RESUMEN
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are members of the voltage-gated cation channel family known to be expressed in the heart and central nervous system. Ivabradine, a small molecule HCN channel-blocker, is FDA-approved for clinical use as a heart rate-reducing agent. We found that HCN2 and HCN3 are overexpressed in breast cancer cells compared with normal breast epithelia, and the high expression of HCN2 and HCN3 is associated with poorer survival in breast cancer patients. Inhibition of HCN by Ivabradine or by RNAi, aborted breast cancer cell proliferation in vitro and suppressed tumour growth in patient-derived tumour xenograft models established from triple-negative breast cancer (TNBC) tissues, with no evident side-effects on the mice. Transcriptome-wide analysis showed enrichment for cholesterol metabolism and biosynthesis as well as lipid metabolism pathways associated with ER-stress following Ivabradine treatment. Mechanistic studies confirmed that HCN inhibition leads to ER-stress, in part due to disturbed Ca2+ homeostasis, which subsequently triggered the apoptosis cascade. More importantly, we investigated the synergistic effect of Ivabradine and paclitaxel on TNBC and confirmed that both drugs acted synergistically in vitro through ER-stress to amplify signals for caspase activation. Combination therapy could suppress tumour growth of xenografts at much lower doses for both drugs. In summary, our study identified a new molecular target with potential for being developed into targeted therapy, providing scientific grounds for initiating clinical trials for a new treatment regimen of combining HCN inhibition with chemotherapy.
Asunto(s)
Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Línea Celular/efectos de los fármacos , Línea Celular/fisiología , Femenino , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/uso terapéutico , Ivabradina/metabolismo , Ivabradina/uso terapéuticoRESUMEN
Purpose: Adjuvant tamoxifen treatment revolutionized the management of estrogen receptor (ER)-positive breast cancers to prevent cancer recurrence; however, drug resistance compromises its clinical efficacy. The mechanisms underlying tamoxifen resistance are not fully understood, and no robust biomarker is available to reliably predict those who will be resistant. Here, we study BQ323636.1, a novel splice variant of the NCOR2 gene, and evaluate its efficacy in predicting tamoxifen resistance in patients with breast cancer.Experimental Design: A monoclonal anti-BQ323636.1 antibody that specifically recognizes the unique epitope of this splice variant was generated for in vitro mechanistic studies and for in vivo analysis by immunohistochemistry on tissue microarrays of two independent cohorts of 358 patients with more than 10 years clinical follow-up data, who had ER-positive primary breast cancer and received adjuvant tamoxifen treatment. An orthotopic mouse model was also used.Results: Overexpression of BQ323636.1 conferred resistance to tamoxifen in both in vitro and in an orthotopic mouse model. Mechanistically, coimmunoprecipitation showed BQ323636.1 could bind to NCOR2 and inhibit the formation of corepressor complex for the suppression of ER signaling. Nuclear BQ3232636.1 overexpression in patients samples was significantly associated with tamoxifen resistance (P = 1.79 × 10-6, sensitivity 52.9%, specificity 72.0%). In tamoxifen-treated patients, nuclear BQ323636.1 overexpression was significantly correlated with cancer metastasis and disease relapse. Nuclear BQ323636.1 was also significantly associated with poorer overall survival (P = 1.13 × 10-4) and disease-specific survival (P = 4.02 × 10-5).Conclusions: These findings demonstrate that BQ323636.1 can be a reliable biomarker to predict tamoxifen resistance in patients with ER-positive breast cancer. Clin Cancer Res; 24(15); 3681-91. ©2018 AACRSee related commentary by Jordan, p. 3480.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Co-Represor 2 de Receptor Nuclear/genética , Isoformas de Proteínas/genética , Tamoxifeno/administración & dosificación , Adulto , Anciano , Animales , Antineoplásicos Hormonales/administración & dosificación , Antineoplásicos Hormonales/efectos adversos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Quimioterapia Adyuvante , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Células MCF-7 , Ratones , Persona de Mediana Edad , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Receptores de Estrógenos/genética , Tamoxifeno/efectos adversos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously demonstrated that the overexpression of homeoprotein Six1 in hepatocellular carcinoma (HCC) patients is associated with venous infiltration, advanced pathologic tumor metastasis (pTNM) stage and poor overall survival rate (Ng et al. Br J Cancer 2006;95:1050-5). In this study, short hairpin RNA (shRNA) interference approach was used to suppress the expression of Six1 in a metastatic HCC cell line MHCC97L. Stable transfectant MHCC97L-shSix1 carrying Six1-specific shRNA plasmid was established to downregulate Six1 expression to about 40% when compared with MHCC97L-Control. In vitro functional assays demonstrated that the growth rate and proliferation ability of MHCC97L-shSix1 cells were markedly decreased. Moreover, significant decrease of cell motility and invasiveness were observed in MHCC97L-shSix1 cells. Data from in vivo xenograft tumorigenesis model demonstrated that the size of tumor in MHCC97L-shSix1 group was dramatically reduced. Experimental and spontaneous metastasis models indicated that targeting Six1 suppression noticeably reduced the pulmonary metastasis in MHCC97L-shSix1 group. To identify Six1-regulated targets, cDNA microarray was employed to compare the expression profiles of MHCC97L-Control and MHCC97L-shSix1 cells. Twenty-eight downregulated and 24 upregulated genes with known functions were identified in MHCC97L-shSix1. The functions of these target genes are involved in diverse biological activities. Our data suggest that Six1 may be involved in regulation of proliferation and invasiveness of HCC; thus targeting suppression of Six1 is a viable option for treating HCC patients.
Asunto(s)
Carcinoma Hepatocelular/prevención & control , Proteínas de Homeodominio/antagonistas & inhibidores , Neoplasias Renales/prevención & control , Neoplasias Hepáticas Experimentales/prevención & control , ARN Interferente Pequeño/farmacología , Neoplasias del Bazo/prevención & control , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/secundario , Adhesión Celular , Ciclo Celular , Movimiento Celular , Proliferación Celular , Ensayo de Unidades Formadoras de Colonias , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Neoplasias Renales/genética , Neoplasias Renales/secundario , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias del Bazo/genética , Neoplasias del Bazo/secundario , Cicatrización de HeridasRESUMEN
BACKGROUND AND AIMS: The peptidyl prolyl isomerase Pin1 frequently is overexpressed in hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) is the most common etiologic agent in HCC, and its encoded X-protein (HBx) is oncogenic and possesses a serine-proline motif that may bind Pin1. The role of Pin1 in hepatocarcinogenesis, particularly in HBV-related HCC, was investigated. METHODS: Immunohistochemical staining was performed to evaluate the prevalence of Pin1 overexpression in HCCs of different etiologies. Glutathione S-transferase pull-down and co-immunoprecipitation experiments were used to validate the physical interaction between Pin1 and HBx. Reporter assay, cell proliferation assay, and xenotransplantation experiments were used to show the functional consequence and importance of Pin1-HBx interaction in hepatocarcinogenesis. RESULTS: We showed preferential Pin1 overexpression in HBV-related tumors and confirmed the interaction between Pin1 and HBx at the specific serine-proline motif. Pin1 overexpression increased the protein stability of HBx. Furthermore, HBx-mediated transactivation was enhanced by co-expression of Pin1. HepG2 expressing Pin1 and HBx showed a synergistic increase in cellular proliferation, as compared with cells expressing Pin1 or HBx alone. Furthermore, concomitant expression of Pin1 and HBx in the nontumorigenic human hepatocyte cell line MIHA led to a synergistic increase in tumor growth. Finally, in Hep3B cells with suppressed Pin1 expression, HBx-enhanced tumor growth in nude mice was abrogated. CONCLUSIONS: Pin1 binds HBx to enhance hepatocarcinogenesis in HBV-infected hepatocytes. The discovery of an interaction between Pin1 and HBx will further our understanding of the molecular pathogenic mechanism of HBV-related HCC in human beings.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Transformación Celular Neoplásica/metabolismo , Hepatitis B/complicaciones , Neoplasias Hepáticas/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Transactivadores/metabolismo , Secuencias de Aminoácidos , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Hepatitis B/genética , Hepatitis B/metabolismo , Hepatitis B/patología , Hepatitis B/virología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/virología , Ratones , Ratones Desnudos , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/genética , Fosforilación , Prolina , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Serina , Transactivadores/genética , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Transcripción Genética , Activación Transcripcional , Transfección , Trasplante Heterólogo , Regulación hacia Arriba , Proteínas Reguladoras y Accesorias Virales , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
The peptidyl-proplyl-isomerase, PIN1, upregulates beta-catenin by inhibiting its interaction with APC. beta-catenin accumulation occurs in about 70% of hepatocellular carcinoma (HCC), of which only 20% are due to beta-catenin mutations. The role of PIN1 in beta-catenin upregulation in HCC was investigated. PIN1 was shown to be overexpressed in more than 50% of HCC. All cases with PIN1 overexpression also showed beta-catenin accumulation, with 68% of cases showing concomitant beta-catenin and cyclin D1 accumulation. PIN1 was shown to contribute to beta-catenin and cyclin D1 overexpression directly by in vitro cell-line transfection experiments. Finally, we showed that PIN1 overexpression and beta-catenin gene mutations appeared to be mutually exclusive events, leading to beta-catenin accumulation in HCC. These results showed that PIN1 overexpression leading to beta-catenin accumulation might be a critical event in hepatocarcinogenesis, and that PIN1 is a potential target for therapeutic intervention in HCC.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteínas del Citoesqueleto/genética , Isomerasa de Peptidilprolil/genética , Transactivadores/genética , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/genética , Ciclina D1/biosíntesis , Ciclina D1/genética , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , beta CateninaRESUMEN
The helix-loop-helix protein Id-1 has been suggested to play a positive role in cell proliferation and tumorigenesis of many types of human cancers. However, little is known about the molecular mechanism involved in the function of Id-1. In this study, using four stable Id-1 transfectant clones, we investigated the involvement of MAPK signaling pathway in the Id-1 induced serum independent prostate cancer cell growth. Our results demonstrated that both transient and stable ectopic Id-1 expression in prostate cancer LNCaP cells led to activation of the Raf/MEK1/2 signaling pathway. In addition, inhibition of MEK1/2 phosphorylation by one of its inhibitors, PD098059, resulted in the decreased cell cycle S phase fraction and cell growth rate, suggesting that activation of MAPK signaling pathway is essential for Id-1 induced prostate cancer cell proliferation. Furthermore, treatment with antisense oligonucleotide complementary to Id-1 mRNA in PC-3 and DU145 cells resulted in a decreased Id-1 expression which was accompanied by decreased Egr-1 protein. Our results suggest for the first time that the function of Id-1 is associated with MAPK signaling pathway activation and indicate a possible novel mechanism in which Id-1 regulates prostate cancer cell growth and tumorigenesis.
Asunto(s)
Proteínas Inmediatas-Precoces , Sistema de Señalización de MAP Quinasas/fisiología , Neoplasias de la Próstata/patología , Proteínas Represoras , Factores de Transcripción/fisiología , División Celular , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Secuencias Hélice-Asa-Hélice , Humanos , Proteína 1 Inhibidora de la Diferenciación , Masculino , Proteínas Recombinantes/metabolismo , Factores de Transcripción/genética , Transfección , Células Tumorales CultivadasRESUMEN
Gene expression profiling with cDNA array allows simultaneous analysis of the gene expression pattern of a large number of genes and may enhance the investigation of the molecular mechanisms involved in the treatment of hepatocellular carcinoma with cisplatin. We used cDNA array technology to assess the gene expression profiles related to cell cycle regulation and apoptosis in human hepatoma Hep3B cells in response to cisplatin treatment. In Hep3B cells, apoptosis induced by cisplatin was p53-independent, and was associated with up-regulation of cell cycle regulators, pro-apoptotic genes, growth receptors, and genes involved in signal transduction. These included p33ING1, c-Abl, Bax, insulin-like growth factor binding protein 3, Siva, cyclin D1, RhoA, and Raf-1. Down-regulation of cell cycle regulator CDC2 was observed. Semi-quantitative reverse transcription-polymerase chain reaction and/or Western blot analysis performed on seven of these genes confirmed their upregulation of gene expression. Such global analysis of the cytotoxic response to chemotherapeutic drugs may yield insight into the mechanisms of drug action and allow rational design of more effective treatment strategies.