Date on Master's Thesis/Doctoral Dissertation

5-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Degree Program

Biochemistry and Molecular Biology, PhD

Committee Chair

Klinge, Carolyn

Committee Co-Chair (if applicable)

Cheng, Alan

Committee Member

Cheng, Alan

Committee Member

Kalbfleisch, Ted

Committee Member

Mitchell, Robert

Committee Member

Samuelson, David

Committee Member

Schaner-Tooley, Christine

Author's Keywords

miRNA-29b-1; miRNA-29a; breast cancer; endocrine-resistance; estrogen receptor; tamoxifen; RNA-seq

Abstract

Therapies targeting estrogen receptor α (ERα) including selective estrogen receptor modulators (SERMs), e.g., tamoxifen (TAM); selective estrogen receptor downregulators (SERDs), e.g., fulvestrant (ICI 182,780); and aromatase inhibitors (AI), e.g., letrozole, are successfully used in treating breast cancer patients whose initial tumor expresses ERα. Unfortunately, the effectiveness of endocrine therapies is limited as ~ 40% of breast cancer patients will eventually acquire resistance to them. The role of miRNAs in the progression of endocrine-resistant breast cancer is of keen interest in developing biomarkers and therapies to counter metastatic disease. This dissertation begins with a review on miRNAs implicated in breast cancer, their bona fide gene targets, and associated pathways promoting endocrine resistance. Although microRNAs are dysregulated in breast cancer, their contribution to endocrine-resistance is not yet fully understood. Previous microarray analysis identified miR-29a and miR-29b-1 as repressed by TAM in MCF-7 endocrine-sensitive breast cancer cells but stimulated by TAM in LY2 endocrine-resistant breast cancer cells. Here we examined the mechanism for the differential regulation of these miRs by TAM in MCF-7 versus TAM-resistant LY2 and LCC9 breast cancer cells and the functional role of these microRNAs in these cells. Knockdown studies revealed that ERα is responsible for TAM regulation of miR-29b-1/a transcription. Transient overexpression of miR-29b-1/a decreased MCF-7, LCC9, and LY2 proliferation and inhibited LY2 cell migration and colony formation but did not sensitize LCC9 or LY2 cells to TAM. Furthermore, TAM reduced DICER1 mRNA and protein in LY2 cells, a known target of miR-29. Supporting this observation, anti-miR-29b-1 or anti-miR-29a inhibited the suppression of DICER by 4-OHT. These results suggest that miR-29b-1/a have tumor suppressor activity in TAM-resistant cells and do not appear to play a role in mediating TAM resistance. The target genes mediating miR-29b-1/a tumor suppressor activity were unknown. Here, using RNA sequencing, we identify miR-29b-1 and miR-29a target transcripts in both MCF-7 and LCC9 cells. We find that miR-29b-1 and miR-29a regulate common and unique transcripts in each cell line. The cell-specific and common downregulated genes were characterized using the MetaCore Gene Ontology (GO) enrichment analysis algorithm. LCC9-sepecific miR-29b-1/a-regulated GO processes include oxidative phosphorylation, ATP metabolism, and apoptosis. Extracellular flux analysis of cells transfected with anti- or pre- miR-29a confirmed that miR-29a inhibits mitochondrial bioenergetics in LCC9 cells. qPCR and luciferase reporter assays also verified the ATP synthase subunit genes ATP5G1 and ATPIF1 as bona fide miR29b-1/a targets. Our results suggest that miR-29 repression of TAM-resistant breast cancer cell proliferation is mediated in part through repression of genes important in mitochondrial bioenergetics. There is a critical need to develop sensitive circulating biomarkers that accurately identify signaling pathways altered in breast cancer patients resistant to endocrine therapies. Serum miRNAs have the potential to serve as biomarkers of the progression of endocrine-resistant breast cancer due to their cancer-specific expression and stability. Exosomal transfer of miRNAs has been implicated in metastasis and endocrine-resistance. This dissertation ends with a review on miRNAs in breast tumors and in serum, including exosomes, from breast cancer patients that are associated with resistance to tamoxifen.

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