RESUMEN
BACKGROUND: Glucocorticoid receptor (GR) activation has been associated with breast cancer cell survival in vitro. Glucocorticoid (GC)-dependent protection against tumor necrosis factor (TNF)-induced cell death has been well characterized in MCF7 luminal A breast cancer cells. The GR activates a variety of protective mechanisms, such as inhibitors of apoptosis proteins (IAPs). However, the relative contribution of the GR-dependent expression of IAPs in the protection of cell death has not, to our knowledge, been evaluated. METHODS: MCF7 cells were used for all experiments. GR was activated with cortisol (CORT) or dexamethasone (DEX) and inhibited with mifepristone (RU486). Cell viability was determined in real-time with the xCELLigence™ RTCA System and at specific endpoints using crystal violet stain. The mRNA levels of the eight members of the IAP family were measured by qRT-PCR. The protein levels of GR, PR, ERα, HER2, PARP1, c-IAP1 and XIAP were evaluated by Western blot analysis. The knockdown of c-IAP1 and XIAP was accomplished via transient transfection with specific siRNAs. GR activation was verified by a gene reporter assay. Via the cBioportal interphase we queried the mRNA levels of GR and IAPs in breast cancer tumors. RESULTS: RU486 significantly inhibited the anti-cytotoxic effect of both GCs. PARP1 processing was diminished in the presence of both GCs. The combined treatments of GCs + TNF increased the relative mRNA levels of Survivin>c-IAP1 > NAIP>Apollon>XIAP>Ts-IAP > ML-IAP > c-IAP2. Additionally, GR mRNA content increased with the combined treatments of GCs + TNF. Sustained levels of the proteins c-IAP1 and XIAP were observed after 48 h of the combined treatments with GCs + TNF. With c-IAP1 and XIAP gene silencing, the GC-mediated protection was diminished. In the breast tumor samples, the GR mRNA was coexpressed with Apollon and XIAP with a Pearson coefficient greater than 0.3. CONCLUSIONS: The effect of GCs against TNF-mediated cytotoxicity involves increased mRNA expression and sustained protein levels of c-IAP1 and XIAP. The antagonist effects of RU486 and the qRT-PCR results also suggest the role of the GR in this process. This finding may have clinical implications because the GR and IAPs are expressed in breast tumor samples.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Proteínas Inhibidoras de la Apoptosis/genética , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Biomarcadores , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Genes Reporteros , Humanos , Células MCF-7 , ARN Mensajero/genéticaRESUMEN
In this study, we analyzed soluble factors secreted by two Estrogen Receptor Positive (ER-α) human breast cancer cell lines, ZR 75.30 (luminal B) and MCF7 (luminal A), and evaluated their effect on endothelial activation. The composition of tumoral soluble factors (TSFs) was analyzed by ELISA (Bio-Plex). TSFs from ZR 75.30 cells expressed higher levels of TNF, IFN-γ, IL-6, and IL-8 compared to TSFs from MCF-7 cells. TSFs from ZR 75.30 cells induced a pro-adhesive phenotype in human umbilical vein endothelial cells (HUVECs), as characterized by increased monocytic cell adhesion, adhesion molecule expression and NF-κB activation and decreased IκB-α expression. Conversely, TSFs from MCF-7 cells exerted none of these effects on HUVECs. We then added TNF, IFN-γ, IL-6 or IL-8 alone or in combination with TSFs from MCF-7 cells to HUVECs. Only the combinations that included TNF induced endothelial activation. A neutralizing antibody against IL-1ß (this cytokine was not measured in the ELISA) had a modest blocking effect on cellular adhesion or the expression of adhesion molecules induced by TSFs from ZR 75.30 cells in HUVECs. However neutralizing antibodies against TNF, IFN-γ, IL-6 or IL-8 had no effect. Our results suggest that although TNF is an inducer of endothelial cell activation, it is not the only molecule that is responsible for this effect in TSFs from ZR 75.30 cells.
RESUMEN
Tumor microenvironment is an important promoter of tumorigenesis in all forms of breast cancer and has been associated with the risk of metastasis in the different breast cancer subtypes including the more frequent luminal subtypes that encompass 60% of cancer patients. Adhesive properties of endothelial cells (ECs) are strikingly affected during cancer cell dissemination and are related to functional changes of adhesion receptors. The contribution of tumor secreted factors to tumorEC adhesion represents a therapeutic opportunity for breast cancer metastasis. Conditioned medium (CM) of tumor cells can be used as a model to study the role of the secreted molecules to the tumor microenvironment. We explored transcriptomic changes associated to a proadhesive phenotype in primary human umbilical vein endothelial cells (HUVECs) treated with CM of the breast cancer cell line ZR75.30 or with TNF for 3 h. Selected genes were used to validate the microarray through RTqPCR. The bioinformatic analysis identified NFκB as the main regulator of the pro-adhesive phenotype and this was confirmed by pharmacological inhibition of NFκB pathway with BAY 117085. The changes induced by ZR75.30CM mimic those promoted by TNF and display changes in the expression of genes related to inflammatory response, wound healing, extracellular matrix, cytokines, metabolism and cell communication. Despite the abundance of GCSF, IL8, IL6 and VEGF in the ZR75.30CM and the confirmed activation of STAT3 and VEGFR2 pathways, our results suggest dominance of NFκB as a central controller of the transcriptomic response of ECs to breast cancer cells leading to expression of cell adhesion receptors.
Asunto(s)
Neoplasias de la Mama/patología , Adhesión Celular/fisiología , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/patología , FN-kappa B/metabolismo , Transcriptoma , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , FN-kappa B/genética , Metástasis de la Neoplasia , Fenotipo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of developing breast cancer. Epithelial-mesenchymal-transition (EMT) is a process, by which epithelial cells are transdifferentiated to a mesenchymal state, and it has been implicated in cancer progression, including invasion and metastasis. Linoleic acid (LA) induces proliferation and invasion in breast cancer cells. However, the role of LA on the EMT process in human mammary epithelial cells remains to be studied. In the present study, we demonstrate that LA induces a transient down-regulation of E-cadherin expression, accompanied with an increase of Snail1, Snail2, Twist1, Twist2 and Sip1 expressions. Furthermore, LA induces FAK and NFκB activation, MMP-2 and -9 secretions, migration and invasion. In summary, our findings demonstrate, for the first time, that LA promotes an EMT-like process in MCF10A human mammary epithelial cells.