The identification of biomarkers of chemotherapy resistance in breast cancer using comparative proteomics

Hodgkinson, Victoria C.

September 2011

Thesis or dissertation

© 2011 Victoria C Hodgkinson. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.

Background:Chemotherapy resistance is a major obstacle in effective neoadjuvant treatment for locally advanced breast cancer. The ability to predict tumour response would allow chemotherapy administration to be directed towards only those patients who would benefit, thus maximising treatment efficiency. This project aimed to identify predictive protein biomarkers associated with chemotherapy resistance, using proteomic analysis of fresh breast cancer tissue samples.Materials and Methods:Chemotherapy-sensitive (CS) and chemotherapy-resistant (CR) tumour samples were collected from breast cancer patients who received neoadjuvant therapy consisting of epirubicin with cyclophosphamide followed by docetaxel. Comparative proteomic analysis was performed, to identify differentially expressed proteins (DEPs) between CS and CR invasive ductal carcinoma samples, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with MALDI-TOF/TOF mass spectrometry and antibody microarray analysis. DEPs were submitted to Ingenuity Pathway Analysis (IPA) to identify any canonical pathway links, confirmed using western blotting and clinically validated in a pilot series of archival breast cancer samples, from patients treated with neoadjuvant chemotherapy.Results:Five datasets were generated by antibody microarray analysis, revealing 38 targets. Of these, 7 DEPs were identified in at least 2 datasets and these included 14-3-3 theta/tau, BID and Bcl-xL. Three datasets were generated using 2D-PAGE with MALDI-TOF/TOF MS, containing 132 unique DEPs. These included several isoforms of 14-3-3 proteins. The differential expression of 14-3-3, BID and Bcl-xL was confirmed by immunoblotting in samples used for the discovery phase. Clinical validation using immunohistochemical analysis of archival breast cancers revealed 14-3-3 theta/tau and tBID to be significantly associated with chemotherapy resistance.Discussion:The use of comparative proteomic techniques using fresh clinical tumour samples, for the search for putative biomarkers of chemotherapy resistance has been successful. Two DEPs; 14-3-3 theta/tau and tBID have passed through all stages of the biomarker discovery pipeline, and present themselves as putative predictive biomarkers of neoadjuvant chemotherapy resistance in breast cancer.

Postgraduate Medical School, The University of Hull
Cawkwell, Lynn; Lind, Michael
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