Date on Master's Thesis/Doctoral Dissertation

12-2012

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Committee Chair

Klinge, Carolyn Muriel

Author's Keywords

Breast cancer; endocrine; microRNA; resistance; Tamoxifen

Subject

Breast--Cancer--Genetic aspects

Abstract

MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level by repressing translation or stimulating mRNA degradation. In this study, I tested the hypothesis that miRNAs are differentially expressed in antiestrogen-sensitive MCF-7 versus -resistant L Y2 human breast cancer cells. Microarray analyses identified 97 miRNAs that are differentially expressed between two estrogen receptor alpha (ERa) -positive human breast cancer cell lines: endocrine-sensitive MCF-7 versus -resistant L Y2 cells under basal conditions. Opposite expression of miRs-lOa,-21, -22, -12Sb, -181, -200a, -200b, -200c, -221, and -222 was confirmed between MCF-7 and L Y2 cells. The ER antagonist ICI 182,780 (fulvestrant or Faslodex) generally blocked the effect of estradiol E2 and 4-hydroxytamoxifen (4-OHT) regulated miRs, i.e .. , miR-lOa, miR-21, miR-22, miR-200a, miR-221, and miR-222, indicating that these responses in MCF-7 cells are ER-mediated. Time dependent variation in basal (ethanol, the vehicle), E2, and 4-0HT regulation of the top 8 miRNAs was detected in MCF-7 cells. Bioinformatic analyses to impute the biological significance of the identified miRNAs by identifying their computationally predicted target genes in the human genome using TargetScan, Pic Tar, and the Sanger miRBase Targets databases was performed. Thirty six putative mRNA targets were identified. Agreement in the direction of anticipated regulation was detected for 12 putative targets. These miRNAs showing opposite expression between these two breast cancer cell lines may be involved in endocrine resistance. MiR-200 family includes two clusters i.e. miR-200 a/200 b/ 429 and miR-200c/141 encoded on chromosome 1 and chromosome 12, respectively. Lower miR-200a, miR-200 b and miR-200c expression was observed in estrogen-independent LCC1 and endocrine-resistant LCC2, LCC9, and LY2 compared to the parental, endocrine-sensitive MCF-7 human breast cancer cell line. ZEB 1 protein was found to be expressed in endocrine-resistant LY2 cells but not in endocrine-sensitive MCF-7 cells. L Y2 cells did not express E-cadherin, a ZEB 1 target which is a marker for epithelial phenotype. This is the first demonstration that L Y2 cells have undergone EMT as part of their endocrine-resistant phenotype. Concomitant with miR-200 decrease, there was an increase in ZEB 1 mRNA expression m L Y2 cells. Overexpression of miR-200b or miR-200c in LY2 cells changed the cellular morphology from a mesenchymal to an epithelial appearance and sensitized cells to inhibition by 4-0HT and fulvestrant. These studies indicate that reduced expression of miR-200 and a corresponding increase in ZEB 1 protein is an indicator of endocrine-resistance in breast cancer cells.

Download

Share

COinS