Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Inibidores da Aromatase/farmacologia , Inibidores da Aromatase/uso terapêutico , Receptores de Estrogênio/genética , Receptores Androgênicos/genética , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
OBJECTIVES: To compare the PathVysion fluorescence in-situ hybridisation assay with the INFORM HER2 Dual in-situ hybridisation assay on 104 invasive breast cancers with a broad spectrum of immunohistochemistry scores. METHODS: This case series involved consecutive patients diagnosed with invasive breast carcinoma with equivocal immunohistochemistry score and referred for further HER2 assessment from the departments of Surgery and/or Clinical Oncology of the two hospitals between January 2013 and February 2014. An additional 10 cases with negative HER2 immunohistochemistry and 11 cases with positive HER2 immunohistochemistry were further included. RESULTS: The results of both fluorescence in-situ hybridisation and dual in-situ hybridisation were available in 99 of 104 cases, respectively. Student'st test showed no statistically significant difference in the mean number of HER2 count, CEP17 copies, or HER2/CEP17 ratio between that obtained by fluorescence in-situ hybridisation and that obtained by dual in-situ hybridisation. Pearson's correlation of results for the two assays was strong for HER2/CEP17 signal ratio (R=0.963, P<0.001) and mean HER2 copies per nucleus (R=0.897, P<0.001). Overall agreement was 96.0% (95 out of 99 cases, ĸ0.882). Three of the four discordant cases were equivocal for either fluorescence in-situ hybridisation or dual in-situ hybridisation. The results of immunohistochemistry 0/1+ and 3+ cases showed 100% concordance between the two assays. The failure rate was 0.96% for fluorescence in-situ hybridisation and 3.85% for dual in-situ hybridisation. Cases that failed for fluorescence in-situ hybridisation were successful for dual in-situ hybridisation and vice versa. CONCLUSIONS: Our study showed that dual in-situ hybridisation is a reliable and useful option for HER2 testing in breast cancer.
Assuntos
Neoplasias da Mama/patologia , Hibridização in Situ Fluorescente/métodos , Hibridização In Situ/métodos , Receptor ErbB-2/genética , Neoplasias da Mama/genética , Feminino , Amplificação de Genes , Hong Kong , Humanos , Imuno-Histoquímica , Invasividade Neoplásica , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
BACKGROUND: The BRCA1 gene is an important breast-cancer susceptibility gene. Promoter polymorphisms can alter the binding affinity of transcription factors, changing transcriptional activity and may affect susceptibility to disease. METHODS AND RESULTS: Using direct sequencing of the BRCA1 promoter region, we identified four polymorphisms c.-2804T-->C (rs799908:T-->C), c.-2265C-->T (rs11655505:C-->T), c.-2004A-->G (rs799906:A-->G) and c.-1896(ACA)(1)-->(ACA)(2) (rs8176071:(ACA)(1)-->(ACA)(2)) present in Hong Kong Chinese. Each polymorphism was studied independently and in combination by functional assays. Although all four variants significantly altered promoter activity, the c.-2265T allele had stronger binding than the C allele, and the most common mutant haplotype, which contains the c.-2265T allele, increased promoter activity by 70%. Risk association first tested in Hong Kong Chinese women with breast cancer and age-matched controls and replicated in a large population-based study of Shanghai Chinese, together totalling >3000 participants, showed that carriers of the c.-2265T allele had a reduced risk for breast cancer (combined odd ratio (OR) = 0.80, 95% CI 0.69 to 0.93; p = 0.003) which was more evident among women aged >or=45 years at first diagnosis of breast cancer and without a family history of breast cancer (combined OR = 0.75, 95% CI 0.61 to 0.91; p = 0.004). The most common haplotype containing the c.-2265T allele also showed significant risk association for women aged >or=45 years without a family history of breast cancer (OR = 0.64, 95% CI 0.46 to 0.89; p = 0.008). CONCLUSION: This comprehensive study of BRCA1 promoter polymorphisms found four variants that altered promoter activity and with the most significant contribution from c.-2265C-->T, which could affect susceptibility to breast cancer in the Chinese population. Its significance in other populations remains to be investigated.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Polimorfismo Genético/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica , Povo Asiático/genética , Sítios de Ligação , Neoplasias da Mama/epidemiologia , Estudos de Casos e Controles , China/epidemiologia , Estudos de Coortes , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Predisposição Genética para Doença , Genótipo , Hong Kong/epidemiologia , Humanos , Fatores de Risco , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In the published version of this article, the images for cytoplasmic and nuclear FGF7 in MDA-MB-231 cells were duplicated and mistaken for total FGF7 in SKBR-3 and MDA-MB-231 cells.
RESUMO
INTRODUCTION: Germline mutations of BRCA1 and BRCA2 account for the majority of hereditary breast cancers, many of which are classified as variants of unknown significance (VUS). We report the identification of a novel BRCA2 variant (c.7806-9T > G) in a Chinese family with multiple breast cancers and document it as a pathogenic mutation. METHODS: The proband in this family was diagnosed with breast cancer at age 50 with a strong family history of breast cancer. DNA and RNA were extracted from the blood of the proband and her family, and was used for BRCA gene mutation/deletion screening and RNA splicing analysis. RESULTS: BRCA2 c.7806-9T > G was identified in the proband, which was suggestive of a variant. This change was also found in two sisters of the proband with a history of breast cancer, as well as from the proband's maternal gastric cancer. The only sibling free of breast cancer did not carry the BRCA2 variant, thus demonstrating that the mutation segregates with the clinical phenotype in this family. RNA analysis on the proband blood sample revealed three aberrant splicing variants: c.7806_7874del, c.7806_7976del, and c.7806-8_7806-1ins. The latter causes a frameshift and creates a truncated protein, whilst the other two splicing variants resulted in shorter forms of the protein. CONCLUSIONS: The identified BRCA2 c.7806-9T > G [Genbank: DQ889340] was found to be pathogenic, based on aberrant splicing events resulting in the formation of truncated protein products. Thus, better understanding and classification of BRCA variants as neutral or disease causing has important implications for genetic counseling so that appropriate management can be given.
Assuntos
Povo Asiático/genética , Neoplasias da Mama/genética , Genes BRCA2 , Mutação , Adulto , Sequência de Bases , Neoplasias da Mama/patologia , Feminino , Genes BRCA1 , Mutação em Linhagem Germinativa , Humanos , Pessoa de Meia-Idade , Linhagem , Mutação Puntual , Fatores de Risco , Análise de Sequência de DNARESUMO
BACKGROUND: hRad9 is a cell cycle checkpoint gene that is up-regulated in breast cancer. We have previously shown that the mRNA up-regulation correlated with tumor size and local recurrence. Immunohistochemical studies were made to better define the role of hRad9 in breast carcinogenesis. METHODS: Localisation of hRad9 protein were performed on paired tumor and normal breast tissues. Immunoblotting with and without dephosphorylation was used to define the protein isolated from breast cancer cells. RESULTS: Increased hRad9 protein was observed in breast cancer cells nucleus compared to non-tumor epithelium. This nuclear protein existed in hyperphosphorylated forms which may be those of the hRad9-hRad1-hHus1 complex. CONCLUSION: Finding of hyperphosphorylated forms of hRad9 in the nucleus of cancer cells is in keeping with its function in ameliorating DNA instability, whereby it inadvertently assists tumor growth.
Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Proteínas de Ciclo Celular/biossíntese , Neoplasias da Mama/patologia , Carcinoma/patologia , Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Reparo do DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Glândulas Mamárias Humanas/metabolismo , Especificidade de Órgãos , Fosforilação , Fosfotransferases/genética , Fosfotransferases/metabolismo , Isoformas de Proteínas/biossíntese , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
OBJECTIVES: To evaluate the prevalence of human epidermal growth factor receptor 2 (HER2) gene overexpression in breast cancer patients encountered in Hong Kong and the concordance of HER2 findings from primary immunohistochemistry assays and confirmatory in-situ hybridisation assays. DESIGN: Retrospective study. SETTING: Department of Clinical Oncology in a public hospital in Hong Kong. PATIENTS: All patient referrals between July 2006 and June 2007 with newly diagnosed invasive breast cancer (for prevalence evaluation), and all patients treated at our unit with confirmatory in-situ hybridisation tests performed within the study period (for concordance evaluation). RESULTS: There were 272 consecutive breast cancer patients eligible for prevalence evaluation. The distribution for immunohistochemistry staining in 249 cases for scores 0, 1+, 2+, and 3+ were 99 (40%), 40 (16%), 58 (23%), and 52 (21%) respectively. In the remaining 23 patients, four and 19 breast cancers were unscored and reported by immunohistochemistry to be HER2-positive and -negative, respectively. The overall HER2 overexpression rate (3+ or reported as positive) was 21%. HER2 overexpression was associated with grade 3 histology (P<0.001) and negative hormonal receptor status (P<0.001). However, it was not associated with age (P=0.525), T-classification (P=0.740), N-classification (P=0.691), nor group stages (P=0.433). Of the 37 patients with confirmatory in-situ hybridisation tests performed, 10 (71%) of 14 with immunohistochemistry staining of 3+ and 1 (4%) of 23 with immunohistochemistry staining of 2+ were found to have HER2 gene amplification. CONCLUSIONS: More than 25% of HER2 overexpression identified by immunohistochemistry assays in this Hong Kong cohort could not be verified by confirmatory in-situ hybridisation assays. Compliance with the latest guidelines for HER2 testing should improve the future accuracy and concordance.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal/genética , Carcinoma Lobular/genética , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Hibridização In Situ/métodos , Receptor ErbB-2/genética , Adulto , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/patologia , Carcinoma Ductal/epidemiologia , Carcinoma Ductal/patologia , Carcinoma Lobular/epidemiologia , Carcinoma Lobular/patologia , Estudos Transversais , Feminino , Amplificação de Genes/genética , Regulação Neoplásica da Expressão Gênica/genética , Hong Kong , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Estudos RetrospectivosRESUMO
1. A genetic risk-association study involving more than 1200 subjects showed individuals homozygous for L-SIGN tandem repeats are less susceptible to SARS infection. 2. This was supported by in vitro binding studies that demonstrated homozygous L-SIGN, compared to heterozygous, had higher binding capacity for SARS coronavirus (SARS-CoV), with higher proteasome-dependent viral degradation. In contrast, homozygous L-SIGN demonstrated lower binding capacity for HIV1-gp120.3. Genetic-association studies for single nucleotide polymorphisms of the inflammatory response genes, namely TNF-alpha, INF-alpha, INF-beta, INF-gamma, IL1-alpha, IL1-beta, IL-4, IL-6 and iNOS, failed to show a significant association with SARS clinical outcomes or susceptibility.
Assuntos
Moléculas de Adesão Celular/genética , Predisposição Genética para Doença , Lectinas Tipo C/genética , Polimorfismo Genético , Receptores de Superfície Celular/genética , Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Adulto , Alelos , Análise de Variância , Estudos de Casos e Controles , Doenças Transmissíveis/genética , Doenças Transmissíveis/fisiopatologia , Intervalos de Confiança , Citocinas/genética , Citocinas/metabolismo , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Probabilidade , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/fisiopatologia , Sequências de Repetição em Tandem , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: All-trans retinoic acid (ATRA) is a natural vitamin A derivative that has a profound effect on the regulation of cell growth, differentiation and death. AIM: To investigate the tissue dynamic and cellular invasion effects of ATRA in choriocarcinoma (CCA), an aggressive trophoblastic tumour, by using a three-dimensional organotypic culture model system and cell invasion assay, respectively. METHODS: An organotypic culture model of two CCA cell lines, JAR and JEG, was established. The effects of 1 microM ATRA on proliferation, differentiation and apoptosis on this CCA model were assessed by morphological assessment of the mitotic and apoptotic figures as well as by Ki-67 and caspase-related M30 cytoDeath antibody immunohistochemistry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay. The effect of ATRA on p53 and its regulated protein product, WAF1/Cip1, was also evaluated with DO7 and p21(WAF1) antibodies, respectively. Moreover, the effect of ATRA on cellular (CCA) invasion was also investigated with Cell Invasion Kit on the JEG cell line. RESULTS: ATRA was found to induce marked apoptosis in organotypic cultures of both cell lines, as evidenced by increased M30-positive cells (p<0.0001) and increased TUNEL-positive cells (p<0.0001) in treated cultures; to decrease proliferation, as evidenced by decreased Ki-67-positive cells (p<0.0001); and to decrease p53-DO7 immunoreactivity (p<0.0001) and increase p21(WAF1) (p<0.0001) immunoreactivity. 1.5 microM ATRA was found to effectively inhibit JEG cell invasion in the cell invasion assay. CONCLUSION: ATRA treatment was found to inhibit invasion and proliferation and enhance apoptosis, probably by the activation of caspases and induction of differentiation. ATRA and synthetic retinoids may be alternative agents for the treatment of CCA.
Assuntos
Antineoplásicos/farmacologia , Coriocarcinoma/patologia , Tretinoína/farmacologia , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Coriocarcinoma/metabolismo , Relação Dose-Resposta a Droga , Humanos , Marcação In Situ das Extremidades Cortadas/métodos , Proteína Supressora de Tumor p53/metabolismoRESUMO
FOXM1 has been implicated in taxane resistance, but the molecular mechanism involved remains elusive. In here, we show that FOXM1 depletion can sensitize breast cancer cells and mouse embryonic fibroblasts into entering paclitaxel-induced senescence, with the loss of clonogenic ability, and the induction of senescence-associated ß-galactosidase activity and flat cell morphology. We also demonstrate that FOXM1 regulates the expression of the microtubulin-associated kinesin KIF20A at the transcriptional level directly through a Forkhead response element (FHRE) in its promoter. Similar to FOXM1, KIF20A expression is downregulated by paclitaxel in the sensitive MCF-7 breast cancer cells and deregulated in the paclitaxel-resistant MCF-7Tax(R) cells. KIF20A depletion also renders MCF-7 and MCF-7Tax(R) cells more sensitive to paclitaxel-induced cellular senescence. Crucially, resembling paclitaxel treatment, silencing of FOXM1 and KIF20A similarly promotes abnormal mitotic spindle morphology and chromosome alignment, which have been shown to induce mitotic catastrophe-dependent senescence. The physiological relevance of the regulation of KIF20A by FOXM1 is further highlighted by the strong and significant correlations between FOXM1 and KIF20A expression in breast cancer patient samples. Statistical analysis reveals that both FOXM1 and KIF20A protein and mRNA expression significantly associates with poor survival, consistent with a role of FOXM1 and KIF20A in paclitaxel action and resistance. Collectively, our findings suggest that paclitaxel targets the FOXM1-KIF20A axis to drive abnormal mitotic spindle formation and mitotic catastrophe and that deregulated FOXM1 and KIF20A expression may confer paclitaxel resistance. These findings provide insights into the underlying mechanisms of paclitaxel resistance and have implications for the development of predictive biomarkers and novel chemotherapeutic strategies for paclitaxel resistance.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição Forkhead/fisiologia , Cinesinas/genética , Mitose , Paclitaxel/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Feminino , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Cinesinas/metabolismo , Camundongos , Mitose/efeitos dos fármacos , Regiões Promotoras Genéticas , Fuso Acromático/fisiologia , Células Tumorais CultivadasRESUMO
The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Cisteína Endopeptidases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Epirubicina/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cicloeximida/farmacologia , Dano ao DNA/genética , Reparo do DNA/genética , Enzimas Desubiquitinantes , Feminino , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Inibidores da Síntese de Proteínas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Ubiquitinação/genéticaRESUMO
The forkhead box M1 (FOXM1) transcription factor has a central role in genotoxic agent response in breast cancer. FOXM1 is regulated at the post-translational level upon DNA damage, but the key mechanism involved remained enigmatic. RNF168 is a ubiquitination E3-ligase involved in DNA damage response. Western blot and gene promoter-reporter analyses showed that the expression level and transcriptional activity of FOXM1 reduced upon RNF168 overexpression and increased with RNF168 depletion by siRNA, suggesting that RNF168 negatively regulates FOXM1 expression. Co-immunoprecipitation studies in MCF-7 cells revealed that RNF168 interacted with FOXM1 and that upon epirubicin treatment FOXM1 downregulation was associated with an increase in RNF168 binding and conjugation to the protein degradation-associated K48-linked polyubiquitin chains. Consistently, RNF168 overexpression resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide. Conversely, RNF168, knockdown significantly enhanced the half-life of FOXM1 in both absence and presence of epirubicin. Using a SUMOylation-defective FOXM1-5x(K>R) mutant, we demonstrated that SUMOylation is required for the recruitment of RNF168 to mediate FOXM1 degradation. In addition, clonogenic assays also showed that RNF168 mediates epirubicin action through targeting FOXM1, as RNF168 could synergise with epirubicin to repress clonal formation in wild-type but not in FOXM1-deficient mouse embryo fibroblasts (MEFs). The physiological relevance of RNF168-mediated FOXM1 repression is further emphasized by the significant inverse correlation between FOXM1 and RNF168 expression in breast cancer patient samples. Moreover, we also obtained evidence that RNF8 recruits RNF168 to FOXM1 upon epirubicin treatment and cooperates with RNF168 to catalyse FOXM1 ubiquitination and degradation. Collectively, these data suggest that RNF168 cooperates with RNF8 to mediate the ubiquitination and degradation of SUMOylated FOXM1 in breast cancer genotoxic response.
RESUMO
BRCA1 mutation or depletion correlates with basal-like phenotype and poor prognosis in breast cancer but the underlying reason remains elusive. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with downregulation of the expression of the pleiotropic tumour suppressor FOXO3. Knockdown of BRCA1 by small interfering RNA (siRNA) resulted in downregulation of FOXO3 expression in the BRCA1-competent MCF-7, whereas expression of BRCA1 restored FOXO3 expression in BRCA1-defective HCC70 and MDA-MB-468 cells, suggesting a role of BRCA1 in the control of FOXO3 expression. Treatment of HCC70 and MDA-MB-468 cells with either the DNA methylation inhibitor 5-aza-2'-deoxycitydine, the N-methyltransferase enhancer of zeste homologue 2 (EZH2) inhibitor GSK126 or EZH2 siRNA induced FOXO3 mRNA and protein expression, but had no effect on the BRCA1-competent MCF-7 cells. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNMT1/3a/b and histone H3 lysine 27 trimethylation (H3K27me3) are recruited to the endogenous FOXO3 promoter, further advocating that these proteins interact to modulate FOXO3 methylation and expression. In addition, ChIP results also revealed that BRCA1 depletion promoted the recruitment of the DNA methyltransferases DNMT1/3a/3b and the enrichment of the EZH2-mediated transcriptional repressive epigenetic marks H3K27me3 on the FOXO3 promoter. Methylated DNA immunoprecipitation assays also confirmed increased CpG methylation of the FOXO3 gene on BRCA1 depletion. Analysis of the global gene methylation profiles of a cohort of 33 familial breast tumours revealed that FOXO3 promoter methylation is significantly associated with BRCA1 mutation. Furthermore, immunohistochemistry further suggested that FOXO3 expression was significantly associated with BRCA1 status in EZH2-positive breast cancer. Consistently, high FOXO3 and EZH2 mRNA levels were significantly associated with good and poor prognosis in breast cancer, respectively. Together, these data suggest that BRCA1 can prevent and reverse FOXO3 suppression via inhibiting EZH2 and, consequently, its ability to recruit the transcriptional repressive H3K27me3 histone marks and the DNA methylases DNMT1/3a/3b, to induce DNA methylation and gene silencing on the FOXO3 promoter.
RESUMO
Inherited mutations in the BRCA1 gene confer increased susceptibility to breast and ovarian cancer. Its role in sporadic carcinogenesis is not well defined. Somatic mutations in breast cancers have not been reported and to date there are only three reports of somatic mutations in sporadic ovarian cancers. To investigate the contribution of BRCA1 mutations to sporadic breast and ovarian cancer in the Chinese population, we analysed 62 samples from Chinese women using the protein truncation test. There were 40 cases of breast cancer under age 50 and 22 cases of ovarian cancer, all unselected for family history. There was no age selection for the ovarian cancers. We found two somatic BRCA1 mutations in exon 11, one in a breast cancer and the other in an ovarian cancer, both of which result in truncated proteins. Our results indicate that somatic BRCA1 mutations, like somatic mutations in the BRCA2 gene, though very rare, can be found in both breast and ovarian cancers and support a tumor suppressor function for BRCA1 in sporadic tumors.
Assuntos
Povo Asiático/genética , Proteína BRCA1/genética , Neoplasias da Mama/genética , Mutação , Neoplasias Ovarianas/genética , Adulto , China , Feminino , Humanos , Pessoa de Meia-IdadeAssuntos
Antígenos HLA/genética , Fenômenos Imunogenéticos , Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome Respiratória Aguda Grave/epidemiologia , Síndrome Respiratória Aguda Grave/imunologia , Adulto JovemAssuntos
Moléculas de Adesão Celular/genética , Lectinas Tipo C/genética , Receptores de Superfície Celular/genética , Síndrome Respiratória Aguda Grave/fisiopatologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Estudos de Casos e Controles , Predisposição Genética para Doença , Humanos , L-Lactato Desidrogenase/metabolismo , Polimorfismo de Nucleotídeo Único , Prognóstico , Regiões Promotoras Genéticas/genética , Estudos Retrospectivos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/isolamento & purificação , Síndrome Respiratória Aguda Grave/virologia , Índice de Gravidade de DoençaRESUMO
FOXA1 expression correlates with the breast cancer luminal subtype and patient survival. RNA and protein analysis of a panel of breast cancer cell lines revealed that BRCA1 deficiency is associated with the downregulation of FOXA1 expression. Knockdown of BRCA1 resulted in the downregulation of FOXA1 expression and enhancement of FOXA1 promoter methylation in MCF-7 breast cancer cells, whereas the reconstitution of BRCA1 in Brca1-deficent mouse mammary epithelial cells (MMECs) promoted Foxa1 expression and methylation. These data suggest that BRCA1 suppresses FOXA1 hypermethylation and silencing. Consistently, the treatment of MMECs with the DNA methylation inhibitor 5-aza-2'-deoxycitydine induced Foxa1 mRNA expression. Furthermore, treatment with GSK126, an inhibitor of EZH2 methyltransferase activity, induced FOXA1 expression in BRCA1-deficient but not in BRCA1-reconstituted MMECs. Likewise, the depletion of EZH2 by small interfering RNA enhanced FOXA1 mRNA expression. Chromatin immunoprecipitation (ChIP) analysis demonstrated that BRCA1, EZH2, DNA methyltransferases (DNMT)1/3a/3b and H3K27me3 are recruited to the endogenous FOXA1 promoter, further supporting the hypothesis that these proteins interact to modulate FOXA1 methylation and repression. Further co-immunoprecipitation and ChIP analysis showed that both BRCA1 and DNMT3b form complexes with EZH2 but not with each other, consistent with the notion that BRCA1 binds to EZH2 and negatively regulates its methyltransferase activity. We also found that EZH2 promotes and BRCA1 impairs the deposit of the gene silencing histone mark H3K27me3 on the FOXA1 promoter. These associations were validated in a familial breast cancer patient cohort. Integrated analysis of the global gene methylation and expression profiles of a set of 33 familial breast tumours revealed that FOXA1 promoter methylation is inversely correlated with the transcriptional expression of FOXA1 and that BRCA1 mutation breast cancer is significantly associated with FOXA1 methylation and downregulation of FOXA1 expression, providing physiological evidence to our findings that FOXA1 expression is regulated by methylation and chromatin silencing and that BRCA1 maintains FOXA1 expression through suppressing FOXA1 gene methylation in breast cancer.
Assuntos
Neoplasias da Mama/genética , Cromatina/genética , Metilação de DNA , Inativação Gênica , Genes BRCA1 , Fator 3-alfa Nuclear de Hepatócito/genética , Regiões Promotoras Genéticas , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Complexo Repressor Polycomb 2/metabolismo , DNA Metiltransferase 3BRESUMO
The forkhead transcription factor FOXK2 has recently been implicated in cancer cell proliferation and survival, but a role in cancer chemotherapeutic drug resistance has hitherto not been explored. Here we demonstrate that FOXK2 has a central role in mediating the cytotoxic drug response in breast cancer. Clonogenic and cell viability assays showed that enhanced FOXK2 expression sensitizes MCF-7 breast cancer cells to paclitaxel or epirubicin treatment, whereas FOXK2 depletion by small interfering RNAs (siRNAs) confers drug resistance. Our data also showed that the activation of the tumour suppressor FOXO3a by paclitaxel and epirubicin is mediated through the induction of FOXK2, as depletion of FOXK2 by siRNA limits the induction of FOXO3a by these drugs in MCF-7 cells. Chromatin immunoprecipitation (ChIP) analysis showed that in response to drug treatment, FOXK2 accumulates and binds to the proximal FOXO3a promoter region in MCF-7 cells. Furthermore, we also uncovered that FOXK2 is deregulated and, therefore, can express at high levels in the nucleus of both the paclitaxel and epirubicin drug-resistant MCF-7 cells. Our results showed that ectopically overexpressed FOXK2 accumulates in the nuclei of drug-resistant MCF-7 cells but failed to be recruited to target genes, including FOXO3a. Crucially, we found that FOXO3a is required for the anti-proliferative and epirubicin-induced cytotoxic function of FOXK2 in MCF-7 cells by sulphorhodamine and clonogenic assays. The physiological importance of the regulation of FOXO3a by FOXK2 is further confirmed by the significant correlations between FOXO3a and FOXK2 expression in breast carcinoma patient samples. Further survival analysis also reveals that high nuclear FOXK2 expression significantly associates with poorer clinical outcome, particularly in patients who have received conventional chemotherapy, consistent with our finding that FOXK2 is deregulated in drug-resistant cells. In summary, our results suggest that paclitaxel and epirubicin target the FOXK2 to modulate their cytotoxicity and deregulated FOXK2 confers drug resistance.
RESUMO
Germline mutations in the BRCA1 and BRCA2 genes predispose women to breast and ovarian cancer. An incidence of 5% and 3.3% respectively has been reported of BRCA1 and BRCA2 mutations in women with ovarian cancer unselected for family history. The contribution of BRCA1 and BRCA2 mutations to ovarian cancer in Chinese women is unknown. A total of 60 samples of ovarian cancer diagnosed in Chinese unselected for age or family history were analyzed for BRCA mutations using the protein truncation test. The entire coding exon of BRCA1 of 53 cases and that of exon 11 of BRCA2 of 43 cases were successfully screened. Six germline (11.3%) mutations (633C>T, 1080delT, 1129delA, 2371-2372delTG, 3976-3979delGTGA, and IVS 22+7 A>G) were detected in BRCA1. One germline mutation (3337C>T) (2.1%) was detected in BRCA2. None of these seven cases were associated with strong family history of breast and/or ovarian cancer. Five out of our six BRCA1 mutations and the one BRCA2 mutation identified are novel. Our 11.3% incidence of BRCA1 mutations in ovarian cancer found amongst Chinese with insignificant family history is apparently higher than that previously reported in other populations. It suggests that BRCA1 mutation may play a significant role in the development of sporadic ovarian cancer in Chinese women.
Assuntos
Proteína BRCA1/genética , Mutação em Linhagem Germinativa/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína BRCA2 , China , Análise Mutacional de DNA , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
HAM56 (human alveolar macrophage) is a monoclonal antibody that reacts with macrophages and endothelial cells. There has been controversy as to its usefulness in differentiating adenocarcinomas of ovarian or gastrointestinal origin. The aim of this study is to test the specificity of HAM56 in identifying the origin of metastatic adenocarcinomas. Ninety-two adenocarcinomas of known primary site, metastatic to omentum or lymph nodes were used. Immunostaining for HAM56 was performed on formalin-fixed, paraffin-embedded tissue after antigen retrieval with microwave pretreatment. Positive immunostaining was shown as membrane or cytoplasmic staining with luminal accentuation. Nonspecific staining in necrotic debris or mucin was excluded. Immunoreactivity for HAM56 was found in 37 metastatic adenocarcinomas; 22 of 31 cases (71%) of ovarian origin, 7 of 33 (21%) of colonic origin, 4 of 16 (25%) of gastric origin, 3 of 6 (50%) of biliary origin; and 1 of uterine origin (100%). Negative staining was found in adenocarcinomas from the pancreas (n = 2), cervix (n = 2), and fallopian tube (n = 1). These findings suggest that HAM56 reacts with adenocarcinomas arising from several origins though with a higher frequency in ovarian tumors. It is thus not specific for ovarian carcinomas and is, therefore, not a useful tool to help distinguish adenocarcinomas of unknown origin.