HEXIM1 Regulates 17 -Estradiol/Estrogen Receptor- -Mediated Expression of Cyclin D1 in Mammary Cells via Modulation of P-TEFb

Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
Cancer Research (Impact Factor: 9.33). 10/2008; 68(17):7015-24. DOI: 10.1158/0008-5472.CAN-08-0814
Source: PubMed


Estrogen receptor alpha (ERalpha) plays a key role in mammary gland development and is implicated in breast cancer through the transcriptional regulation of genes linked to proliferation and apoptosis. We previously reported that hexamethylene bisacetamide inducible protein 1 (HEXIM1) inhibits the activity of ligand-bound ERalpha and bridges a functional interaction between ERalpha and positive transcription elongation factor b (P-TEFb). To examine the consequences of a functional HEXIM1-ERalpha-P-TEFb interaction in vivo, we generated MMTV/HEXIM1 mice that exhibit mammary epithelial-specific and doxycycline-inducible expression of HEXIM1. Increased HEXIM1 expression in the mammary gland decreased estrogen-driven ductal morphogenesis and inhibited the expression of cyclin D1 and serine 2 phosphorylated RNA polymerase II (S2P RNAP II). In addition, increased HEXIM1 expression in MCF-7 cells led to a decrease in estrogen-induced cyclin D1 expression, whereas down-regulation of HEXIM1 expression led to an enhancement of estrogen-induced cyclin D1 expression. Studies on the mechanism of HEXIM1 regulation on estrogen action indicated a decrease in estrogen-stimulated recruitment of ERalpha, P-TEFb, and S2P RNAP II to promoter and coding regions of ERalpha-responsive genes pS2 and CCND1 with increased HEXIM1 expression in MCF-7 cells. Notably, increased HEXIM1 expression decreased only estrogen-induced P-TEFb activity. Whereas there have been previous reports on HEXIM1 inhibition of P-TEFb activity, our studies add a new dimension by showing that E(2)/ER is an important regulator of the HEXIM1/P-TEFb functional unit in breast cells. Together, these studies provide novel insight into the role of HEXIM1 and ERalpha in mammary epithelial cell function.

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Available from: Koh Fujinaga, Apr 04, 2014
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    • "CDK9 activity is upregulated in hypertrophic cardiomyopathy [38]. Moreover, Mixed Lineage Leukemia [39] and certain ER-positive breast cancer [40,41] exhibit deregulation of P-TEFb activity. Thus, understanding the consequences of inhibiting CDK9 activity on gene expression over time is important. "
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    ABSTRACT: The Positive Transcription Elongation Factor b (P-TEFb) is a complex of Cyclin Dependent Kinase 9 (CDK9) with either cyclins T1, T2 or K. The complex phosphorylates the C-Terminal Domain of RNA polymerase II (RNAPII) and negative elongation factors, stimulating productive elongation by RNAPII, which is paused after initiation. P-TEFb is recruited downstream of the promoters of many genes, including primary response genes, upon certain stimuli. Flavopiridol (FVP) is a potent pharmacological inhibitor of CDK9 and has been used extensively in cells as a means to inhibit CDK9 activity. Inhibition of P-TEFb complexes has potential therapeutic applications. It has been shown that Lipopolysaccharide (LPS) stimulates the recruitment of P-TEFb to Primary Response Genes (PRGs) and proposed that P-TEFb activity is required for their expression, as the CDK9 inhibitor DRB prevents localization of RNAPII in the body of these genes. We have previously determined the effects of FVP in global gene expression in a variety of cells and surprisingly observed that FVP results in potent upregulation of a number of PRGs in treatments lasting 4-24 h. Because inhibition of CDK9 activity is being evaluated in pre-clinical and clinical studies for the treatment of several pathologies, it is important to fully understand the short and long term effects of its inhibition. To this end, we determined the immediate and long-term effect of FVP in the expression of several PRGs. In exponentially growing normal human fibroblasts, the expression of several PRGs including FOS, JUNB, EGR1 and GADD45B, was rapidly and potently downregulated before they were upregulated following FVP treatment. In serum starved cells re-stimulated with serum, FVP also inhibited the expression of these genes, but subsequently, JUNB, GADD45B and EGR1 were upregulated in the presence of FVP. Chromatin Immunoprecipitation of RNAPII revealed that EGR1 and GADD45B are transcribed at the FVP-treatment time points where their corresponding mRNAs accumulate. These results suggest a possible stress response triggered by CDK9 inhibition than ensues transcription of certain PRGs. We have shown that certain PRGs are transcribed in the presence of FVP in a manner that might be independent of CDK9, suggesting a possible alternative mechanism for their transcription when P-TEFb kinase activity is pharmacologically inhibited. These results also show that the sensitivity to FVP is quite variable, even among PRGs.
    Full-text · Article · Mar 2012 · Cell Division
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    • "A Pol II promoter-driven miRNA expression vector system (Invitrogen, CA) was used. To make pcDNA-HEXIM1 miRNA, miRNA oligos (Ogba et al 2008) were annealed and cloned into the pcDNA 6.2 GW/EmGFP vector (Invitrogen) according to the manufacturer's instructions. MCF-7 cells were transfected with pcDNA 6.2-GW/EmGFP-miR expression vectors containing either the HEXIM1 miRNA insert or a control miRNA insert. "
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    ABSTRACT: Tamoxifen resistance is a major problem in the treatment of estrogen receptor (ER)-positive patients. We have previously reported that hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) inhibits ERα activity by competing with ERα for binding to cyclin T1, a subunit of positive transcription elongation b (P-TEFb). This results in the inhibition of the phosphorylation of RNA polymerase II (RNAPII) at serine 2 and the inhibition of transcription elongation of ERα target genes. As HEXIM1 can inhibit ER activity, we examined whether it has a critical role in the inhibitory effects of tamoxifen on ER. We observed that tamoxifen-induced HEXIM1 recruitment to the promoter region of ER target genes and decreased the recruitment of cyclin T1 and serine 2 phosphorylated RNAPII to the coding regions of these genes. Conversely, in cells wherein HEXIM1 expression has been downregulated we observed attenuation of the inhibitory effects of tamoxifen on estrogen-induced cyclin T1 recruitment to coding regions of ER target genes. As a consequence, downregulation of HEXIM1 resulted in the attenuation of the repressive effects of tamoxifen on estrogen-induced gene expression and proliferation. Conferring clinical relevance to our studies is our analysis of human breast cancer tissue samples that indicated association of lower expression of HEXIM1 with tumor recurrence in patients who received tamoxifen. Our studies provide a better understanding of the mechanistic basis for the inhibitory effect of tamoxifen on ER activity and may suggest new therapeutic targets for the treatment of tamoxifen-resistant breast cancer.
    Full-text · Article · Mar 2011 · Oncogene
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    • "This suggests that the regulation of VEGF expression by HEXIM1 in the mouse mammary gland could be due to a dysregulation of ERα, which would be consistent with our breast epithelial cell studies, and indicate that HEXIM1 inhibits the actions of ERα and HIF-1α, two positive regulators of VEGF gene transcription. We deduced that this regulation is independent of P-TEFb, given that HEXIM1 1-312 was found to inhibit P-TEFb activity but did not appear to have an effect on the expression levels of cyclin D1 and Serine 2 phosphorylated RNAP II in the mammary gland, which have been shown to be reflective of changes in P-TEFb activity in previous studies (Ogba et al., 2008). However, we cannot completely rule out a role for P-TEFb in the regulation of VEGF transcription, as many genes contribute to VEGF transcriptional activity but are beyond the scope of our current study. "
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    ABSTRACT: Recently, we found that mutation of the C-terminus of transcription factor hexamethylene bisacetamide-inducible protein 1 (HEXIM1) in mice leads to abnormalities in cardiovascular development because of aberrant vascular endothelial growth factor (VEGF) expression. HEXIM1 regulation of some genes has also been shown to be positive transcription elongation factor b (P-TEFb) dependent. However, it is not known whether HEXIM1 regulates VEGF in the mammary gland. We demonstrate that HEXIM1 regulates estrogen-induced VEGF transcription through inhibition of estrogen receptor-alpha recruitment to the VEGF promoter in a P-TEFb-independent manner in MCF-7 cells. Under hypoxic conditions, HEXIM1 inhibits estrogen-induced hypoxia-inducible factor-1 alpha (HIF-1alpha) protein expression and recruitment of HIF-1alpha to the hypoxia-response element in the VEGF promoter. In the mouse mammary gland, increased HEXIM1 expression decreased estrogen-driven VEGF and HIF-1alpha expression. Conversely, a mutation in the C-terminus of HEXIM1 (HEXIM1(1-312)) led to increased VEGF and HIF-1alpha expression and vascularization in mammary glands of heterozygous HEXIM1(1-312) mice when compared with their wild-type littermates. In addition, HEXIM1(1-312) mice have a higher incidence of carcinogen-induced mammary tumors with increased vascularization, suggesting an inhibitory role for HEXIM1 during angiogenesis. Taken together, our data provide evidence to suggest a novel role for HEXIM1 in cancer progression.
    Full-text · Article · Jun 2010 · Oncogene
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