Author: David Kaiwen Lung

Publisher:

Published: 2020

Total Pages: 0

ISBN-13:

Estrogen receptor [alpha] (ER) plays a critical role in the growth and survival of breast cancer, which has made it an important target for endocrine therapies that ultimately inhibit its transcriptional function. However, in advanced stages of breast cancer, endocrine therapies decline in effectiveness, despite the majority of breast cancers maintaining their ER-positive status during disease progression. Two potentially key contributors to endocrine therapy resistance are the tumor microenvironment (TME) and the emergence of [ESR1] mutations that confer constitutive ER activity. To mediate endocrine therapy resistance, the TME and [ESR1] mutations affect the expression and function of the receptor, but it is unclear how these extrinsic and intrinsic factors co-exist to ultimately affect breast cancer cell behavior. In the work presented in this thesis, my goal focused on determining how the bone marrow microenvironment, the most common site of breast cancer metastasis, regulates ER expression and activity, and how these paracrine interactions affect cells with [ESR1] mutations. I determined that conditioned media (CM) from cancer-associated bone marrow stromal cells (BMSCs) and the BMSC cell line HS5 primarily transcriptionally repress [ESR1] expression to decrease overall ER expression in ER-positive breast cancer cell models MCF7 and T47D. Transcriptional repression of [ESR1] by HS5-CM involved rapid eviction of RNA polymerase II (Pol II) and potential inhibition of p300 activation on two major regulatory elements of [ESR1], the proximal promoter and a distal enhancer (ENH1). Additionally, HS5-CM treatment decreased the active enhancer mark H3K27Ac on ENH1, implicating ENH1 as a central regulatory element for driving [ESR1] transcriptional repression. BMSC-CM also caused co-repression of several neighboring genes within a 300 kb locus in addition to [ESR1]. Further studies assessed the impact of ER downregulation on the ER transactivation pathway by BMSCs. Despite detection of ER phosphorylation at serine 118 (pS118-ER) by HS5-CM, no increase in ER occupancy above basal levels was observed on strong ER binding sites nor changes in ERE activity. HS5-CM also repressed activated ER target genes, suggesting BMSCs have an overall repressive effect on ER transcriptional activity. In MCF7 cells expressing the [ESR1] mutations D538G or Y537S, HS5-CM was also able to significantly downregulate ER expression. However, activation of ER target genes remained significantly higher in cells expressing these mutations relative to cells expressing wild-type ER, despite treatment with HS5-CM. Furthermore, knockdown of a central co-activator p300 produced similar results with maintenance of significantly elevated ER target gene expression relative to cells expressing wild-type receptor. Together, these findings suggest that the TME affects breast cancer cell behavior by decreasing ER expression, potentially allowing other stimulated signaling pathways to control cell growth and survival. However, [ESR1] mutations appear to overcome the repressive effects of the TME on ER expression and transcriptional activity as well as the need for the co-activator p300 to mediate its transcriptional activity, demonstrating these mutations allow ER to maintain control over cancer cell behavior. These results ultimately contribute to our limited knowledge of the relationship between the TME and ER and provide the basis for our understanding on how [ESR1] mutations are affected by the metastatic TME.