University of Louisville
The Effect of COUP-TFII-nucleolin Interaction on RARβ2 Expression in Human Breast Cancer Cells
Institution
University of Louisville
Faculty Advisor/ Mentor
Carolyn Klinge
Abstract
Tamoxifen resistance remains a major problem in the treatment of breast cancer patients. COUP-TFII, an orphan nuclear receptor, expression is reduced in tamoxifen-resistant human breast cancer cells. The mechanism of the involvement of COUP-TFII in tamoxifen resistance is currently not well understood. Nucleolin is a protein that interacts with COUP-TFII in MCF-7 human breast cancer cells. The goal of the current study was to examine COUP-TFII-nucleolin interaction in two human breast cancer cell lines: MCF-7 and T47D, and to determine how this interaction regulates retinoic acid receptor β2 (RARβ2) expression. Interaction of COUP-TFII with nucleolin was confirmed in the nuclear extract of both cell lines by coimmunoprecipitation. To examine how nucleolin impacts COUP-TFII-regulated RARβ2 expression, MCF-7 cells were transiently transfected with a luciferase reporter bearing the RARβ2 gene promoter and increasing amounts of COUP-TFII and/or nucleolin. COUP-TFII increased RARβ2-luc promoter activity and nucleolin inhibited COUP-TFII induced luciferase activity. However, nucleolin inhibited both Renilla and firefly luciferase activities, suggesting that nucleolin inhibits transcription factor-promoter interactions. Next, we optimized siRNA knockdown of nucleolin in MCF-7 cells and confirmed nucleolin knockdown at the mRNA and protein levels 48 h after siNucleolin transfection. The results showed a 70% and 20% reduction in nucleolin mRNA and protein. Future experiments will determine the effect of nucleolin knockdown on COUP-TFII regulated RARβ2 expression in MCF-7 and T47D breast cancer cells. We anticipate that experiments with siNucleolin will elucidate the role of COUP-TFII-nucleolin interaction in regulating expression of the tumor suppressor RARβ2 in breast cancer cells.
The Effect of COUP-TFII-nucleolin Interaction on RARβ2 Expression in Human Breast Cancer Cells
Tamoxifen resistance remains a major problem in the treatment of breast cancer patients. COUP-TFII, an orphan nuclear receptor, expression is reduced in tamoxifen-resistant human breast cancer cells. The mechanism of the involvement of COUP-TFII in tamoxifen resistance is currently not well understood. Nucleolin is a protein that interacts with COUP-TFII in MCF-7 human breast cancer cells. The goal of the current study was to examine COUP-TFII-nucleolin interaction in two human breast cancer cell lines: MCF-7 and T47D, and to determine how this interaction regulates retinoic acid receptor β2 (RARβ2) expression. Interaction of COUP-TFII with nucleolin was confirmed in the nuclear extract of both cell lines by coimmunoprecipitation. To examine how nucleolin impacts COUP-TFII-regulated RARβ2 expression, MCF-7 cells were transiently transfected with a luciferase reporter bearing the RARβ2 gene promoter and increasing amounts of COUP-TFII and/or nucleolin. COUP-TFII increased RARβ2-luc promoter activity and nucleolin inhibited COUP-TFII induced luciferase activity. However, nucleolin inhibited both Renilla and firefly luciferase activities, suggesting that nucleolin inhibits transcription factor-promoter interactions. Next, we optimized siRNA knockdown of nucleolin in MCF-7 cells and confirmed nucleolin knockdown at the mRNA and protein levels 48 h after siNucleolin transfection. The results showed a 70% and 20% reduction in nucleolin mRNA and protein. Future experiments will determine the effect of nucleolin knockdown on COUP-TFII regulated RARβ2 expression in MCF-7 and T47D breast cancer cells. We anticipate that experiments with siNucleolin will elucidate the role of COUP-TFII-nucleolin interaction in regulating expression of the tumor suppressor RARβ2 in breast cancer cells.