The role of Bilirubin in Colorectal Cancer and Cardiovascular Disease Development

Cardiovascular diseases (CVD) are the most common chronic diseases globally, and colorec- tal cancer (CRC) is the third most commonly diagnosed malignancy and the fourth leading cause of cancer-related deaths in the world and its burden is expected to increase by 60% to more than 2.2 million new cases and 1.1 million cancer deaths by 2030. Established environmental CVD and CRC risk factors include life style such as a typical west- ern diet rich in animal products, smoking or physical inactivity. Bile pigments are end products of haem catabolism and had long been thought to possess little or no physiological function. The main pigments, called bilirubin and biliverdin, are in- tensely colored and can been seen in the skin during jaundice (bilirubin) and in the green (bili- verdin) and yellow (bilirubin) color of bruises However, a compelling body of evidence from experimental and clinical studies, some of them performed by us, has demonstrated that the bile pigments exhibit substantial anti-inflammatory and antioxidant properties, interact with and neutralize mutagens, and they may also have unique mechanistic effects that regulate cell apoptosis and carcinogenesis. In a human case-control-study, subjects with increased blood bilirubin levels (also known as Gilberts Syndrome, 5-19% prevalence in the Caucasian pop- ulation) showed an improved lipid metabolism (with lower risk factors such as total- and LDL- cholesterol or triglycerides), lower mediators for inflammation and a lower body mass index. Interestingly, the effects were more pronounced in older subjects. However, data linking subjects with higher bilirubin concentrations to CRC but also to underly- ing mechanisms regarding CVD protection such as lipid metabolism or body composition from large prospective studies are missing. With this proposal we want to close this gap by having the unique possibility to have access to three very large studies/datasets (EPIC: European Prospective Investigation into Cancer and Nutrition; UK Biobank and GECCO: the Genetics and Epidemiology of Colorectal Cancer Consortium) to explore key questions around bilirubin metabolism and colorectal cancer as well as CVD and total mortality in more than 35.000 subjects. The study is the largest so far linking prospective CRC cases with bilirubin metabolism. We will further investigate the link between already existing genetic data which are strongly asso- ciated with elevated circulating total bilirubin levels to CRC and CVD, CVD risk marker as well as total mortality. These studies with the large data sets will help to understand the role of bilirubin metabolism in CVD and CRC development and establish a new preclinical biomarker for disease predic- tion. Specific focus will be given on gender, aging and age related effects.

The aims of the research project were based on the questions whether bilirubin as biomarker is associated to the risk of cancer and cardiovascular diseases. For cancer we investigated in a case-control study nested in the European Prospective Investigation into Cancer and Nutrition (EPIC), pre-diagnostic unconjugated bilirubin (UCB, the main component of total bilirubin) concentrations were measured in plasma samples of 1386 colorecal cancer (CRC) cases and their individually matched controls. Additionally, 115 single-nucleotide polymorphisms (SNPs) associated with circulating total bilirubin were instrumented in a Mendelian randomization (MR) study to test for a potential causal effect of bilirubin on CRC risk in 52,775 CRC cases and 45,940 matched controls in the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), the Colon Cancer Family Registry (CCFR), and the Colorectal Transdisciplinary (CORECT) study and the Prospective Cooperative Health Research in the Region of Augsburg (KORA) Study. Among men, higher levels of UCB were positively associated with CRC risk. In women however, an inverse association was observed. In the MR analysis of the main UGT1A1 SNP (rs6431625), genetically predicted higher levels of total bilirubin were associated with a 7% increase in CRC risk in men, while there was no association in women. Raised bilirubin levels, predicted by instrumental variables excluding rs6431625, were suggestive of an inverse association with CRC in men, but not in women. In the UK Biobank we applied two-sample MR to investigate whether genetically raised bilirubin levels are causally associated with the risk of ten cancers (pancreas, kidney, endometrium, ovary, breast, prostate, lung, Hodgkin's lymphoma, melanoma, and neuroblastoma). The number of cases and their matched controls of European descent ranged from 122,977 and 105,974 for breast cancer to 1200 and 6417 for Hodgkin's lymphoma, respectively. Here we could show that genetically raised bilirubin levels were inversely associated with risk of squamous cell lung cancer as well as Hodgkin's lymphoma and positively associated with risk of breast cancer. For the CVD direction we could summarize the state of the art knowledge how to diagnose Gilberts Syndrome (GS) (a mild hyperbilirubinaemic condition) and how this condition is linked to a lower risk for CVD such as reducing circulating cholesterol, oxidative lipid/protein modifications, and blood pressure. In addition, bilirubin inhibits platelet activation and protects the heart from ischemia-reperfusion injury. These effects attenuate multiple stages of the atherosclerotic process in addition to protecting the heart during resultant ischemic stress, likely underpinning the profound reduction in cardiovascular mortality in hyperbilirubinemic GS. We could further show that bilirubin considerably decreases lipid accumulation in skeletal muscle and liver cells, which suggests that mildly elevated bilirubin levels could lower ectopic lipid deposition, a major key element in the pathogenesis of type 2 diabetes.