Colorectal cancer (CRC) is a common malignant tumor that is associated with an increased incidence of morbidity and mortality. Nasopharyngeal carcinoma-associated gene 6 (NGX6) is a novel candidate suppressor gene of tumor metastasis, which is down-regulated in CRC. In the present study, we constructed a colorectal tissue microarray to examine the expression profiles of NGX6, phospho-c-Jun N-terminal kinase (p-JNK), and phospho-extracellular signal-regulated kinase (p-ERK ) in CRC tissues. We found that the NGX6 expression was lower in CRC tissues and metastatic lymph nodes, whereas the expressions of p-JNK and p-ERK were higher in CRC tissues, than in normal intestinal mucosa. The expressions of NGX6, p-JNK, and p-ERK were associated with the clinical pathological features of colorectal tissues. NGX6 overexpression inhibited the activation and nuclear translocation of JNK1, which led to an accumulation of p-JNK in the cytoplasm, but did not inhibit the activation and nuclear translocation of ERK1/2. NGX6 also inhibited the expression of the transcription factors AP-1 (c-jun and c-fos) and Ets-1. In addition, NGX6 overexpression decreased the expression of cyclin D1 and dramatically suppressed the transcriptional efficiency of the cyclin D1 promoter. We propose that NGX6 expression is lost in the multi-step process of human colorectal carcinogenesis. Its overexpression can inhibit the expression of transcription factors AP-1 and Ets-1, and down-regulate the transcriptional activity of the cyclin D1 promoter in human CRC.

Previously, we reported that BRCA1 inhibits progesterone receptor (PR) activity and blocks progesterone-stimulated gene expression and cell proliferation. In the present manuscript, we studied the mechanism of BRCA1 inhibition of PR activity, using c-Myc as a model progesterone-regulated promoter. Here, we found that BRCA1 has little or no effect on PR ligand-binding affinity. However, BRCA1 overexpression inhibited the R5020-induced recruitment of PR to the c-Myc and mouse mammary tumor virus progesterone response elements (PREs) and blocked R5020-stimulated c-Myc expression, whereas BRCA1 underexpression did the opposite. In EMSAs, BRCA1 overexpression blocked the R5020-induced complex formation between PR and several radiolabeled PRE-containing oligonucleotides, and in vitro-translated BRCA1 blocked the interaction of full-length PR-A or a fragment containing the DNA-binding domain of PR with a radiolabeled PRE oligonucleotide. In further studies, BRCA1 overexpression inhibited the recruitment of coactivators (steroid receptor coactivator 1 and amplified in breast cancer 1) and enhanced the recruitment of a corepressor (histone deacetylase 1) to the c-Myc PRE, whereas BRCA1 knockdown increased the abundance of AIB1 and decreased the abundance of HDAC1 at the c-Myc PRE. These findings suggest that BRCA1 inhibits progestin-stimulated PR activity, in part, by preventing PR from binding to the PRE and by promoting the formation of a corepressor complex rather than a coactivator complex.

Inherited mutations of the breast cancer susceptibility gene BRCA1 confer a high risk for breast cancer development. The ³⁰⁰RXKK and ²⁶⁶KXK motifs have been identified previously as sites for acetylation of the estrogen receptor- (ER-), and ³⁰²K was also found to be a site for BRCA1-mediated mono-ubiquitination of ER- in vitro. Here we show that ER- proteins with single or double lysine mutations of these motifs (including K303R, a cancer-associated mutant) are resistant to inhibition by BRCA1, even though the mutant ER- proteins retain the ability to bind to BRCA1. We also found that BRCA1 overexpression reduced and knockdown increased the level of acetylated wild-type ER-, without changing the total ER- protein level. Increased acetylation of ER- due to BRCA1 small interfering RNA was dependent upon phosphatidylinositol 3-kinase/Akt signaling and on up-regulation of the coactivator p300. In addition, using an in vitro acetylation assay, we found that in vitro-translated wild-type BRCA1 but not a cancer-associated point mutant (C61G) inhibited p300-mediated acetylation of ER-. Furthermore, BRCA1 overexpression increased the levels of mono-ubiquitinated ER- protein, and a BRCA1 mutant that is defective for ubiquitin ligase activity but retains other BRCA1 functions (I26A) did not ubiquitinate ER- or repress its activity in vivo. Finally, ER- proteins with mutations of the ³⁰⁰RXKK or ²⁶⁶KXK motifs showed modest or no BRCA1-induced ubiquitination. We propose a model in which BRCA1 represses ER- activity, in part, by regulating the relative degree of acetylation vs. ubiquitination of ER-.