RESUMEN
The epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) control the epithelial-to-mesenchymal transition (EMT) splicing program in cancer. However, their role in breast cancer recurrence is unclear. In this study, we report that high levels of ESRP1, but not ESRP2, are associated with poor prognosis in estrogen receptor positive (ER+) breast tumors. Knockdown of ESRP1 in endocrine-resistant breast cancer models decreases growth significantly and alters the EMT splicing signature, which we confirm using TCGA SpliceSeq data of ER+ BRCA tumors. However, these changes are not accompanied by the development of a mesenchymal phenotype or a change in key EMT-transcription factors. In tamoxifen-resistant cells, knockdown of ESRP1 affects lipid metabolism and oxidoreductase processes, resulting in the decreased expression of fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), and phosphoglycerate dehydrogenase (PHGDH) at both the mRNA and protein levels. Furthermore, ESRP1 knockdown increases the basal respiration and spare respiration capacity. This study reports a novel role for ESRP1 that could form the basis for the prevention of tamoxifen resistance in ER+ breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Metabolismo Energético , Redes y Vías Metabólicas , Proteínas de Unión al ARN/metabolismo , Receptores de Estrógenos/metabolismo , Empalme Alternativo , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Modelos de Riesgos Proporcionales , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Deleted in Liver Cancer-1 (DLC1), a member of the RhoGAP family of proteins, functions as a tumor suppressor in several cancers including breast cancer. However, its clinical relevance is unclear in breast cancer. In this study, expression of DLC1 was correlated with prognosis using publicly available breast cancer gene expression datasets and quantitative Reverse Transcription PCR in cohorts of Estrogen Receptor-positive (ER+) breast cancer. Low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer with further decrease in metastatic lesions. The Cancer Genome Atlas (TCGA) data showed that down regulation of DLC1 is not due to methylation or mutations. To seek further insights in understanding the role of DLC1 in ER+ breast cancer, we stably overexpressed DLC1-full-length (DLC1-FL) in T-47D breast cancer cells; this inhibited cell colony formation significantly in vitro compared to its control counterpart. Label-free global proteomic and TiO2 phosphopeptide enrichment assays (ProteomeXchange identifier PXD008220) showed that 205 and 122 phosphopeptides were unique to DLC1-FL cells and T-47D-control cells, respectively, whereas 6,726 were quantified by phosphoproteomics analysis in both conditions. The top three significant clusters of differentially phosphopeptides identified by DAVID pathway analysis represent cell-cell adhesion, mRNA processing and splicing, and transcription regulation. Phosphoproteomics analysis documented an inverse relation between DLC1 expression and several phosphopeptides including epithelial cell transforming sequence 2 (ECT2). Decreased phosphorylation of ECT2 at the residue T359, critical for its active conformational change, was validated by western blot. In addition, the ECT2 T359-containing phosphopeptide was detected in both basal and luminal patient-derived breast cancers breast cancer phosphoproteomics data on the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Assay portal. Together, for the first time, this implicates ECT2 phosphorylation in breast cancer, which has been proposed as a therapeutic target in lung cancer. In conclusion, this data suggests that low expression of DLC1 is associated with poor prognosis. Targeting ECT2 phosphopeptides could provide a promising mechanism for controlling poor prognosis seen in DLC1low ER+ breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Receptores de Estrógenos/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adhesión Celular/fisiología , Línea Celular Tumoral , Metilación de ADN/fisiología , Regulación hacia Abajo/fisiología , Estudios de Evaluación como Asunto , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Fosforilación/fisiología , Pronóstico , Proteómica/métodos , Proteínas Proto-Oncogénicas , ARN Mensajero/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Cancer cells acquire essential characteristics for metastatic dissemination through the process of epithelial-to-mesenchymal transition (EMT), which is regulated by gene expression and chromatin remodeling changes. The enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), catalyzes trimethylation of lysine 27 of histone H3 (H3K27me3) to repress gene transcription. Here we report the functional roles of EZH2-catalyzed H3K27me3 during EMT in ovarian cancer (OC) cells. TGF-ß-induced EMT in SKOV3 OC cells was associated with decreased levels of EZH2 and H3K27me3 (P<0.05). These effects were delayed (~72 h relative to EMT initiation) and coincided with increased (>15-fold) expression of EMT-associated transcription factors ZEB2 and SNAI2. EZH2 knockdown (using siRNA) or enzymatic inhibition (by GSK126) induced EMT-like changes in OC cells. The EMT regulator ZEB2 was upregulated in cells treated with either approach. Furthermore, TGF-ß enhanced expression of ZEB2 in EZH2 siRNA- or GSK126-treated cells (P<0.01), suggesting that H3K27me3 plays a role in TGF-ß-stimulated ZEB2 induction. Chromatin immunoprecipitation assays confirmed that TGF-ß treatment decreased binding of EZH2 and H3K27me3 to the ZEB2 promoter (P<0.05). In all, these results demonstrate that EZH2, by repressing ZEB2, is required for the maintenance of an epithelial phenotype in OC cells.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Histonas/metabolismo , Humanos , Indoles/farmacología , Metilación/efectos de los fármacos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Regiones Promotoras Genéticas/genética , Unión Proteica , Piridonas/farmacología , Interferencia de ARN , Factor de Crecimiento Transformador beta/farmacología , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismoRESUMEN
OBJECTIVES: Epigenetic alterations have been implicated in the development of platinum resistance in ovarian cancer (OC). In this study, we aimed to identify DNA methylation changes in platinum resistant tumors and their functional implications. METHODS: To identify DNA methylation alterations we used the Illumina 450k DNA methylation array and profiled platinum sensitive and resistant OC xenografts. Validation analyses employed RT-PCR and immunohistochemistry (IHC). RESULTS: Genome-wide DNA methylation analysis of OC xenografts identified 6 genes (SSH3, SLC12A4, TMEM88, PCDHGC3, DAXX, MEST) whose promoters were significantly hypomethylated in resistant compared to sensitive (control) xenografts (p<0.001). We confirmed that TMEM88 and DAXX mRNA expression levels were increased in platinum resistant compared to control xenografts, inversely correlated with promoter methylation levels. Furthermore treatment of OC cells with SGI-110 (guadecitabine), a DNA methyl transferase (DNMT) inhibitor, increased TMEM88 mRNA expression levels, supporting that TMEM88 is transcriptionally regulated by promoter methylation. TMEM88 was detectable by IHC in all histological types of ovarian tumors and its knock-down by using siRNA promoted OC cell proliferation and colony formation and re-sensitized cells to platinum. Furthermore, TMEM88 knock down induced upregulation of cyclin D1 and c-Myc, known Wnt target genes, supporting that TMEM88 inhibits Wnt signaling. CONCLUSIONS: Overall, our results support that OC platinum resistance was correlated with TMEM88 overexpression regulated through decreased promoter methylation. Our data suggest that TMEM88 functions as an inhibitor of Wnt signaling, contributing to the development of platinum resistance.
Asunto(s)
Metilación de ADN , Proteínas de la Membrana/genética , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/genética , Compuestos Organoplatinos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Animales , Carcinoma Epitelial de Ovario , Línea Celular Tumoral , Proliferación Celular/genética , Resistencia a Antineoplásicos , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Glandulares y Epiteliales/metabolismo , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Regiones Promotoras Genéticas , Vía de Señalización Wnt , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-ß-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-ß stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-ß-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-ß stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-ß treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-ß-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-ß induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-ß-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-ß-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.
Asunto(s)
Metilación de ADN/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Ováricas/genética , Factor de Crecimiento Transformador beta/farmacología , Antígenos CD , Azacitidina/análogos & derivados , Azacitidina/farmacología , Cadherinas/genética , Línea Celular Tumoral/efectos de los fármacos , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Islas de CpG/efectos de los fármacos , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Inhibidores Enzimáticos/farmacología , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Regiones Promotoras Genéticas , Factor de Crecimiento Transformador beta/metabolismo , ADN Metiltransferasa 3BRESUMEN
The breast-specific antigen alpha-lactalbumin is expressed in >60% of breast cancer tissues. To evaluate the effect of gene therapy for breast cancer by controlling adenovirus replication with human alpha-lactalbumin promoter, we investigated the activity of a 762-bp human alpha-lactalbumin promoter. Alpha-lactalbumin promoter showed significantly higher activity in MDA-MB-435S and T47D breast cancer cells than in normal breast cell lines or other tumor cell lines. We then developed two novel breast cancer-restricted replicative adenoviruses, AdALAE1a and AdE1aALAE1b. In AdALAE1a, expression of adenoviral E1a gene is under the control of alpha-lactalbumin promoter, and in AdE1aALAE1b, expression of both E1a and E1b genes is under the control of a single alpha-lactalbumin promoter. Both breast cancer-restricted replicative adenoviruses showed viral replication efficiency and tumor cell-killing capability similar to wild-type adenovirus in MDA-MB-435S and T47D cells. The replication efficiency and tumor cell-killing capability of both viruses were attenuated significantly in cells that did not support alpha-lactalbumin promoter. AdE1aALAE1b showed better breast cancer-restricted replication than AdALAE1a, suggesting that a transcriptional targeting modality with alpha-lactalbumin promoter controlling both E1a and E1b gene expression is superior to alpha-lactalbumin promoter controlling only E1a gene expression. Importantly, we found that AdE1aALAE1b could be used to target hormone-independent breast tumors in vivo by inhibiting the growth of MDA-MB-435S s.c. tumors. These data showed that alpha-lactalbumin promoter could regulate the replication of adenovirus to target hormone-independent breast cancers, suggesting that alpha-lactalbumin promoter can be used to develop a novel therapeutic modality for hormone-independent breast cancer.