The anticipatory unfolded protein response and estrogen receptor mutations in breast cancer

Yu, Liqun

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https://hdl.handle.net/2142/104998 Copy

Description

Title

The anticipatory unfolded protein response and estrogen receptor mutations in breast cancer

Author(s)

Yu, Liqun

Issue Date

2019-04-10

Director of Research (if dissertation) or Advisor (if thesis)

Shapiro, David J.

Doctoral Committee Chair(s)

Shapiro, David J.

Committee Member(s)

• Chen, Lin-Feng
• Nelson, Erik R.
• Zhang, Kai

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Estrogen receptor mutations
• unfolded protein response
• epidermal growth factor
• Src
• Myc

Abstract

Estrogen, acting via estrogen receptor α (ERα), stimulates cancer cell proliferation and metastasis. Endocrine therapy targeting E2:ERα activity often leads to development of antiestrogen resistance. Approximately 30% of patients who have metastatic endocrine therapy resistant breast cancer express ERα mutations. Two of the most common and therefore widely studied mutations are ERαY537S and ERαD538G. Patients whose metastatic breast tumors express Y537S or D538G mutations have 1 year and 6 months shorter median survival time than patients whose metastatic tumors express wild-type ERα. To better characterized the aggressive phenotypes of the ERα mutations in breast cancer cells, we used CRISPR-Cas9 technology to replace wild-type ERα in T47D, human breast cancer cells, with the most common mutations, ERαY537S and ERαD538G. The mutant cells exhibit partially estrogen-independent and antiestrogen resistant gene expression and cell proliferation. A novel invasion-dissociation-rebinding (IDR) assay demonstrated that the mutant cells have a higher tendency to dissociate from invasion sites and rebind to a second site. Compared to wild type breast tumors, mutant tumors exhibited dramatic increases in lung metastasis. The ERαY537S mutation further enhanced the metastatic capability of the breast tumors. Gene set enrichment analysis (GSEA) showed Myc target pathways are highly induced in mutant cells. Moreover, chromatin immunoprecipitation showed constitutive, fulvestrant-resistant, recruitment of ERα mutants to the Myc enhancer region, resulting in estrogen-independent Myc overexpression in mutant cells and tumors. Knockdown and overexpression experiments showed Myc is necessary and sufficient for ligand-independent proliferation of the mutant cells but had no effect on metastasis-related phenotypes. Other than gain-of-function mutations, breast cancer cells can develop therapy resistance via recently described pathways of hormone action. Our laboratory recently revealed that estrogen acting through ERα, rapidly phosphorylates and activates phospholipase C γ (PLCγ) resulting in transient calcium release from endoplasmic reticulum and mild activation of the unfolded protein response. I identified that this anticipatory UPR is a conserved pathway shared by different mitogenic hormones including epidermal growth factor, estrogen and progesterone. EGF rapidly induced a calcium increase in the cytosol and moderate activation of the IRE1α and ATF6α arms of the UPR, resulting in induction of BiP chaperone. Knockdown or inhibition of EGF receptor (EGFR), PLCγ or IP3 receptor (IP3R) blocks the increase in intracellular Ca2+. While blocking the increase in intracellular Ca2+ by locking the IP3R calcium channel with 2-APB had no effect on EGF activation of the ERK or AKT signaling pathways, it abolished EGF-mediated immediate early gene expression, suggesting EGF stimulated calcium efflux and signaling transduction are two independent pathways and are both essential for EGF regulated gene expression. Knockdown of ATF6α or XBP1, which regulate UPR-induced chaperone production, inhibited EGF stimulated cell proliferation. These data highlight the importance of anticipatory UPR pathway in the normal actions of mitogenic hormones. Unlike EGFR which functions as a receptor tyrosine kinase, ERα cannot directly phosphorylate PLCγ. Using the small molecule ERα biomodulator, BHPI, which uses the same pathway as E2 and induces toxic hyperactivation of the anticipatory UPR, we applied unbiased long-term-selection on ERα positive breast cancer cells and isolated T47D and MCF-7 cells that proliferate in the presence of a lethal concentration of BHPI. We showed that 4 out of 11 T47D and almost all MCF-7 BHPI resistant clones have reduced Src expression. Src overexpression by virus transduction in resistant clones restored sensitivity to BHPI. Furthermore, in wild-type cells, several-fold knockdown of Src, but not of ERα, strongly blocked BHPI-mediated UPR activation and subsequent HMGB1 release and necrotic cell death. Supporting Src kinase linking estrogen and progesterone to activation of the anticipatory UPR, we identified extranuclear complexes of ERα:Src:PLCγ and progesterone receptor:Src:PLCγ. Thus, Src plays a previously undescribed pivotal role in activation of the tumor protective anticipatory UPR, thereby increasing the resilience of breast cancer cells.

Graduation Semester

2019-05

Type of Resource

text

Permalink

http://hdl.handle.net/2142/104998

Copyright and License Information

Copyright 2019 Liqun Yu

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