RESUMO
Our previous works have indicated that extracellular ATP is an important prometastasis factor. However, the molecular mechanism involved needs to be further studied. We demonstrated that extracellular ATP treatment could upregulate the expression of connective tissue growth factor (CTGF) in both triple-negative breast cancer (TNBC) cells and endothelial cells (ECs). Extracellular ATP stimulated the migration of TNBC cells and ECs, and angiogenesis of ECs via the P2Y2--YAP-CTGF axis. Furthermore, we demonstrated that adenosine triphosphate (ATP) stimulated TNBC cell adhesion to ECs and transmigration through the EC layer via CTGF by upregulation of integrin ß1 on TNBC cells and VCAM-1 on ECs. Both apyrase (ATP-diphosphohydrolase) and CTGF shRNA treatments could inhibit the metastasis of inoculated tumors to lung and liver in a mouse model, and these treated tumors had fewer blood vessels. Collectively, our data indicated that extracellular ATP promotes tumor angiogenesis and the interactions between TNBC cells and ECs through upregulation of CTGF, thereby stimulating TNBC metastasis. The pleiotropic effects of ATP in angiogenesis and cell adhesion suggest that extracellular ATP or CTGF could be an effective target for TNBC therapy.
Assuntos
Trifosfato de Adenosina , Fator de Crescimento do Tecido Conjuntivo , Neoplasias de Mama Triplo Negativas , Trifosfato de Adenosina/metabolismo , Animais , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Endoteliais/metabolismo , Humanos , Camundongos , Neovascularização Patológica/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Regulação para CimaRESUMO
PURPOSE: This study aimed to investigate whether genetic polymorphisms in TGFB1 contribute to breast cancer (BC) susceptibility, and explore the mechanism of action. METHODS: A total of 7 tagging SNPs (tSNPs) were genotyped in 1161 BC cases and 1337 age-matched controls among Chinese Han population. Bioinformatics analysis was used to predict functional SNP closely linked to tSNPs. Luciferase gene reporter assay was performed to determine the effect of genetic variants on promoter activity. DNA pull-down assay and mass spectrometry were used to identify the differentially binding proteins to genetic variants. RESULTS: Genotyping analysis showed that rs1800469 (C>T) in the 5' regulatory region of TGFB1 was associated with reduced BC risk. Bioinformatics analysis predicted that rs11466313 (-2389_-2391 Del/AGG) in the 5' regulatory region of TGFB1, was closely linked to tSNP rs1800469 and could be functional. The genotyping of rs11466313 by PCR-SSCP showed that rs11466313 also conferred decreased BC risk. Luciferase assays demonstrated that rs11466313 minor allele reduced over ninefold of promoter activity compared with its major allele (p < 0.001). DNA pull-down assay and mass spectrometry revealed that rs11466313 minor allele lost the binding ability with FAM98B and HSP90B. Knocking down FAM98B but not HSP90B, the enhanced promoter activity driven by TGFB1 rs11466313 major allele was attenuated. CONCLUSIONS: This study elucidates the impact of functional polymorphism rs11466313 in the regulatory region of TGFB1 on breast cancer susceptibility and gene expression, and could be helpful for future research to determine the value of this TGFB1 variant in the clinical setting.
Assuntos
Neoplasias da Mama , Alelos , Neoplasias da Mama/genética , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Haplótipos , Humanos , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fator de Crescimento Transformador beta1/genéticaRESUMO
Extracellular ATP has been shown to play an important role in invasion and the epithelial-mesenchymal transition (EMT) process in breast cancer; however, the mechanism is unclear. Here, by using a cDNA microarray, we demonstrated that extracellular ATP could stimulate hypoxia-inducible factor (HIF) signaling and upregulate hypoxia-inducible factor 1/2α (HIF-1/2α) expression. After knocking down HIF-1/2α using siRNA, we found that ATP-driven invasion and EMT were significantly attenuated via HIF2A-siRNA in breast cancer cells. By using ChIP assays, we revealed that the biological function of extracellular ATP in invasion and EMT process depended on HIF-2α direct targets, among which lysyl oxidase-like 2 (LOXL2) and matrix metalloproteinase-9 (MMP-9) mediated ATP-driven invasion, and E-cadherin and Snail mediated ATP-driven EMT, respectively. In addition, using silver staining and mass spectrometry, we found that phosphoglycerate kinase 1 (PGK1) could interact with HIF-2α and mediate ATP-driven HIF-2α upregulation. Furthermore, we demonstrated that expressions of HIF-2α and its target proteins could be regulated via ATP by AKT-PGK1 pathway. Using a Balb/c mice model, we illustrated the function of HIF-2α in promoting tumor growth and metastasis in vivo. Moreover, by exploring online databases, we found that molecules involved in ATP-HIF-2α signaling were highly expressed in human breast carcinoma tissues and were associated with poor prognosis. Altogether, these findings suggest that extracellular ATP could promote breast carcinoma invasion and EMT via HIF-2α signaling, which may be a potential target for future anti-metastasis therapy.
Assuntos
Trifosfato de Adenosina/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/fisiologia , Hipóxia/patologia , Invasividade Neoplásica/patologia , Aminoácido Oxirredutases/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Células MCF-7 , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologiaRESUMO
Extracellular adenosine 5'-triphosphate (ATP), secreted by living cancer cells or released by necrotic tumor cells, plays an important role in tumor invasion and metastasis. Our previous study demonstrated that ATP treatment in vitro could promote invasion in human prostate cancer cells via P2Y2, a preferred receptor for ATP, by enhancing EMT process. However, the pro-invasion mechanisms of ATP and P2Y2 are still poorly studied in breast cancer. In this study, we found that P2Y2 was highly expressed in breast cancer cells and associated with human breast cancer metastasis. ATP could promote the in vitro invasion of breast cancer cells and enhance the expression of ß-catenin as well as its downstream target genes CD44, c-Myc and cyclin D1, while P2Y2 knockdown attenuated above ATP-driven events in vitro and in vivo. Furthermore, iCRT14, a ß-catenin/TCF complex inhibitor, could also suppress ATP-driven migration and invasion in vitro. These results suggest that ATP promoted breast cancer cell invasion via P2Y2-ß-catenin axis. Thus blockade of the ATP-P2Y2-ß-catenin axis could suppress the invasive and metastatic potential of breast cancer cells and may serve as potential targets for therapeutic interventions of breast cancer.
Assuntos
Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/patologia , Invasividade Neoplásica/patologia , Receptores Purinérgicos P2Y2/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Feminino , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that there appeared to be two instances of overlapping data panels comparing between the cell migration and invasion assay data shown in Figs. 4 and 6 on p. 143 and 145, respectively, such that data which were intended to represent the results from differently performed experiments had apparently been derived from the same original sources. In addition, the authors themselves realized that incorrect western blotting data for Snail protein in Fig. 10A on p. 147 had been included in the figure. The authors were able to reexamine their original data files, and realized that the affected data panels in these figures had inadvertently been incorporated into them incorrectly. The revised versions of Figs. 4, 6, and 10, featuring the correct data for the 'NC / Control' panels in Fig. 4B and C and the 'siRNA2 / ATP 12 h' panels in Fig. 4A and B, a replacement data panel for the 'siRNA1 / Control' experiment in Fig. 6, and the correct western blotting data for Snail protein in Fig. 10A (together with a revised histogram for the MCF7 cell line relating to Fig. 10A) are shown on the next three pages. The authors wish to emphasize that the errors made in compiling these figures did not affect the overall conclusions reported in the paper, and they are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish this corrigendum. All the authors agree to the publication of this corrigendum, and also apologize to the readership for any inconvenience caused. [Oncology Reports 39: 138150, 2018; DOI: 10.3892/or.2017.6081].
RESUMO
Centrosome aberrations have been suggested to cause chromosomal instability and aneuploidy, and eventually promote cancer development. The Centrobin and Nek2 proteins interact with each other and both are involved in centrosome duplication and chromosome segregation. This study aimed to investigate whether genetic polymorphisms in these two genes may affect breast cancer susceptibility in Chinese Han population using a haplotype-based analysis. Five single nucleotide polymorphisms (SNPs) in centrobin and four SNPs in Nek2 were genotyped in 1,215 cases of infiltrating ductal breast cancer and 1,215 age-matched cancer-free controls from Chinese Han population. The results showed that CATCG haplotype of centrobin was strongly associated with decreased breast cancer risk (adjusted OR = 0.14, 95 % CI = 0.09-0.22), which was mainly driven by the C allele of SNP rs11650083 (A>C, located in exon 12, resulting in Pro578Gln). None of the individual SNPs in Nek2 was associated with breast cancer risk. However, haplotype GTAT of Nek2 was associated with increased risk of breast cancer (adjusted OR = 1.56, 95 % CI = 1.18-2.06) and its risk was significantly elevated among women with both family history of cancer and a longer menarche-first full-term pregnancy (FFTP) interval (>11 years) (adjusted OR = 5.31, 95 % CI = 1.97-14.32). Furthermore, women harboring both at-risk haplotype GTAT of Nek2 and protective haplotype CATCG of centrobin were linked with decreased breast cancer risk, suggesting that the association between genetic variants of Nek2 and increased breast cancer risk was modified by genetic variants of centrobin. Our results indicate that genetic polymorphisms of centrobin and Nek2 are related to breast cancer susceptibility in Chinese Han women.
Assuntos
Neoplasias da Mama , Proteínas de Ciclo Celular/genética , Estudos de Associação Genética , Proteínas Serina-Treonina Quinases/genética , Adulto , Alelos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , China , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Pessoa de Meia-Idade , Quinases Relacionadas a NIMA , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
Centrosome defects can result in aneuploidy and genomic instability, and have important implications for breast cancer development. The Aurora-A and BRCA1 proteins interact and both are strongly involved in centrosome regulation. Genetic variants in these two genes may have an effect on breast cancer development. Here, we report a comprehensive single nucleotide polymorphism (SNP) and haplotype-tagging association study on these two genes in 1334 breast cancer cases and 1568 unaffected controls among the Chinese Han population. Apart from a missense SNP, rs2273535 (Phe31Ile), and a probable risk SNP, rs2064863, six htSNPs were analysed in three high-LD blocks of AURKA spanning from 10 kb upstream to 2 kb downstream of AURKA. For BRCA1, six htSNPs were analysed in a large high-LD region covering 98 kb (10 kb was extended to each end of BRCA1). The results showed that four SNPs in AURKA (data in recessive model, rs2273535: OR = 2.19, 95% CI = 1.03-4.66, p = 0.0422; rs2298016: OR = 0.38, 95% CI = 0.18-0.82, p = 0.0141; rs6024836: OR = 1.54, 95% CI = 1.18-2.00, p = 0.0014; rs10485805: OR = 0.68, 95% CI = 0.47-0.98, p = 0.0380) and one SNP in BRCA1 (rs3737559, dominant model OR = 1.35, 95% CI = 1.11-1.64, p = 0.0030) were associated with breast cancer susceptibility. After correction for multiple comparisons (FDR = 0.05), only rs6024836 and rs3737559 remained significant. Two haplotypes (CC of block 2, OR = 20.74, 95% CI = 4.35-98.88, p = 0.0001; GG of block 3, OR = 1.32, 95% CI = 1.12-1.56, p = 0.0010) and one diplotype (AG-GG of block 3, OR = 1.63, 95% CI = 1.18-2.26, p = 0.0031) within AURKA showed strong associations with breast cancer risk. One haplotype of BRCA1 (CTGTTG, OR = 1.30, 95% CI = 1.06-1.59, p = 0.0118) was also associated with breast cancer risk. However, women harbouring both at-risk genotypes of Aurora-A and BRCA1 were at a slightly increased risk compared with those harbouring either at-risk variant alone. Common genetic variants in the AURKA and BRCA1 genes may contribute to breast cancer development.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Povo Asiático , Aurora Quinase A , Aurora Quinases , Neoplasias da Mama/etnologia , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/etnologia , Carcinoma Ductal de Mama/patologia , Estudos de Casos e Controles , China/etnologia , Feminino , HumanosRESUMO
We have previously demonstrated that extracellular adenosine 5'-triphosphate (ATP) promotes breast cancer cell chemoresistance. However, the underlying mechanism remains unclear. Using a cDNA microarray, we demonstrated that extracellular ATP can stimulate hypoxia-inducible factor (HIF) signaling. In this study, we report that hypoxia-inducible factor 1α (HIF-1α) was upregulated after ATP treatment and mediated the ATP-driven chemoresistance process. We aimed to investigate the mechanisms and identify potential clinically relevant targets that are involved. Using mass spectrometry, we found that aldolase A (ALDOA) interacts with HIF-1α and increases HIF-1α expression. We then demonstrated that STAT3-ALDOA mediates ATP-HIF-1α signaling and upregulates the HIF-1 target genes adrenomedullin (ADM) and phosphoinositide-dependent kinase-1 (PDK1). Moreover, we show that PI3K/AKT acts upstream of HIF-1α in ATP signaling and contributes to chemoresistance in breast cancer cells. In addition, HIF-1α-knockdown or treatment with direct HIF inhibitors combined with the ATP hydrolase apyrase in MDA-MB-231 cells induced enhanced drug sensitivity in nude BALB/c mice. We then used in vitro spheroid formation assays to demonstrate the significance of ATP-HIF-1α in mediating chemoresistance. Furthermore, considering that indirect HIF inhibitors are effective in clinical cancer therapy, we treated tumor-bearing BALB/c mice with STAT3 and PI3K/AKT inhibitors and found that the dual-targeting strategy sensitized breast cancer to cisplatin. Finally, using breast cancer tissue microarrays, we found that ATP-HIF-1α signaling is associated with cancer progression, poor prognosis, and resistance to chemotherapy. Taken together, we suggest that HIF-1α signaling is vital in ATP-driven chemoresistance and may serve as a potential target for breast cancer therapies.
Assuntos
Neoplasias da Mama , Trifosfato de Adenosina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Most genome-wide association study (GWAS)-identified breast cancer-associated causal variants remain uncharacterized. To provide a framework of understanding GWAS-identified variants to function, we performed a comprehensive study of noncoding regulatory variants at the NTN4 locus (12q22) and NTN4 gene in breast cancer etiology. We find that rs11836367 is the more likely causal variant, disrupting enhancer activity in both enhancer reporter assays and endogenous genome editing experiments. The protective T allele of rs11837367 increases the binding of GATA3 to the distal enhancer and up-regulates NTN4 expression. In addition, we demonstrate that loss of NTN4 gene in mice leads to tumor earlier onset, progression, and metastasis. We discover that NTN4, as a tumor suppressor, can attenuate the Wnt signaling pathway by directly binding to Wnt ligands. Our findings bridge the gaps among breast cancer-associated single-nucleotide polymorphisms, transcriptional regulation of NTN4, and breast cancer biology, which provides previously unidentified insights into breast cancer prediction and prevention.
Assuntos
Estudo de Associação Genômica Ampla , Neoplasias , Netrinas/metabolismo , Alelos , Animais , Predisposição Genética para Doença , Camundongos , Neoplasias/genética , Netrinas/genética , Polimorfismo de Nucleotídeo Único , Via de Sinalização Wnt/genéticaRESUMO
OBJECTIVE: To investigate the effect of over expression of human hyperplasia suppressor gene (HSG) on proliferation, invasion, apoptosis and cell cycle of human breast cancer cells and to determine the relationship between HSG and Ras-dependent signaling pathway. METHODS: Full length HSG coding sequences were cloned into plasmid pcDNA3.0. The recombinant plasmids were transfected into MDA-MB-231, a highly malignant breast cancer cell line. Vacant pcDNA3.0 was used as the control. MTT, Matrigel transwell assay and flow cytometric analysis were used to test for proliferation, invasion, cell cycle distribution and apoptosis of tumor cells after transient transfection of HSG.GST-pulldown and Western blotting assays were performed to investigate the activity of Ras protein. RESULTS: HSG transfection inhibited proliferation of MDA-MB-231 cells, and significantly decreased the number of invading cells in Matrigel transwell assay compared with the vector/231 group (78.5 +/- 5.8 vs. 131.1 +/- 14.5) cells. FACS analyses demonstrated that compared with the vector/231 group, up-regulation of HSG promoted breast cancer cell apoptosis [(35.8 +/- 4.8)% vs. (25.6 +/- 3.5%)] and induced G(0)/G(1) phase arrest [(56.3 +/- 2.3)% vs. (50.4 +/- 1.9%)] after transfection for 18 hours. Furthermore, GST-pulldown assay showed that over-expression of HSG remarkably decreased the activity of Ras (about 65% lower than control). CONCLUSIONS: HSG exibits multiple anticancer functions in breast cancer cells including inhibition of proliferation and in vitro invasion, G(0)/G(1) arrest and promotion of apoptosis. Besides, inhibition of Ras-dependent signaling pathway may be involved in these processes.
Assuntos
Apoptose , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Neoplasias da Mama/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Humanos , Proteínas de Membrana/genética , Camundongos , Proteínas Mitocondriais/genética , Células NIH 3T3 , Invasividade Neoplásica , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transfecção , Regulação para Cima , Proteínas ras/metabolismoRESUMO
Our previous research demonstrated that extracellular adenosine 5'-triphosphate (ATP) could promote breast cancer cell invasion. However, the impact of extracellular ATP on chemoresistance and the mechanisms behind ATP pro-invasion and pro-chemoresistance remain unclear. Here we aimed to determine the molecules or signaling pathways involved. cDNA microarray was performed to identify the differentially expressed genes before and after ATP treatment. As a result, Sex-determining region Y-box 9 (SOX9) was up-regulated after ATP treatment in breast cancer cells. In vitro invasion and migration assays demonstrated that knocking down SOX9 attenuated ATP-driven invasive capability. Mass spectrometry and co-IP revealed that SOX9 interacted with Janus kinase 1 (JAK1). Afterward, IL-6-JAK1-STAT3 signaling was demonstrated to promote SOX9 expression and invasion following ATP treatment. Notably, ATP-IL-6-SOX9 signaling was shown to stimulate chemoresistance in breast cancer cells. ChIP assays identified some potential SOX9 target genes, among which carcinoembryonic antigen-related cell adhesion molecule 5/6 (CEACAM5/6) was demonstrated to mediate ATP pro-invasive function, while ATP-binding cassette subfamily B member 1 (ABCB1) and ATP-binding cassette subfamily G member 2 (ABCG2) mediated ATP-driven chemoresistance. In addition, SOX9-knockdown and apyrase (an ATP hydrolase)-treated MDA-MB-231 cells illustrated decreased tumor growth and enhanced drug sensitivity in nude mice. In vitro spheroid formation assays also proved the significance of ATP-SOX9 in mediating chemoresistance. Moreover, molecules involved in ATP-SOX9 signaling were up-regulated in human breast carcinoma specimens and were associated with poor prognosis. Altogether, SOX9 signaling is vital in ATP-driven invasion and chemoresistance, which may serve as a potential target for breast cancer therapies.
Assuntos
Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Fatores de Transcrição SOX9/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Transdução de Sinais , TransfecçãoRESUMO
MAGE-D1, also known as NRAGE or Dlxin-1, is a member of the MAGE family of proteins. It interacts with multiple adaptors and mediates various cellular functions such as regulation of apoptosis, transcription, cell cycle, cell adhesion and angiogenesis. In this study, we evaluated the effect of MAGE-D1 plasmid transfection on the growth, migration and invasion of breast cancer cells. MTT assay and cell counting consistently showed that MAGE-D1 transfection could effectively inhibit the proliferation of breast cancer cells. However, further FACS analyses failed to demonstrate any alterations in cell cycle distribution and apoptosis after MAGE-D1 transfection. In vitro scratch wound healing assay exhibited that MAGE-D1 suppressed cell migration. In addition, Boyden chamber invasion assay showed that MAGE-D1 significantly inhibited cell invasion. Furthermore, in an attempt to elucidate the mechanism of MAGE-D1 in suppressing cellular growth and invasion, the protein expressions of p53, p21, E-cadherin, beta-catenin, MMP-2 and MMP-9 were assessed. Western blotting showed that MAGE-D1 up-regulated the expression of p53, p21 and E-cadherin, whereas down-regulated beta-catenin expression. Taken together, this study suggests that MAGE-D1 play important roles in the regulation of cell proliferation, migration and invasion of breast cancer cells. Enhanced expression of MAGE-D1 by gene transfer could reverse the malignant phenotypes of breast cancer cells. MAGE-D1 may be a potential therapeutic target for breast cancer.
Assuntos
Antígenos de Neoplasias/fisiologia , Neoplasias da Mama/prevenção & controle , Proteínas de Neoplasias/fisiologia , Antígenos de Neoplasias/genética , Apoptose , Neoplasias da Mama/patologia , Ciclo Celular , Movimento Celular , Proliferação de Células , Feminino , Humanos , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Plasmídeos , TransfecçãoRESUMO
Inhibition of epidermal growth factor receptor (EGFR) signaling is a promising treatment strategy for malignant tumors. In this study, we evaluated the effectiveness of tyrphostin AG1478, a potent and specific inhibitor of EGFR tyrosine kinase, on the growth, apoptosis and invasion of breast cancer cells. Western blotting demonstrated that AG1478 inhibited the phosphorylation of EGFR, ERK1/2 and AKT in a dose-dependent manner. Three proliferation analyses, MTT, cell counting, and clone formation assay, consistently showed that AG1478 significantly inhibited cell proliferation in a dose-dependent manner. FACS analysis demonstrated that AG1478 promoted cell apoptosis. In addition, TRAP assay exhibited that AG1478 significantly suppressed telomerase activity of tumor cells, which was parallel with growth inhibition. Semi-qantitative RT-PCR revealed that the suppression of telomerase activity was correlated with the decreased expression of human telomerase catalytic subunit (hTERT) mRNA, the rate-limiting determinant of its enzyme activity. These data suggest that AG1478 suppressed cellular growth by inhibiting cellular proliferation, inducing apoptosis and inhibiting telomerase activity. Furthermore, we also examined the effects of AG1478 on cellular invasion. Boyden chamber invasion assay showed that AG1478 significantly inhibited cell invasion in a dose-dependent manner. Western blotting revealed that AG1478 could down-regulate the expression of MMP-9, which may be one of the mechanisms by which AG1478 suppressed cellular invasion. In conclusion, this study demonstrated that Tyrphostin AG1478 effectively inhibited the proliferation and invasion of breast cancer cells. Tyrphostin AG1478 may be a potential EGFR-targeted therapeutic agent for breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Quinazolinas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/biossínteseRESUMO
Adenosine 5'-triphosphate (ATP) is one of the most abundant biochemical constituents within the tumor microenvironment and is postulated to play critical roles in the progression of a number of types of tumors via interaction with the P2Y2 receptor. In the present study, we demonstrated that the P2Y2 receptor was highly expressed in MCF7 and Hs578T breast cancer cells. Downregulation of the P2Y2 receptor by small interfering RNA (siRNA) significantly attenuated ATP- or UTP-driven migration and invasion of the breast cancer cells as well as expression of EMT-related genes Snail and E-cadherin. Consistent with the observations in vitro, the P2Y2 receptor was found to be abundantly expressed at the invasive edge of the tumor, in infiltrating tumor cells in breast adipose tissues and/or the cancer embolus in the lymphatic sinuses compared with the tumor core areas. Furthermore, high Snail expression and weak or negative expression of E-cadherin were observed at the invasive edge of tumors. Taken together, these data indicate that the P2Y2 receptor promoted cell migration and invasion in breast cancer cells via EMT-related genes Snail and E-cadherin.
Assuntos
Neoplasias da Mama/patologia , Caderinas/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Adulto , Idoso , Antígenos CD , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Fatores de Transcrição da Família Snail/genética , Regulação para Cima , Adulto JovemRESUMO
Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis.
Assuntos
Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/metabolismo , Movimento Celular/efeitos dos fármacos , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Animais , Apirase/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Niclosamida/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100/genética , Regulação para CimaRESUMO
The cadherin 1 (CDH1) gene plays critical roles in the epithelial-mesenchymal transition process, potentially offering us a glimpse into the development of endometrial carcinoma (EC). The present study aimed to identify whether genetic variants in CDH1 affect EC susceptibility in Chinese Han women, using a strategy combining haplotype-tagging single nucleotide polymorphisms (htSNPs) association analysis with fine-scale mapping. A total of 9 htSNPs in CDH1 were genotyped among 516 cases and 706 age-matched cancer-free controls. Logistic regression analyses revealed 3 htSNPs (rs17715799, rs6499199 and rs13689) to be associated with increased EC risk and 3 htSNPs (rs12185157, rs10431923 and rs4783689) with decreased EC risk. Furthermore, 14 newly imputed SNPs of CDH1 were identified to be associated with EC risk (P<0.05) using genotype imputation analysis. Notably, multivariate logistic analysis demonstrated that rs13689, rs10431923 and rs10431924 could affect EC susceptibility independently (P≤0.001). Subsequent Generalized Multifactor Dimensionality Reduction analysis revealed several best fitting models for predicting EC risk, including SNP-SNP interactions among rs7100190, rs12185157, rs10431923, rs7186053, rs6499199, rs4783689, rs13689, rs6499197 and rs10431924, and SNP-environment interactions between related SNPs and number of childbirth. Moreover, functional annotations suggest that the majority of these susceptible variants may carry potential biological functions that affect certain gene regulatory elements. In summary, this study suggested that the genetic polymorphisms of CDH1 were indeed associated with EC susceptibility on several levels. If further additional functional studies could verify these findings, these genetic variants may serve as future personalized markers for the early prediction of endometrial cancer in Chinese Han women.
RESUMO
Extensive evidence suggests that the genetic etiologies of breast cancer (BC) and ovarian cancer (OC) show a certain degree of similarity. This study aimed to find out whether the single nucleotide polymorphisms (SNPs) of genes SNAI1 and TWIST1 may affect BC and OC susceptibility. A total of 7 taggingSNPs (tSNPs) were directly genotyped in 1,161 BC cases, 286 OC cases and 1,273 cancerfree controls among Chinese Han women. Twentyeight variants in these 2 genes were genotyped by 'in silico' genotype imputation. Logistic regression (LR) revealed that tSNPs SNAI1 rs6125849, TWIST1 rs4721746 and TWIST1 rs4721745 were protective genetic variants for BC/OC. Allelic association tests of genewide SNPs demonstrated that the minor alleles of SNAI1 rs6125849, TWIST1 rs4721745 and TWIST1 rs11973396 were strongly associated with BC/OC susceptibility. Multivariate LR presented that SNAI1 rs6125849, TWIST1 rs4721745, rs4721746 and rs11973396 affected BC/OC susceptibility independently, and women harboring all four protective genoytpes had the lowest risk. Multifactor dimensionality reduction analysis further showed that SNAI1 rs6125849 and TWIST1 rs4721745 had the strongest synergistic interaction. Functional annotation predicted that the minor alleles of SNAI1 rs6125849 and TWIST1 rs4721745 altered their binding affinities with transcription factors E2F6 and TCF11MafG respectively. These results indicate that genetic variants in SNAI1 and TWIST1, most probably SNAI1 rs6125849 and TWIST1 rs4721745, may modulate BC and OC susceptibility.
Assuntos
Povo Asiático/genética , Neoplasias da Mama/genética , Proteínas Nucleares/genética , Neoplasias Ovarianas/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição da Família Snail/genética , Proteína 1 Relacionada a Twist/genética , Adulto , Povo Asiático/etnologia , Estudos de Casos e Controles , China/etnologia , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Transcrição da Família Snail/metabolismo , Proteína 1 Relacionada a Twist/metabolismoRESUMO
In this study, we examined aberrant methylation of the E-cadherin, estrogen receptor, RB1 , p16, p15, p14, and MGMT genes by the methylation-specific polymerase chain reaction method in 101 gastric carcinomas. Hypermethylation was detected in E-cadherin, estrogen receptor, RB1, p16, p14, p15, and MGMT at the rates of 27.7%, 44.6%, 44.6%, 30.7%, 19.2%, 7.7%, and 6.9%, respectively. A total of 82.2% cases had methylation in at least 1 of these genes, and 44.6% had methylation in 2 or more of these genes. Methylation of RB1 was associated with absence of lymph node metastasis. Methylation of estrogen receptor was associated with age and tumor location. Methylation of E-cadherin coincided with methylation of p16 or estrogen receptor. Moreover, loss of p16 protein was strongly associated with its gene methylation. This study indicates that aberrant methylation of multiple genes is involved in gastric carcinogenesis.
Assuntos
Adenocarcinoma/genética , Metilação de DNA , DNA de Neoplasias/metabolismo , Genes Neoplásicos , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Caderinas/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores de Estrogênio/genética , Proteína do Retinoblastoma/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p14ARF/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: E-cadherin, beta-catenin, cathepsin D, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 are all invasion-related proteins. The expression patterns of these proteins in invasive ductal breast carcinomas, and their associations with known clinicopathological parameters, tumor recurrence and expressions of estrogen receptor (ER), progesterone receptor (PR), PS2 and c-erbB2 were not well studied in Chinese patients. METHODS: In a set of 94 invasive ductal breast carcinomas, protein expressions of these molecular markers were investigated by immunohistochemistry, and their associations with known clinicopathological parameters, tumor recurrence and expressions of ER, PR, PS2 and c-erbB2 were also examined. In addition, the interrelationship between the expressions of these proteins were studied. RESULTS: Preserved membrane E-cadherin expression was associated with late tumor stage and tumor recurrence, whereas the reduced junctional beta-catenin associated with positive lymph node status and c-erbB2 overexpression. Positive staining of cathepsin D in tumor stromal cells displayed a significant association with late tumor stage. High expression of MMP-2 in cancer cells was associated with large tumor size and PR positive expression. TIMP-2 expression was positively associated with tumor recurrence. In addition, inter-relationship between the expressions of these biomarkers was also assessed. Cathepsin D staining in cancer cells was inversely correlated with its staining in stromal cells, and also inversely correlated with MMP-2 staining in tumor stromal cells. MMP-2 expression in stromal cells displayed an inverse correlation with TIMP-2 expression. MMP-9 expression displayed parallel associations with TIMP-1 and TIMP-2 expression. CONCLUSION: Evaluation of E-cadherin, beta-catenin, cathepsin D, MMP-2 and TIMP-2 expression may be of some help in more accurately predicting the prognosis of invasive ductal breast carcinomas.
Assuntos
Neoplasias da Mama/química , Caderinas/análise , Carcinoma Ductal de Mama/química , Catepsina D/análise , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 9 da Matriz/análise , beta Catenina/análise , Adulto , Idoso , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Inibidor Tecidual de Metaloproteinase-1/análise , Inibidor Tecidual de Metaloproteinase-2/análiseRESUMO
Germ line mutations in BRCA2 gene predispose women to early-onset familial breast and ovarian cancer. BRCA2 is a protein of multiple functions. In addition to its role in DNA double-strand break repair, BRCA2 also plays a role in stabilization of stalled DNA replication forks, cytokinesis, transcription regulation, mammalian gametogenesis, centrosome duplication, and suppression of cell proliferation. However, how BRCA2 mutations predispose women specifically to breast and ovarian cancer remains undefined. Here we found that BRCA2 binds and stabilizes MAGE-D1, a member of the MAGE gene family of proteins. Expression of BRCA2 and MAGE-D1 synergistically suppresses cell proliferation independently of the p53 pathway. Using two MAGE-D1 RNA interferences and two cell lines expressing low or undetectable levels of MAGE-D1, we further showed that the expression of MAGE-D1 is required for BRCA2-mediated suppression of cell proliferation, indicating that MAGE-D1 is a downstream target of BRCA2 and that BRCA2 suppresses cell proliferation via stabilizing MAGE-D1. Importantly, MAGE-D1 protein expression was reduced in 6 of 16 breast carcinoma cell lines tested as compared with untransformed immortal mammary epithelial cell lines, suggesting that suppression of MAGE-D1 expression may be involved in the tumorigenesis of a subset of sporadic breast cancers.