Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and Cell Cycle Progression in MCF-7 Breast Cancer Cells

Abstract

Sp1 is a member of the Sp and Krü ppel-like family of transcription factors that bind GC and CACCC boxes to regulate gene expression (1-3). Sp1 is widely expressed in multiple tissues (4), and targeted disruption of Sp1 in mice results in retarded development and embryo-lethality (5). Sp1 interacts with GC-rich Sp1 binding sites in multiple promoters to regulate gene expression, and there are an increasing number of studies showing that Sp1 interacts with other nuclear proteins, including promoter-bound transcription factors, to attenuate tissue-specific expression of selected genes (1-3). For example, Sp1 and NFY cooperatively interact to regulate multiple genes through NFY-GC-rich motifs, and both proteins also physically interact (6 -10). Sp1 also binds estrogen receptor ␣ (ER␣) 1 and other members of the nuclear receptor family of transcription factors (11)(12)(13)(14)(15)(16)(17)(18)(19), and research in our laboratory has focused on the molecular mechanisms of the ligand-dependent activation of ER␣/Sp1 in breast and endometrial cancer cell lines (20 -31). Promoter analysis studies in breast cancer cells have identified GC-rich sites required for hormone activation of several genes including E2F1, DNA polymerase ␣, cyclin D1, insulin-like growth factor-binding protein 4, retinoic acid receptor ␣1, cathepsin D, vascular endothelial growth factor, c-fos, heat shock protein 27, bcl-2, thymidylate synthase, and adenosine deaminase (20 -27, 29 -31). Studies in other cell lines have also demonstrated a role for ER␣/Sp1 activation of the progesterone receptor, epidermal growth factor receptor, telomerase, and receptor for advanced glycation end products (32-35). Activation of ER␣/Sp1 does not require the DNA binding domain of ER␣ (promoter DNA-independent) and is primarily dependent on the activation function-1 (AF1) of ER␣ (30), whereas DNAdependent activation through ER binding to estrogen response elements (EREs) is primarily dependent on AF2 of ER␣. Recent studies have demonstrated that RNA interference through small inhibitory RNAs (iRNAs) targeted to endogenous or heterologous genes can be used to suppress intracellular expression of these genes in mammalian cells, and this technique is well suited for mechanistic studies on gene/protein function (36 -42). This study investigates the role of Sp1 protein in mediating hormone-responsiveness in MCF-7 cells using sequence-specific duplexes of 21 nucleotides targeted to Sp1 mRNA as well as Lamin B1 and the heterologous firefly luciferase gene (GL2) mRNAs. Transfection of iRNA for Sp1 (iSp1) decreases (40 -60%) the expression of nuclear Sp1 protein in ER-positive MCF-7 and ZR-75 human breast cancer cell extracts. In transfected cells, Sp1 protein is barely detectable by immunofluorescence, and both basal and estrogen-inducible transactivation is decreased in cells transfected with iSp1 and a GC-rich construct. These data, combined with results showing that iSp1 inhibits hormone-induced MCF-7 cell cycle progression from G 0 /G 1 to S phase, demonstrate that ER␣/Sp1...

Role of specificity protein transcription factors in estrogen-induced gene expression in MCF-7 breast cancer cells

“…In this study, we have also used RNA interference and small inhibitory RNAs (siRNAs) for Sp1 (iSp1), Sp3 (iSp3), and Sp4 (iSp4) to investigate the role of individual Sp proteins in ERa/Sp-mediated transactivation in MCF-7 cells. Previous studies in cancer cell lines have demonstrated the specificity and effectiveness of iSp1, iSp3, and iSp4 in Sp protein knockdown (Abdelrahim et al 2002, Higgins et al 2006a, and results in Fig. 1A-C illustrate that transfected iSp1, iSp3, and iSp4 significantly decreased Sp1, Sp3, and Sp4 mRNA levels in whole cell extracts from MCF-7 cells.…”

“…Cells were transfected with iNS1 (non-specific RNA), iSp1, iSp3 or iSp4 for 55-60 h, then treated with Me 2 SO or 10 nM E 2 for an additional 12 h and mRNA levels were determined in whole cell extracts by real-time PCR. Previous studies show that within this time period, there is a maximal decrease of Sp proteins using these siRNAs (Abdelrahim et al 2002, Higgins et al 2006a. Basal E 2 F1 mRNA levels were significantly decreased by transfecting iSp1, iSp3, and iSp4 by 40, 86, and 77% respectively, demonstrating the role of Sp proteins in mediating E 2 F1 mRNA expression.…”

“…Some of the genes that fall into this category are important for cell proliferation, angiogenesis, and nucleotide metabolism and include E 2 F1, retinoic acid receptor a (RARa), carbamoylphosphate synthetase/ aspartate transcarbamylase/dihydroorotase (CAD), bcl-2, DNA polymerase a, vascular endothelial growth factor (VEGF), VEGFR receptor 2 (VEGFR2), progesterone receptor, creatine kinase B, thymidylate synthase, insulinlike growth factor binding protein 4, epidermal growth factor receptor, receptor for advanced glycation end products, low density lipoprotein receptor, vitamin D receptor, pS2, LRP16, metastasis associated protein 3, and SK3 (Porter et al 1996, Duan et al 1998, Sun et al 1998, 2005, Qin et al 1999, Xie et al 1999, 2000, Byrne et al 2000, Salvatori et al 2000, Saville et al 2000, Stoner et al 2000, Tanaka et al 2000, Castro-Rivera et al 2001, Li et al 2001, Bruning et al 2003, Jacobson et al 2003, Khan et al 2003, Ngwenya & Safe 2003, Schultz et al 2003, 2005, Fujita et al 2004, Bardin et al 2005, Zhao et al 2005, Higgins et al 2006b). In addition, RNA interference studies with a small inhibitory RNA (siRNA) for Sp1 (iSp1) show that after transfection with iSp1, there was a significant decrease in hormone-induced G 0 /G 1 to S-phase progression, suggesting that ERa/Sp1 plays an important role in this process (Abdelrahim et al 2002).…”

The Aryl Hydrocarbon Receptor Mediates Degradation of Estrogen Receptor α through Activation of Proteasomes

“…The final concentration of siRNAs in each well was 140 nM. Thirty-six hours after transfection, cells were treated with DMSO, 10 nM E2, or 10 nM TCDD for 5 h, and nuclear extracts were obtained and analyzed by Western blot analysis for AhR, ER␣, and Sp1 proteins essentially as described elsewhere (1). Replicate (three) experiments were carried out to quantitate the effects of siRNA for the AhR on TCDD-induced downregulation of ER␣.…”

Specific downregulation of bcl-2 and xIAP by RNAi enhances the effects of chemotherapeutic agents in MCF-7 human breast cancer cells

“…19,20 Along this line, several recent studies have documented: (a) successful downregulation of BCR-ABL expression, resulting in increased apoptosis and decreased proliferation, in chronic myeloid leukemia cells; 21,22 (b) the MDR1 downregulation followed by chemosensitization of human pancreatic and gastric cell lines; 23 (c) combined inhibition of Bcl-2 and Raf-1 in human myeloid leukemia cells resulting in induction of apoptosis and chemosensitization. 24 Bearing in mind that RNAi is feasible in MCF-7 cell line [25][26][27] and that these cells are known to be relatively resistant to etoposide and doxorubicin, 28,29 the purpose of this study was to investigate if specific downregulation of bcl-2 or xIAP gene expression by RNAi sensitized MCF-7 cells to those chemotherapeutic drugs.…”