Oestrogen-dependent regulation of gene expression by cohesin in breast cancer
Two-thirds of human breast carcinomas test positive for oestrogen receptor α (ERα), which predominantly mediates oestrogenic actions in the normal breast as well in breast tumour cells. Treatment of hormone-sensitive tumours has generally relied on anti-oestrogen therapy. However, ~30% of such tumours do not respond to hormone therapy, and a considerable proportion of tumours develop treatment resistance, ultimately leading to cancer recurrence. Therefore, understanding the regulatory mechanisms controlling tumour cell proliferation downstream of oestrogen is crucial for development of new treatment strategies. Oestrogen Receptors (ERs) function as part of multi-protein complexes, and the overall cellular response to oestrogen is dependent on cell type-specific functional partners. A previous study identified that ERα shares coincident chromatin binding with the cohesin protein complex in oestrogen-stimulated breast cancer cells. The overlap was striking at oestrogen-regulated genes. The genome-wide concordance in ERα and cohesin binding led to the hypothesis that cohesin facilitates oestrogen-dependent transcription. Following on from this, my research focused on investigating the transcriptional role of cohesin in ER-positive MCF7 breast cancer cells. Cohesin’s role in ER-transcription was tested by cDNA microarray analysis of cohesin (RAD21)-depleted MCF7 cells. Microarray analysis revealed that cohesin (RAD21) depletion affected transcription of a subset of oestrogen-responsive genes, either activating or impeding transcription depending on the gene target. Oestrogen-responsive genes most significantly influenced by cohesin were over-represented in ErbB1 and PI3K/mTOR, pathways associated with breast cancer progression and survival. I then went on to investigate the mechanism of cohesin’s divergent transcriptional control for two of the microarray-identified candidate genes. Oestrogen activation of SOX4 was robustly enhanced whereas expression of IL20 was significantly reduced in cohesin (RAD21)-depleted cells. Chromatin immunoprecipitation followed by quantitative PCR analyses revealed that RAD21 depletion enriched ERα binding to putative enhancers of SOX4, whereas ERα and RNA Polymerase II binding were weakened at the promoter of IL20, corresponding with the direction of expression of these genes. I next investigated if a cohesin inhibitor PCI-34051, which is predicted to target cohesin by blocking SMC3 deacetylation, was able to recapitulate transcriptional dysregulations observed upon RAD21-mediated depletion of cohesin. Despite accumulation of acetylated-SMC3, oestrogen responsiveness of selected cohesin-dependent genes remained unaffected. This highlighted that chemical inhibition of SMC3 deacetylation was not comparable with RNAi-mediated depletion of RAD21. Rather, PCI-34051 treatment led to concentration-dependent cell cycle delay, suppression of cell proliferation and induction of cell death in MCF7 cells. Taken together, these studies have revealed that cohesin can both positively and negatively influence ER-dependent transcription in a gene-dependent manner. Since cohesin-dependent genes participate in breast cancer signalling networks, results from this study suggest that cohesin function might be important for ER-positive breast cancer. PCI-34051 was unable to target the oestrogen-specific transcriptional role of cohesin in MCF7 cells; however, PCI-34051 was significantly oncostatic. Thus, thorough functional characterisation in additional ER-positive model systems would be necessary before considering PCI-34051 as a potential anti-cancer agent for ER-positive breast cancers.
Advisor: Horsfield, Julia; Eccles, Michael
Degree Name: Doctor of Philosophy
Degree Discipline: Pathology
Publisher: University of Otago
Keywords: Cohesin; Transcription; ER; Breastcancer
Research Type: Thesis