The Influence of Biological Ageing in the Pathogenesis of Colorectal Cancer

Despite improvements in risk factor awareness, diagnosis and enhanced management strategies, the incidence and five year survival of colorectal cancer, has remained largely unchanged over the last twenty years. As with many epithelial cancers, a preponderance of new colorectal cancer diagnoses occur in the over sixty five age group, making chronological age a strong risk factor. Given this indelible link between ageing and cancer in general, genetic pathways which are implicated in one process could influence the other. Thus, an understanding of the biology of ageing and factors which regulate it may provide insight into cancer pathogenesis. Telomeres are nucleo-protein complexes sited at the ends of all chromosomes and have a critical function in the protection of the genome. Telomeres are implicated in the ageing process as a result of the inadequacies of the DNA replication machinery in somatic cells meaning that a section of telomeric DNA sequence is lost during each round of cell division, thus telomere length reduces with age and is a putative marker of biological ageing. Control of telomere length is complex and involves interplay between a number of genetic and environmental factors, of which oxidative stress is particularly important. However, critically short and hence dysfunctional telomeres have been implicated in cancer development through an inability to maintain genomic intergrity and an effect on senescence. Telomeres play an integral role in the sensing and repair of DNA damage, however, cells must possess a finely tuned mechanism through which they can sense DNA damage and initiate a response. This usually involves the activation of cell cycle checkpoints, either temporarily to allow repair, or on an irreversible basis to prevent the clonal expansion of cells with deleterious mutations. If the damage is deemed irrepairable apoptotic pathways are initiated. The sirtuins are a group of genes first discovered and shown to control longevity in saccharomyces cerevisiae. Intense work has defined seven mammalian homologs termed SIRT1-7 which vary in their sub-cellular localisation, and have critical cellular functions ranging from the control of apoptosis, mitochondrial biogenesis, glucose and lipid metabolism, maintenance of genomic integrity and cell cycle control. Given these functions it is therefore no surprise that aberrancy of sirtuin expression is implicated in ageing and its commonly related diseases, particularly cancer. The aim of this study was therefore, to determine if patients with colorectal cancer display aberrancy of ageing related factors, namely telomere biology and sirtuin expression. This study was undertaken using two sources of material for investigation. Quantitative-PCR was utilised to measure telomere length in the peripheral blood leucocytes of 64 colorectal cancer patients and 1348 controls. In addition, telomere length was similarly measured in colorectal cancer tumour and normal adjacent tissue. Telomere length was then correlated with a number of clinical and pathological parameters to determine diagnostic or prognostic utility. Furthermore, an attempt was made to establish whether telomere lengths were reflected in circulating mediators of inflammation and redox control factors, including fetuin-A a circulating modulator of calcium homeostasis. Sirtuin relative transcriptional expression (SIRT1-7) was then measured in the tumour and normal tissue samples. Clinically relevant information was derived by analysing the SIRT1-7 transcriptional data in terms of clinico-pathological, inflammatory and outcome variables. Finally, sirtuin expression was correlated with other factors known to be involved with biological ageing to determine any potential association. Colorectal cancer patients had significantly shorter telomeres in their peripheral blood leucocytes (adjusted mean RelT/S=0.61) compared with chronologically older controls (mean age 75, adjusted mean RelT/S=0.70) (ANCOVA, p=0.004), indicating colorectal cancer patients were biologically older than their control counterparts. In addition, telomere length in tumour tissue (median=0.43, IQR=0.40) was significantly shorter than adjacent normal tissue (median=0.65, IQR=0.28) (p=0.004). Patients with low plasma fetuin-A levels were shown to have significantly shorter telomeres (p=0.041) and patients with rectal tumours had significantly higher levels of fetuin-A than those with colonic tumours (p=0.045). There was no correlation between telomere length and other redox factors, namely anti-oxidant vitamins, micronutrients and divalent cations. There was, however, a significant association between telomere length and systemic inflammation as determined by the neutrophil to lymphocyte ratio. SIRT 1-7 were differentially expressed between tumour and normal tissue, with significant attenuation evident in tumour samples when compared with normal tissue (p<0.0001 except SIRT2 p=0.003). SIRT2 (p=0.021) and SIRT4 (p=0.027) expression in tumour samples, was significantly associated with anatomical tumour site and pathologically determined nodal status respectively. Whilst, SIRT3 expression in normal tissue correlated with pro-inflammatory status, indicated by higher serum CRP levels. Finally, there was a significant inverse relationship with colorectal cancer tissue telomere length and SIRT3. When overall survival was considered, Kaplan-Maier analysis revealed a significant difference in survival in relation to SIRT4 expression levels. We have observed that patients with colorectal cancer display clear evidence of telomere attrition compared with controls. This is congruent with accelerated biological ageing in the pathogenesis of colorectal cancer and indicates cancer patients have ‘more miles on the clock’. An imbalance in redox control mechanisms and calcium homeostasis may be a contributing factor to telomere dynamics in these patients. The demonstration of attenuated sirtuin expression in colorectal cancer suggests a role as potential tumour suppressors and provides further evidence implicating biological ageing in the oncogenic process. Furthermore, plasma fetuin-A and tissue SIRT2 expression levels can be used to distinguish between colon and rectal cancers, providing further information regarding the molecular characteristics of these tumours. Telomere biology and the sirtuins could both play a pivotal role in the MTR (Mitochondria Telomere Ribosome biogenesis) paradigm, aberrancy of which could explain the apparent link between biological ageing and cancer. Enhancement of the understanding of the determinants of telomere length could mean that manipulation could lead to reduced colorectal cancer risk at the population level. In addition, the data provided in this thesis strengthens the evidence base which suggests that targeting individual sirtuins could be a future chemotherapeutic strategy, or indeed prove useful as markers of prognosis.