Date of Award

2014

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Dr Caoimhín Concannon

Second Supervisor

Professor Jochen Prehn

Funder/Sponsor

HRB in Ireland who funded this project under grant No PHD/2007/11

Abstract

The deleterious effects o f physiological and environmental stresses which provoke cell damage is defended by a fundamental cellular mechanism, the heat shock response (HSR), mediated by heat shock factor 1 (HSF1). Evidence suggests th a t genetic depletion of HSF1 dramatically protects mice from tumor formation mediated by RAS over-activation or p53 inactivation both commonly mutated in colorectal cancer. Here we studied the effects of silencing HSF1 in HCT116 wt, HCT p53 - /- and HKE3 colon cancer cell lines with varying p53 and KRAS statuses. Targeting HSF1 expression using shRNA increased cell death and decreased clonogenic survival o f HCT116 cells independent o f th e ir genetic background.

Constitutively active HSF1 significantly protected HCT116 w t cells from treatment with 5-FU/Oxal and increased cell viability in a normal colon epithelial cell line when treated with chemotherapeutic agents. Using both a monolayer and spheroid culture systems we could demonstrate th a t the pharmacological inhibitor of HSF1, triptolide, significantly reduced the cell viability in all the colon cancer cell lines examined in a predominately HSF1 dependent manner. In the clinical setting, a patient cohort examined exhibited a trend of increased levels of HSF1 being associated

with a favourable outcome. Current literature has highlighted th a t HSF1 is involved in regulating genes outside of its classical role as a mediator of the HSR. To identify genes regulated by HSF1 in the different genetic backgrounds, RNA sequencing was performed after a viral knockdown of HSF1 for 48 h. In total 169 genes were upregulated and 233

downregulated irrespective of genetic background including 6 upregulated and 20 downregulated across all 3 cell lines, most of whom were novel genes not previously known to be dependent on HSF1 signalling. Genes upregulated were predominantly linked to histone modification, whilst those downregulated included genes involved in metabolism and pH regulation. Collectively our data supports a hypothesis th a t HSF1 is involved in the malignant transformation o f colon cancer. In light of the differential gene expression identified, HSF1 gene signatures could be targeted fo r the treatment of colon cancer and fo r the prediction of patient outcome.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

16.0 MB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2014.

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