RESUMEN
Triple-negative breast cancer (TNBC) is the most aggressive subtype of the disease with lack of recognized molecular targets for therapy. TNBC cells are known to secrete high levels of the proinflammatory cytokines interleukin-6 (IL-6) and IL-8, which promote angiogenesis and favor the growth and spread of the disease. In the present study, we have shown that the humanized anti-IL-6 receptor tocilizumab (Actemra) is also a potent inhibitor of IL-8 in TNBC cells. Similar effect was also obtained by specific IL-6 inhibition either by small interfering RNA or by neutralizing antibody. Likewise, neutralizing IL-8 with specific antibody downregulated IL-8 and inhibited the IL-6/signal transducer and activator of transcription 3 and nuclear factor-κB pathways. Interestingly, simultaneous co-inhibition of IL-6 and IL-8 did not increase the effects of the single inhibitors. Additionally, we present clear evidence that tocilizumab has potent antiangiogenic effect. Indeed, tocilizumab abolished the ability of TNBC cells to induce the differentiation of endothelial cells into network-like tubular structures in vitro and impaired neovascularization in humanized breast orthotopic tumor xenografts. This was associated with tocilizumab-dependent downregulation of the main proangiogenic factor vascular endothelial growth factor A and its coactivator hypoxia-inducible factor 1 both in vitro and in vivo. Therefore, tocilizumab could be of great therapeutic value for TNBC patients through targeting angiogenesis.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos Inmunológicos/farmacología , Interleucina-8/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Antineoplásicos Inmunológicos/uso terapéutico , Línea Celular Tumoral , Femenino , Humanos , Interleucina-8/inmunología , Ratones Endogámicos NOD , Ratones SCID , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/inmunología , Neovascularización Patológica/patología , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
BACKGROUND: Overexpressed vascular endothelial growth factor A (VEGFA) and phosphorylated signal transducer and activator of transcription 3 (P-STAT3) cause unrestricted tumor growth and angiogenesis of breast cancer (BRCA), especially triple-negative breast cancer (TNBC). Hence, novel treatment strategy is urgently needed. RESULTS: We found sphingosine 1 phosphate receptor 1 (S1PR1) can regulate P-STAT3/VEGFA. Database showed S1PR1 is highly expressed in BRCA and causes the poor prognosis of patients. Interrupting the expression of S1PR1 could inhibit the growth of human breast cancer cells (MCF-7 and MDA-MB-231) and suppress the angiogenesis of human umbilical vein endothelial cells (HUVECs) via affecting S1PR1/P-STAT3/VEGFA axis. Siponimod (BAF312) is a selective antagonist of S1PR1, which inhibits tumor growth and angiogenesis in vitro by downregulating the S1PR1/P-STAT3/VEGFA axis. We prepared pH-sensitive and tumor-targeted shell-core structure nanoparticles, in which hydrophilic PEG2000 modified with the cyclic Arg-Gly-Asp (cRGD) formed the shell, hydrophobic DSPE formed the core, and CaP (calcium and phosphate ions) was adsorbed onto the shell; the nanoparticles were used to deliver BAF312 (BAF312@cRGD-CaP-NPs). The size and potential of the nanoparticles were 109.9 ± 1.002 nm and - 10.6 ± 0.056 mV. The incorporation efficacy for BAF312 was 81.4%. Results confirmed BAF312@cRGD-CaP-NP could dramatically inhibit tumor growth and angiogenesis in vitro and in MDA-MB-231 tumor-bearing mice via downregulating the S1PR1/P-STAT3/VEGFA axis. CONCLUSIONS: Our data suggest a potent role for BAF312@cRGD-CaP-NPs in treating BRCA, especially TNBC by downregulating the S1PR1/P-STAT3/VEGFA axis.
Asunto(s)
Inductores de la Angiogénesis/farmacología , Azetidinas/farmacología , Compuestos de Bencilo/farmacología , Nanopartículas/química , Factor de Transcripción STAT3/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Azetidinas/química , Compuestos de Bencilo/química , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Factor de Transcripción STAT3/genética , Receptores de Esfingosina-1-Fosfato/genética , Neoplasias de la Mama Triple Negativas/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Triple-negative breast cancer (TNBC) is an aggressive breast tumor subtype characterized by poor clinical outcome. In recent years, numerous advancements have been made to better understand the biological landscape of TNBC, though appropriate targets still remain to be determined. In the present study, we have determined that the expression levels of FGF2 and S100A4 are higher in TNBC with respect to non-TNBC patients when analyzing "The Invasive Breast Cancer Cohort of The Cancer Genome Atlas" (TCGA) dataset. In addition, we have found that the gene expression of FGF2 is positively correlated with S100A4 in TNBC samples. Performing quantitative PCR, Western blot, CRISPR/Cas9 genome editing, promoter studies, immunofluorescence analysis, subcellular fractionation studies, and ChIP assays, we have also demonstrated that FGF2 induces in TNBC cells the upregulation and secretion of S100A4 via FGFR1, along with the ERK1/2-AKT-c-Rel transduction signaling. Using conditioned medium from TNBC cells stimulated with FGF2, we have also ascertained that the paracrine activation of the S100A4/RAGE pathway triggers angiogenic effects in vascular endothelial cells (HUVECs) and promotes the migration of cancer-associated fibroblasts (CAFs). Collectively, our data provide novel insights into the action of the FGF2/FGFR1 axis through S100A4 toward stimulatory effects elicited in TNBC cells.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/fisiología , Proteínas de Neoplasias/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/fisiología , Proteína de Unión al Calcio S100A4/fisiología , Transducción de Señal/fisiología , Neoplasias de la Mama Triple Negativas/fisiopatología , Antígenos de Neoplasias/fisiología , Movimiento Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Neovascularización Patológica/fisiopatología , Comunicación Paracrina , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-rel/fisiología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Células Tumorales CultivadasRESUMEN
The up-regulation of EMT regulator Twist1 has been implicated in vasculogenic mimicry (VM) formation in human triple-negative breast cancer (TNBC). Twist1 targets the Claudin15 promoter in hepatocellular carcinoma cells. Claudin family members are related with TNBC. However, the relationship between Claudin15 and VM formation is not clear. In this study, we first found that Claudin15 expression was frequently down-regulated in human TNBC, and Claudin15 down-regulation was significantly associated with VM and Twist1 nuclear expression. Claudin15 down-regulation correlated with shorter survival compared with high levels. Claudin15 silence significantly enhanced cell motility, invasiveness and VM formation in the non-TNBC MCF-7 cells. Conversely, an up-regulation of Claudin15 remarkably reduced TNBC MDA-MB-231 cell migration, invasion and VM formation. We also showed that down-regulation of Claudin15 was Twist1-dependent, and Twist1 repressed Claudin15 promoter activity. Furthermore, GeneChip analyses of mammary glands of Claudin15-deficient mice indicated that Claudin18 and Jun might be downstream factors of Twist1-Claudin15. Our results suggest that Twist1 induced VM through Claudin15 suppression in TNBC, and Twist1 inhibition of Claudin15 might involve Claudin18 and Jun expression.
Asunto(s)
Claudinas , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares , Neoplasias de la Mama Triple Negativas , Proteína 1 Relacionada con Twist , Animales , Femenino , Humanos , Antígenos CD/metabolismo , Cadherinas/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Núcleo Celular/metabolismo , Claudinas/deficiencia , Claudinas/genética , Claudinas/metabolismo , Estimación de Kaplan-Meier , Glándulas Mamarias Animales/patología , Invasividad Neoplásica , Proteínas Nucleares/metabolismo , Fenotipo , Transcripción Genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Proteína 1 Relacionada con Twist/metabolismo , Regulación hacia Arriba/genética , RatonesRESUMEN
BACKGROUND: Limited information is available on biomarker(s) for triple-negative breast cancer (TNBC) that can address the higher incidence and aggressiveness of TNBC in African-American (AA) women. Our previous studies have demonstrated annexin A2 (AnxA2) association with exosomes which promotes angiogenesis and metastasis. Therefore, our goal was to examine the expression and function of exosomal-annexin A2 (exo-AnxA2) derived from the serum samples of breast cancer patients. METHODS: The expression of serum exo-AnxA2 and its association with clinicopathological features of the breast cancer patients were determined. The role of serum exo-AnxA2 to promote angiogenesis was determined by an in vivo Matrigel plug assay. RESULTS: Our results show that the expression of serum exo-AnxA2 in breast cancer patients (n = 169; 83.33 ± 2.040 ng/mL, P < 0.0001) is high compared to non-cancer females (n = 68; 34.21 ± 2.238 ng/mL). High expression of exo-AnxA2 levels in breast cancer was significantly associated with tumor grade (P < 0.0001), poor overall survival (hazard ratio (HR) 2.802; 95% confidence intervals (CI) = 1.030-7.620; P = 0.0353), and poor disease-free survival (HR 7.934; 95% CI = 1.778-35.398; P = 0.0301). The expression of serum exo-AnxA2 levels was significantly elevated in TNBC (n = 68; 109.1 ± 2.905 ng/mL; P < 0.0001) in comparison to ER+ (n = 50; 57.35 ± 1.545 ng/mL), HER2+ (n = 59; 78.25 ± 1.146 ng/mL), and non-cancer females (n = 68; 34.21 ± 2.238 ng/mL). Exo-AnxA2 showed diagnostic values with a maximum AUC as 1.000 for TNBC, 0.8304 for ER+, and 0.9958 for HER2+ compared to non-cancer females. The expression of serum exo-AnxA2 was significantly elevated in AA women with TNBC (n = 29; 118.9 ± 4.086 ng/mL, P < 0.0001) in comparison to Caucasian-American TNBC (n = 27; 97.60 ± 3.298 ng/mL) patients. Our in vivo results suggest a role of serum exo-AnxA2 in angiogenesis and its association with aggressiveness of TNBC in AA women. CONCLUSIONS: Our results demonstrated that the expression of serum exo-AnxA2 is high in AA women with TNBC and promotes angiogenesis. These findings suggest that exo-AnxA2 holds promise as a potential prognosticator of TNBC and may lead to an effective therapeutic option.
Asunto(s)
Anexina A2/sangre , Biomarcadores de Tumor/sangre , Negro o Afroamericano/estadística & datos numéricos , Exosomas/metabolismo , Neovascularización Patológica/patología , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Animales , Femenino , Humanos , Ratones , Ratones Desnudos , Clasificación del Tumor , Neovascularización Patológica/metabolismo , Curva ROC , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)--a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)--is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44(high)CD24(low) population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factores de Transcripción/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Antígeno CD24/metabolismo , Hipoxia de la Célula/genética , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Silenciador del Gen , Humanos , Receptores de Hialuranos/metabolismo , Ratones , Invasividad Neoplásica , Recurrencia Local de Neoplasia , Pronóstico , ARN Polimerasa II/metabolismo , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/genética , Respuesta de Proteína Desplegada , Proteína 1 de Unión a la X-BoxRESUMEN
Rapid sequestration and prolonged retention of intravenously injected nanoparticles by the liver and spleen (reticuloendothelial system (RES)) presents a major barrier to effective delivery to the target site and hampers clinical translation of nanomedicine. Inspired by biological macromolecular drugs, synthesis of ultrasmall (diameter ≈12-15 nm) porous silica nanoparticles (UPSNs), capable of prolonged plasma half-life, attenuated RES sequestration, and accelerated hepatobiliary clearance, is reported. The study further investigates the effect of tumor vascularization on uptake and retention of UPSNs in two mouse models of triple negative breast cancer with distinctly different microenvironments. A semimechanistic mathematical model is developed to gain mechanistic insights into the interactions between the UPSNs and the biological entities of interest, specifically the RES. Despite similar systemic pharmacokinetic profiles, UPSNs demonstrate strikingly different tumor responses in the two models. Histopathology confirms the differences in vasculature and stromal status of the two models, and corresponding differences in the microscopic distribution of UPSNs within the tumors. The studies demonstrate the successful application of multidisciplinary and complementary approaches, based on laboratory experimentation and mathematical modeling, to concurrently design optimized nanomaterials, and investigate their complex biological interactions, in order to drive innovation and translation.
Asunto(s)
Nanopartículas/química , Neovascularización Patológica/patología , Tamaño de la Partícula , Dióxido de Silicio/química , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Animales , Línea Celular Tumoral , Radioisótopos de Cobre/farmacocinética , Femenino , Humanos , Ratones Endogámicos BALB C , Modelos Biológicos , Nanopartículas/ultraestructura , Porosidad , Distribución Tisular , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Neoplasias de la Mama Triple Negativas/patología , Microambiente TumoralRESUMEN
PURPOSE: To investigate associations between the clinicopathologic features and CT perfusion parameters of triple-negative breast cancer (TNBC) and non-TNBC using low-dose computed tomography perfusion imaging (LDCTPI), and to find potential clinical applications in the prognosis assessment of TNBC. MATERIALS AND METHODS: A total of 60 patients with breast cancer confirmed by pathological examination were studied prospectively using LDCTPI on a 64-slice spiral CT scanner. The acquired volume data were used for calculations, mapping, and analysis by using a tumor perfusion protocol in the CT perfusion software package to measure 2 parameters namely, blood flow (BF), and permeability surface (PS) area product. Patients were grouped into TNBC (nâ=â27) and non-TNBC (nâ=â33) subtypes. Associations between these two subtypes and clinicopathologic characteristics were evaluated by both univariate and multivariate logistic regression. CT perfusion parameters values were compared for clinicopathologic characteristics using independent 2-sample t test. RESULTS: TNBC displayed higher CT perfusion parameters values (BF: 57.56±10.94 vs 52.70±7.79âmL/100âg/min, pâ=â0.006; PS: 38.98±9.46 vs 33.39±8.07âmL/100âg/min, pâ=â0.001) than non-TNBC. In addition, breast cancer with poorly histologic grade or positive Ki-67 expression showed higher BF and PS values than those with well and moderately histologic grade or negative Ki-67 expression (pâ<â0.05). TNBC had poorer histologic grade (Pâ=â0.032) and higher Ki-67 expression (Pâ=â0.013) than non-TNBC. CONCLUSION: LDCTPI is a functional imaging technology from the perspective of hemodynamics with potential of clinical applications. The BF and PS values were higher in TNBC patient group than non-TNBC group. TNBC patients also have poorer clinicopathologic outcome.
Asunto(s)
Imagen de Perfusión/métodos , Tomografía Computarizada Espiral/métodos , Neoplasias de la Mama Triple Negativas/diagnóstico por imagen , Adulto , Anciano , Femenino , Humanos , Persona de Mediana Edad , Proyectos Piloto , Pronóstico , Interpretación de Imagen Radiográfica Asistida por Computador , Programas Informáticos , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Antiangiogenic tyrosine kinase inhibitors (TKIs) target vascular endothelial growth factor receptors and other receptor tyrosine kinases. As a result of toxicity, the clinical failures or the modest benefits associated with antiangiogenic TKI therapy may be related in some cases to suboptimal drug dosing and scheduling, thereby facilitating resistance. Most antiangiogenic TKIs, including pazopanib, are administered on a continuous daily basis. Here, instead, we evaluated the impact of increasing the dose and administering the drug intermittently. The rationale is that using such protocols, antitumor efficacy could be enhanced by direct tumor cell targeting effects in addition to inhibiting tumor angiogenesis. To test this, we employed two human tumor xenograft models, both of which manifest intrinsic resistance to pazopanib when it is administered continuously: the VHL-wildtype SN12-PM6-1 renal cell carcinoma (RCC) and the metastatic MDA-MB-231/LM2-4 variant breast cancer cell line, when treated as distant metastases. We evaluated four different doses and schedules of pazopanib in the context of primary tumors and advanced metastatic disease, in both models. The RCC model was not converted to drug sensitivity using the intermittent protocol. Using these protocols did not enhance the efficacy when treating primary LM2-4 tumors. However, one of the high-dose intermittent pazopanib protocols increased median survival when treating advanced metastatic disease. In conclusion, these results overall suggest that primary tumors showing sensitivity to continuous pazopanib treatment may predict response to this drug when given at high doses intermittently in the context of advanced metastatic disease, that are otherwise resistant to the conventional protocol.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Carcinoma de Células Renales , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Renales , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Sulfonamidas/farmacología , Neoplasias de la Mama Triple Negativas , Animales , Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/enzimología , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Femenino , Humanos , Indazoles , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/enzimología , Neoplasias Renales/patología , Ratones , Ratones SCID , Ratones Transgénicos , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/enzimología , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Triple negative breast cancer (TNBC) is among the most clinically aggressive subtypes of breast cancer. Despite the availability of new drugs, patients suffering TNBC bear disheartening prognosis. Vasculogenic mimicry (VM) is a malignant tumor specific non-endothelial vascular network, which provide oxygen and nutrients to tumor cells and facilitate tumor progression. Therefore targeting TNBC VM formation may contribute to tumor treatment. In this study, we found that long non-coding RNA TP73 antisense RNA 1 (TP73-AS1) was upregulated in VM positive TNBC tissues. Knockdown of TP73-AS1 suppressed TNBC cell line MDA-MB-231â¯cell VM formation in vitro. In addition, RNA immunoprecipitation assay and dual luciferase reporter assay showed that TP73-AS1 bound to miR-490-3p in a sequence-specific manner. miR-490-3p was downregulated in VM positive tissues and was involved in TP73-AS1-mediated MDA-MB-231â¯cell VM formation. Furthermore, TWIST1 was a target of miR-490-3p and participated in TP73-AS1/miR-490-3p-modulated MDA-MB-231â¯cell VM formation. Findings in this study may provide insight in TNBC management.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Interferencia de ARN , ARN Largo no Codificante/genética , Neoplasias de la Mama Triple Negativas/genética , Proteína 1 Relacionada con Twist/genética , Regiones no Traducidas 3'/genética , Secuencia de Bases , Línea Celular Tumoral , Femenino , Humanos , MicroARNs/metabolismo , Neovascularización Patológica/genética , ARN Largo no Codificante/metabolismo , Homología de Secuencia de Ácido Nucleico , Transducción de Señal/genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/metabolismo , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
It is believed that hypoxia stimulates triple-negative breast cancers (TNBCs) metastasis, which is associated with a poor prognosis. However, the underlying mechanism remains unclear. Here, we demonstrated that hypoxia up-regulates both the levels of Orai1 and Notch1, and the increase in Orai1 is mediated by Notch1 signaling in TNBCs. Functionally, Orai1 caused a sustained elevation of intracellular Ca2+ via Store-operated Ca2+ entry (SOCE), then activated the calcineurin-nuclear factor of activated T-cell 4 (NFAT4, also named NFATc3) in hypoxic TNBCs. Furthermore, pharmacologic inhibition or gene-silencing studies showed that the aggressiveness mediated by Orai1 during hypoxia is dependent on the Notch1/Orai1/SOCE/NFAT4 signaling. Moreover, Orai1 signaling also mediated hypoxia-induced angiogenesis in TNBCs. Thus, our results revealed a novel role of Orai1 as an inducer of aggression and angiogenesis under hypoxic conditions, and this suggests a novel mechanism of hypoxia-induced invasion. It may be worthwhile to further explore the potential of using Orai1 signaling as new target for anti-tumor therapy in TNBCs.
Asunto(s)
Señalización del Calcio , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Proteína ORAI1/metabolismo , Receptor Notch1/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Femenino , Humanos , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteínas de Neoplasias/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Proteína ORAI1/genética , Receptor Notch1/genética , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
A hallmark of breast tumors is its spatial heterogeneity that includes its distribution of cancer stem cells and progenitor cells, but also heterogeneity in the tumor microenvironment. In this study we focus on the contributions of stromal cells, specifically macrophages, fibroblasts, and endothelial cells on tumor progression. We develop a computational model of triple-negative breast cancer based on our previous work and expand it to include macrophage infiltration, fibroblasts, and angiogenesis. In vitro studies have shown that the secretomes of tumor-educated macrophages and fibroblasts increase both the migration and proliferation rates of triple-negative breast cancer cells. In vivo studies also demonstrated that blocking signaling of selected secreted factors inhibits tumor growth and metastasis in mouse xenograft models. We investigate the influences of increased migration and proliferation rates on tumor growth, the effect of the presence on fibroblasts or macrophages on growth and morphology, and the contributions of macrophage infiltration on tumor growth. We find that while the presence of macrophages increases overall tumor growth, the increase in macrophage infiltration does not substantially increase tumor growth and can even stifle tumor growth at excessive rates.
Asunto(s)
Fibroblastos/patología , Macrófagos/patología , Células Madre Neoplásicas/patología , Neovascularización Patológica/genética , Neoplasias de la Mama Triple Negativas/patología , Animales , Comunicación Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Humanos , Macrófagos/metabolismo , Ratones Desnudos , Modelos Biológicos , Células Madre Neoplásicas/metabolismo , Neovascularización Patológica/metabolismo , Trasplante Heterólogo , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/genética , Carga Tumoral/genética , Microambiente Tumoral/genéticaRESUMEN
Triple negative breast cancer (TNBC) is highly aggressive and has a few therapeutic treatments, so new targeted therapy and biomarkers are required to provide alternative choices for treating TNBC patients. Recent studies showed that vasculogenic mimicry (VM), the formation of blood channels by aggressive cancer cells that mimic endothelial cells, is a factor contributing to poor prognosis in TNBC. Wilms' tumor 1 (WT1) gene has been found to be highly expressed in TNBC, and has 4 major distinct isoforms; isoform A (-17AA/-KTS; -/-), isoform B (+17AA/-KTS; +/-), isoform C (-17AA/+KTS; -/+) and isoform D (+17AA/+KTS; +/+). The involvement of each WT1 isoform in TNBC progression remains largely unclear. In this study, WT1 isoform-overexpressing cell sublines were established from a TNBC cell line, MDA-MB-231, by stable transfection, and the aggressive behavior of the cell sublines were evaluated. Only the WT1 isoform B- and isoform C-overexpressing cell sublines showed the significant increase in VM forming capability compared to the parental cell line and other isoform cell sublines. qRT-PCR was used to explore the change in expression level of two VM-related genes, EphA2 and VE-cadherin. All WT1 isoform cell sublines showed up-regulation of EphA2 but the levels detected in the isoform B- and isoform C-cell sublines were higher than those observed in other cell sublines. In contrast, significant up-regulation of VE-cadherin was found only in isoform A- and isoform D-cell sublines. Isoform B- and isoform C-cell sublines showed higher rates of cell migration compared to those of other cell sublines, as determined by both wound healing and Transwell assays. Gelatin zymography revealed increased MMP-9 enzyme production in isoform D-cell subline compared to the parental cell line, but this change was not observed in other cell sublines. Western blot analysis showed significantly increased expression of ß-catenin in isoform B- and isoform C-cell sublines, compared to parental cell line and other isoform cell sublines. In conclusion, our findings demonstrate that WT1 isoforms play different roles in modulating the VM-forming capacity and metastatic potential of TNBC cells.
Asunto(s)
Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Proteínas WT1/metabolismo , Línea Celular Tumoral , Movimiento Celular , Progresión de la Enfermedad , Efrina-A2/metabolismo , Femenino , Genes del Tumor de Wilms , Humanos , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Imitación Molecular , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/fisiopatología , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor EphA2 , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Proteínas WT1/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND: Endoplasmic reticulum disulfide oxidase 1-α (ERO1-α) is an oxidase that exists in the endoplasmic reticulum and has a role in the formation of disulfide bonds of secreted proteins and cell-surface proteins. Recently, we reported that ERO1-α is present in high levels in various types of tumours, and that ERO1-α is a novel factor of poor prognosis. However, how ERO1-α affects a tumour in vivo and why patients who have a tumour with a high expression level of ERO1-α have a poor prognosis are still unknown. Therefore, to clarify the mechanism, we investigated the effect of ERO1-α on a tumour from the point of view of angiogenesis. METHODS: The effect of ERO1-α on tumour growth and angiogenesis was analysed by using non-obese diabetic-severe combined immunodeficient mice. The production of vascular endothelial growth factor (VEGF) in MDA-MB-231 cells with ERO1-α- overexpression or with ERO1-α knockdown was measured. The role of ERO1-α on VEGF expression was investigated. In triple-negative breast cancer cases, the relationship between expression of ERO1-α and angiogenesis was analysed. RESULTS: We found that the expression of ERO1-α promoted tumour growth in a mouse study and angiogenesis. The effects of ERO1-α on angiogenesis were mediated via oxidative protein folding of VEGF and enhancement of VEGF mRNA expression by using MDA-MB-231. In triple-negative breast cancer cases, the expression of ERO1-α related to the number of the blood vessel. Furthermore, we found that ERO1-α was a poor prognosis factor in triple-negative breast cancer. CONCLUSIONS: Our study has established a novel link between expression of ERO1-α and secretion of VEGF, providing new evidence for the effectiveness of ERO1-α-targeted therapy in patients with ERO1-α-expressed cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glicoproteínas de Membrana/fisiología , Proteínas de Neoplasias/fisiología , Neovascularización Patológica/enzimología , Oxidorreductasas/fisiología , Pliegue de Proteína , Neoplasias de la Mama Triple Negativas/enzimología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/patología , Línea Celular Tumoral , Disulfuros/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Oxidación-Reducción , Oxidorreductasas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Epithelial-mesenchymal transition (EMT) programmes provide cancer cells with invasive and survival capacities that might favour metastatic dissemination. Whilst signalling cascades triggering EMT have been extensively studied, the impact of EMT on the crosstalk between tumour cells and the tumour microenvironment remains elusive. We aimed to identify EMT-regulated soluble factors that facilitate the recruitment of host cells in the tumour. Our findings indicate that EMT phenotypes relate to the induction of a panel of secreted mediators, namely IL-8, IL-6, sICAM-1, PAI-1 and GM-CSF, and implicate the EMT-transcription factor Snail as a regulator of this process. We further show that EMT-derived soluble factors are pro-angiogenic in vivo (in the mouse ear sponge assay), ex vivo (in the rat aortic ring assay) and in vitro (in a chemotaxis assay). Additionally, conditioned medium from EMT-positive cells stimulates the recruitment of myeloid cells. In a bank of 40 triple-negative breast cancers, tumours presenting features of EMT were significantly more angiogenic and infiltrated by a higher quantity of myeloid cells compared to tumours with little or no EMT. Taken together, our results show that EMT programmes trigger the expression of soluble mediators in cancer cells that stimulate angiogenesis and recruit myeloid cells in vivo, which might in turn favour cancer spread.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Quimiotaxis , Citocinas/metabolismo , Transición Epitelial-Mesenquimal , Células Mieloides/metabolismo , Neovascularización Patológica , Comunicación Paracrina , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/metabolismo , Microambiente Tumoral , Proteínas Angiogénicas/genética , Animales , Línea Celular Tumoral , Medios de Cultivo Condicionados/metabolismo , Citocinas/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones SCID , Células Mieloides/patología , Fenotipo , Interferencia de ARN , Ratas , Transducción de Señal , Transfección , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
We previously demonstrated the capacity of GAS1 (Growth Arrest Specific 1) to inhibit the growth of gliomas by blocking the GDNF-RET signaling pathway. Here, we show that a soluble form of GAS1 (tGAS1), decreases the number of viable MDA MB 231 human breast cancer cells, acting in both autocrine and paracrine manners when secreted from producing cells. Moreover, tGAS1 inhibits the growth of tumors implanted in female nu/nu mice through a RET-independent mechanism which involves interfering with the Artemin (ARTN)-GFRα3-(GDNF Family Receptor alpha 3) mediated intracellular signaling and the activation of ERK. In addition, we observed that the presence of tGAS1 reduces the vascularization of implanted tumors, by preventing the migration of endothelial cells. The present results support a potential adjuvant role for tGAS1 in the treatment of breast cancer, by detaining tumor growth and inhibiting angiogenesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Neovascularización Patológica/prevención & control , Proteínas del Tejido Nervioso/metabolismo , Neoplasias de la Mama Triple Negativas/prevención & control , Animales , Apoptosis , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular/genética , Movimiento Celular , Medios de Cultivo Condicionados/farmacología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Proteínas del Tejido Nervioso/genética , Fosforilación , ARN Mensajero/genética , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To study the expression and clinical significance of MTDH and VEGF in triple-negative breast cancer (TNBC). METHODS: Tissue samples of 168 breast cancers (including 112 TNBC tissue and 56 non-TNBC tissue), 10 breast fibroadenomas and 15 normal breast tissues were collected. Postoperative specimens were examined by immunohistochemistry for MTDH and VEGF expression. The correlation between the expression of MTDH and VEGF and clinicopathological features was analyzed. RESULTS: MTDH and VEGF were expressed in 57.1% and 49.4% of breast cancer patients, 64.3% and 56.3% in TNBC patients, respectively, significantly higher than that in the non-TNBC tissues, breast fibroadenomas and normal breast tissues (P<0.05 for all). Statistically significant correlation was found between the MTDH and VEGF expressions (r=0.356, P<0.001). Moreover, MTDH expression was correlated with tumor size, BMI index, lymph node metastasis, pathological stage, recurrence and metastasis, and the expression of p53 and Ki-67 proteins (P<0.05 for all). The VEGF protein expression was correlated with lymph node metastasis, pathological staging, recurrence and metastasis, and the expression of Ki-67 protein (P<0.05 for all). The patients with high expression of MTDH and VEGF showed a lower DFS and OS (P<0.05 for both). CONCLUSIONS: MTDH and VEGF expression may be correlated with tumor angiogenesis and progression and has the potential to be valuable prognostic factors in patients with TNBC.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Moléculas de Adhesión Celular/metabolismo , Fibroadenoma/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Mama/metabolismo , Supervivencia sin Enfermedad , Femenino , Fibroadenoma/irrigación sanguínea , Fibroadenoma/patología , Humanos , Inmunohistoquímica , Metástasis Linfática , Proteínas de la Membrana , Neovascularización Patológica , Pronóstico , Proteínas de Unión al ARN , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
AIMS: Exchange protein directly activated by cAMP 1 (EPAC1), a major isoform of guanine nucleotide exchange factors, is highly expressed in vascular endothelia cells and regulates angiogenesis in the retina. High intratumor microvascular densities (MVD) resulting from angiogenesis is responsible for breast cancer development. Downregulation of EPAC1 in tumor cell reduces triple-negative breast cancer (TNBC)-induced angiogenesis. However, whether Epac1 expressed in vascular endothelial cells contributes to angiogenesis and tumor development of TNBC remains elusive. MAIN METHODS: We employed NY0123, a previously identified potent EPAC inhibitor, to explore the anti-angiogenic biological role of EPAC1 in vitro and in vivo through vascular endothelial cells, rat aortic ring, Matrigel plug, and chick embryo chorioallantoic membrane (CAM) and yolk sac membrane (YSM) assays, as well as the in vivo xenograft tumor models of TNBC in both chick embryo and mice. KEY FINDINGS: Inhibiting EPAC1 in vascular endothelial cells by NY0123 significantly suppresses angiogenesis and tumor growth of TNBC. In addition, NY0123 possesses a better inhibitory efficacy than ESI-09, a reported specific EPAC inhibitor tool compound. Importantly, inhibiting EPAC1 in vascular endothelia cells regulates the typical angiogenic signaling network, which is associated with not only vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor-2 (VEGFR2) signaling, but also PI3K/AKT, MEK/ERK and Notch pathway. CONCLUSIONS: Our findings support that EPAC1 may serve as an effective anti-angiogenic therapeutic target of TNBC, and EPAC inhibitor NY0123 has the therapeutic potential to be developed for the treatment of TNBC.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido , Neovascularización Patológica , Neoplasias de la Mama Triple Negativas , Animales , Embrión de Pollo , Humanos , Ratones , Ratas , Células Endoteliales/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasas , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neovascularización Patológica/tratamiento farmacológicoRESUMEN
PURPOSE: Vascular mimicry (VM) tubules are lumen structures comprised of malignant tumor cells without the participation of endothelial cells. VM simulates blood vessel function in tumors to deliver a sufficient blood supply for proliferation, invasion, and metastasis of malignant tumors, thereby reducing the clinical effects of anti-angiogenic treatments. The elimination or prevention of malignant tumor VM development therefore represents an urgent research goal as a therapeutic strategy to and cut off nutrients required for tumor growth. The GATA transcription factor TRPS1 is abnormally up-regulated in breast cancer, osteosarcoma, prostate cancer, and other tumor tissues, and is instrumental in regulating cell proliferation, differentiation, and tissue growth and development. METHODS: Here, we explored the effects of TRPS1 knockdown on VM and the proteins underlying its development in triple-negative breast cancer cell line MDA-MB-231. RESULTS: We found that TRPS1 knockdown resulted in obvious inhibition of VM development. Fluorescence microscopy of F-actin and tubulin revealed that loss of TRPS1 function resulted in disruption of cytoskeleton and microtubule formation, respectively. In addition, TRPS1-suppressed cells exhibited reduced accumulation of VM-associated proteins EphA2, MMP-2, MMP-9, VEGF, and VE-cadherin. Moreover, it is interesting to know that the capacity for migration and invasion were limited in MDA-MB-231cells after TRPS1 knockdown and that the average number of VM tubules, their length, and number of intersections were also significantly decreased. CONCLUSIONS: Based on our results, and in light of previous studies, we thus proposed that TRPS1 suppression negatively affects vascular mimicry possibly through reduced TRPS1-mediated transcriptional regulation of VM-related protein VEGF-A.
Asunto(s)
Neovascularización Patológica , Proteínas Represoras/fisiología , Neoplasias de la Mama Triple Negativas/irrigación sanguínea , Neoplasias de la Mama Triple Negativas/patología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Represoras/genéticaRESUMEN
Breast cancer cells (BCCs) preferentially metastasize to bone. It is known that BCCs remotely primes the distant bone site prior to metastasis. However, the reciprocal influence of bone cells on the primary tumor is relatively overlooked. Here, to study the bone-tumor paracrine influence, a tri-cellular 3-D vascularized breast cancer tissue (VBCTs) model is engineered which comprised MDA-MB231, a triple-negative breast cancer cells (TNBC), fibroblasts, and endothelial cells. This is indirectly co-cultured with osteoblasts (OBs), thereby constituting a complex quad-cellular tumor progression model. VBCTs alone and in conjunction with OBs led to abnormal vasculature and reduced vessel density but enhanced VEGF production. A total of 1476 significantly upregulated and 775 downregulated genes are identified in the VBCTs exposed to OBs. HSP90N, CYCS, RPS27A, and EGFR are recognized as upregulated hub-genes. Kaplan Meier plot shows HSP90N to have a significant outcome in TNBC patient survivability. Furthermore, compared to cancer tissues without vessels, gene analysis recognized 1278 significantly upregulated and 566 downregulated genes in VBCTs. DKK1, CXCL13, C3 protein and BMP4 are identified to be downregulated hub genes in VBCTs. Together, a multi-cellular breast cancer model and culture protocols are established to study pre-metastatic events in the presence of OBs.