The present study provides novel evidence that HEXIM1 is critical for tamoxifen inhibition of ERα activity. Our studies also suggest that tamoxifen-liganded ER has activities on its own and a more proactive role beyond competitively inhibiting the interaction of ERα with estrogens. Tamoxifen-liganded ERα induced recruitment of HEXIM1 to ER target genes, resulting in the inhibition of recruitment of cyclin T1 and serine 2 phosphorylated RNAP II to the coding regions of these ER target genes. We also demonstrated a functional role for HEXIM1 in the inhibition of ER-mediated gene expression and proliferation by tamoxifen. Most importantly, our immunohistochemical studies indicate a correlation between HEXIM1 expression and disease recurrence in patients who have been treated with tamoxifen.
Coactivator and corepressor proteins play critical roles in mediating transcriptional activation by the ER, and enhancement of coactivator or inhibition of corepressor expression may contribute to the tamoxifen resistant phenotype (Ring and Dowsett 2004
, Scott et al 2007
). Overexpression of the coactivators Amplified in Breast Cancer 1 (AIB1) or Nuclear Coactivator-3 (NCoA3) in breast cancer has been correlated with tamoxifen resistance and decreased overall survival (Lahusen et al 2009
). Corepressors have been reported to interact with tamoxifen-liganded ER (Lavinsky et al 1998
, Webb et al 2003
). In vitro
studies have indicated that reduced levels of corepressors N-COR and SMRT resulted in attenuation of the inhibitory effects of tamoxifen (Lavinsky et al 1998
). However modulation of the recruitment of AIB1, NCoA3, N-COR, or SMRT to ER target genes by tamoxifen has not been reported. Correlations between the levels of known corepressors and tamoxifen resistance in human breast tissue samples have also not been reported. We have previously reported that HEXIM1 interacts with tamoxifen-liganded ER (Wittmann et al 2005
). We now report on the consequences of this interaction and show that tamoxifen enhances the recruitment of HEXIM1 to the promoters of ER target genes that, in turn, inhibits the recruitment of cyclin T1 and phosphorylated RNAP II.
To demonstrate the functional relevance of tamoxifen-induced recruitment of HEXIM1 in the inhibitory effects of tamoxifen, we stably downregulated HEXIM1 expression and showed that decreased HEXIM1 recruitment to the promoter region of pS2 and CCND1 resulted in attenuation of tamoxifen inhibition of E2-induced recruitment of cyclin T1. Moreover, downregulation of HEXIM1 resulted in the enhancement of E2-induced cell proliferation, and attenuated the inhibitory effects of tamoxifen on cyclin D1 expression and cell proliferation. Therefore, our studies provide evidence for a critical role of HEXIM1 in the inhibitory effects of tamoxifen.
binds to ER at the hydrophobic pocket of ligand binding domain (LBD) that is sealed by helix 12 providing a platform for recruiting coactivator proteins. Tamoxifen also binds to ER at LBD and repositions helix 12. A key difference between E2
-liganded ER and SERM- or tamoxifen- ER complex is that helix 12 is prevented from sealing the ligand binding pocket (Shiau et al 1998
). HEXIM1 has been shown to interact with the E/F domain of ERα in vitro
(Wittmann et al 2005
). The binding of tamoxifen to ER resulted in several residues exposing different side chains than those observed in E2
-liganded ER (Shiau et al 1998
), perhaps allowing for an interaction with HEXIM1 to be more energetically favored. Based on our current studies, we speculate that tamoxifen can enhance HEXIM1 recruitment to ER target genes because of conformational changes that occur after tamoxifen binds to ER that allows for higher affinity binding to HEXIM1 than E2
-liganded ER. Further studies should be done to gain a better understanding of structural basis for the interaction of HEXIM1 with tamoxifen-liganded ER.
The clinical relevance of our findings was supported by our immunohistochemical analysis of HEXIM1 expression in human breast cancer specimens. Our studies indicated that lower expression of HEXIM1 was significantly associated with increased risk of tumor recurrence in patients who received tamoxifen treatment. Thus we speculate that loss of HEXIM1 may be involved in the development of tamoxifen resistance. Therefore, therapies aimed at increasing HEXIM1 expression can be developed for tamoxifen-resistant patients to improve breast cancer survival. We are currently testing this possibility using mouse models generated in our laboratory.