundance subclones, demonstrating that primary tumor clones harbor properties enabling them to seed and thrive in multiple organ sites. Further, clones that dominate multi-organ metastases share a genomic lineage. Thus, intrinsic properties of rare primary tumor subclones enable the seeding and colonization of metastases in secondary organs in these models.
Asunto(s)
Metástasis de la Neoplasia/genética , Neoplasias de la Mama Triple Negativas/complicaciones , Neoplasias de la Mama Triple Negativas/genética , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundario , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Ratones , Ratones SCID , Metástasis de la Neoplasia/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Studies of fracture repair have revealed that paracrine endothelial-mesenchymal interactions direct bone formation that restores osseous integrity. Angiogenic growth factors and specific members of the bone morphogenetic protein (BMP) family mediate these interactions. Recently, these same signals have been shown to be critical in the vascular pathobiology of hypertension, diabetes, and atherosclerosis. In the arterial vasculature, mechanical and inflammatory redox signals, characteristic of hypertension and diabetes have emerged as a secretagogues for BMP production-with downstream activation of endothelial NADPH oxidases (Nox). Preliminary data now indicate that the paracrine signals provided by BMP and reactive oxygen species augment aortic myofibroblast Msx2-Wnt signaling and matrix turnover. The net mural response to these stimuli promotes osteogenic differentiation of calcifying vascular cells, moreover, oxidation of vascular LDL cholesterol generates oxysterols that trigger Runx2 activity via hedgehog pathways. Thus, BMP, Wnt, and hedgehog gene expression programs-osteogenic pathways highly familiar to the bone biologist-are elaborated in the arterial vasculature via redox-regulated mechanisms. In the brief review, we recount mounting evidence that points to oxidative stress as a major contributor to the pathobiology of diabetic arterial calcification.
Asunto(s)
Arterias/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Estrés Oxidativo , Proteínas Wnt/metabolismo , Animales , Vasos Sanguíneos/metabolismo , Diferenciación Celular , Diabetes Mellitus/metabolismo , Humanos , Inflamación , Modelos Biológicos , Osteogénesis , Oxidación-Reducción , Especies Reactivas de OxígenoRESUMEN
Vascular calcification increasingly afflicts our aging and dysmetabolic population. Once considered a passive process, it has emerged as an actively regulated form of calcified tissue metabolism, resembling the mineralization of endochondral and membranous bone. Executive cell types familiar to bone biologists, osteoblasts, chondrocytes, and osteoclasts, are seen in calcifying macrovascular specimens. Lipidaceous matrix vesicles, with biochemical and ultrastructural "signatures" of skeletal matrix vesicles, nucleate vascular mineralization in diabetes, dyslipidemia, and uremia. Skeletal morphogens (bone morphogenetic protein-2 (BMP) and BMP4 and Wnts) divert aortic mesoangioblasts, mural pericytes (calcifying vascular cells), or valve myofibroblasts to osteogenic fates. Paracrine signals provided by these molecules mimic the epithelial-mesenchymal interactions that induce skeletal development. Vascular expression of pro-osteogenic morphogens is entrained to physiological stimuli that promote calcification. Inflammation, shear, oxidative stress, hyperphosphatemia, and elastinolysis provide stimuli that: (1) promote vascular BMP2/4 signaling and matrix remodeling; and (2) compromise vascular defenses that limit calcium deposition, inhibit osteo/chondrogenic trans-differentiation, and enhance matrix vesicle clearance. In this review, we discuss the biology of vascular calcification. We highlight how aortic fibrofatty tissue expansion (adventitia, valve interstitium), the adventitial-medial vasa, vascular matrix, and matrix vesicle metabolism contribute to the regulation of aortic calcium deposition, with greatest emphasis placed on diabetic vascular disease.
Asunto(s)
Aorta/metabolismo , Calcinosis/etiología , Calcio/metabolismo , Angiopatías Diabéticas/complicaciones , Angiopatías Diabéticas/metabolismo , Enfermedades Vasculares/etiología , Animales , Válvula Aórtica , Enfermedades de las Válvulas Cardíacas/etiología , HumanosRESUMEN
Vascular calcification increasingly afflicts our aging and dysmetabolic population, predisposing patients to cardiovascular mortality and lower extremity amputation. Active osteogenic processes are evident in most histoanatomic variants, including elaboration of BMP2-Msx2 signals required for craniofacial bone formation. We developed an animal model of diet-induced diabetes, dyslipidemia, and vascular calcification. High-fat diets promote vascular calcification in male low-density lipoprotein receptor (LDLR)-deficient mice, with concomitant upregulation of aortic BMP2 and Msx2 gene expression. We wished to test if Msx2 exerts pro-calcific actions during vascular calcification, as it does in craniofacial bone. We studied CMV-Msx2Tg+;LDLR+ transgenic mice (C57Bl/6), a model previously demonstrated to recapitulate features of Msx2 signaling during craniosynostosis. After 16 weeks of fatty diets, vascular calcification was studied in CMV-Msx2Tg+ versus nontransgenic sibs. Only CMV-Msx2Tg+ mice fed high-fat diets exhibited vascular calcium accumulation by alizarin red staining, noted in the tunica media of coronary arteries and the aorta. Gene expression studies revealed that while Msx2 was expressed primarily in adventitial cells, alkaline phosphatase (ALP) expression and calcification occurred primarily in the tunica media. Msx2 promotes the elaboration of a pro-osteogenic milieu by upregulating expression of Wingless type (Wnt) ligands while downregulating the canonical antagonist, Dickkopf (Dkk1). Msx2 upregulates aortic Wnt signaling in vivo, revealed by the analysis of TOPGAL+ (Wnt reporter) versus CMV-Msx2Tg+; TOPGAL+ mice. Aortic Msx2 exerts pro-osteogenic signaling in vivo and in vitro, mediated in part via the enhancement of paracrine Wnt signaling. Strategies that selectively inhibit aortic Msx2-Wnt cascades may help diminish the initiation and progression of diabetic vascular disease.
Asunto(s)
Vasos Sanguíneos/patología , Calcinosis/fisiopatología , Osteogénesis/fisiología , Animales , Homeostasis , Humanos , Ratones , Ratones Noqueados , Osteoblastos/fisiología , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de LDL/fisiologíaRESUMEN
Intense effort has been devoted to understanding predisposition to chronic systemic inflammation because it contributes to cardiometabolic disease. We demonstrate that deletion of the macrophage vitamin D receptor (VDR) in mice (KODMAC) is sufficient to induce insulin resistance by promoting M2 macrophage accumulation in the liver as well as increasing cytokine secretion and hepatic glucose production. Moreover, VDR deletion increases atherosclerosis by enabling lipid-laden M2 monocytes to adhere, migrate, and carry cholesterol into the atherosclerotic plaque and by increasing macrophage cholesterol uptake and esterification. Increased foam cell formation results from lack of VDR-SERCA2b interaction, causing SERCA dysfunction, activation of ER stress-CaMKII-JNKp-PPARγ signaling, and induction of the scavenger receptors CD36 and SR-A1. Bone marrow transplant of VDR-expressing cells into KODMAC mice improved insulin sensitivity, suppressed atherosclerosis, and decreased foam cell formation. The immunomodulatory effects of vitamin D in macrophages are thus critical in diet-induced insulin resistance and atherosclerosis in mice.
Asunto(s)
Aterosclerosis/metabolismo , Colesterol/metabolismo , Resistencia a la Insulina , Monocitos/metabolismo , Receptores de Calcitriol/metabolismo , Animales , Aterosclerosis/terapia , Transporte Biológico , Trasplante de Médula Ósea , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Estrés del Retículo Endoplásmico , Células Espumosas/metabolismo , Eliminación de Gen , Hígado/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , PPAR gamma/metabolismo , Receptores de Calcitriol/genética , Receptores Depuradores/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
When fed high-fat diets, male LDLR(-/-) mice develop obesity, hyperlipidemia, hyperglycemia, and arteriosclerotic calcification. An osteogenic Msx-Wnt regulatory program is concomitantly upregulated in the vasculature. To better understand the mechanisms of diabetic arteriosclerosis, we generated SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice, assessing the impact of Msx1+Msx2 gene deletion in vascular myofibroblast and smooth muscle cells. Aortic Msx2 and Msx1 were decreased by 95% and 34% in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) animals versus Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) controls, respectively. Aortic calcium was reduced by 31%, and pulse wave velocity, an index of stiffness, was decreased in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice vs. controls. Fasting blood glucose and lipids did not differ, yet SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) siblings became more obese. Aortic adventitial myofibroblasts from SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR(-/-) mice exhibited reduced osteogenic gene expression and mineralizing potential with concomitant reduction in multiple Wnt genes. Sonic hedgehog (Shh) and Sca1, markers of aortic osteogenic progenitors, were also reduced, paralleling a 78% reduction in alkaline phosphatase (TNAP)-positive adventitial myofibroblasts. RNA interference revealed that although Msx1+Msx2 supports TNAP and Wnt7b expression, Msx1 selectively maintains Shh and Msx2 sustains Wnt2, Wnt5a, and Sca1 expression in aortic adventitial myofibroblast cultures. Thus, Msx1 and Msx2 support vascular mineralization by directing the osteogenic programming of aortic progenitors in diabetic arteriosclerosis.
Asunto(s)
Aorta/metabolismo , Arteriosclerosis/genética , Diabetes Mellitus Experimental/genética , Proteínas de Homeodominio/genética , Factor de Transcripción MSX1/genética , Miocitos del Músculo Liso/metabolismo , Miofibroblastos/metabolismo , Calcificación Vascular/genética , Rigidez Vascular/genética , Animales , Antígenos Ly/metabolismo , Arteriosclerosis/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa , Eliminación de Gen , Perfilación de la Expresión Génica , Proteínas Hedgehog/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Osteogénesis/genética , Receptores de LDL/genética , Calcificación Vascular/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5a , Proteína wnt2/metabolismoRESUMEN
In LDLR(-/-) mice fed high-fat diabetogenic diets, osteogenic gene-regulatory programs are ectopically activated in vascular myofibroblasts and smooth muscle cells that promote arteriosclerotic calcium deposition. Msx2-Wnt signaling pathways previously identified as important for craniofacial skeletal development are induced in the vasculature by TNF, a prototypic cytokine mediator of the low-grade systemic inflammation of diabesity. To better understand this biology, we studied TNF actions on Msx2 in aortic myofibroblasts. TNF up-regulated Msx2 mRNA 4-fold within 3 h but did not regulate Msx1. Although IL-1ß could also induce Msx2 expression, TNF-related apoptosis inducing ligand, receptor activator of nuclear factor-κB ligand, and IL-6 were inactive. Inhibition of nicotinamide adenine dinucleotide phosphate oxidase (Nox) activity and genetically induced Nox deficiency (p47phox(-/-)) reduced Msx2 induction, indicating contributions of reactive oxygen species (ROS) and redox signaling. Consistent with this, rotenone, an antagonist of mitochondrial complex I, inhibited TNF induction of Msx2 and Nox2, whereas pyruvate, an anapleurotic mitochondrial metabolic substrate, enhanced induction. Moreover, the glutathione peroxidase-mimetic ebselen abrogated this TNF response. Treatment of aortic myofibroblasts with hydrogen peroxide up-regulated Msx2 mRNA, promoter activity, and DNA-protein interactions. In vivo, SM22-TNF transgenic mice exhibit increased aortic Msx2 with no change in Msx1. Dosing SM22-TNF mice with either 20 ng/g Nox1 + 20 ng/g Nox2 antisense oligonucleotides or low-dose rotenone reduced arterial Msx2 expression. Aortic myofibroblasts from TNFR1(-/-) mice expressed levels of Msx2 that were 5% that of wild-type and were not inducible by TNF. Wnt7b and active ß-catenin levels were also reduced. By contrast, TNF-inducible Msx2 expression was not reduced in TNFR2(-/-) cells. Finally, when cultured under mineralizing conditions, TNFR1(-/-) aortic myofibroblasts exhibited reduced calcification compared with wild-type and TNFR2(-/-) cells. Thus, ROS metabolism contributes to TNF induction of Msx2 and procalcific responses in myofibroblasts via TNFR1. Strategies that reduce vascular Nox- or mitochondrially activated ROS signals may prove useful in mitigating arteriosclerotic calcification.