Lifestyle, Diet, and Colorectal Cancer Risk According to (Epi)genetic Instability: Current Evidence and Future Directions of Molecular Pathological Epidemiology

The Traditional Adenoma-Carcinoma Pathway

Tumors arising via the traditional adenoma-carcinoma pathway begin as premalignant lesions comprising of conventional, tubular or tubulovillous adenomas [11•], and account for approximately 60–90% of sporadic CRCs [2]. They are characterized by CIN, which describes a condition of aneuploidy that is caused by an accelerated rate of gains and losses of entire or large portions of the chromosome during cell division [14, 15]. CIN is associated with inactivating mutations or losses in the Adenomatous Polyposis Coli (APC) tumor suppressor gene, which occurs as an early event in this sequence [16]. Mutations in the KRAS oncogene, as well as TP53, SMAD4, and PIK3CA genes are also frequently observed [2]. With CIN, there is an increased rate of heterozygosity, which may contribute to the inactivation of tumor suppressor genes or activation of tumor oncogenes [17]. Descriptively, tumors that arise from this pathway are more often associated with male sex, and observed in the distal colon [11•].

Serrated Neoplasia Pathway

Approximately 10–30% of sporadic CRC tumors arise via the serrated neoplasia pathway [11•] and have distinctly different histology compared to tumors derived from the traditional adenoma-carcinoma sequence. They are characterized by MSI, a form of genetic instability characterized by length alterations within simple repeated microsatellite sequences of DNA. This is the result of strand slippage during DNA replication, which is not repaired due to a defective postreplication mismatch repair system [18]. An early event of these tumors is mutation of the BRAF proto-oncogene, which inhibits normal apoptosis of colonic epithelial cells [19]. The driving force of the serrated neoplasia pathway is the CpG methylator phenotype (CIMP), a form of epigenetic instability responsible for silencing a range of tumor suppressor genes, including MLH1 [2]. Loss of MLH1 is thought to cause microsatellite instability (MSI) and once MLH1 is inactivated, the rate of progression to malignant transformation is rapid [19]. Descriptively, these tumors are more frequently associated with female sex, and are observed in the proximal colon [11•].

Insights from the Cancer Genome Atlas Study

The Cancer Genome Atlas study, a collaboration between the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI), has generated a comprehensive, multi-dimensional map of the key genomic changes in CRC [20]. As recently summarized by Bae et al. [11•], the Cancer Genome Atlas study reports that CIN and MSI are mutually exclusive. CIMP, on the other hand, overlaps with the MSI pathway because of sporadic MSI-high CRCs, which are also usually CIMP-high, but does not appear to be in an exclusive relationship with the CIN pathway [11•, 20]. CIMP-high tumors can exist in the absence of MSI-high, and these tumors show some copy number variations across the genome, but the degree of CIN is less pronounced than CIMP-negative, MSI-low tumors. This suggests that CIMP alone may not be enough for the malignant transformation of serrated polyps, and requires collaboration with either CIN or MSI to promote successful malignant transformation [11•, 20].

In an MPE paradigm, a potential etiological factor, such as diet or lifestyle, is assessed with risk of an outcome across strata of molecular characteristics for the disease of interest [12••]. For purposes of this review, focus is on MPE studies that have considered diet and lifestyle factors in conjunction with primary molecular markers of (epi)genetic instability. For the traditional adenoma-carcinoma pathway, these include CIN, APC mutation, KRAS mutation, and TP53 mutation. For the serrated neoplasia pathway, these include BRAF mutation, MSI, hypermethylation of MLH1, and CIMP.

The Current Review

We reviewed the literature by searching combinations of key words (molecular pathological epidemiology, prospective cohort study, case-control study, KRAS mutation, APC mutation, Microsatellite Instability, CpG Island Methylator Phenotype, CIMP, BRAF mutation) in Pubmed and EMBASE databases, as well as by analyzing proceedings and participants of the International Molecular Pathological Epidemiology Meeting Series. Eight prospective cohort studies, five case-control studies, and one cross-sectional study that explicitly presented data on molecular markers of (epi)genetic instability were identified (Table 1). However, one cohort study did not further consider associations with diet and lifestyle factors [71], so for purposes of this review, was excluded from discussion. Of the remaining studies, associations have been published on molecular endpoints of CRC in relation to smoking, alcohol consumption; body mass index (BMI); waist:hip ratio; adult attained height; physical activity; early life energy restriction; ethnicity; dietary acrylamide, fiber, fat, methyl donors, omega 3 fatty acids; meat intake, including total protein, processed meat, and heme iron; and vegetable intake. For purposes of comparison and discussion, statistical associations are summarized in Tables 2 and 3, according to markers of the traditional adenoma-carcinoma and serrated neoplasia pathways, respectively, and the impact of these findings on advancing knowledge of CRC etiology is described in further detail below.

Smoking

Smoking has been studied in relation to both the traditional adenoma-carcinoma pathway [25, 41, 42, 58, 70, 72] and the serrated neoplasia pathway [30, 58, 60–62, 65]. As described in the proceedings of the third international MPE meeting, smoking provides one of the best examples of how MPE research can better predict CRC compared to epidemiological studies without molecular classification [12••]. Meta-analysis of traditional epidemiological studies showed only a modest link between smoking and CRC (i.e., a RR usually below 1.2) [73], which may lead one to believe that smoking is not a convincing risk factor for CRC. However, with the advent of MPE, it can be seen that once CRC cases are stratified by MSI or CIMP status, this risk increases up to two-fold for MSI-H and CIMP-H tumors in prospective cohort studies, while there are null associations for tumors not exhibiting these phenotypes (i.e., tumors of the traditional adenoma-carcinoma pathway). These data supports the premise that traditional epidemiological studies may mask true associations between some risk factors and cancer, and that MPE studies can shed light on true patterns of association.

Alcohol Intake

The association between alcohol intake and CRC has been studied separately by tumor markers related to the traditional carcinoma-adenoma pathway [21, 38, 43, 66] and the serrated neoplasia pathway [22, 38, 44, 63, 67]. Although considered by the WCRF as a convincing risk factor for CRC in menand women, MPE data is conflicting. Acetaldehyde in alcoholic beverages is a highly toxic substance that is carcinogenic to humans. In one of the earliest case-control studies considering alcohol in relation to risk of APC mutations, Diergaarde et al. found that alcohol intake only increased the risk of APC wildtype tumors [66]. In 2006, Bongaerts et al. concluded that alcohol was not associated with tumors harboring mutations in the KRAS gene [43]; however, in 2016, Jayasekra et al. concluded that alcohol intake is associated with an increased risk of KRAS mutated and BRAF wildtype/KRAS wildtype tumors originating via the traditional adenoma-carcinoma pathway but not with BRAF mutated tumors originating via the serrated pathway [38]. This is in contrast to case-control data from Slattery et al., who was the first to report that alcohol intake is associated with MSI [63]. Some reasons for these discrepancies may include heterogeneity between the way that alcohol intake was measured (i.e. lifetime exposure, highest vs. lowest intake, continuous intake), and the inability to consider men and women separately in data analysis due to limitations with sample size. Another layer of complexity in the association between alcohol and CRC risk is that there are susceptibility genes in relation to alcohol metabolism not accounted for in MPE studies. This may also explain some of the observed heterogeneity.

Indicators of Energy Balance

Indicators of energy balance include lifestyle factors that play a role in the development of body growth and obesity. These include body mass index (BMI), waist and hip circumference, adult-attained height, caloric intake and physical activity. The majority of MPE research on these factors has been conducted with respect to markers of the serrated neoplasia pathway [26, 39•, 45, 46, 59, 62, 64]. Although associations with APC, KRAS, and CIN have not been directly considered, the fact that BMI and waist measurements are positively associated with BRAF mutations and BRAF-wildtype, MSI and microsatellite stable tumors, and CIMP-H and non-CIMP tumors, is in accordance with WCRF evidence showing that overweight is a strong risk factor for CRC in general.

On the other hand, studies on adult-attained height and early life energy restriction suggest that timing of exposure may be important for influencing CRC risk. Height is a marker of aggregated fetal and childhood experience, and can be considered a proxy measure for important nutritional exposures, which affect several hormonal and metabolic axes [3]. Like body weight, adult-attained height is also an established risk factor for CRC in general; however, observations tend to be stronger for tumors demonstrating BRAF mutation and MSI [39•, 45]. One study on early life energy restriction showed that exposure to famine during childhood and adolescence decreased the risk of developing a tumor characterized by CIMP [46]. Taken together, this suggests that early life exposures may influence risk of epigenetic instability and CRC risk through the serrated neoplasia pathway, but data are scarce and more research is needed in this area.

Dietary Factors

Because the majority of MPE studies are derived from larger cohort and case-control studies that were designed to consider outcomes between diet and cancer, and therefore have validated food frequency questionnaires in place, it is not uncommon for multiple dietary exposures to be presented in the same publication.

Red meat intake was identified by the WCRF as a probable risk factor for CRC, and MPE research supports that this may especially be true for tumors of the traditional adenoma-carcinoma pathway; dietary heme intake shows stronger associations with KRAS.mutated tumors than KRAS wildtype tumors. It has been hypothesized that heme can enhance the endogenous formation of carcinogenic N-nitroso compounds [51•]. The study by Gilsing et al. is important because it is the first human observational study providing evidence, as expected, for an association between heme and tumors with specific point mutations [51•].

Similarly, the first observational study showing that dietary acrylamide might be associated with CRC with specific somatic mutations, such as G > C or G > T mutations, was recently published [47], which supports the a priori hypothesis that metabolites of acrylamide are human carcinogens.

With respect to dietary fat, a high intake of polyunsaturated fat, in particular linoleic acid, has also been linked to KRAS mutations [49]. Intriguingly, and in contrast, it was recently reported that high marine omega-3 polyunsaturated fatty acid intake is associated with lower risk of MSI-high CRC but not MSS tumors, suggesting a potential role of omega-3 fatty acids in protection against CRC through DNA mismatch repair [31]. Calcium, milk, and garlic were not significantly associated with specific tumor subtypes in the reviewed publications [21, 22, 63, 64, 51•].

Alcohol is often considered in conjunction with dietary methyl donors such as folate, because folate may influence promoter methylation at gene promoters, and is depleted with alcohol intake. It has been hypothesized that methyl donors such as folate and methionine influence CRC through the serrated neoplasia pathway because of their role in methyl transport (i.e. a deficient status may result in a decrease in promotor hyper methylation, as observed in CIMP). Folate intake is associated with BRAF mutations, suggesting that it does play a role in epigenetic aberrations [52]. However, high folate consumption also appears to reduce the risk of APC wildtype colon tumors, while being positively associated with APC mutated colon tumors in men [50], indicating that folate may also enhance colorectal carcinogenesis through a distinct APC mutated pathway. More research, with attention to sample size, is needed to replicate and clarify these associations.