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GLYATL1 PROMOTES ENDOCRINE THERAPY RESISTANCE IN BREAST CANCER

Sofyali, Emre

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Abstract

Breast cancer is the most frequently diagnosed malignancy among women. Due to its molecular heterogeneity, a generalized therapy is not possible. Nuclear estrogen receptor-α (ER-α) is overexpressed in the majority of breast tumors. ER-positive patients benefit from endocrine therapy that abrogates estrogen-induced tumor growth. However, approximately half of the patients do not respond or relapse due to de novo or acquired resistance against therapy. Occurrence of resistance is a drawback for long-term efficacy of endocrine therapy and leads to poor prognosis. The mechanisms underlying acquisition of endocrine therapy resistance, however, remain elusive. Recently, epigenetic reprogramming has been proposed as a means to render the tumor cells refractory to treatment. Therefore, the aims of this project were to uncover novel targets that confer resistance and to elucidate the involvement of epigenetics in this process. To this end, two ER-positive cell lines (MCF7 and T47D) were utilized to recapitulate endocrine therapy resistance in vitro by treating them either with tamoxifen (TAMR) or depriving them of estrogen (LTED). I identified GLYATL1 (glycine-N-acyltransferase like 1) as a highly de-regulated gene as revealed by RNA-seq and ATAC-seq integrative analysis comparing resistant cell lines to the sensitive parental. GLYATL1 encodes for an enzyme that catalyzes the transfer of an acyl group to glutamine. I showed that knockdown of GLYATL1 sensitizes resistant cell lines while GLYATL1 overexpression renders sensitive luminal cells resistant to endocrine therapy. Furthermore, I found GLYATL1 is involved in acetylation of histone residues H3K9 and H3K14 since the knockdown of GLYATL1 led to a decrease in these two histone marks in resistant MCF7 cells. Moreover, I showed the expression of GLYATL1 to be regulated in these cells by methylation, growth factor receptor HER2, and transcription factors ERα, GATA3 and p300. CRISPR/dCas9-mediated epigenetic editing method was adopted to validate the involvement of methylation in GLYATL1 regulation. This method is a repurposed version of CRISPR/Cas9 system where Cas9 is catalytically inactive and fused to catalytic domain of epigenetic enzymes. Combined with sgRNAs, these effectors can be recruited to target regions to modulate the epigenetic landscape, thereby altering gene expression. Furthermore, I utilized this method of epigenetic editing to investigate endocrine therapy resistance involvement of other genes such as CD44 and BAMBI, expression of which were also found to be elevated in resistant cells compared to parental. I showed that targeting promoter regions of CD44 and BAMBI with dCas9-p300 yielded upregulation of both genes whereas dCas9-G9a combination led to a downregulation which resulted in retarded proliferation in LTED cells. Moreover, altering expression of BAMBI elicited similar changes in CD44 expression further proving CD44 as a direct target gene of the Wnt signaling pathway, for which BAMBI acts as an activator. In conclusion, my results demonstrate the importance of GLYATL1 in initiation and maintenance of endocrine therapy resistance and identify its involvement in H3K9 and H3K14 acetylation. This study demonstrates the potential of epigenetic reprogramming mediated regulation of target gene expression as a novel method of therapeutic intervention.

Item Type: Dissertation
Supervisor: Wiemann, Prof. Dr. Stefan
Date of thesis defense: 9 September 2019
Date Deposited: 25 Sep 2019 08:02
Date: 2020
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > German Cancer Research Center (DKFZ)
Subjects: 500 Natural sciences and mathematics
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