Date of Award

10-2009

Document type

Thesis

Degree Name

MD (Medical Doctor)

First Supervisor

Dr. C.G. Concannon

Second Supervisor

Professor J.H.M. Prehn

Third Supervisor

Professor E.W. Kay

Keywords

Proteins - therapy, Colonic Neoplasms, Ubiquitins

Abstract

The ubiquitin-proteasome pathway (UPP) facilitates intracellular protein degradation, and as such has become the target of scientific work as many of the substrates of the proteasome are mediators of cell cycle pathways which become dysregulated during tumourigenesis. Modulation of this pathway through proteasomal inhibition represents a novel approach to the targeted treatment of malignant disease.

We investigated the expression of proteins involved in the UPP, using fresh frozen colonic tumour tissue and patient - matched normal adjacent tissue from a cohort of patients with Dukes' C colonic adenocarcinoma. (n=18) Subsequently, expression levels of ubiquitin and proteasome subunits were assessed in a large tissue microarray (TMA) of patients with Dukes' B and C colon cancer. (n=240)

Immunoblotting analysis demonstrated increased expression of ubiquitinylated proteins in tumour specimens compared with normal colonic specimens. Furthermore, parallel up-regulation in expression of regulatory subunits of the proteasome, Rpt4 and 20s core were observed.

Furthermore, we investigated treatment of colon cancer HCTl16 cells with theproteasome inhibitor, bonezomib and demonstrated caspase dependent apoptosis maximal at 24 hours. Moreover, flow cytometry analysis of a panel of five colon cancer cell lines treated with bortezomib, revealed that these cells were highly sensitive to proteasome inhibition.(p<0.05) In addition, we demonstrated that siRNA silencing of Rpt4 induces apoptosis and prevents proliferation of colon cancer cells.(p<0.05)

Analysis of our TMA, yielded interesting results. Most notably, Rpt4 was determined to be an independent prognostic marker for reduced disease free and overall survival in Dukes B patients. (Disease free survival :*p=0.0217, hazard ratio 0.6) Overall survival: *p=0.0467, hazard ratio 0.65)

Our data supports an overstressed UPP with increased levels of ubiquitinylatedproteins despite elevated proteasomal levels. Moreover, proteasomal inhibition induces caspase dependent apoptosis in vitro. Herein, proteasomal inhibition represents a novel chemotherapeutic entry point for the treatment of colon cancer.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

File Size

10,119 KB

Comments

A thesis submitted to Royal College of Surgeons in Ireland, and presented to the Faculty of Medicine for the degree of Doctor of Medicine October, 2009.

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