Generation and Characterization of Tamoxifen-resistant ER+ Breast Cancer Cells Using Functional Genomics Strategy

Zahra Barati

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological and Environmental Sciences

Date of Award

Spring 2018

Abstract

Despite the success of tamoxifen as a targeted therapy in ER+ breast cancer, approximately 40% of patients are intrinsically resistant or acquire resistance during treatment. Signaling pathways conferring tamoxifen resistance has not been completely elucidated. Although genomic strategies for gene identification and validation--such as antisense blocking agent, small regulatory RNA, and ribozyme--were used for identifying antiestrogen resistant gene, prior knowledge of the target gene is prerequisite in such strategies. To address the issue, Dr.Cheriyath designed a novel high-throughput genome wide functional genomic strategy called GRIP (genome wide random insertion of promoter). Upon random integration into the host genome, GRIP vector links to a downstream endogenous gene and the splice donor of GRIP exon adopts the poly (A) signal of the downstream gene. Survival in selection media as well as the diverse phenotypes of GRIP-integrated MCF-7 cell clones suggested random activation of gene products by GRIP. Based upon preliminary results, we hypothesized that elevated expression of the gain-of-function genes that complement estrogen-signaling pathways confers antiestrogen resistance in ER+ breast cancer cells. To test this hypothesis, a GRIP library of 1 million clones was screened and 2 tamoxifen-resistant clones were identified. While parental MCF-7 cells failed to survive in estrogen-free media with 600 nM tamoxifen, GRIP-induced tamoxifen-resistant clones survived and proliferated in estrogen-free media with tamoxifen for 6 months. Tamoxifen-resistant sublines were more loosely packed and exhibited a high level of cell membrane projections. In immunofluorescence studies, compared to parental MCF- cells, estrogen receptor (ER) expression was significantly higher in GRIP-TAM-R (p < 0.0164). On the contrary, expression of intermediate filament vimentin and homophilic cell-cell adhesion receptor E-cadherin were significantly downregulated (p < 0.0001). Consequently, in migration assays, GRIP-TAM-R cells migrated two-fold faster than the MCF-7 cells (p < 0.0012) and had significantly higher numbers of migratory structures such as filopodia and lamellipodia (p < 0.0001). In summary, our results validate the ability of GRIP functional genomics strategy to establish TAM-R breast cancer cell clones and migratory characteristics of GRIP-TAM-R clones. By generating the tamoxifen-resistant cell line and understanding the characteristics of resistance and identifying the genes in future studies, we can provide therapeutic strategies to overcome tamoxifen resistance.

Advisor

Venu Cheriyath

Subject Categories

Biology | Life Sciences

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