Molecular effects of aspirin on NFkB in colorectal cancer

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

Development of a customised data management system for a COVID-19-adapted colorectal cancer pathway

Objectives A customised data management system was required for a rapidly implemented COVID-19-adapted colorectal cancer pathway in order to mitigate the risks of delayed and missed diagnoses during the pandemic. We assessed its performance and robustness.Methods A system was developed using Microsoft Excel (2007) to retain the spreadsheets’ intuitiveness of direct data entry. Visual Basic for Applications (VBA) was used to construct a user-friendly interface to enhance efficiency of data entry and segregate the data for operational tasks.Results Large data segregation was possible using VBA macros. Data validation and conditional formatting minimised data entry errors. Computation by the COUNT function facilitated live data monitoring.Conclusion It is possible to rapidly implement a makeshift database system with clinicians’ regular input. Large-volume data management using a spreadsheet system is possible with appropriate data definition and VBA-programmed data segregation. The described concept is applicable to any data management system construction requiring speed and flexibility in a resource-limited situation

Statin use and association with colorectal cancer survival and risk:Case control study with prescription data linkage

Background: In Scotland colorectal cancer (CRC) is the third most common cancer and a leading cause of cancer death. Epidemiological studies have reported conflicting associations between statins and CRC risk and there is one published report of the association between statins and CRC survival.Methods: Analysis was carried out on 309 cases and 294 controls from the Scottish Study of Colorectal Cancer (SOCCS). Cox's hazard and logistic regression models were applied to investigate the association between statin use and CRC risk and survival.Results: In an adjusted logistic regression model, statins were found to show a statistically significant association for three of the four statin variables and were found to not show a statistically significant association with either all-cause or CRC-specific mortality (OR 0.49; 95%CI 0.49-1.36; p-value = 0.17 and OR 0.33; 95%CI 0.08-1.35; P-value = 0.12, respectively).Conclusion: We did find a statistically significant association between statin intake and CRC risk but not statin intake and CRC-specific mortality. However, the study was insufficiently powered and larger scale studies may be advisable.</p

Implementation of a risk mitigating COVID-adapted colorectal cancer pathway

No abstract available

Aspirin in the 21st century-common mechanisms of disease and their modulation by aspirin: a report from the 2015 scientific conference of the international aspirin foundation, 28 August, London, UK.

Professor Peter Rothwell of Oxford University chaired the annual Scientific Conference of the International Aspirin Foundation in London on 28 August 2015. It took the form of four sessions. Aspirin has more than one action in its effects on disease. Its acetylation of cyclooxygenase 2 (COX-2) in platelets leads to the blockade of pro-inflammatory chemicals and generation of anti-inflammatory mediators and increase in nitrous oxide (NO) production, which helps to preserve arterial endothelium. But platelets are not its only target. There is now evidence that aspirin has a direct antitumour effect on intestinal mucosal cells that block their potential transformation into cancer cells. Randomised placebo-controlled trials (RCTs) in people with histories of colorectal neoplasia have shown that aspirin reduces the risk of recurrent adenomas and reduces long-term cancer incidence in patients with Lynch syndrome. Among women given aspirin for cardiovascular disease, there were fewer cancers than in those given placebo. Epidemiological evidence has suggested that aspirin treatment after cancer is diagnosed reduces the incidence of metastases and prolongs survival, and long-term studies of anticancer treatment with aspirin are under way to confirm this. Apart from cancer studies, aspirin use is now firmly established as treatment for antiphospholipid syndrome (Hughes syndrome) and is being used to prevent and treat the heightened risk of cardiovascular disease in diabetes mellitus and in patients with HIV

Real-world practice and outcomes in pilonidal surgery:Pilonidal Sinus Treatment Studying the Options (PITSTOP) cohort

Background: Numerous surgical approaches exist for the treatment of pilonidal disease. Current literature on treatment is of poor quality, limiting the ability to define optimal intervention. The aim of this study was to provide real-world data on current surgical practice and report patient and risk-Adjusted outcomes, informing future trial design. Methods: This UK-wide multicentre prospective cohort study, including patients (aged over 16 years) who had definitive treatment for symptomatic pilonidal disease, was conducted between May 2019 and March 2022. Patient and disease characteristics, and intervention details were analysed. Data on patient-reported outcomes, including pain, complications, treatment failure, wound issues, and quality of life, were gathered at various time points up to 6 months after surgery. Strategies were implemented to adjust for risk influencing different treatment choices and outcomes. Results: Of the 667 participants consenting, 574 (86.1%) were followed up to the study end. Twelve interventions were observed. Broadly, 59.5% underwent major excisional surgery and 40.5% minimally invasive surgery. Complications occurred in 45.1% of the cohort. Those who had minimally invasive procedures had better quality of life and, after risk adjustment, less pain (score on day 1: mean difference 1.58, 95% c.i. 1.14 to 2.01), fewer complications (difference 17.5 (95% c.i. 9.1 to 25.9)%), more rapid return to normal activities (mean difference 25.9 (18.4 to 33.4) days) but a rate of higher treatment failure (difference 9.6 (95% c.i. 17.3 to 1.9)%). At study end, 25% reported an unhealed wound and 10% had not returned to normal activities. Conclusion: The burden after surgery for pilonidal disease is high and treatment failure is common. Minimally invasive techniques may improve outcomes at the expense of a 10% higher risk of treatment failure.</p