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MECHANISMS OF RESISTANCE IN ESTROGEN RECEPTOR ALPHA POSITIVE BREAST CANCER

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Abstract

Outcomes from recent studies have led to the development of novel therapeutic treatments for endocrine resistant estrogen receptor alpha positive (ER+) breast cancer (BC) patients. While new therapies, such as cyclin D1-CDK4/6 inhibitors, are extremely beneficial to combat endocrine resistance, a better understanding of endocrine resistance mechanisms is needed to identify new therapeutic options that will provide additional benefit to patients. For this reason, the goal of my thesis work was to contribute to the knowledge of endocrine resistance mechanisms by validating and better characterizing several novel resistance mechanisms that we have identified in the Coonrod lab. Previous studies (Horibata, Rice, Mukai, et al., 2018; Horibata, Rice, Zheng, et al., 2018; Morandi et al., 2013; Plaza-Menacho et al., 2010), revealed that GDNF-RET signaling and early growth response 1 (EGR1) may play a role in the development of endocrine resistance. However, to our knowledge, the mechanism of how this signaling pathway promotes resistance was unknown. Outcomes from my studies found that GDNF-RET signaling utilizes the MAPK signaling pathway, where the transcription factor ELK-1 is phosphorylated, binds to the EGR1 promoter, and induces target gene transcription. I then found, that following EGR1 upregulation, EGR1 directly binds to the GDNF promoter and induces its transcription. Furthermore, I found that EGR1 also binds to the CCND1 promoter and upregulates transcription. Together, this work suggests that a sustained GDNF-RET-EGR1 positive feedback loop promotes resistance to the endocrine therapy tamoxifen (TAM) by upregulating CCND1, which is important for cell cycle progression, therefore leading to tumor progression. The second major goal of my thesis work was to identify and validate currently unknown mechanism(s) of resistance using a genome-wide screen; whereby protein-coding genes were individually upregulated in individual tamoxifen sensitive (TamS) cells using CRISPRa (inducing) sgRNA constructs. These cells underwent prolonged TAM treatment to enrich for TamS cells that contained individual upregulated genes and continued to proliferate in the presence of TAM. Enrichment of these cells identified genes potentially important in TAM resistance. We found multiple enriched genes and signaling pathways that have been previously implicated in other cancers as well as BC. One signaling pathway, c-KIT signaling, was further investigated due to the observations that (a) c-KIT inhibitors are currently used to treat other cancers, and (b) the lack of information on c-KIT signaling in BC resistance. After demonstrating that upregulating c-KIT promoted TAM resistance, we next showed that c-KIT signaling upregulated three ABC efflux transporters, of which two correlated with a worse prognosis in BC patients undergoing endocrine therapy (Lánczky & Győrffy, 2021). This result led us to investigate whether dual TAM and Gleevec (a KIT inhibitor) would provide additional therapeutic benefit. We found that TAM and Gleevec treatments results in both additive and synergistic inhibitory effects, suggesting that a combination therapy could be beneficial in patients that become resistant to endocrine therapy. At a mechanistic level, our results suggest that c-KIT signaling promotes TAM resistance through upregulation of ABC efflux transporters, which then facilitate TAM efflux, thereby reinitiating ER signaling and promoting cell proliferation in the presence of TAM.

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168 pages

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Date Issued

2022-05

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Keywords

Breast Cancer; C-KIT; CRISPRa Screen; Endocrine Resistance; Estrogen receptor alpha; GDNF-RET signaling

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Union Local

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Committee Chair

Coonrod, Scott A.

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Committee Member

Cerione, Richard A.
Danko, Charles G.

Degree Discipline

Biomedical and Biological Sciences

Degree Name

Ph. D., Biomedical and Biological Sciences

Degree Level

Doctor of Philosophy

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Government Document

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dissertation or thesis

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