TReP-132 Controls Cell Proliferation by Regulating the Expression of the Cyclin-Dependent Kinase Inhibitors p21^WAF1/Cip1 and p27^Kip1

Abstract

The transcriptional regulating protein of 132 kDa (TReP-132) has been identified in steroidogenic tissues, where it acts as a coactivator of steroidogenic factor 1 (SF-1). We show here that TReP-132 plays a role in the control of cell proliferation. In human HeLa cells, TReP-132 knockdown by using small interfering RNA resulted in increased G 1 3S cell cycle progression. The growth-inhibitory effects of TReP-132 was further shown to be mediated by induction of G 1 cyclin-dependent kinase inhibitors p21 WAF1 (p21) and p27 KIP1 (p27) expression levels. As a consequence, G 1 cyclin/cyclin-dependent kinase activities and pRB phosphorylation were markedly reduced, and cell cycle progression was blocked in the G 1 phase. The stimulatory effect of TReP-132 on p21 and p27 gene transcription involved interaction of TReP-132 with the transcription factor Sp1 at proximal Sp1-binding sites in their promoters. Moreover, in different breast tumor cell lines, endogenous TReP-132 expression was positively related with a lower proliferation rate. In addition, TReP-132 knockdown resulted in enhanced cell proliferation and lowered p21 and p27 mRNA levels in the steroidresponsive and nonresponsive T-47D and MDA-MB-231 cell lines, respectively. Finally, a statistic profiling of human breast tumor samples highlighted that expression of TReP-132 is correlated with p21 and p27 levels and is associated with lower tumor incidence and aggressiveness. Together, these results identify TReP-132 as a basal cell cycle regulatory protein acting, at least in part, by interacting with Sp1 to activate the p21 and p27 gene promoters.

Functional identification of genes causing estrogen independence of human breast cancer cells

“…1c). Sequence analysis of full-length cDNAs revealed two major splice variants, one (BCAR2b) encoding a 1,200 amino acid protein identical to TREP-132/ TRERF1 and one unique variant containing 20 additional amino acids due to alternative splicing at the end of exon 8 (BCAR2a) [23,Text S1]. Rabbit polyclonal antibodies directed against BCAR2 revealed increased TRERF1 protein levels (approximately 130 kDa) in cell line VIII-24 (carrying the integration in the cVIS2 locus) compared with other ZR-75-1-derived cell lines (Fig.…”

“…Within the nuclear compartment, NCOR2 participates in a co-repressor complex resulting in chromatin condensation and may also modulate ligand dependency of hormone receptors and contribute to estrogen independency [34][35][36]. TRERF1 was reported to negatively modulate breast cancer cell proliferation by upregulation of G1 cyclin-dependent kinase inhibitors and through co-activation of the progesterone receptor [23,37]. Whether the transcription regulators CITED2 and TLE3 fit into the same pathways as the cytoplasmic targets or represent alternative signaling routes, remains an intriguing question.…”

“…In breast disease, RHOBTB3 belongs to a gene set capable of classifying tumors with evidence of hypoxia [38] and TRERF1 appears to be down-modulated in breast cancer in comparison to normal epithelium [23]. High expression of EGFR is associated with poor response to tamoxifen treatment [39].…”

TReP-132 Is a Novel Progesterone Receptor Coactivator Required for the Inhibition of Breast Cancer Cell Growth and Enhancement of Differentiation by Progesterone

“…The TReP-132 expression vector subcloned in pcDNA3 and the wild-type or mutated glutathione S-transferase (GST)-TReP-132 fusion protein were constructed as previously described (24). The p21 WAF1/Cip1 and the p27 Kip1 promoter luciferase reporter constructs were kindly provided by X. Wang (Dept.…”

“…Notably, in human adrenal carcinoma NCI-H295 cells, TReP-132 acts as a coactivator of the nuclear receptor steroidogenic factor 1 to enhance cytochrome P450 side chain cleavage (P450scc) and c17 (P450c17) gene promoter activity (23). Recently, we identified a new function for TReP-132 as a growth suppressor protein in HeLa and mammary cancer cells (24). As with PR, the antiproliferative activity of TReP-132 involves its interaction with Sp1 to activate the p21 and p27 promoters, resulting in the inhibition of G 1 CDK-mediated phosphorylation of pRB and histone H1.…”