Identification of predictive biomarkers of resistance to radiotherapy in rectal cancer

Mehmood, Sajid

Medicine
December 2013

Thesis or dissertation


Rights
© 2013 Sajid Mehmood. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.
Abstract

Introduction and Aims:
Neoadjuvant radiotherapy (RT) provides local control of disease in rectal cancer, however, the ability to predict response to RT is limited. The aim of this study was to establish a novel radioresistant (RR) rectal cancer cell line model and identify putative biomarkers of radioresistance using a microarray-based comparative proteomic platform.

Methods:
The inherent radiosensitivity of SW-837 and HRA-19 rectal adenocarcinoma parental cells was initially assessed. Following irradiation at 0, 2, 4, 6, 8 and 10 Grey (Gy) single doses using a linear accelerator, modified colony counting assays were performed. Dose response curves (DRCs) were plotted from calculated survival fraction (SF) values. To induce radioresistance, parental SW-837 and HRA-19 cells were irradiated in 8 and 4 Gy fractions, respectively, to a total dose of 48 Gy to generate novel sub-lines (termed SW-837 RR and HRA-19 RR). Following comparison of the DRCs from untreated parental cells and novel RR cells, total protein was extracted from radiosensitive parental (PN) cells and novel RR cells. Protein lysates from respective PN and RR cells were co-incubated onto Panorama antibody microarray (AbMa) slides containing 725 antibodies. Proteins which demonstrated at least 1.8-fold difference in expression between PN and RR cells were considered significant. The proteins with consistent differential expression and a putative role in radioresistance were selected for validation by immunohistochemistry (IHC), as a pilot study, in an archival series of 33 rectal cancer tissues categorised into ‘good response’ and ‘poor response’ groups based on tumour regression grading following long course chemoradiotherapy.

Results:
The comparison of DRCs revealed SW-837 RR cells to be significantly more radioresistant at 4, 6, 8 and 10 Gy and HRA-19 RR cells at 4, 6 and 8 Gy single doses than their respective parental cells (p<0.05). Comparative AbMa analyses of respective PN and RR cells demonstrated 62 differentially expressed proteins (DEPs) common to both RR cell lines. Of these, two apoptosis related proteins - DR4 and Bcl-2 were chosen for preliminary immunohistochemical validation for their putative role in radioresistance. The DR4 protein was found to be equally expressed whereas Bcl-2 demonstrated a trend towards reduced expression in 'poor-responder' rectal cancers compared with 'good-responders' that was not statistically significant.

Conclusions:
A radioresistant rectal cancer model consisting of novel SW-837 RR and HRA-19 RR cell sublines was successfully established and comparative proteomic analysis revealed a number of DEPs. In a small pilot IHC study, initial validation experiments showed no significant expression difference for DR4 and Bcl-2 proteins between ‘poor-responder’ and ‘good-responder’ rectal cancer patients. A panel of novel biomarkers from antibody array data would require further validation to determine their association with radioresistance before any firm conclusions can be drawn.

Publisher
Postgraduate Medical Institute, The University of Hull
Supervisor
Cawkwell, Lynn; Hunter, Andy
Qualification level
Doctoral
Qualification name
MD
Language
English
Extent
4 MB
Identifier
hull:10110
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