CtBP1 was initially recognized as an adenoviral E1A-binding protein and its over-activation, in combination with a mutant Ras, leads to tumorigenesis and metastasis, suggesting CtBP1 plays a critical role in oncogenesis [22
]. The underlying molecular mechanisms of CtBP1 in oncogenesis could be linked to its function as a transcriptional co-repressor of tumor suppressors, including E-cadherin, p16INK4a, and p15INK4b [1
]. To assess the potential involvement of CtBP1 in breast cancer development, we stained a human breast cancer array with an anti-CtBP1 antibody. Positive nuclear CtBP1 staining was found in 92% of invasive ductal breast cancer cases (BR1502, Biomax) (, right panels); in contrast, only 4% of normal breast tissue (BRN801, Biomax) stained positive for CtBP1 (, left panels).
CtBP1 positive staining in invasive ductal breast cancers. Normal breast tissue array US Biomax BRN801 (left) and human malignant breast tissue array US Biomax BR1502 (right) were stained for CtBP1. Scale bar = 40 μm.
Because positive CtBP1 staining was detected in human breast cancer cells, we examined if CtBP1 can repress the tumor suppressor Brca1.
CtBP1 serves a key role in cellular regulation by binding to a variety of transcriptional repressors critical for development and tumorigenesis [25
]. The expression of these transcription repressors might be cell- or tissue- specific, resulting in different responses to CtBP1-mediated repression. CtBP1 potentially interacts with many components assembled at the Brca1
promoter including direct association with DNA binding proteins or indirect interactions with co-repressor complexes containing the CtBP1 binding adaptor protein, CtIP [1
]. Therefore, we assessed if CtBP1 was recruited to the Brca1
gene to repress transcription in breast cancer cells. We performed chromatin immunoprecipitation (ChIP) to identify CtBP1 binding sites in the Brca1
promoter region in MDA-MB-231 cells (). A CtBP1 binding site was found surrounding the transcriptional start site of the Brca1
promoter, similar to the CtBP1 binding site in head and neck squamous cell carcinoma (HNSCC) cells [19
]. Our previous study demonstrated this CtBP1 binding site confers transcriptional repression to the Brca1
mRNA levels increased 3 fold when CtBP1 was abrogated in MDA-MB-231 cells (). Accordingly, Brca1 protein was upregulated when CtBP1 was knocked down in MDA-MB-231 cells ().
Figure 2 CtBP1 represses Brca1 expression. A. CtBP1 binding to the Brca1 regulatory element. Human breast cancer MDA-MB-231 cells were used for ChIP assay with an anti-CtBP1 antibody. Primers encompassing the Brca1 promoter were used to q-PCR-amplify the ChIP (more ...)
To further assess if restoration of Brca1
expression by CtBP1 knockdown confers rescue of Brca1 function, we examined Brca1-mediated DNA repair foci formation by immunofluorescence staining in MDA-MB-231 cells and MDA-MB-231-siCtBP1 cells treated with mitomycin C (MMC). Under normal conditions, Brca1 translocates to sites of MMC-induced DNA damage with other members of the Fanc/Brca pathway to form DNA repair nuclear foci [27
]. 24 h after 10 ng/ml MMC treatment, only about 10% of MDA-MB-231 cells were able to form Brca1 foci, whereas 32% of MDA-MB-231 cells with siCtBP1 48 h knockdown were able to form MMC-induced DNA repair foci (). These data show that CtBP1-mediated Brca1
repression abrogates Brca1 function.
Our study suggests that under normal conditions, Brca1 expression in breast cells is not repressed by low levels of CtBP1. However, during breast cancer carcinogenesis, increased levels of CtBP1 induce repression of Brca1. To determine the relative expression of Brca1 and CtBP1 in breast cancer, we performed IHC for CtBP1 and Brca1 on invasive ductal breast cancer cases using serial slides of a breast cancer array (BR1502, Biomax). Brca1 loss was detected in 62% (34/55) of the CtBP1-positive invasive ductal breast cancer cases; 0% (0/5) of the Brca1 loss cases with CtBP1-negative staining, suggesting an inverse correlation between Brca1 and CtBP1 in these lesions ( and ). These data strongly support CtBP1 repression of Brca1 expression in vivo; therefore CtBP1 might serve as a biomarker, alone or in combination with Brca1 loss, for breast cancer development.
Figure 3 Correlation between CtBP1 up-regulation and Brca1 down-regulation in invasive ductal breast cancers. Immunohistochemical staining was performed using antibodies against Brca1 and CtBP1 to stain consecutive tissue sections as we previously described [ (more ...)
Correlation between CtBP1 positive staining and Brca1 down-regulation in invasive ductal breast cancers.
The ability of tumors to metastasize is a hallmark of malignancy. A critical event during metastasis is adhesion reduction, facilitating tumor cell invasion into surrounding tissues and vascular channels, ultimately leading to cancer progression. E-cadherin-mediated cell–cell adhesion is essential for maintaining homeostasis and architecture of epithelial tissues. Down-regulation of E-cadherin expression occurs concomitantly with dedifferentiation and invasion of epithelial cells during tumorigenesis [8
]. Consequently, E-cadherin and its associated complex are thought to be key mediators of tumor cell invasion [28
]. To explore functional consequences of CtBP1 up-regulation in breast cancer, we assessed if the E-cadherin
gene is suppressed by CtBP1 in breast cancer cells.
We performed a ChIP assay using the anti-CtBP1 antibody to identify CtBP1 binding sites in the E-cadherin
promoter region of MDA-MB-231 cells (). Compared to the normal IgG, the anti-CtBP1 antibody pulled down 2–3 fold more E-cadherin promoter, suggesting CtBP1 was recruited to the E-cadherin
promoter. Furthermore, E-cadherin mRNA levels increased 6 fold when CtBP1 was abrogated in MDA-MB-231 cells (), demonstrating CtBP1 transcriptionally represses E-cadherin
in human breast cancer cells. E-cadherin protein was increased by CtBP1 knockdown as well (). A similar effect has been observed in MCF7 cells [29
Figure 4 CtBP1 represses E-cadherin expression. A. CtBP1 binding to the E-cadherin regulatory element. MDA-MB-231 cells were used for ChIP assay with an anti-CtBP1 antibody. Primers encompassing the E-cadherin promoter were used to q-PCR-amplify the ChIP sample. (more ...)
Next, we examined E-cadherin expression by immunofluorescence staining using MDA-MB-231 and MDA-MB-231-siCtBP1 cells. Weak E-cadherin staining was observed in a punctate pattern along the cell edge in MDA-MB-231 cells, whereas MDA-MB-231 cells with siCtBP1 48 h knockdown for exhibited stronger E-cadherin staining (). These data show CtBP1-mediated E-cadherin transcriptional repression abrogates E-cadherin expression in breast cancer cells, suggesting CtBP1 might contribute to metastatic potential during breast cancer development.
To explore the potential regulation of E-cadherin by CtBP1 in breast cancer, we performed IHC for CtBP1 and E-cadherin using serial slides of a breast cancer array. E-cadherin loss was detected in 83% (45/57) of the invasive ductal breast cancer cases with CtBP1-positive staining; whereas E-cadherin loss was not detected in the malignant breast cancer cases lacking CtBP1 staining (0/5), suggesting an inverse correlation between E-cadherin and CtBP1 in these lesions as well ( and ). These data strongly support CtBP1 repression of E-cadherin expression in vivo.
Figure 5 Correlation between CtBP1 up-regulation and E-cadherin down-regulation in invasive ductal breast cancers. Immunohistochemical staining was performed using antibodies against E-cadherin and CtBP1 to stain consecutive tissue sections as we previously described (more ...)
Correlation between CtBP1 positive staining and E-cadherin down-regulation in invasive ductal breast cancers.