RESUMEN
BACKGROUND: Cutaneous melanoma is an aggressive disease. S100beta is an established biomarker of disease progression; however, the mechanism of its regulation in melanoma is undefined. METHODS: Expression of HOXC11 and SRC-1 was examined by immunohistochemistry and immunofluorescence. Molecular and cellular techniques were used to investigate regulation of S100beta, including, western blot, qPCR, ChIP and migration assays. RESULTS: Expression levels of the transcription factor HOXC11 and its coactivator SRC-1 were significantly elevated in malignant melanoma in comparison with benign nevi (P<0.001 and P=0.017, respectively, n=80), and expression of HOXC11 and SRC-1 in the malignant tissue associated with each other (P<0.001). HOXC11 recruitment to the promoter of S100beta was observed in the primary melanoma cell line SKMel28. S100beta expression was found to be dependant on both HOXC11 and SRC-1. Treatment with the Src/Abl inhibitor, dasatinib, reduced HOXC11-SRC-1 interaction and prevented recruitment of HOXC11 to the S100beta promoter. Dasatinib inhibited both mRNA and protein levels of S100beta and reduced migration of the metastatic cell line MeWo. CONCLUSION: We have defined a signalling mechanism regulating S100beta in melanoma, which can be modulated by dasatinib. Profiling patients for expression of key markers of this network has the potential to increase the efficacy of dasatinib treatment.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Factores de Crecimiento Nervioso/genética , Coactivador 1 de Receptor Nuclear/metabolismo , Pirimidinas/farmacología , Proteínas S100/genética , Tiazoles/farmacología , Western Blotting , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Dasatinib , Técnica del Anticuerpo Fluorescente , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Melanoma/genética , Factores de Crecimiento Nervioso/metabolismo , Coactivador 1 de Receptor Nuclear/genética , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Células Tumorales CultivadasRESUMEN
Treatment with tyrosine kinase inhibitors (TKIs) including trastuzumab has revolutionized the management of HER2-positive breast cancer. Recent evaluation of clinical trial data suggests that a subset of HER2/ER double-positive cancers may not receive significant benefit from the TKI therapy. Here we investigate the cross talk between HER2 and ER in breast cancer and monitor the effect of trastuzumab on the tyrosine kinase effector transcription factor Myc. In HER2-positive breast cancer patients treated with neoadjuvant trastuzumab, steroid receptor-negative status (ER and PR negative) of pre-treatment biopsies predicted pathological complete response (pCR) (n=31 patients, P=0.0486), whereas elevated Myc protein inversely associated with pCR (P=0.0446). Liquid chromatography mass spectrometry identified the corepressor SMRT as a novel Myc-interacting protein. Trastuzumab treatment enhanced Myc-SMRT interactions in HER2-overexpressing breast cancer cells (LCC1) and inhibited expression of the Myc target gene survivin. In HER2-low, ER-positive steroid-dominant cells (MCF7), trastuzumab therapy repressed Myc-SMRT interactions and upregulated survivin expression. Trastuzumab treatment induced ER-CBP interactions, enhanced ER transcriptional activity and upregulated expression of the ER target gene pS2. The absence of pS2 expression in pre-treatment biopsies predicted pCR to neoadjuvant trastuzumab in breast cancer patients (n=25, P=0.0089) and pS2 expression associated with residual cancer burden (P=0.0196). Furthermore, metastatic tissues from patients who had failed trastuzumab therapy were pS2 positive. In HER2-overexpressing cells, trastuzumab treatment can repress Myc transcriptional activity and clinical response is favorable. However, with co-expression of the steroid pathway, this inhibition is lost and response to treatment is often poor.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor Cross-Talk/fisiología , Receptor ErbB-2/fisiología , Receptores de Estrógenos/fisiología , Neoplasias de la Mama/química , Femenino , Humanos , Células MCF-7 , Co-Represor 2 de Receptor Nuclear/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Receptor ErbB-2/análisis , Receptores de Estrógenos/análisis , Trastuzumab , Factor Trefoil-1 , Proteínas Supresoras de Tumor/análisisRESUMEN
Breast cancer resistance to endocrine therapies such as tamoxifen and aromatase inhibitors is a significant clinical problem. Steroid receptor coactivator-1 (SRC-1), a coregulatory protein of the oestrogen receptor (ER), has previously been shown to have a significant role in the progression of breast cancer. The chromatin protein high mobility group box 2 (HMGB2) was identified as an SRC-1 interacting protein in the endocrine-resistant setting. We investigated the expression of HMGB2 in a cohort of 1068 breast cancer patients and found an association with increased disease-free survival time in patients treated with endocrine therapy. However, it was also verified that HMGB2 expression could be switched on in endocrine-resistant tumours from breast cancer patients. To explore the function of this poorly characterized protein, we performed HMGB2 ChIPseq and found distinct binding patterns between the two contexts. In the resistant setting, the HMGB2, SRC-1 and ER complex are enriched at promoter regions of target genes, with bioinformatic analysis indicating a switch in binding partners between the sensitive and resistant phenotypes. Integration of binding and gene expression data reveals a concise set of target genes of this complex including the RNA helicase DDX18. Modulation of DDX18 directly affects growth of tamoxifen-resistant cells, suggesting that it may be a critical downstream effector of the HMGB2:ER complex. This study defines HMGB2 interactions with the ER complex at specific target genes in the tamoxifen-resistant setting.
Asunto(s)
Neoplasias de la Mama/metabolismo , ARN Helicasas DEAD-box/metabolismo , Proteína HMGB2/metabolismo , Receptores de Estrógenos/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular/genética , ARN Helicasas DEAD-box/genética , Resistencia a Antineoplásicos/genética , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteína HMGB2/genética , Humanos , Estimación de Kaplan-Meier , Células MCF-7 , Ratones Endogámicos BALB C , Ratones SCID , Coactivador 1 de Receptor Nuclear/genética , Coactivador 1 de Receptor Nuclear/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , Receptores de Estrógenos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Associations between p160 coactivator proteins and endocrine resistance have been described. Though thought to primarily interact with steroid receptors, the p160 proteins can also interact with non-nuclear receptor transcription factors including the MAP kinase effector proteins Ets. Here, we observed that in breast cancer cells resistant and insensitive to endocrine treatment, the growth factor EGF induced Ets-2 but not Ets-1 transcriptional regulation of the oncogene myc. Ets-2 regulation of myc was found to be reliant on the p160 proteins SRC-1 and SRC-3. In support of these molecular observations, strong associations were observed between the transcription factor, Ets-2 and its coactivator SRC-1 (P<0.01) and the target gene myc (P<0.0001) in a cohort of breast cancer patients with locally advanced disease. Expression of Ets-2, SRC-1 and c-Myc individually all associated with reduced disease-free survival (P<0.001, P<0.001 and P=0.002 respectively). There was no association between SRC-3 and disease-free survival (P=0.707). SRC-1 can utilize MAP kinase effector transcription factor Ets-2 to regulate the production of the oncogene myc. These signalling mechanisms may be important in the development of steroid resistant/independent breast cancer.