Molecular mechanisms for aberrant expression of the human breast cancer specific gene 1 in breast cancer cells: control of transcription by DNA methylation and intronic sequences

Lü, Aiping; Gupta, Anu; Liu, Cong; Ahlborn, Thomas E.; Ma, Yanju; Shi, Eric; Liu, Jingwen

doi:10.1038/sj.onc.1204668

Cited by 55 publications

(65 citation statements)

References 16 publications

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“…Since BubR1 is a critical component of the mitotic checkpoint control, it could be a potential cellular target of oncogenic proteins such as BCSG1 that induces tumor progression potentially through mechanisms of disturbing genome stability. To confirm BCSG1 and BubR1 interaction in mammalian cells, initially we performed immunoprecipitation (IP) assay using total cell lysates isolated from a BCSG1-positive cell line T47D and a BCSG1-negative cell line HepG2 (Lu et al, 2001). Using BCSG1-specific antibody to IP, BubR1 was only detected in the IP complex of T47D cells ( Figure 1a, lane 2) but not in the IP complex formed with the HepG2 cell lysate (Figure 1a, lane 4), despite the detection of BubR1 protein in the total cell lysate of HepG2 (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Breast cancer-specific gene 1 interacts with the mitotic checkpoint kinase BubR1

et al. 2003

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The abnormal expression of breast cancer-specific gene 1 (BCSG1) in malignant mammary epithelial cells is highly associated with the development and progression of breast cancer. A series of in vitro and in vivo studies performed in our laboratory and others have demonstrated that BCSG1 expression significantly stimulates proliferation, invasion, and metastasis of breast cancer cells. However, currently little is known about how BCSG1 exerts its oncogenic functions. To elucidate the cellular mechanisms underlying the effects of BCSG1 in breast cancer cells, we used a yeast two-hybrid system to screen for proteins that could associate with BCSG1. Through this screening, we identified the mitotic checkpoint protein BubR1 as a novel binding partner of BCSG1. The specific association of BCSG1 with BubR1 in breast cancer cells was demonstrated by immunoprecipitation and GST pull-down assays. Intriguingly, experiments conducted in four different cell lines all showed that exogenous expressions of BCSG1 consistently reduce the cellular levels of the BubR1 protein without affecting BubR1 mRNA expression. The tendency of endogenous BCSG1 expression coinciding with lower BubR1 protein levels was also observed in seven out of eight breast cancer cell lines. We further showed that the reducing effect of BCSG1 on BubR1 protein expression could be prevented by treating BCSG1-transfected cells with MG-132, a selective 26S proteasome inhibitor, implying that the proteasome machinery may be involved in the BCSG1-induced reduction of the BubR1 protein. Accompanied with a reduction of BubR1 protein level, BCSG1 expression resulted in multinucleation of breast cancer cells upon treatment with spindle inhibitor nocodazole, indicating an impaired mitotic checkpoint. Taken together, our novel findings suggest that BCSG1 may accelerate the progression of breast cancer at least in part by compromising the mitotic checkpoint control through inactivation of BubR1.

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Section: Resultsmentioning

confidence: 99%

Breast cancer-specific gene 1 interacts with the mitotic checkpoint kinase BubR1

et al. 2003

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“…Sequence analysis indicates that the exon 1 region contains a CpG island. In vivo genomic bisulfite sequencing demonstrates that the BCSG1 CpG island is totally unmethylated in BCSG1-positive SKBR-3 and T47D cells but partially methylated in BCSG1-negative MCF-7 and HepG2 cells (7). This suggests that DNA demethylation may be an important factor contributing to the aberrant expression of BCSG1 in breast cancer cells.…”

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confidence: 90%

“…However, the promoter reporter activity was markedly increased by inclusion of the intron 1 region of the BCSG1 gene. Further deletion analysis localized a consensus AP1 binding site (TGACTCA) in the intron that was largely responsible for the increased promoter activity (7). These previous studies suggest that the activator protein AP1 may regulate BCSG1 transcription, possibly through the intronic AP1 binding site.…”

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confidence: 95%

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Blockade of AP1 Transactivation Abrogates the Abnormal Expression of Breast Cancer-specific Gene 1 in Breast Cancer Cells

Lü

Zhang

Gupta

et al. 2002

Journal of Biological Chemistry

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Breast cancer-specific gene 1 (BCSG1) is not expressed in normal breast tissue but is highly expressed in the vast majority of invasive and metastatic breast carcinomas. When over-expressed, BCSG1 significantly stimulates the proliferation and invasion of breast cancer cells. The accumulated evidence suggests that the aberrant expression of BCSG1 in breast carcinomas is caused by transcriptional activation of the BCSG1 gene. However, the transcription factors that activate BCSG1 transcription have not been identified. In this study, we extensively investigated the role of AP1 in BCSG1 expression in breast cancer cells. We demonstrate that there are two closely located AP1 binding sites residing in the first intron of the BCSG1 gene. Mutation of either AP1 motif on the BCSG1 promoter constructs markedly reduces the promoter activity. We further show that 12-O-tetradecanoylphorbol-13-acetate (TPA) increases BCSG1 mRNA expression and up-regulates BCSG1 promoter activity through the intronic AP1 sites. The effect of TPA on BCSG1 transcription is also demonstrated under in vivo conditions in intact cells by using chromatin immunoprecipitation assays that show the TPA-induced binding of c-Jun to the chromatin region encompassing the intronic AP1 sites. Finally, to examine the direct effect of AP1 transactivation on BCSG1 expression, we established stable cell lines of T47D that express the dominant negative mutant of c-Jun, TAM67. RT-PCR and Western blot analyses demonstrated that levels of BCSG1 mRNA and protein in TAM67 transfectants were drastically reduced as compared with mocktransfected cells. Furthermore, inhibition of BCSG1 expression by blocking AP1 transactivation produced a similar repressive effect on cell growth as that by expressing BCSG1 antisense mRNA. We show that the anchorage-independent growth of T47D cells expressing either TAM67 or BCSG1 antisense mRNA is significantly inhibited. Taken together, we provide strong evidence that AP1 plays an overriding role in the transcription of the BCSG1 gene and that blockade of AP1 transactivation down-regulates BCSG1 expression and suppresses tumor phenotype.Previous studies conducted by differential DNA sequencing and in situ hybridization have identified BCSG1 1 (1), also referred to as synuclein ␥ (2) or persyn (3), as a breast cancerspecific gene because of its distinct expression pattern in breast tissues. BCSG1 is not expressed in normal breast tissue or tissues with benign breast diseases but is highly expressed in the vast majority of stage III/IV breast carcinomas (1, 4). BCSG1 expression in advanced breast carcinomas is not merely adventitious but plays a positive role in the process of invasion and metastasis. It has been demonstrated that exogenous expression of BCSG1 in breast cancer cells leads to a significant increase in cell motility and cell proliferation in cell culture (5, 6), as well as a profound augmentation of metastasis in nude mice (5). Thus far, BCSG1 gene mutations or amplifications have not been found by examination of breast carc...

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“…The SNCG expression in lung tumors shows a correlation with stage progression and metastasis. The exon 1 of SNCG gene contains a CpG island with 15 CpG dinucleotides (Lu et al, 2001). By performing genomic sequencing and methylation-specific polymerase chain reaction (MSP) assays, we have shown that the SNCG CpG island is completely unmethylated in lung tumor samples as well as in other types of tumors, which is in sharp contrast to the predominantly methylated CpG island of SNCG in the majority of normal tissues adjacent to tumors (Liu et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Cigarette smoke induces demethylation of prometastatic oncogene synuclein-γ in lung cancer cells by downregulation of DNMT3B

et al. 2007

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The prometastatic oncogene synuclein-c (SNCG) is not expressed in normal lung tissues, but it is highly expressed in lung tumors. Here, we show that cigarette smoke extract (CSE) has strong inducing effects on SNCG gene expression in A549 lung cancer cells through demethylation of SNCG CpG island. CSE treatment also augments the invasive capacity of A549 cells in an SNCG-dependent manner. To elucidate the mechanisms underlying the demethylating effects of CSE, we examined expression levels of DNA methyltransferases (DNMTs), 1, 3A and 3B in CSE-treated cells. We show that the mRNA expression of DNMT3B is specifically downregulated by CSE with a kinetics concurrent to SNCG reexpression. Utilizing siRNA to knockdown DNMT3B expression, we show that inhibition of DNMT3B directly increases SNCG mRNA expression. We further show that exogenous overexpression of DNMT3B in an SNCG-positive lung cancer cell line H292 suppresses SNCG mRNA and protein expression and induces de novo methylation of SNCG CpG island, whereas overexpression of DNMT1 or DNMT3A has no effects. Taken together, these new findings demonstrate that tobacco exposure induces the abnormal expression of SNCG in lung cancer cells through downregulation of DNMT3B. This work sheds light on the molecular understanding of demethylation of this oncogene during cancer progression.

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Molecular mechanisms for aberrant expression of the human breast cancer specific gene 1 in breast cancer cells: control of transcription by DNA methylation and intronic sequences

Cited by 55 publications

References 16 publications

Breast cancer-specific gene 1 interacts with the mitotic checkpoint kinase BubR1

Breast cancer-specific gene 1 interacts with the mitotic checkpoint kinase BubR1

Blockade of AP1 Transactivation Abrogates the Abnormal Expression of Breast Cancer-specific Gene 1 in Breast Cancer Cells

Cigarette smoke induces demethylation of prometastatic oncogene synuclein-γ in lung cancer cells by downregulation of DNMT3B

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