INTRODUCTION
Estrogen is implicated as an important factor in stimulating breast cancer cell proliferation, and presence of estrogen receptor
(ER) is an indication of a good prognosis in breast cancer patients. Mcl-1 is an anti-apoptotic Bcl-2 family member that is often
overexpressed in breast tumors, correlating with poor survival. Estrogen has been previously shown to regulate Bcl-2 family
members, leading to an evasion of apoptosis, however the role of estrogen in regulating Mcl-1 expression is unclear. I
hypothesize that estrogen increases the expression of anti-apoptotic gene Mcl-1 through binding of ERα to a half estrogen
response element (ERE) site within the promoter of Mcl-1 gene. This leads to increased Mcl-1 expression in breast cancer cells,
ultimately contributing to an evasion of apoptosis.
METHODS
Four distinct breast cancer cell lines: MCF-7 and ZR-75, which both express ERα, and SKB-BR-3 and MDA-MB-231, which
do not express ERα, to investigate the role of estrogen plays in regulating Mcl-1 expression. Cells were grown in serum-starved
white media with charcoal-stripped FBS for five days prior to treatment with estrogen. Cells were treated with ERα antagonists
Tamoxifen and Fulvestrant in combination with estrogen. Also, siRNA knockdown of ERα was performed and mRNA
expression was evaluated. Chromatin immunoprecipitation (ChIP) was used to investigate if ERα binds to a specific ERE halfsite
within the Mcl-1 promoter. To further validate this data, a streptavidin pull-down assay was performed using a biotinlabeled
probe specific to this region.
RESULTS
In ERα positive cell lines, estrogen treatment increased Mcl-1 expression at both the protein and mRNA level. In two ERα
negative cell lines, SK-BR-3 and MDA-MB-231, estrogen failed to increase in Mcl-1 protein expression. ERα antagonists
decreased estrogen mediated Mcl-1 expression at both the protein and message level. Upon knockdown of ERα, Mcl-1 mRNA
expression after estrogen treatment was also decreased. ChIP showed an enrichment of ERα to the Mcl-1 promoter at a region
3683 bp upstream of the translation start site containing a half ERE site. Streptavidin-pull down assay showed both ERα and
transcription factor Sp1 bind to this region and mutation of the half ERE site eliminated this binding.
CONCLUSIONS
These results suggest that estrogen is involved in regulating Mcl-1 expression specifically through a mechanism
involving ERα. Ultimately, a better understanding of the role of estrogen in regulating Mcl-1 expression will determine
whether Mcl-1 is a valid molecular target for breast cancer therapy.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:MWU.1993/23211 |
| Date | 14 January 2014 |
| Creators | Schacter, Jennifer Leah |
| Contributors | Gibson, Spencer (Biochemistry and Medical Genetics), McManus, Kirk (Biochemistry and Medical Genetics) Myal, Yvonne (Pathology) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Detected Language | English |
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