MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERalpha) in regulation of the MUC1 gene expression

Abstract

Background: The MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter.

Results: Using TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERalpha, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed.

Conclusion: Analysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERalpha in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy.

Figure 1

Expression of the MUC1 isoform specific mRNA in mouse DA3 cells transfected with plasmids pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 and ppolyIII. DA3 cells were transfected with plasmid DNA. Cells transfected with ppolyIII were used as negative controls. DA3 cells stably transfected with MUC1/SEC, MUC1/TM and MUC1/Y cDNA were used as positive control. Total RNA was extracted 48 hrs after transfection and cDNA was synthesized. PCR amplification of MUC1 isoform specific fragments were performed using isoform specific primers. PCR products were separated by electrophoresis on 1.2% agarose gel and stained with ethidium bromide. A – Schematic structure of the pDpr, pDprΔ2154, pDprΔ2446, pDprΔ2839 plasmids and the table of the MUC1/SEC, MUC1/TM and MUC1/Y mRNA expression in transfected DA3 cells. B – RT-PCR of the MUC1 isoform specific RNA extracted from DA3 cells transiently transfected with indicated plasmids. Lane M-DNA marker; lanes 1, 4, 7, 10 and 13 (negative control) – PCR performed with MUC1/SEC specific primers; lanes 2, 5, 8, 11 and 14 (negative control) – PCR performed with MUC1/TM specific primers; lanes 3, 6, 9, 12 and 15 (negative control) – PCR performed with MUC1/Y specific primers. Positive control: DA3 cells stably transfected with MUC1 isoform specific cDNA. Lane SEC – cells expressed MUC1/SEC; lane TM – cells expressed MUC1/TM and lane Y – cells expressed MUC1/Y RNAs.

Figure 2

Expression of the MUC1 isoform specific mRNA in human mammary epithelial cells detected by RT-PCR. Total RNA from human MCF-7, T47D and MDA-231 cells and RNA from mouse DA3 cells stably transfected with MUC1/SEC (lane – SEC), MUC1/TM (lane – TM) and MUC1/Y (lane – Y) cDNA (positive controls) were extracted and RT-PCR amplification of the MUC1 isoform specific fragments were performed. Lanes 1, 4, 7, 10, 13, 16 and SEC – PCR performed with MUC1/SEC specific primers; lanes 2, 5, 8, 11, 14, 17 and TM – PCR performed with MUC1/TM specific primers; lane 3, 6, 9, 12, 15, 18 and Y – PCR performed with MUC1/Y specific primers.

Figure 3

Effect of estrogen and 4-hydroxytamoxifen (4-OHT) on expression of MUC1 isoform specific mRNA in T47D and MCF7 cells analyzed by RT-PCR. Human T47D (clone 10) and MCF7 cells were cultured in DMEM medium with or without estrogen and 4-OHT supplements as described in "Materials and Methods". Total RNA was extracted and MUC1 isoform specific RT-PCR was performed. Lane M-DNA marker; lane 1 – cells grown in the medium without estrogen; lanes 2, 3 and 4 – cells grown with 0.1 nM, 1 nM and 10 nM estrogen, respectively; lanes 5, 6 and 7 – cells grown with 10 nM estrogen and with 10 nM, 100 nM and 1 μM 4-OHT supplements, respectively.

Figure 4

ERα binding to EREs detected in the MUC1 promoter. The EMSA of the complexes (C1, C2 and C3) developed by ERα from T47D nuclear lysate and ³²P-labeled oligonucleotides containing the MUC1 promoter EREs (for details see "Materials and Methods"). A: Binding of ERα to ³²P-labeled oligonucleotides ERE1, ERE2, ERE3, ERE4 and ERE5, containing 1/2 consensus core sequence of the classical ERE, and to palindrome like ERE (putative ERE-6 and Vit ERE). Lanes 1, 3, 5, 7, 10, 12, 19 and 26 – complexes developed in absence of anti-ERα Ab; lanes 2, 4, 6, 8, 11, 13, 20 and 27 – partial or complete inhibition of complex formation by anti-ERα Ab; lanes 14, 21 and 28 – binding of ERα to ³²P-ERE2, Vit-ERE and ERE6, respectively, in the presence of "cold" Oct1 oligonucleotide; lanes 15–18 – binding of ERα to ³²P-ERE2 in presence of decreasing amount of the "cold" ERE2; lanes 22–25 – binding of ERα to ³²P-Vit ERE in presence of decreasing amount of the "cold" Vit ERE (* indicates an intermediate complex developed when binding reaction was kept on ice); lanes 29–32 – binding of ERα to ³²P-ERE6 in presence of decreasing amount of the "cold" ERE6. B: Effects of anti-ERα Ab and normal rabbit serum (NRS) on ERα binding to MUC1 specific and mutated EREs. Lanes 1, 6 and 11 – binding of ERα in absence of anti-ERα Ab; lanes 2, 7 and 12 – partial or complete inhibition of binding complexes by anti-ERα Ab; lanes 3, 8 and 13 – binding of ERα in presence of NRS; lanes 4, 9 and 14 – binding of ERα to mutated ERE in absence of anti-ERα Ab; lanes 5, 10 and 15 – binding of ERα to mutated ERE in presence of anti-ERα Ab.