Din, Farhat Vanessa Nasim

2018-03-29T12:16:06Z

2018-03-29T12:16:06Z

2006

Colorectal cancer (CRC) is the most common fatal malignancy in the non-smoking population and Scotland is a high incidence country with an average number of 3445 annual registrations (1997-2001). Despite research and development in clinical practice, the overall 5-year survival is only 51%. Curative surgical resection can increase survival to 70%, but this is only applicable to patients with localised disease. One approach that holds promise in reducing overall disease mortality involves primary prevention using chemopreventive agents. Substantial evidence indicates that non-steroidal anti¬ inflammatory drugs (NSAIDs) protect against colorectal cancer, but the molecular basis is not fully elucidated. The host laboratory has previously demonstrated that aspirin-induced apoptosis in colorectal cancer cells is associated with modulation of the NFkB signalling pathway. The transcription factor NFkB regulates expression of genes involved in proliferation, apoptosis and carcinogenesis. Aspirin-induced apoptosis in colorectal cancer cells was found to be dependent on aspirin-induced degradation of IkBoi, the NFkB inhibitory protein, and NFkB nuclear translocation. Epidemiological data indicates that the protective effect of NSAIDs is greater in colorectal cancer compared to other cancer types, suggesting the possibility that aspirin might target distinct molecular pathways in colonic epithelial cells.

The aims of this thesis were to investigate the specificity of this response for colorectal cancer compared to other cancer cell types and to identify any potential molecular markers for the response; to determine the influence of mismatch repair (MMR) and p53 status on the NFkB apoptotic response; to examine whether any detectable modulation of NFkB signalling occurs within the constrained environment of clinical studies and finally to analyse NFkB pathway genes Rel A and IkBa for mutations in colorectal cancer.

To investigate the question of specificity, the effects of aspirin on cell viability and NFkB signalling were studied in a panel of colorectal cancer cell lines compared to cancer cell lines of non-colonic origin. Aspirin induced a concentration-dependent decrease in viable cell number, paralleled by proportionate increases in apoptosis in six colorectal cancer cell lines. There was no consistent change in apoptosis in the five non-colorectal cancer cell lines studied. In colorectal cancer cell lines, aspirin-mediated apoptosis was associated with aspirin-induced IkBoi degradation and NFkB nuclear translocation. In contrast, these changes in NFkB signalling were absent in non-colorectal cancer cell lines, paralleling the lack of changes in cell viability and apoptosis observed. This work establishes the degree of specificity of the aspirin-induced NFkB apoptotic response in colorectal cancer. Further work to elucidate the molecular mechanism of this differential sensitivity may lend insight into the mode of action of NSAIDs, and identify molecular markers of response. Basal expression levels of COX-2 or of P-catenin did not relate to susceptibility to NSAID-induced apoptosis. This suggests that COX-2 and P-catenin are unlikely to play a predominant role in aspirin-mediated apoptosis via the NFkB pathway. However, this work does emphasise the generality of the aspirin-induced NFkB apoptotic response with respect to colorectal cancer genotype.

The influence of MMR and p53 status on the NFkB apoptotic response was determined, as these pathways are deranged in colorectal cancer, and are implicated as NSAID targets. Furthermore, this may potentially identify which subsets of colorectal cancer, with respect to genotype, are susceptible to aspirin-mediated chemoprevention. This was investigated by studying the effects of aspirin on HCT-116 (MMR deficient & wild-type p53), HCT 116⁺ᶜʰ³ (MMR proficient & wild-type p53) and HCT-116ᵖ⁵³⁻/⁻ (p53 null) colorectal cancer lines. Aspirin induced a dose-dependent decrease in viable cell number, paralleled by proportionate increases in apoptosis in all three cell lines, which was accompanied by IkBα degradation and NFkB nuclear translocation in each case. These results show that the aspirin-induced NFkB apoptotic response occurs irrespective of MMR and p53 status.

It is important to investigate the in vivo relevance of these molecular observations. Hence, the effects of aspirin and the COX-2 selective inhibitor rofecoxib were studied in normal mucosa and cancer of rectal cancer patients, and in normal mucosa of genetically predisposed patients before and after ingestion for 7 days. The preliminary results show that there was wide variability, in terms of ficBa expression, between patients and no consistent change was demonstrated between baseline IkBcc protein expression and posttreatment levels, in normal mucosa and rectal cancers of patients treated with NSAIDs.

The NFkB pathway plays a central role in death signalling and since altered regulation of NFkB has been observed in colorectal cancers, it is possible that deranged NFkB signalling in colorectal cancer may be due to mutations in the NFkB pathway genes, Rel A and TkBa. Polymorphic DNA sequence variations, or mutations, in Rel A or IkBa genes could account for the variability in NFkB response observed in colorectal cancer patients and also the cell-type specific nature of the NFkB apoptotic response. Flence, mutation analysis of Rel A and IkBa genes was performed. This has identified a number of interesting variants that merit further investigation.

The work in this thesis underscores the importance of NFkB as a key target for the antitumour activity of NSAIDs in colorectal cancer. The results provide evidence of a molecular rationale for the greater specificity of NSAID-mediated protection observed in colorectal cancer compared to other cancers. Furthermore, the lack of association with COX-2 and P-catenin expression and independence from MMR and p53 mutation status emphasises the generality of the aspirin-induced NFkB apoptotic response in colorectal cancer. This is clinically relevant when considering NSAIDs for chemoprevention in genetically predisposed individuals and as adjuvant therapy for sporadic colorectal cancers. The clinical studies demonstrate, for the first time, that changes in IkBcc protein levels are detectable in normal mucosa and tumour biopsies from patients. However, the intra- and inter-patient variability in IkBα levels before and after NSAID treatment precludes any conclusion pending further experimentation. The variability requires more detailed controls than the pre- and post- sampling protocols shown here in pilot studies. Overall, this thesis contributes to the understanding of NSAID-mediated apoptosis via modulation of NFkB signalling in colorectal cancer, and may inform chemoprevention trials and novel drug design targeting the signalling pathways involved.

http://hdl.handle.net/1842/29088

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 17

Already catalogued

Molecular effects of aspirin on NFkB in colorectal cancer

Thesis or Dissertation

Doctoral

MD Doctor of Medicine