RESUMO
FOXO1 transcription factor not only limits the cell cycle progression but also promotes cell death as a tumor suppressor protein. Though the expression of FOXO1 is largely examined in breast cancer, the regulation of FOXO1 by miRNA is yet to be explored. In the current study, self-assembled branched DNA (bDNA) nanostructures containing oncogenic miRNAs were designed and transfected to the MCF7 cell line to decipher the FOXO1 expression. bDNA containing oncogenic miRNAs 27a, 96, and 182 synergistically downregulate the expression of FOXO1 in MCF7 cells. The down-regulation is evident both in mRNA and protein levels suggesting that bDNA having miRNA sequences can selectively bind to mRNA and inhibit translation. Secondly, the downstream gene expression of p21 and p27 was also significantly downregulated in presence of miR-bDNA nanostructures. The cell proliferation activity was progressively increased in presence of miR-bDNA nanostructures which confirms the reduced tumor suppression activity of FOXO1 and the downstream gene expression. This finding can be explored to design novel bDNA structures which can downregulate the tumor suppressor proteins in normal cells and induce cell proliferation activity to identify early-phase markers of cancer.
Assuntos
MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Regulação para Baixo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , DNA , RNA MensageiroRESUMO
BACKGROUND: Orphan nuclear receptors ERRα, ERRß and ERRγ that belong to NR3B or type IV nuclear receptor family are well studied for their role in breast cancer pathophysiology. Their homology with the canonical estrogen receptor dictates their possible contributing role in mammary gland development and disease. Although function and regulation of ERRα, ERRγ and less about ERRß is reported, role of histone methylation in their altered expression in cancer cells is not studied. Transcriptional activity of nuclear receptors depends on co-regulatory proteins. The present study for the first time gives an insight into regulation of estrogen-related receptors by histone methylation specifically through methyltransferase EZH2 in breast cancer. METHODS: Expression of ERRα, ERRß, ERRγ and EZH2 was assessed by immunohistochemistry in four identical tissue array slides that were prepared as per the protocol. The array slides were stained with ERRα, ERRß, ERRγ and EZH2 simultaneously. Array data was correlated with expression in MERAV expression dataset. Pearson correlation coeficient r was calculated from the partial matrix expression values available at MERAV database to study the strength of association between EZH2 and three orphan nuclear receptors under study. By western blot and real time PCR, their correlated expression was studied in breast cancer cell lines MCF-7, MDA-MB-231, T47D and MDA-MB-453 including normal breast epithelial MCF-10A cells at both protein and RNA level. Regulation of ERRα, ERRß, ERRγ by EZH2 was further investigated upon overexpression and silencing of EZH2. The interaction between ERRs and EZH2 was validated in vivo by CHIP-qPCR. RESULTS: We found a negative correlation between estrogen-related receptors and Enhancer of Zeste Homolog 2, a global repressor gene. Immunohistochemistry in primary breast tumors of different grades showed a correlated expression of estrogen-related receptors and EZH2. Their correlated expression was further validated using online MERAV expression dataset where a negative correlation of variable strengths was observed in breast cancer. Ectopic expression of EZH2 in low EZH2-expressing normal breast epithelial cells abrogated their expression and at the same time, its silencing enhanced the expression of estrogen-related receptors in cancerous cells. Global occupancy of EZH2 on ERRα and ERRß was observed in-vivo. CONCLUSION: Our findings identify EZH2 as a relevant coregulator for estrogen-related receptors in breast carcinoma.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/fisiologia , Receptores de Estrogênio/fisiologia , Proteína Potenciadora do Homólogo 2 de Zeste/análise , Feminino , Humanos , Células MCF-7 , Receptores de Estrogênio/análise , Receptores de Estrogênio/genética , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
BACKGROUND: Breast cancer (BC) is highly heterogeneous with ~ 60-70% of estrogen receptor positive BC patient's response to anti-hormone therapy. Estrogen receptors (ERs) play an important role in breast cancer progression and treatment. Estrogen related receptors (ERRs) are a group of nuclear receptors which belong to orphan nuclear receptors, which have sequence homology with ERs and share target genes. Here, we investigated the possible role and clinicopathological importance of ERRß in breast cancer. METHODS: Estrogen related receptor ß (ERRß) expression was examined using tissue microarray slides (TMA) of Breast Carcinoma patients with adjacent normal by immunohistochemistry and in breast cancer cell lines. In order to investigate whether ERRß is a direct target of ERα, we investigated the expression of ERRß in short hairpin ribonucleic acid knockdown of ERα breast cancer cells by western blot, qRT-PCR and RT-PCR. We further confirmed the binding of ERα by electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), Re-ChIP and luciferase assays. Fluorescence-activated cell sorting analysis (FACS) was performed to elucidate the role of ERRß in cell cycle regulation. A Kaplan-Meier Survival analysis of GEO dataset was performed to correlate the expression of ERRß with survival in breast cancer patients. RESULTS: Tissue microarray (TMA) analysis showed that ERRß is significantly down-regulated in breast carcinoma tissue samples compared to adjacent normal. ER + ve breast tumors and cell lines showed a significant expression of ERRß compared to ER-ve tumors and cell lines. Estrogen treatment significantly induced the expression of ERRß and it was ERα dependent. Mechanistic analyses indicate that ERα directly targets ERRß through estrogen response element and ERRß also mediates cell cycle regulation through p18, p21cip and cyclin D1 in breast cancer cells. Our results also showed the up-regulation of ERRß promoter activity in ectopically co-expressed ERα and ERRß breast cancer cell lines. Fluorescence-activated cell sorting analysis (FACS) showed increased G0/G1 phase cell population in ERRß overexpressed MCF7 cells. Furthermore, ERRß expression was inversely correlated with overall survival in breast cancer. Collectively our results suggest cell cycle and tumor suppressor role of ERRß in breast cancer cells which provide a potential avenue to target ERRß signaling pathway in breast cancer. CONCLUSION: Our results indicate that ERRß is a negative regulator of cell cycle and a possible tumor suppressor in breast cancer. ERRß could be therapeutic target for the treatment of breast cancer.
Assuntos
Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Estrogênio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Proliferação de Células/genética , Conjuntos de Dados como Assunto , Receptor alfa de Estrogênio/genética , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Células MCF-7 , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genética , Análise Serial de Tecidos , Regulação para CimaRESUMO
BACKGROUND: Well-known anti-malarial drug artemisinin exhibits potent anti-cancerous activities. In-vivo and in-vitro studies showed its anti-tumor and immunomodulatory properties signifying it as a potent drug candidate for study. The studies of mechanisms of cell movement are relevant which can be understood by knowing the involvement of genes in an effect of a drug. Although cytotoxicity and anti-proliferative activity of artemisinin is evident, the genes participating in its anti-migratory and reduced invasive effect are not well studied. The present study reports the alteration in the expression of 84 genes involved in cell motility upon artemisinin treatment in MCF-7 breast cancer cells using pathway focused gene expression PCR array. In addition, the effect of artemisinin on epigenetic modifier HDACs is studied. METHODS: We checked the functional stimulus of artemisinin on cell viability, migration, invasion and apoptosis in breast cancerous cell lines. Using qRT-PCR and western blot, we validated the altered expression of relevant genes associated with proliferation, migration, invasion, apoptosis and mammary gland development. RESULTS: Artemisinin inhibited cell proliferation of estrogen receptor negative breast cancer cells with fewer efficacies in comparison to estrogen receptor positive ones. At the same time, cell viability and proliferation of normal breast epithelial MCF10A cells was un-affected. Artemisinin strongly inhibited cancer cell migration and invasion. Along with orphan nuclear receptors (ERRα, ERRß and ERRγ), artemisinin altered the ERα/ERß/PR/Her expression status of MCF-7 cells. The expression of genes involved in the signaling pathways associated with proliferation, migration, invasion and apoptosis was significantly altered which cooperatively resulted into reduced growth promoting activities of breast cancer cells. Interestingly, artemisinin exhibited inhibitory effect on histone deacetylases (HDACs). CONCLUSIONS: Upregulated expression of tumor suppressor genes along with reduced expression of oncogenes significantly associated with growth stimulating signaling pathways in response to artemisinin treatment suggests its efficacy as an effective drug in breast cancer treatment.
Assuntos
Antineoplásicos/farmacologia , Artemisininas/farmacologia , Neoplasias da Mama/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Transcriptoma/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Artemisininas/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Genes Supressores de Tumor/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Humanos , Células MCF-7 , Invasividade Neoplásica/prevenção & controle , Oncogenes/efeitos dos fármacosRESUMO
Silent mating type information regulation 2 homolog 1 (SIRT1) is a class III histone deacetylase (HDAC) that is NAD + dependent and essential for metabolism, senescence, and cell survival. SIRT1 is overexpressed in several cancers, including breast cancer. SIRT1 is a well-known target gene of the estrogen receptor alpha (ER alpha) and is closely related to ER alpha deacetylation. Transcription factor Estrogen-related receptors (ERRs) share sequence homology with ERs in the DNA-binding domain, therefore, the possibility of sharing target genes between them is high. Our current research aims to gain insight into the function of ERRß in regulating the activity of SIRT1 during the progression of breast cancer. ER-positive (ER + ve) breast cancer cells and tissues had considerably enhanced SIRT1 expression. Six potential ERRE sites were identified by analysis of the 5' upstream region of SIRT1, and both in vitro and in vivo experiments supported their presence. We found SIRT1 to be up-regulated in ERRß overexpressed ER + ve breast cancer cells. Furthermore, our findings suggested that ectopic production of ERR and PCAF would increase SIRT1 activity. Our findings also indicated that ectopic production of ERRß and PCAF increased SIRT1 activity. With sufficient evidence demonstrating the substantial involvement of SIRT1 in cell proliferation, migration, and colony formation capability, we were also able to illustrate the tumorigenic role of SIRT1. Overall, our findings highlight SIRT1's tumorigenic influence on breast cancer and suggest that SIRT1 inhibitors might serve as potential therapeutic drugs for the treatment of breast cancer.
Assuntos
Neoplasias da Mama , Regulação Neoplásica da Expressão Gênica , Receptores de Estrogênio , Sirtuína 1 , Feminino , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Células MCF-7 , Receptores de Estrogênio/metabolismo , Sirtuína 1/metabolismo , Sirtuína 1/genéticaRESUMO
Breast invasive carcinoma (BRCA) is the most malignant and leading cause of death in women. Global efforts are ongoing for improvement in early detection, prevention, and treatment. In this milieu, a comprehensive analysis of RNA-sequencing data of 1097 BRCA samples and 114 normal adjacent tissues is done to identify dysregulated genes in major molecular classes of BRCA in various clinical stages. Significantly enriched pathways in distinct molecular classes of BRCA have been identified. Pathways such as interferon signaling, tryptophan degradation, granulocyte adhesion & diapedesis, and catecholamine biosynthesis were found to be significantly enriched in Estrogen/Progesterone Receptor positive/Human Epidermal Growth Factor Receptor 2 negative, pathways such as RAR activation, adipogenesis, the role of JAK1/2 in interferon signaling, TGF-ß and STAT3 signaling intricated in Estrogen/Progesterone Receptor negative/Human Epidermal Growth Factor Receptor 2 positive and pathways as IL-1/IL-8, TNFR1/TNFR2, TWEAK, and relaxin signaling were found in triple-negative breast cancer. The dysregulated genes were clustered based on their mutation frequency which revealed nine mutated clusters, some of which were well characterized in cancer while others were less characterized. Each cluster was analyzed in detail which led to the identification of NLGN3, MAML2, TTN, SYNE1, ANK2 as candidate genes in BRCA. They are central hubs in the protein-protein-interaction network, indicating their important regulatory roles. Experimentally, the Real-Time Quantitative Reverse Transcription PCR and western blot confirmed our computational predictions in cell lines. Further, immunohistochemistry corroborated the results in ~ 100 tissue samples. We could experimentally show that the NLGN3 & ANK2 have tumor-suppressor roles in BRCA as shown by cell viability assay, transwell migration, colony forming and wound healing assay. The cell viability and migration was found to be significantly reduced in MCF7 and MDA-MB-231 cell lines in which the selected genes were over-expressed as compared to control cell lines. The wound healing assay also demonstrated a significant decrease in wound closure at 12 h and 24 h time intervals in MCF7 & MDA-MB-231 cells. These findings established the tumor suppressor roles of NLGN3 & ANK2 in BRCA. This will have important ramifications for the therapeutics discovery against BRCA.
Assuntos
Neoplasias da Mama , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Redes Reguladoras de Genes , Transdução de Sinais , Perfilação da Expressão Gênica , Linhagem Celular Tumoral , Invasividade NeoplásicaRESUMO
Breast cancer morbidity is surging towards the peak in females across the globe. An inherent property of cancer cells is enhanced cell proliferation rate and migration capability, leading to deregulated cell signaling cascades. G-protein-coupled receptors (GPCRs) have recently emerged as a hot-spot target in cancer research. We identify aberrant expression of G-protein-coupled receptor 141 (GPR141) in different breast cancer subtypes that correlate with poor prognosis. However, the molecular mechanism via which GPR141 advances breast cancer remains elusive. Increased GPR141 expression enhances the migratory behavior of breast cancer, driving oncogenic pathways both in vitro and in vivo through activation of epithelial to mesenchymal transition (EMT), oncogenic mediators and regulation of p-mTOR/p53 signaling. Our study unveils a molecular mechanism for p53 downregulation and activation of p-mTOR1 and its substrates in GPR141 overexpressed cells, accelerating breast tumorigenesis. We find that an E3 ubiquitin ligase, Cullin1, partly mediates p53 degradation via proteasomal pathway. Co-immunoprecipitation result shows that the phosphorylated form of 40S ribosome protein S6 (ps6., a p-mTOR1 substrate) forms a complex with Cullin1. These findings suggest an interplay between Cullin1 and p-mTOR1 in GPR141 overexpressed cells that downregulates p53 expression, thus inducing tumor growth. GPR141 silencing restores p53 expression and attenuates p-mTOR1 signaling events, thereby impeding proliferation and migration in breast cancer cells. Our findings describe the role of GPR141 in breast cancer proliferation, and metastasis, as well as in influencing the tumor microenvironment. Modulating GPR141 expression could pave the way for a better therapeutic approach to regulating breast cancer progression and metastasis.
Assuntos
Neoplasias da Mama , Receptores Acoplados a Proteínas G , Feminino , Humanos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Serina-Treonina Quinases TOR/metabolismo , Microambiente Tumoral , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
BACKGROUND: Acquired resistance to drug involves multilayered genetic and epigenetic regulation. Inhibition of EZH2 has proven to reverse the tamoxifen resistance back to the sensitive state in breast cancer. However, the molecular players involved in EZH2-mediated effects on tamoxifen-resistant MCF-7 cells are unknown. This study was conducted to understand the global change in proteome profile of tamoxifen-resistant MCF-7 breast cancer cells upon EZH2 knockdown. METHODS: Tamoxifen resistance MCF-7 breast cancer cells were established using increasing concentrations of 4-hydroxy tamoxifen. Using label free proteomics approach, we studied the alteration in total proteome in resistant cells as well as cells transfected with siEZH2 in comparison to sensitive and cells transfected with non-targeting siRNA. RESULTS: Here, we report list of proteins that were previously not recognized for their role in tamoxifen resistance and hold a close association with breast cancer patient survival. Proteins Annexin A2, CD44, nucleosome assembly protein 1, and lamin A/C were among the most upregulated protein in tamoxifen-resistant cells that were found to be abrogated upon EZH2 knockdown. The study suggests the involvement for various proteins in acquiring resistance towards tamoxifen and anticipates further research for investigating their therapeutic potentials. CONCLUSION: Overall, we propose that targeting EZH2 or the molecules down the cascade might be helpful in reacquiring sensitivity to tamoxifen in breast cancer.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Técnicas de Silenciamento de Genes/métodos , Proteoma/metabolismo , Tamoxifeno/farmacologia , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteína Potenciadora do Homólogo 2 de Zeste/deficiência , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteômica/métodos , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Transfecção , Regulação para Cima/genéticaRESUMO
Sulfatase enzymes catalyze sulfate ester hydrolysis, thus deficiencies of sulfatases lead to the accumulation of biomolecules resulting in several disorders. One of the important sulfatases is estrone sulfatase that converts inactive estrone sulfate to active estradiol. Posttranslational modification of highly conserved cysteine residue leads to unique formylglycine in the active site of sulfatases being critical for its catalytic activity. The essential factor responsible for this modification of sulfatase is Sulfatase-Modifying Factor 1 (SUMF1). The role of estrone sulfatase is well evident in breast cancer progression. However, the function and regulation of SUMF1 in cancer are not studied. In the present study, for the first time, we have assessed the expression of SUMF1 in breast cancer and report the oncogenic behavior upon overexpression of SUMF1. Although increased expression or activity of SUMF1 is anticipated based on its function, the expression of SUMF1 was found to be reduced in breast cancer cells at both mRNA and protein levels. An estrogen receptor (ER) dependent expression of SUMF1 was observed and higher SUMF1 expression is associated with improved breast cancer patient survival in ER-positive cases. However, high SUMF1 expression leads to reduced median survival in ER-negative breast cancer patients. Putative binding sites for miRNAs-106b-5p, 128-3p and 148b-3p were found at 3'-UTR of SUMF1. Since self-assembled branched DNA (bDNA) structures have emerged as a highly efficient strategy for targeting multiple miRNAs simultaneously, we studied the alteration in SUMF1 expression using bDNA nanostructures with a complementary sequence to miRNAs. The findings suggest the involvement of co-regulators and repressors in miRNA-mediated SUMF1 expression in breast cancer cells and reveal the therapeutic potential of SUMF1 in endocrine-related malignancies.
RESUMO
The major antioxidant enzyme catalase is downregulated and the enzyme activity is compromised in various disease conditions such as malarial and cancer. Hence, the restoration and protection of catalase is a promising therapeutic strategy in disease management. In the present study, for the first time we have demonstrated the protective role of well-known anti-malarial drug Artemisinin (ART) on the time and temperature-induced degradation of bovine liver catalase (BLC) activity. The findings at different time intervals and at higher temperature showed the protective role of ART on BLC activity. Molecular docking studies suggested specific binding of ART on BLC through heme group interface which was further supported by cyclic voltammetry and dynamic light scattering study. The stabilization of BLC in presence of ART was mediated through forming a BLC-ART complex with reduced and shifted electrochemical peak and increased hydrodynamic diameter. ART substantially prevents the temperature-induced reduction in α-helical content with simultaneous increment in other secondary structures like antiparallel, parallel, ß-turn and random coils. Nevertheless, the protective role of ART was accepted from the enhanced thermal stability and increased Tm value of BLC in presence of ART at higher temperatures. Our results uncover the mechanism of interaction between ART with BLC and suggest the protective role of ART towards spatiotemporal alteration of BLC by preventing the structural and molecular change in BLC. Thus, the findings advocate ART as a potential therapeutic drug for diseases associated with reduced catalase activity.
Assuntos
Antioxidantes/química , Artemisininas/química , Catalase/química , Animais , Antioxidantes/metabolismo , Artemisininas/metabolismo , Catalase/isolamento & purificação , Catalase/metabolismo , Domínio Catalítico , Bovinos , Humanos , Ligação de Hidrogênio , Fígado/química , Fígado/enzimologia , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Estrogen-related receptor beta (ERRß) is downregulated in breast cancer cells and its overexpression in breast cancer patients is positively correlated with an improved prognosis and prolonged relapse-free survival. Here, we unravelled a molecular mechanism for ERRß downregulation in breast cancer. We found that ERRß is a key substrate of the SCF complex and that NEDDylation can activate the Cullin subunits of the SCF complex to target ERRß for degradation in breast cancer. Consistently, using in vitro and in vivo models, we demonstrated that MLN4924, a specific small molecule inhibitor of NEDDylation, can restore ERRß expression and culminate in a reduction in cell proliferation and migration of breast cancer cells. We also showed that increased ERRß expression promotes the upregulation of its target genes, including the tumour suppressors p21Cip1/Waf1 and E-cadherin, involved in cell proliferation and migration arrest at the gene promoter level. Interestingly, this tumour suppressive role of ERRß does not depend on the expression of ERα in breast cancer. Moreover, our data revealed that the ERRß recruits the transcription co-activator p300 to its targeted gene promoters to upregulate their expression. Collectively, our work revealed that restoration of ERRß expression using the NEDDylation inhibitor MLN4924 can be a novel and effective strategy for breast cancer treatment.
Assuntos
Neoplasias da Mama/metabolismo , Receptor beta de Estrogênio/metabolismo , Proteína NEDD8/antagonistas & inibidores , Neoplasias da Mama/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas Culina/metabolismo , Ciclopentanos/farmacologia , Progressão da Doença , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Recidiva Local de Neoplasia/genética , Pirimidinas/farmacologia , Receptores de Estrogênio/metabolismo , Ubiquitinas/metabolismoRESUMO
Understanding the mechanism underlying p53's ability to induce cell cycle arrest versus apoptosis is critical to treating human gliomas, 70% of which contain wild-type p53. Although N-terminal phosphorylation results in activation of p53, the role of N-terminal phosphorylation, particularly at serines 15 and 20, in p53's ability to induce cell cycle arrest versus apoptosis remains controversial. Here we test the hypothesis that phosphorylation of serine 15 and/or 20 is causally related to p53-mediated apoptosis in human gliomas. Introduction of p53 plasmids containing alanine mutations at serine 15 or/and serine 20 (which block phosphorylation) or aspartate mutations (which mimic phosphorylation) at the same sites, implicated simultaneous phosphorylation of both sites in the induction of apoptosis. When a double phosphorylation-mimicking adenoviral p53 vector (Ad-p53-15D20D) was compared with an unphosphorylated p53 vector (Ad-p53), treatment with Ad-p53 resulted in G1-arrest, whereas Ad-p53-15D20D induced apoptosis. These effects occurred independent of phosphorylation of other N-terminal serine (i.e., serines 6, 9, 33, 37, 46) indicating that phosphorylation of S15 and S20 is sufficient for inducing apoptosis. Mechanistically, Ad-p53 was capable only of increasing the expression of p21/CIP, whereas Ad-p53-15D20D increased the binding to and expression of the pro-apoptotic genes Fas, Puma and PIG3. However, Ad-p53-15D20D did not alter the expression of Noxa, Bid, IGFBP3, PERP and Killer/DR5, suggesting that phosphorylation of S15 and S20 resulted in the expression of specific pro-apoptotic gene. In conclusion, simultaneous phosphorylation of S15 and S20 is causally associated with apoptosis, resulting in increased expression of specific p53-responsive pro-apoptotic genes.
Assuntos
Apoptose/fisiologia , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Citometria de Fluxo , Expressão Gênica , Glioma/genética , Glioma/patologia , Humanos , Imunoprecipitação , Mutação , Fosforilação , Serina/genética , Serina/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Several pioneering work have established that apart from genetic alterations, epigenetic modifications contribute significantly in tumor progression. Remarkable role of EZH2 in cancer highlights the importance of identifying its targets. Although much emphasis has been placed in recent years in designing drugs and inhibitors targeting EZH2, less effort has been given in exploring its existing targets that will help in understanding the oncogenic role of EZH2 in turn which may provide a more stringent method of targeting EZH2. In the present study, we validated six direct targets of EZH2 that are GPNMB, PMEPA1, CoL5A1, VGLL4, POMT2 and SUMF1 associated with cancer related pathways. Upon EZH2 knockdown, more than two fold increase in the target gene expression was evident. CHIP-qPCR performed in both MCF-7 and MDA-MDA-231 confirmed the in-vivo binding of EZH2 on its identified target. Thirty invasive breast carcinoma cases with their adjacent normal tissues were included in the study. Immunohistochemistry in primary breast tumor tissue array showed tumor dependent expression of EZH2. Array of MERAV expression database revealed the strength of association of EZH2 with its target genes. Real time PCR performed with RNA extracted from breast tumor tissues further authenticated the existing negative correlation between EZH2 and its target genes. Pearson correlation coefficient & statistical significance computed using the matrix provided in the database strengthened the negative correlation between identified target genes and EZH2. KM plotter analysis showed improved relapse-free survival with increased expression of PMEPA1, POMT2, VGLL4 and SUMF1 in breast cancer patients indicating their therapeutic potential. While investigating the relevance of these target genes, different mutations of them were found in breast cancer patients. Seeking the clinical relevance of our study, following our recent publication that reports the role of EZH2 in nicotine-mediated breast cancer development and progression, we observed significant reduced expression of SUMF1 in breast cancer patient samples with smoking history in comparison to never-smoked patient samples.
Assuntos
Neoplasias da Mama/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Manosiltransferases/genética , Proteínas de Membrana/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Fatores de Transcrição/genética , Mama/patologia , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Manosiltransferases/biossíntese , Proteínas de Membrana/biossíntese , Recidiva Local de Neoplasia/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/biossíntese , Receptores de Estrogênio/metabolismo , Fumar/efeitos adversos , Fatores de Transcrição/biossínteseRESUMO
Recent studies show substantial growth-promoting properties of nicotine (NIC) in cancer, which is a combined outcome of genetic and epigenetic alterations. However, the role of epigenetic modifiers in response to NIC in breast cancer is less studied. In the present study, for the first time we have shown NIC-induced enhanced EZH2 expression. Six pairs of smoking-associated breast cancer patient tissues were analyzed. Samples from smoking breast cancer patients showed distinguished enhanced EZH2 expression in comparison to non-smoking ones. The upregulation in EZH2, which is due to NIC, was further confirmed in breast carcinoma cell lines using 10 µM NIC, 1 µM DZNepA, and EZH2si. The upregulation of EZH2 was concomitant with upregulation in Myc and α9-nAChR. The xenograft of breast cancer cells in BALB/c nude mice in the presence or absence of NIC showed significantly higher tumor uptake in the NIC injected group, which clearly demonstrates the effect of NIC in breast cancer progression. Interestingly, DZNepA considerably suppressed the NIC-mediated tumor growth. CHIP-qPCR assay confirmed the increased Myc enrichment on EZH2 promoter upon NIC treatment, thereby strengthening our findings that there exists an association between NIC, Myc, and EZH2. Overall, the present study identifies a strong association between NIC and EZH2 particularly in the progression of breast cancer in smokers through a novel axis involving nAChR and Myc. Moreover, the findings provide preliminary evidence suggesting potential of high level of EZH2 expression as a prognostic marker in smoking-associated breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Inibidores Enzimáticos/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Metiltransferases/antagonistas & inibidores , Nicotina/efeitos adversos , Fumantes , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Humanos , Antígeno Ki-67/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação para Cima/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer (BC) is one of the most common types of cancer in women worldwide. Several factors including genetic and environmental have been linked with susceptibility to development of BC. Her2 is a transmembrane protein with tyrosine kinase activity, overexpressed in several cancers including BC. Various studies in different populations have shown association of Her2 variants with susceptibility to BC, however these results were inconsistent, inconclusive and controversial. To obtain a common conclusive finding, we performed meta-analysis of 35 case-control studies reported earlier including 19, 220 cases and 22, 306 controls. We observed significant association of Her2 Ile655Val polymorphism with susceptibility to development of breast cancer (Overall allele Val vs Ile: OR = 1.130, 95% CI = 1.051-1.216, p = 0.001; Ile-Val vs Ile-Ile: OR = 1.100, 95% CI = 1.016-1.192, p = 0.019; Val-Val+Ile-Val vs Ile-Ile: OR = 1.127, 95% CI = 1.038-1.223, p = 0.004). Subgroup analysis indicated a significant association with susceptibility to breast cancer in African and Asian populations. However, such association was not observed in other ethnic groups. Our findings suggested that Her2 Ile655Val polymorphism is associated with breast cancer risk in overall, Asian and African populations, and can be used as diagnostic marker for BC.
Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único , Receptor ErbB-2/genética , Estudos de Casos e Controles , HumanosRESUMO
The transcriptional activity of estrogen receptor alpha (ER-alpha) is modified by regulatory action and interactions of coactivators and corepressors. Recent studies have shown that the metastasis-associated protein 1 (MTA1) represses estrogen receptor element (ERE)-driven transcription in breast cancer cells. With a yeast two-hybrid screen to clone MTA1-interacting proteins, we identified a known nuclear receptor coregulator (NRIF3) as an MTA1-binding protein. NRIF3 interacted with MTA1 both in vitro and in vivo. NRIF3 bound to the C-terminal region of MTA1, while MTA1 bound to the N-terminal region of NRIF3, containing one nuclear receptor interaction LXXLL motif. We showed that NRIF3 is an ER coactivator, hyperstimulated ER transactivation functions, and associated with the endogenous ER and its target gene promoter. MTA1 repressed NRIF3-mediated stimulation of ERE-driven transcription and interfered with NRIF3's association with the ER target gene chromatin. In addition, NRIF3 deregulation enhanced the responsiveness of breast cancer cells to estrogen-induced stimulation of growth and anchorage independence. Furthermore, we found that NRIF3 is an estrogen-inducible gene and activated ER associated with the ER response element in the NRIF3 gene promoter. These findings suggest that NRIF3, an MTA1-interacting protein, is an estrogen-inducible gene and that regulatory interactions between MTA1 and NRIF3 might be important in modulating the sensitivity of breast cancer cells to estrogen.
Assuntos
Histona Desacetilases/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas Repressoras/metabolismo , Motivos de Aminoácidos , Northern Blotting , Western Blotting , Divisão Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA Complementar/metabolismo , Receptor alfa de Estrogênio , Estrogênios/farmacologia , Deleção de Genes , Glutationa Transferase/metabolismo , Humanos , Modelos Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Transativadores , Transcrição Gênica , Ativação Transcricional , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Forkhead box protein A1 (FOXA1) is essential for the growth and differentiation of breast epithelium, and has a favorable outcome in breast cancer (BC). Elevated FOXA1 expression in BC also facilitates hormone responsiveness in estrogen receptor (ESR)-positive BC. However, the interaction between these two pathways is not fully understood. FOXA1 and GATA binding protein 3 (GATA3) along with ESR1 expression are responsible for maintaining a luminal phenotype, thus suggesting the existence of a strong association between them. The present study utilized the Oncomine™ microarray database to identify FOXA1:ESR1 and FOXA1:ESR1:GATA3 co-expression co-regulated genes. Oncomine™ analysis revealed 115 and 79 overlapping genes clusters in FOXA1:ESR1 and FOXA1:ESR1:GATA3 microarrays, respectively. Five ESR1 direct target genes [trefoil factor 1 (TFF1/PS2), B-cell lymphoma 2 (BCL2), seven in absentia homolog 2 (SIAH2), cellular myeloblastosis viral oncogene homolog (CMYB) and progesterone receptor (PGR)] were detected in the co-expression clusters. To further investigate the role of FOXA1 in ESR1-positive cells, MCF7 cells were transfected with a FOXA1 expression plasmid, and it was observed that the direct target genes of ESR1 (PS2, BCL2, SIAH2 and PGR) were significantly regulated upon transfection. Analysis of one of these target genes, PS2, revealed the presence of two FOXA1 binding sites in the vicinity of the estrogen response element (ERE), which was confirmed by binding assays. Under estrogen stimulation, FOXA1 protein was recruited to the FOXA1 site and could also bind to the ERE site (although in minimal amounts) in the PS2 promoter. Co-transfection of FOXA1/ESR1 expression plasmids demonstrated a significantly regulation of the target genes identified in the FOXA1/ESR1 multi-arrays compared with only FOXA1 transfection, which was suggestive of a synergistic effect of ESR1 and FOXA1 on the target genes. In summary, the present study identified novel FOXA1, ESR1 and GATA3 co-expressed genes that may be involved in breast tumorigenesis.
RESUMO
The estrogen receptor plays an important role in breast cancer progression. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1), also called modulator of nongenomic activity of estrogen receptor (MNAR), a novel coactivator of estrogen receptor, modulates estrogen receptor transactivation functions. The mechanisms by which PELP1 modulates estrogen receptor genomic functions is not known. Here, using biochemical and scanning confocal microscopic analysis, we have demonstrated nuclear localization and functional implications of PELP1. Subnuclear fractionation showed PELP1 association with chromatin and nuclear matrix fractions. Ligand stimulation promoted recruitment of PELP1 to 17beta-estradiol responsive promoters, its colocalization with acetylated H3, and increased PELP1-associated histone acetyltransferase enzymatic activity. Far Western analysis revealed that PELP1 interacts with histone 1 and 3, with more preference toward histone 1. Using deletion analysis, we have identified the PELP1 COOH-terminal region as the histone 1 binding site. The PELP1 mutant lacking histone 1-binding domain acts as a dominant-negative and blocks estrogen receptor alpha-mediated transcription. Chromatin immunoprecipitation analysis showed a cyclic association and dissociation of PELP1 with the promoter, with recruitment of histone 1 and PELP1 occurring in opposite phases. PELP1 overexpression increased the micrococcal nuclease sensitivity of estrogen response element-containing nucleosomes. Our results provide novel insights about the transcription regulation of PELP1 and suggest that PELP1 participates in chromatin remodeling activity via displacement of histone 1 in cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Histonas/metabolismo , Transativadores/fisiologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteínas Correpressoras , Citosol/metabolismo , Epitopos/biossíntese , Epitopos/genética , Estradiol/farmacologia , Ácido Glutâmico/metabolismo , Humanos , Matriz Nuclear/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Plasmídeos/genética , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Transativadores/genética , Transativadores/imunologia , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
Recent studies have shown that metastasis-associated protein-1 short form (MTA1s) - metastatic tumor antigen 1 short form sequesters estrogen receptor-alpha (ER-alpha) in the cytoplasm of breast cancer cells. Using a yeast two-hybrid screening to clone MTA1s-interacting proteins, we identified casein kinase I-gamma 2 (CKI-gamma2, a ubiquitously expressed cytoplasmic kinase) as an MTA1s-binding protein. We show that MTA1s interacts with CKI-gamma2 both in vitro and in vivo and colocalizes in the cytoplasm. In addition, we found that CKI-gamma2 can phosphorylate MTA1s, but not ER, in an antiestrogen-dependent manner and that estrogen stimulates CKI-gamma2 activity that could be effectively blocked by a specific inhibitor of CKI. CKI-gamma2 could further potentiate the ER corepressive function of MTA1s. Kinase dead CK1-gamma2 could not repress estrogen-induced ER transactivation functions. Results from mutagenesis studies suggest that substitution of the serine residue at 321 to alanine, which is a possible CKI-gamma2 phopshorylation site in MTA1s, results in a significant reduction in the ability of MTA1s to repress ER transactivation. These findings identified MTA1s as a target of CKI-gamma2, and provided new evidence to suggest that CKI-gamma2 phosphorylates and modulates the functions of MTA1s, and that these extranuclear effects of estrogen might have important implications in regulating the functions of MTA1s in human mammary epithelial and cancer cells.
Assuntos
Histona Desacetilases/metabolismo , Proteínas Quinases/fisiologia , Proteínas Repressoras/metabolismo , Animais , Mama/citologia , Mama/metabolismo , Neoplasias da Mama/patologia , Caseína Quinases , Linhagem Celular Tumoral , Citoplasma/metabolismo , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Receptor alfa de Estrogênio , Feminino , Glutationa Transferase/genética , Histona Desacetilases/química , Humanos , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Fosforilação , Ligação Proteica , Inibidores de Proteínas Quinases , Proteínas Quinases/química , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Receptores de Estrogênio/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Transativadores , Ativação Transcricional/efeitos dos fármacos , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Cytosolic inorganic pyrophosphatase plays an important role in the cellular metabolism by hydrolyzing inorganic pyrophosphate (PPi) formed as a by-product of various metabolic reactions. Inorganic pyrophosphatases are known to be associated with important functions related to the growth and development of various organisms. In humans, the expression of inorganic pyrophosphatase (PPA1) is deregulated in different types of cancer and is involved in the migration and invasion of gastric cancer cells and proliferation of ovarian cancer cells. However, the transcriptional regulation of the gene encoding PPA1 is poorly understood. To gain insights into PPA1 gene regulation, a 1217 bp of its 5'-flanking region was cloned and analyzed. The 5'-deletion analysis of the promoter revealed a 266 bp proximal promoter region exhibit most of the transcriptional activity and upon sequence analysis, three putative Sp1 binding sites were found to be present in this region. Binding of Sp1 to the PPA1 promoter was confirmed by Electrophoretic mobility shift assay (EMSA) and Chromatin immunoprecipitation (ChIP) assay. Importance of these binding sites was verified by site-directed mutagenesis and overexpression of Sp1 transactivates PPA1 promoter activity, upregulates protein expression and increases chromatin accessibility. p300 binds to the PPA1 promoter and stimulates Sp1 induced promoter activity. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor induces PPA1 promoter activity and protein expression and HAT activity of p300 was important in regulation of PPA1 expression. These results demonstrated that PPA1 is positively regulated by Sp1 and p300 coactivates Sp1 induced PPA1 promoter activity and histone acetylation/deacetylation may contribute to a local chromatin remodeling across the PPA1 promoter. Further, knockdown of PPA1 decreased colony formation and viability of MCF7 cells.