RESUMEN
17beta-hydroxysteroid dehydrogenase type 7 (17ß-HSD7) promotes breast cancer cell growth via dual-catalytic activity by modulating estradiol and DHT. Here, we clarified the expression pattern of 17ß-HSD7 in postmenopausal luminal A type breast cancer with The Cancer Genome Atlas (TCGA) cohort. The impact of 17ß-HSD7 inhibition on the proteome of MCF-7 cells was investigated and on cell apoptosis was revealed. MCF-7 cells were treated with an efficient inhibitor of 17ß-HSD7 (INH7) or with vehicle, and a differential proteomics study was performed using two-dimensional (2D) gel electrophoresis followed by mass spectrometry and ingenuity pathway analysis (IPA). Cell apoptosis was analyzed by flow cytometry, followed by reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot to investigate the expression of apoptosis-related genes. Our data showed 17ß-HSD7 is amplified in primary and progressive breast cancer, inhibition of 17ß-HSD7 in MCF-7 cells modulated 104 proteins primarily involved in cell death/survival, cell growth and DNA processing. The expression of 78kDa glucose-regulated protein (GRP78) and anti-apoptosis factor Bcl-2 were significantly suppressed via 17ß-HSD7 inhibition with INH7, consequently induced MCF-7 cell apoptosis. However, INH7 treatment of T47D, another widely used epithelial ER+ breast cancer cell line, led to an up-regulation of GRP78 expression, resulting in a limited increase in apoptosis. These results suggest cell-specific effects of INH7 in the breast cancer, which is interesting for further study. An combinatory effect on apoptosis by INH7 and Letrozole (aromatase inhibitor) was further demonstrated in MCF-7. Down-regulation of GRP78 via 17ß-HSD7 inhibition enhances cell apoptosis in response to Letrozole. This study highlights GRP78 as a key regulator related to 17ß-HSD7 inhibition and effect. Taken together, results from the present study suggest a hypothesis that inhibition of 17ß-HSD7 would be a complementary strategy to Letrozole by suppression of GRP78 in ER+ breast cancer. However, from a research perspective, further studies have to be carried out with more breast cancer cell lines as well as in vivo model to assess the efficacy of inhibitor combination.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Inhibidores de la Aromatasa/farmacología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Chaperón BiP del Retículo Endoplásmico , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Perfilación de la Expresión Génica , Proteínas de Choque Térmico , Humanos , Letrozol , Anotación de Secuencia Molecular , Nitrilos/farmacología , Especificidad de Órganos , Posmenopausia , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Triazoles/farmacologíaRESUMEN
17beta-hydroxysteroid dehydrogenase type 5 (17ß-HSD5) is an important enzyme associated with sex steroid metabolism in hormone-dependent cancer. However, reports on its expression and its prognostic value in breast cancer are inconsistent. Here, we demonstrate the impact of 17ß-HSD5 expression modulation on the proteome of estrogen receptor-positive (ER+) breast cancer cells. RNA interference technique (siRNA) was used to knock down 17ß-HSD5 gene expression in the ER+ breast cancer cell line MCF-7 and the proteome of the 17ß-HSD5-knockdown cells was compared to that of MCF-7 cells using two-dimensional (2-D) gel electrophoresis followed by mass spectrometry analysis. Ingenuity pathway analysis (IPA) was additionally used to assess functional enrichment analyses of the proteomic dataset, including protein network and canonical pathways. Our proteomic analysis revealed only four differentially expressed protein spots (fold change > 2, p<0.05) between the two cell lines. The four spots were up-regulated in 17ß-HSD5-knockdown MCF-7 cells, and comprised 21 proteins involved in two networks and in functions that include apoptosis inhibition, regulation of cell growth and differentiation, signal transduction and tumor metastasis. Among the proteins are nucleoside diphosphate kinase A (NME1), 78kDa glucose-regulated protein (GRP78) and phosphoglycerate kinase 1 (PGK1). We also showed that expression of 17ß-HSD5 and that of the apoptosis inhibitor GRP78 are strongly but negatively correlated. Consistent with their opposite regulation, GRP78 knockdown decreased MCF-7 cell viability whereas 17ß-HSD5 knockdown or inhibition increased cell viability and proliferation. Besides, IPA analysis revealed that ubiquitination pathway is significantly affected by 17ß-HSD5 knockdown. Furthermore, IPA predicted the proto-oncogene c-Myc as an upstream regulator linked to the tumor-secreted protein PGK1. The latter is over-expressed in invasive ductal breast carcinoma as compared with normal breast tissue and its expression increased following 17ß-HSD5 knockdown. Our present results indicate a 17ß-HSD5 role in down-regulating breast cancer development. We thus propose that 17ß-HSD5 may not be a potent target for breast cancer treatment but its low expression could represent a poor prognosis factor.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/metabolismo , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Choque Térmico/metabolismo , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfoglicerato Quinasa/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , 3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , Miembro C3 de la Familia 1 de las Aldo-Ceto Reductasas , Neoplasias de la Mama/patología , Proliferación Celular , Supervivencia Celular , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática , Femenino , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/antagonistas & inhibidores , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , Hidroxiprostaglandina Deshidrogenasas/química , Hidroxiprostaglandina Deshidrogenasas/genética , Procesamiento de Imagen Asistido por Computador , Células MCF-7 , Nucleósido Difosfato Quinasas NM23/química , Nucleósido Difosfato Quinasas NM23/genética , Nucleósido Difosfato Quinasas NM23/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fosfoglicerato Quinasa/química , Fosfoglicerato Quinasa/genética , Proteómica/métodos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Receptores de Estrógenos/metabolismo , Electroforesis Bidimensional Diferencial en GelRESUMEN
17beta-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a steroidal enzyme which, in breast cancer cells, mainly synthesizes 17-beta-estradiol (E2), an estrogenic hormone that stimulates breast cancer cell growth. We previously showed that the enzyme increased breast cancer cell proliferation via a dual effect on E2 and 5α-dihydrotestosterone (DHT) levels and impacted gene expression and protein profile of breast cancer cells cultured in E2-contained medium. Here, we used RNA interference technique combined with microarray analyses to investigate the effect of 17ß-HSD1 expression on breast cancer cell transcript profile in steroid-deprived condition. Our data revealed that knockdown of 17ß-HSD1 gene, HSD17B1, modulates the transcript profile of the hormone-dependent breast cancer cell line T47D, with 105 genes regulated 1.5 fold or higher (p < 0.05) in estradiol-independent manner. Using Ingenuity Pathway Analysis (IPA), we additionally assessed functional enrichment analyses, including biological functions and canonical pathways, and found that, in concordance with the role of 17ß-HSD1 in cancer cell growth, most regulated genes are cancer-related genes. Genes that primarily involved in the cell cycle progression, such as the cyclin A2 gene, CCNA2, are generally down-regulated whereas genes involved in apoptosis and cell death, including the pro-apoptotic gene XAF1, IFIH1 and FGF12, are on the contrary up-regulated by 17ß-HSD1 knockdown, and 21% of the modulated genes belong to this latter functional category. This indicates that 17ß-HSD1 may be involved in oncogenesis by favoring anti-apoptosis pathway in breast cancer cells and correborates with its previously shown role in increasing breast cancer cell proliferation. The gene regulation occurring in steroid-deprived conditions showed that 17ß-HSD1 can modulate endogenous gene expression in steroid-independent manners. Besides, we tested the ability of estrogen to induce or repress endogenous genes of T47D by microarray analysis. Expression of a total of 130 genes were found to increase or decrease 1.5-fold or higher (p < 0.05) in response to E2 treatment (1 nM for 48 h), revealing a list of potential new estrogen-responsive genes and providing useful information for further studies of estrogen-dependent breast cancer mechanisms. In conclusion, in breast cancer cells, in addition to its implication in the E2-dependent gene transcription, the present study demonstrates that 17ß-HSD1 also modulates gene expression via mechanisms independent of steroid actions. Those mechanisms that may include the ligand-independent gene transcription of estrogen receptor alpha (ERα), whose expression is positively correlated with that of the enzyme, and that may implicate 17ß-HSD1 in anti-apoptosis pathways, have been discussed.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/metabolismo , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Estradiol/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , 17-Hidroxiesteroide Deshidrogenasas/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Medios de Cultivo , Femenino , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
The data presented here are related to the research article entitled "Estradiol-independent modulation of breast cancer transcript profile by 17beta-hydroxysteroid dehydrogenase type 1" (J.A. Aka, E.L. Calvo, S.X. Lin, 2016) [1]. We evaluated the effect of the steroidal enzyme 17ß-HSD1 and its product, the estrogenic hormone 17-beta-estradiol (E2), on gene transcription profile of breast cancer cells. RNA interference technique was used to knock down the 17ß-HSD1 gene (HSD17B1) in the hormone-dependent breast cancer cell line T47D in steroid-deprived medium. Transfected cells were subsequently treated with E2, and microarray analyses (with three contrasts) were used to investigate (i) the effect of 17ß-HSD1 expression on breast cancer cell transcript profile in steroid-deprived condition, (ii) the effect of E2 on breast cancer gene expression and (iii) if E2 affects gene regulation by 17ß-HSD1. Functional enrichments of the differentially expressed genes were assessed using Ingenuity Pathway Analysis (IPA). Here, we showed data on 140 genes that are induced or repressed 1.5 time or higher (p < 0.05) in the HSD17B1-silenced and E2-treated T47D cells revealed by microarray analysis, and presented the 14 functional terms found in the cancer and in the cell death and survival categories revealed by the IPA biological function analysis. Data on IPA Canonical Pathway and network analyses is also presented. Further discussion on gene regulation by 17ß-HSD1 and E2 is provided in the accompanying publication [1].
RESUMEN
Peptide motifs are often used for protein-protein interactions. We have recently demonstrated that ankyrin repeats of ANKRA2 and the paralogous bare lymphocyte syndrome transcription factor RFXANK recognize PxLPxL/I motifs shared by megalin, three histone deacetylases, and RFX5. We show here that that CCDC8 is a major partner of ANKRA2 but not RFXANK in cells. The CCDC8 gene is mutated in 3M syndrome, a short-stature disorder with additional facial and skeletal abnormalities. Two other genes mutated in this syndrome encode CUL7 and OBSL1. While CUL7 is a ubiquitin ligase and OBSL1 associates with the cytoskeleton, little is known about CCDC8. Binding and structural analyses reveal that the ankyrin repeats of ANKRA2 recognize a PxLPxL motif at the C-terminal region of CCDC8. The N-terminal part interacts with OBSL1 to form a CUL7 ligase complex. These results link ANKRA2 unexpectedly to 3M syndrome and suggest novel regulatory mechanisms for histone deacetylases and RFX7.
Asunto(s)
Repetición de Anquirina , Ancirinas/química , Proteínas Portadoras/química , Enanismo/metabolismo , Hipotonía Muscular/metabolismo , Columna Vertebral/anomalías , Secuencia de Aminoácidos , Ancirinas/metabolismo , Sitios de Unión , Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Proteínas del Citoesqueleto/metabolismo , Enanismo/genética , Células HEK293 , Histona Desacetilasas/metabolismo , Humanos , Datos de Secuencia Molecular , Hipotonía Muscular/genética , Unión Proteica , Proteínas Represoras/metabolismo , Columna Vertebral/metabolismoRESUMEN
INTRODUCTION: Human 17beta-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a steroid-converting enzyme that has long been known to play critical roles in estradiol synthesis and more recently in dihydrotestosterone (DHT) inactivation, showing a dual function that promotes breast cancer cell proliferation. Previously, we reported the first observation of the influence of the enzyme on endogenous estrogen-responsive gene expression. Here, we demonstrate the impact of 17ß-HSD1 expression on the breast cancer cell proteome and investigate its role in cell migration. METHODS: 17ß-HSD1 was stably transfected in MCF7 cells and the proteome of the generated cells overexpressing 17ß-HSD1 (MCF7-17ßHSD1 cells) was compared to that of the wild type MCF7 cells. Proteomics study was performed using two-dimensional gel electrophoresis followed by mass spectrometry analysis of differentially expressed protein spots. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to investigate the transcription of individual gene. The effect of 17ß-HSD1 on MCF7 cell migration was verified by a wound-healing assay. RESULTS: Proteomic data demonstrate that the expression of more than 59 proteins is modulated following 17ß-HSD1 overexpression. 17ß-HSD1 regulates the expression of important genes and proteins that are relevant to cell growth control, such as BRCA2 and CDKN1A interacting protein (BCCIP) and proliferating cell nuclear antigen (PCNA) which are down- and upregulated in MCF7-17ßHSD1 cells, respectively. RT-qPCR data reveal that 17ß-HSD1 increases the mRNA levels of estrogen receptors (ER) alpha and beta by 171 and 120%, respectively, while decreasing that of the androgen receptor by 64%. Interestingly, 17ß-HSD1 increases the mRNA transcript (by 3.6 times) and the protein expression of the metastasis suppressor gene nm23-H1 and the expression of the two enzymes are closely correlated. We have further shown that 17ß-HSD1 expression is associated with an increase of MCF7 cell migration. CONCLUSIONS: In addition to the regulation of important genes, we have demonstrated for the first time that 17ß-HSD1 increases breast cancer cell migration, in spite of its positive regulation of the antimetastatic gene NM23. This is also correlated to its stimulation of breast cancer cell growth, further confirming its targeting in ER positive breast cancer. The novel findings in this study suggest several directions for future research on the contribution of 17ß-HSD1 to breast cancer progression and related treatment.
Asunto(s)
Movimiento Celular , Estradiol Deshidrogenasas/metabolismo , Nucleósido Difosfato Quinasas NM23/metabolismo , ARN Mensajero/metabolismo , Proteína BRCA2/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Estradiol Deshidrogenasas/genética , Receptor alfa de Estrógeno/genética , Receptor beta de Estrógeno/genética , Femenino , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Análisis por Matrices de Proteínas , Proteoma/análisis , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño , Receptores Androgénicos/genéticaRESUMEN
The active estrogen estradiol (E2) stimulates breast cancer cell (BCC) growth, whereas the androgen dihydrotestosterone (DHT) has shown an antiproliferative effect. The principal product synthesized by the 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) is E2, although we have demonstrated that the purified enzyme also inactivates DHT. However, the direct roles of 17beta-HSD1 in sex-hormone regulation and BCC proliferation have not been completely established. Here, we show that 17beta-HSD1 inhibition suppresses DHT catabolism by 19%, whereas knockdown of the gene expression increases the concentration of DHT by 41% in the T47D BCC line. The 17beta-HSD1/DHT complex crystal structure reveals that DHT binds in both normal and reverse modes, but the latter mode leading to O3 reduction is preferred with stronger interactions. Using RNA interference and an inhibitor of 17beta-HSD1, we demonstrate that 17beta-HSD1 expression is negatively correlated to DHT levels in BCC but positively correlated to estrone reduction, E2 levels, and cell proliferation. 17beta-HSD1 inhibition reduces DHT inactivation, increasing the antiproliferative effect by DHT in T47D cells after 8 d treatment. Thus, 17beta-HSD1 up-regulates BCC growth by a dual action on estradiol synthesis and DHT inactivation. We have further demonstrated that 17beta-HSD1 can enhance the E2-induced expression of the endogenous estrogen-responsive gene pS2, providing an important information regarding the modulation of the estrogen responsiveness by 17beta-HSD1 that may also contribute to BCC growth. These results strongly support the rationale for inhibiting 17beta-HSD1 in breast cancer therapy to eliminate estrogen activation via the sulfatase pathway while avoiding the deprivation of DHT.
Asunto(s)
Neoplasias de la Mama/metabolismo , Dihidrotestosterona/metabolismo , Estradiol Deshidrogenasas/metabolismo , Estradiol/metabolismo , Western Blotting , Línea Celular , Línea Celular Tumoral , Proliferación Celular , Estradiol Deshidrogenasas/genética , Cromatografía de Gases y Espectrometría de Masas , Humanos , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Estradiol, the most potent estrogen, plays critical roles in tumor cell proliferation and breast cancer development. It can be synthesized via the aromatase pathway or the sulfatase pathway, and the later has been demonstrated to be more significant. Reductive 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the last step in estrogen activation and are thus critical in breast cancer development. 17beta-HSD Type 1 (17beta-HSD1) is of great importance since it efficiently synthesizes the most potent estrogen estradiol, as well as other estrogens as 5-androstene-3beta,17beta-diol and 5alpha-androstane-3beta,17beta-diol, and inactivates the most active androgen dihydrotestosterone (DHT), all contributing to the stimulation and development of breast cancers. Rational inhibitor design based on the new structure information has been developed, yielding interesting compounds and lead chemicals. This was demonstrated by a hybrid inhibitor that interacts with both the substrate and cofactor binding sites and a recently designed inhibitor 3-(3',17'beta-dihydroxyestra-1',3',5'(10')-trien-16'beta-methyl) benzamide which has been crystallized in complex with 17beta-HSD1. Both inhibitors demonstrate nM level K(i)in vitro. New non-steroidal inhibitors have been designed and reported very recently. The Type 7 17beta-HSD, expressed in several tissues including breast and ovary, can also contribute to estrogen synthesis and DHT inactivation in breast cancer cells. The enzyme role in steroid metabolism and cancer cell proliferation needs to be compared to that in cholesterogenesis. Breast cancer cell lines provide an excellent platform for such study. T47D, MCF-7 and MDA-MB-231-luc cells have been used to create xenografts in nude mice as animal models, now with the possibility of bioluminescent imaging to provide rapid, non-invasive, and quantitative analysis of tumor biomass and metastasis. Here we review the roles of the sulfatase and aromatase pathways and the contribution of the reductive 17beta-HSDs for hormone metabolism in breast cancer.