Chapter 13 – Colorectal cancer

Harry Campbell, Steven Hawken, Evropi Theodoratou, Alex Demarsh, Kimberley Hutchings, Candice Y. Johnson, Lindsey Masson, Linda Sharp, Valerie Tait, and Julian Little

Introduction

Colorectal cancer is a major global public health problem, with approximately 950,000 cases newly diagnosed each year (1). Risk of developing colorectal cancer increases steeply with age and incidence is rising in many industrialized countries as life expectancy and the numbers of elderly people increase. Incidence is also rising in many developing countries, as diet and lifestyle become more similar to those in industrialized countries.

Approximately 25% of colorectal cancer cases are associated with a family history; risk is increased two to four times in first-degree relatives of a patient with colorectal cancer. A substantial proportion of the familial aggregation of colorectal cancer results from inherited susceptibility. Excess familial cancer risk can be accounted for by a combination of rare high-penetrance mutations and large numbers of common variants each conferring small genotypic risk (on the order of 1.1– 2.0). These latter variants combine additively or multiplicatively to confer a range of susceptibilities in the population (2).

The relationships of genetic variants with human disease described so far largely reflect the study designs used to identify them. Linkage studies conducted among families with multiple cases of disease were successful in identifying highly penetrant variants with large effects (such as hMLH1, hMSH2, and APC; see below). The discovery of genes responsible for inherited colorectal cancer syndromes has been important in identifying important etiologic pathways such as the beta-catenin/ APC and TGF beta/SMAD pathways. Association studies conducted in general population samples using common genetic markers typically find variants with very small effects (such as SMAD7 and CRAC1; see Section “Common Low-Penetrance Variants Identified from Genome-Wide Association Studies”). Future resequencing studies are expected to identify rarer variants (e.g., prevalence 0.05–5%) with intermediate effects (3). Genome-wide studies of structural variation will likely identify deletions, amplifications, and other copy number variations influencing colorectal cancer risk.

Rare, High-Penetrance Variants

Mismatch Repair Gene Mutations (hMLH1, hMSH2, hMLH6, hPMS1, hPMS2)

The clinical syndrome due to mismatch repair gene deficiency is known as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) and accounts for 2–5% of all colorectal cancer cases. Affected kindreds have an unusually high occurrence of colorectal and certain extracolonic cancers, with a relatively early age of onset.

Evidence to support a role for the mismatch repair genes hMLH1 and hMSH2 in the etiology of colorectal cancer has come from linkage analysis, segregation studies, and molecular–biologic analysis. The mismatch repair genes hMLH1 and hMSH2 are integral components of the DNA mismatch repair pathway. A HuGE review in 2002 identified 259 different pathogenic mutations (and 45 variants) in hMLH1 and 191 different pathogenic mutations (and 55 variants) in hMSH2 (4). In addition, deletions in mismatch repair genes appear to occur relatively commonly, particularly in hMSH2. HNPCC families in which mutations in hMLH1 and hMSH2 are not identified may harbor pathogenic mutations in other mismatch repair genes, such as hMSH6 and hPMS2.

The available data do not suggest any substantial differences in the frequency of hMLH1 or hMSH2 mutations among populations or ethnic groups (5). The penetrance of mutations in hMLH1/hMSH2 is incomplete and is significantly higher in men (approximately 80%) than in women (approximately 40%). A standardized incidence ratio of 68 for colorectal cancer was reported for carriers of hMLH1 or hMSH2 mutations compared with the general population (6). First-degree relatives of mutation carriers had a relative risk of 8.1 compared with first-degree relatives of noncarriers (7).

APC

The adenomatous polyposis coli (APC) gene is a tumor suppressor gene, and mutations resulting in loss of APC protein function are associated with carcinogenesis. APC protein down-regulates the Wnt signaling pathway through its binding to β-catenin and axin (8).

APC germline mutations lead to the highly penetrant, autosomal dominant neoplastic syndrome of Familial Adenomatous Polyposis Coli (FAP). This condition has an annual incidence of about 1:7,000 live births and is characterized by hundreds or thousands of colorectal adenomas, which if untreated can develop into carcinomas (9). FAP accounts for about 1% of all colorectal cancer cases.

Low-penetrance APC mutations have been implicated in familial colorectal cancer cases (10). The most common APC variants associated with inherited susceptibility are I1307K and E1317Q. At least 12 single nucleotide polymorphisms (SNPs) of APC have been identified, 8 of them in exon 15. The most common allele (Asp1822Val, frequency 10–22%) was found not to be associated with development of colorectal cancer in three studies but a positive association has been observed with two others.

MUTYH

Another familial form of colorectal cancer, MUTYH-associated polyposis (MAP), was first described in families with multiple colorectal adenomas or carcinomas who lacked inherited APC mutations (11,12). The MAP phenotype is clinically comparable to the FAP phenotype; however, it is recessively transmitted and generally results in a smaller number of adenomas and later age at onset of colorectal cancer (13).

MUTYH is a base excision repair gene. Approximately 30 mutations that alter the protein product, 52 missense variants, and 3 inframe insertions/deletions have been identified (14). The two most common MUTYH variants in whites account for >80% of disease-causing alleles; additional alleles have been identified in other populations.

OGG1

Base-excision repair maintains genome stability by countering oxidative DNA damage. OGG1 acts together with MYH and MTH1 to identify and remove 8-oxoguanine that has been incorporated into DNA. OGG1 variants have been reported in association with colorectal cancer, alone or in combination with mutations in other genes (15,16).

Other Rare Variants

Several other rare, autosomal dominant disorders include increased risk of colorectal cancer. Juvenile Polyposis Syndrome, caused by mutations in SMAD4, PTEN, or BMPR1A, is associated with early onset colorectal cancer, typically before 20 years of age.

Common, Low-Penetrance Variants

Many studies have investigated associations of colorectal cancer with common variants of low-penetrance genes. Initially, most of these studies were hypothesis-driven, usually focusing on genes thought to be involved in the metabolism of particular environmental risk factors (the “candidate gene” approach). We have organized this section of the chapter around genes that operate in pathways that are thought to play a role in the causation of colorectal cancer.

We identified relevant studies by using the HuGE Navigator and extracted information using approaches we have used in HuGE reviews.

Genetic Variants Affecting Multiple Substrate Metabolism

Many studies have examined associations between colorectal cancer and variants of genes encoding enzymes involved in metabolism of carcinogens that are present in tobacco smoke or produced as a result of cooking meats. The most extensive evidence relates to the glutathione S-transferase genes GSTM1, GSTT1, and GSTP1, the cytochrome P450 1A1 gene CYP1A1, and the N-acteyltranferase genes NAT1 and NAT2.

The glutathione S-transferases (GSTs)

The glutathione S-transferases (GSTs) play a central role in the detoxification of carcinogens by catalyzing the conjugation of glutathione to potentially genotoxic compounds, including polyaromatic hydrocarbons (17). However, GSTs also conjugate isothiocyanates, which are potent inducers of enzymes that detoxify environmental mutagens, thereby diverting the isothiocyanates from the enzyme induction pathway to excretion. These two opposing potential mechanisms suggest that the role of GSTs in cancer risk is complex. GSTs also modulate the induction of the enzymes and proteins important for cellular functions such as DNA repair.

Systematic reviews and meta-analyses have been conducted examining the association between GSTM1, GSTT1, GSTP1, and colorectal cancer susceptibility (17–21).

GSTM1: Twenty-eight studies have investigated the association of GSTM1 with colorectal cancer. Overall, there is evidence of a weak association of the GSTM1 null genotype with colorectal cancer, with substantial heterogeneity.

GSTT1: Twenty-three studies have investigated the association between GSTT1 and colorectal cancer (Table 13.1). Overall, the evidence suggests a weak association of the GSTT1 null genotype with colorectal cancer.

GSTP1: Eleven studies have investigated the association of GSTP1 with colorectal cancer. None of the studies supports an association.

The cytochrome P450 genes

The cytochrome P450 (CYP) family of enzymes includes over 50 characterized genes, of which the CYP1, CYP2, and CYP3 families are involved in phase I metabolism of xenobiotics and drugs and also metabolism of some endogenous compounds.

CYP1A1: The CYP1A1 gene is under the regulatory control of the aryl hydrocarbon receptor, a transcription factor that regulates gene expression. Overall, there is limited evidence for association with the T3801C variant, and the Ile462Val variant. Both studies investigating the Thr461Asn variant found that the Thr/Asn genotype was associated with significantly reduced risk of colorectal cancer compared with the Thr/Thr genotype.

CYP1A2: Five studies from Korea, France, Hungary, Spain, and the United Kingdom have investigated the A164C (rs762551) genotype in colorectal cancer, with negative or inconsistent results.

CYP1B1: Studies of European populations provide no support for association with colorectal cancer for the C1294G (rs1056836) or the N453S (rs1800440) variant.

CYP2C9: Six studies of CYP2C9*2 genotype and colorectal cancer have had inconsistent results. None of the five studies of CYP2C9*3 genotype found an association. Of the four studies that analyzed both CYP2C9*2 and CYP2C9*3 variants, three suggested an inverse association with possession of one or both variant alleles. Because these studies included only small numbers of persons with the variant genotypes and the associations they found were inconsistent, they provide only weak and insufficient evidence.

CYP2C19: Three studies from Spain, Turkey, and the United Kingdom investigated the association of G681A with colorectal cancer, with inconsistent results.

CYP2D6: Three studies from Australia, Spain, and the United Kingdom have investigated variants in this gene; however, because different gene nomenclatures were used, it is difficult to compare the results, which were inconsistent.

CYP2E1: Studies of two variants found on the c2 allele (rs3813867, rs2031920) and a variant of intron 6 (7632T>A/Dra I) conducted in Australia, China, France, Hungary, the Netherlands, Spain, and the United States have been negative or inconclusive.

CYP3A4: Two studies found no association of G20230A (rs2242480), found on the CYP3A4*1G or *1H alleles, with colorectal cancer.

CYP3A5: The intronic variant 6986A>G (rs776746), found on alleles CYP3A5*3A to CYP3A5*3L, has been investigated in two small studies of colorectal cancer. No association was found in either.

Other CYP genes: Variants of CYP2C8, CYP11A1, CYP17A1, CYP19A1, and CYP7A1 have each been investigated in only one study, with no associations found.

The N-acetyltransferases

N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) function as phase II conjugating enzymes, implicated in the activation and detoxification of known carcinogens.

NAT1: The initial report of an OR of 1.9 with 95% confidence interval (95% CI 1.2–3.1) for association with possession of the NAT1*10 allele has not been replicated in most of the subsequent studies.

NAT2: Early studies reported that the NAT2 rapid acetylation phenotype was associated with increased risk of colorectal cancer and a meta-analysis estimated the pooled OR as 1.51 (95% CI 1.07–2.12) (21). In a meta-analysis of 15 studies published before October 2001 (21), the combined risk estimate for rapid acetylators (inferred on the basis of their genotype) was 1.03 (95% CI 0.94–1.12). Most studies published since the meta-analysis have been null.

Genetic Variants Affecting Nutrient Metabolism

Virtually all epidemiologic studies of diet and colorectal cancer have been observational and subject to three potential biases: (i) diet is related to other aspects of lifestyle, which may influence risk, (ii) people eat foods rather than nutrients, and (iii) misclassification of intake, either of the food group or nutrient being investigated or of other food groups or nutrients, could dilute or bias the associations. Studying associations with genetic variants that influence nutrient metabolism might help unravel the relationship of dietary factors with colorectal cancer.

Variants of genes associated with alcohol intake and metabolism

Case-control studies have examined the associations between colorectal cancer and variants of ALDH2, ADH1B (ADH2), or ADH1C (ADH3). Population frequencies of the most commonly studied variants of these genes have been reviewed recently.

ALDH2: The most frequently reported associations are with a variant of ALDH2 most prevalent in Asian populations (Table 13.2). The ALDH2*487Lys (rs671) variant results in reduced activity of the mitochondrial enzyme aldehyde dehydrogenase, leading to high levels of acetylaldehyde, which is thought to be carcinogenic. Because accumulation of acetaldehyde also produces unpleasant symptoms (facial flushing, increased heart rate, and nausea), persons who are homozygous or heterozygous for reduced-activity ALDH2 variants may consume less alcohol than do those without the variant, which could lower the risk of alcohol-related diseases.

We combined the results of six case-control studies in a meta-analysis, which found the following summary ORs and 95% CIs: for heterozygotes (ALDH2 Glu/Lys vs. ALDH2 Glu/Glu), OR 0.87 (95% CI 0.73–1.04); for homozygotes (ALDH2 Lys/Lys vs. ALDH2 Glu/Glu), OR 0.73 (95% CI 0.57–0.95). Thus, the evidence suggests an inverse association of the ALDH2*487Lys allele with colorectal cancer in populations of north-eastern Asian ancestry.

ADH1B: Combining the results of three case-control studies, one in Europe and two in Japan, to test for heterozygous (ADH1B Arg/His vs. ADH1B His/His) or homozygous (ADH1B Arg/Arg vs. ADH1B His/His) genetic effects, we found combined ORs and 95% CIs of 1.29 (1.10, 1.52) for heterozygotes and 1.51 (1.05, 2.16) for homozygotes. It is noteworthy that this association was confined to the two Japanese studies. Although the evidence is limited, the consistency of the results across these two studies suggests that this association deserves further research attention.

ADH1C: None of the four studies of the ADH1C Ile349Val variant and colorectal cancer has supported an association with colorectal cancer. Combining the results of four case-control studies to test for heterozygous (Ile/Val vs. ADH1C Ile/Ile) or homozygous (Val/Val vs. ADH1C Ile/Ile) genetic effects, we found combined ORs and 95% CIs of 1.03 (0.90, 1.19) for heterozygotes and of 1.02 (0.85, 1.23) for homozygotes.

Variants related to folate and one-carbon metabolism

Folate is a B vitamin (B9) found most abundantly in vegetables and fortified grain products. Folate mediates the transfer of one-carbon units in a variety of cellular reactions, most notably in thymidine, purine, and methionine synthesis. Thymidine and purine are required for DNA synthesis and repair, whereas methionine is a precursor in reactions necessary in the maintenance of normal DNA methylation patterns (45). Hypomethylation of DNA is hypothesized to contribute to carcinogenesis through a number of mechanisms, including proto-oncogene activation, genomic instability and chromosomal structural aberrations, or uracil misincorporation during DNA synthesis.

MTHFR: MTHFR is responsible for converting 5,10-methylene-tetrahydrofolate to 5-methylenetetrahydrofolate, the principal circulating form of folate. Several studies have investigated two common variants of MTHFR, C677T and A1298C, in relation to colorectal neoplasia; these include several meta-analyses and a HuGE review (18,19,46–49). So far, few studies have investigated the effects of combinations of variants (48).

MTHFR C677T: Twenty-nine individual studies of MTHFR C677T have been published, together including more than 13,000 colorectal cancer cases. In general, the risk of colorectal cancer appears to be lower in persons with the TT genotype, compared with the CC genotype (Table 13.3). We performed an updated meta-analysis for this chapter and found the summary OR 0.83 (95% CI 0.76–0.91) for persons with the TT versus the CC genotype. Some evidence suggests that the apparently protective effect of the TT genotype may be negated in persons with low folate or methionine intake and in persons who consume large quantities of alcohol.

MTHFR A1298C: Seventeen studies of MTHFR A1298C have been published, together including more than 7,000 colorectal cancer cases. Most studies found that CC homozygotes were at moderately reduced risk of colorectal cancer compared with AA homozygotes. Our updated, random-effects meta-analysis of these 17 studies found a summary OR 0.80 (95% CI 0.7–0.93) for persons with the CC versus the AA genotype. The C677T and A1298C variants appear to be in strong linkage disequilibrium, suggesting that studies of the A1298C and C677T variants are measuring the same association with colorectal cancer.

Methionine synthase (MTR): Ten studies of MTR A2756G have been published, together including more than 9,000 colorectal cancer cases. Our meta-analysis of all the available studies found a null summary effect, as well as evidence of statistical heterogeneity. Further investigation is required to determine whether population differences in environmental exposures (e.g., alcohol, diet) could help explain this heterogeneity.

Methionine synthase reductase (MTRR): The GG genotype of the A66G variant has been inconsistently associated with moderately reduced risk of colorectal cancer.

Cystathionine-β-synthase (CBS): Three studies have examined a 68 base-pair insertion in exon 8 of the CBS gene in relation to colorectal cancer. An Australian study found that the 68bp insertion was less frequent in subjects with proximal tumors, suggesting a possible protective effect. Two additional studies in the United States and United Kingdom found no evidence of an association.

Thymidylate synthase (TS): The TS enhancer region contains a series of 28 base-pair tandem repeats, most commonly 2 repeats (2rpt) or 3 repeats (3rpt); the 3rpt variant produces a nearly threefold increase in TS expression. The five studies comparing 2rpt genotypes with non-2rpt genotypes in relation to colorectal cancer suggest a protective effect for 2rpt variants, although not all reached statistical significance.

Variants of iron metabolism genes

Iron is a key element in cellular processes (58) and may have a role in the etiology of cancer (59), including colorectal cancer (60).

HFE: HFE is an MHC-Class I molecule involved in the uptake of iron in the small intestine. Two HFE mutations, C282Y and H63D, are associated with hereditary hemochromatosis in populations of European origin. Both mutations are associated with elevated transferrin saturation and serum ferritin levels, with variable biochemical penetrance that may be modified by several factors (see Section “Gene– Environment Interaction in the Etiology of Colorectal Cancer”).

We identified seven case-control studies that investigated associations of the C282Y and H63D variants with colorectal cancer. The frequency of the Y282 allele rarely exceeds 0.10 and these studies were underpowered to assess its effect. Two studies also investigated the S142G variant (rs3817672) in the transferrin receptor gene (TFRC) as a potential modifier of association with the C282Y and H63D variants of HFE. These seven studies offer little evidence to support an association of the C282Y variant with colorectal cancer; the four studies that examined association with the H63D variant gave inconsistent results.

Variants influencing vitamin D and calcium metabolism

VDR: 1α, 25-dihydroxy vitamin D₃ [1α,25(OH)₂D₃], the active form of vitamin D, is synthesized from both dietary vitamin D and skin-derived precursors through the action of ultraviolet sunlight. In addition to its role in regulating calcium absorption and blood calcium concentration, vitamin D may have anticarcinogenic activity via its binding to the vitamin D receptor (VDR) (88). Vitamin D could affect colorectal cancer risk by influencing cell proliferation and differentiation, apoptosis, and angiogenesis (89,90) or by affecting insulin resistance (91).

Several variants of the VDR gene have been identified. A poly-A repeat at the 3′ untranslated region of the gene has been found to be associated with increased mRNA expression; it is in linkage disequilibrium with four restriction fragment length variants (RFLPs) known as BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236), and Tru9I. An RFLP (FokI, rs10735810) at the first potential start site of the gene (ATG to ACG) results in a long version of the VDR protein (T-allele or the “f” allele) or a protein shortened by three amino acids (C-allele or the “F” allele).

Several case-control studies have investigated the associations of VDR variants with colorectal cancer (91); some have reported a positive association with the “f” allele (rs10735810). Our meta-analysis of four studies of FokI found OR 0.94 (95% CI 0.58– 1.53); our meta-analysis of three studies of BsmI found OR 1.18 (95% CI 1.04–1.33).

Lipid metabolism

APOE: The apolipoprotein E (APOE) gene has three alleles: APOE ε2, APOE ε3, and APOE ε4. These alleles arise due to two missense SNPs, rs429358 and rs7412, which result in T/C base substitution, and corresponding Cys/ Arg amino acid changes at residues 112 and 158, respectively. APOE may influence colorectal cancer development through three possible pathways: cholesterol and bile metabolism, triglyceride and insulin regulation, and inflammation (92).

Five studies have examined APOE variants in relation to colorectal cancer. One study found that APOE ε4 was associated with significantly reduced risk of proximal colon cancer (OR 0.35, 95% CI 0.14–0.86). A study conducted in the United Kingdom found that persons with the ε2/ε3 genotype had a 90% increased risk of colorectal cancer compared with the ε3/ε3 genotype; no association was found with the ε4 genotype. A U.S. study found that the absence of an APOE ε3 allele significantly increased the risk of colon cancer (OR 1.37 95% CI 1.00–1.87), especially among those diagnosed at greater than 64 years of age; in this study, APOE genotype was not associated with rectal cancer.

Physical activity, obesity, and insulin-related variants

More than 40 case-control or cohort studies have examined physical activity and the risk of colorectal cancer (93). These studies provide consistent evidence that physical activity is associated with a reduced risk of colon cancer, with relative risks for the highest category of activity compared with the lowest in the range 0.4–0.9 (94). The risk decreases in a dose–response fashion with increasing levels of activity (93). Excess weight raises risk of developing colon cancer (but not rectal cancer), with an increase of 15% in risk for an overweight person and 33% for an obese person (95,96). The similarity of risk factors for colon cancer and diabetes, and the observation that insulin promotes the growth of colon cells in vitro and colon tumors in vivo, suggested that hyperinsulinaemia and insulin resistance could lead to colorectal cancer through the growth-promoting effects of elevated levels of insulin, glucose, or triglycerides.

IGF: One mechanism by which raised insulin levels could affect cancer risk is by increasing the bioactivity of insulin-like growth factor-1 (IGF-1) and inhibiting production of two main binding proteins, IGFBP-1 and IGFBP-2 (97). IGF-1 has mitogenic effects on normal and neoplastic cells, inhibiting apoptosis and stimulating cell proliferation (97). The machinery of the IGF complex is comprised of peptide ligands (IGF-I and IGF-II), as well as their respective receptors, binding proteins (IGFBP-1–6), and IGFBP proteases. The combination of a Western-style diet, sedentary lifestyle, and obesity might lead to an increase in circulating insulin levels, which could trigger elevation of IGF-I bioavailability through insulin-mediated changes in IGFBP concentrations (98).

Four prospective studies of colorectal cancer have observed a greater than twofold increased risk amongst those in the highest quintile of circulating IGF-1 levels, compared with those in the lowest quintile. However, one of the four studies looked separately at rectal cancer and found a statistically nonsignificant (p = 0.09) inverse trend, which provides some weak evidence that this relationship may not hold for rectal cancer. One prospective study observed an inverse relationship between IGFBP-1 and IGFBP-2 levels and colorectal cancer, but two others were null.

One study reported that a genetic variant at position 1663 in the human growth hormone-1 (GH1) gene reduced colorectal cancer risk. Another study reported that variants in genes encoding the insulin receptor substrates (IRS-1, IRS-2) increased colon cancer risk; this study also reported that variants in the IGF-1 and IGFBP-3 genes were not independently related to cancer but appeared to act together with IRS-1 to influence risk. IRS-1 and IGF-1 variants have been reported to be associated with an increased risk of colon cancers with specific KRAS2 and TP53 mutations. All of these findings require replication.

Genetic Variants Affecting Inflammation and Immune Response

Prostaglandin-endoperoxide synthase (PTGS), also known as cyclo-oxygenase (COX), is involved in the biosynthesis of the prostanoids. It has two isozymes, a constitutive PTGS1, and an inducible PTGS2. PTGS2 is involved in inflammation and mitogenesis.

PTGS1/COX1. No associations with colorectal cancer were found in three studies of variants in the PTGS1/COX1 gene; however, the rarity of the minor variants necessitates larger, population-based studies.

PTGS2/COX2. A large study in Beijing found positive associations with variants at -1195 and -765; however, two other studies found no association with the -765 variant. Two studies of the Val511Ala variant (rs5273) found no association with colorectal cancer.

PPARδ and PPARγ

Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that function as transcription factors regulating gene expression. PPARγ is expressed in high levels in normal colonic mucosa, colorectal adenoma, and colon cancer cell lines; it has been implicated as a potential mediator of colorectal cancer risk in animal studies. An association study of PPARγ Pro12Ala (rs1801282) reported the following OR (95% CI): 0.83 (0.69–1.01) for proximal tumors, 1.00 (0.83–1.21) for distal tumors, and 1.04 (0.86–1.25) for rectal tumors. No association with this variant was observed in two other studies. Single studies found no association of PPARγ C1431 or an unspecified rare variant of PPARδ with colorectal cancer. A single study of the PPARγ C478T variant suggested that the TT genotype might be associated with reduced risk.

Cytokine genes

Cytokines include the interleukins, lymphokines, and cell signal molecules, such as tumor necrosis factor and the interferons. IL-1, IL-6, and IL-8 proteins are generally considered proinflammatory and IL-4, IL-4R, and IL-10 antiinflammatory in effect.

Interleukin-1β: The three most commonly studied SNPs in IL-1β are T-31C (rs1143627), C-511T (rs16944), and +3954C/T (rs1143634); the first two are in close linkage disequilibrium and may influence gene expression. Three studies of T-31C found no significant association with colorectal cancer. Two studies that examined the C-511T variant in H. pylori positive persons found that risk of colorectal cancer was reduced in those carrying the T-allele.

Interleukin-6: Studies of the IL-6 -174G/C variant (rs1800795) have produced conflicting results.

Interleukin-8: No clear association has been found for the IL-8 T-251A variant (rs4073) with colorectal cancer.

Interleukin-4: In a study reporting a significant inverse association of the IL-4 -584T allele (rs2243250) with colorectal cancer, Hardy-Weinberg equilibrium was violated in the control samples and this association was not replicated in a subsequent study.

Interleukin-10: Two studies found no association with colorectal cancer for any of three variants in the promoter region of the IL-10 gene: -1082G/A (rs1800871), -592C/A (rs1800872), and -819C/T (rs1800871).

Interleukin-1RN: Two small studies investigating the association of an 86 base-pair VNTR variant in intron 2 of the IL-1RN gene (interleukin-1 receptor antagonist) with colorectal cancer reported conflicting results. A population-based study in Germany found no association of the IL-1RN A9589T variant (rs454078) with colorectal cancer.

Interleukins-12A and 18: In a single study of colorectal cancer and variants of IL12A, no association was observed. In a small single study of IL18, a positive association was found with the 607A variant.

Tumor necrosis factor-α (TNFα): None of five studies investigating the association of the -308G/A variant (rs1800629) with colorectal cancer found significant associations. Investigations of a TNFα microsatellite dinucleotide repeat polymorphism have had conflicting results.

Toll-like receptors (TLRs)

Toll-like receptors are a key component of the innate immune system and inflammatory response to pathogens through activation of the NF-κB and mitogen-associated protein (MAP) kinase signaling pathways (99). Toll-like receptor 4 (TLR4) is of particular interest with respect to gastrointestinal malignancies (99,100).

Three studies have examined the association of colorectal cancer with variants in the TLR4 gene. Positive results from a small study in Croatia were not replicated in two larger studies. The Croatian study also reported an association of a GT dinucleotide repeat microsatellite variant (intron 2) in the TLR2 gene but no association with the Arg753Gln variant (rs5743708).

Other inflammation-related or immunoregulatory genes

HRAS (Harvey Rat Sarcoma Virus Proto-oncogene): H-ras1, a proto-oncogene that encodes a protein involved in mitogenic signal transduction and differentiation, is highly polymorphic in humans (19). Several studies have evaluated the association between HRAS1-VNTR rare alleles and colorectal cancer. Two systematic reviews that included pooled analyses of HRAS1-VNTR rare alleles (frequencies in controls ranging from 1% to 6%) reported the following OR (95% CI): 2.5 (1.54–4.05) and 2.67 (1.47–4.85).

NF-kappaB (NFKB1): Nuclear factor-kappaB (NF-kappaB) is an inducible transcription factor that plays a major role in the regulation of genes involved in immune and inflammatory response. Studies of an insertion/deletion variant (-94ins/delATTG) in the promoter region of the NFKB1 gene have had variable results. The insertion/deletion variant was not associated with colorectal cancer survival. Another study reported a significant association with sporadic colorectal cancer in a Swedish study population with ORs and 95% CIs of 7.73 (3.06–19.57) for heterozygote deletion and 6.58 (2.35–18.43) for homozygote deletion; no association was found among Swedish patients with a family history of colorectal cancer or in a Chinese population.

LTA/TNFβ: Lymphotoxin alpha (LTA), a member of the TNF superfamily, is also known as TNFβ. A single study has reported association with colorectal cancer for a haplotype in the major histocompatibility locus region containing SNPs of TNFα, RAGE, HSP70-2, and LTA. Another study suggested an association with the NcoI RFLP of TNFβ.

NOS2: A single study found no association of NOS2 tetra-repeat and penta-repeat polymorphisms with colorectal cancer. Another study found no association with the NOS2A +524T>C variant.

Genetic Variation and Exogenous Hormones

Exogenous estrogens such as hormone replacement therapy (HRT) might be associated with colorectal tumors. In two large randomized controlled trials of the possible health benefits of HRT in postmenopausal women (101), the incidence of colorectal cancer was reduced by about a third (pooled RR = 0.64, 95% CI 0.45–0.92) (102). Information is limited on the potential genetic modifiers of this apparent protective effect (see Section “Gene–Environment Interaction in the Etiology of Colorectal Cancer”).

Estrogen-metabolizing genes: Seven variants in ten estrogen-metabolizing genes were studied for association with colorectal cancer risk: COMT (Val158Met, rs4680), HSD17 (v1V), CYP17 (rs743572), CYP19 (Arg264Cys, rs70051; C1558T), CYP1A1 (Ile462Val, rs1048943; MspI RFLP), CYP1B1 (Leu432Val, rs1056836), and estrogen receptor (ER) α IVSI (C401T, rs2234693). No associations were found.

Estrogen and androgen receptors: A single study investigated the role of variants in the ERα (A351G, rs9340799), ERβ (G1082A, rs1256049, and a CA repeat variant in intron 5), and androgen receptor (AR, CAG repeat variant) genes. The risk of colorectal cancer was increased in women with at least 25 CA repeats on both alleles in ER β (OR 2.13, 95% CI 1.24–3.64) and in men with increasing numbers of AR CAG repeats (OR 1.28, 95% CI 1.06–1.54). These studies have not been replicated.

Genetic Variants Associated with Adhesion Molecules and Extracellular Matrix Remodeling

Tumor cell–stromal cell interactions and remodeling of the extracellular matrix (ECM) have implications for the progression and spread of cancer (103,104).

CDH1: Of the many cell–cell adhesion molecules, E-cadherin (encoded by CDH1l) has so far received the greatest attention in relation to colorectal cancer. A systematic review of colorectal cancer in association with CDH1*160A reported a pooled OR 1.15 (95% CI 0.89–1.5). Two subsequent studies reported that the A-allele was not associated with colorectal cancer. A systematic review of association with the 870A variant reported OR 1.19 (95% CI 1.06–1.34).

ICAM1: A single study reported no associations with colorectal cancer for variants in the ICAM1 gene (G241R, rs1799969; K469E, rs5498).

MMP variants: Matrix metalloproteinases, a family of 23 enzymes in humans, are important for proteolysis of the extracellular matrix but also for cell growth, regulation of apoptosis, and cell motility (105,106). Nine studies have investigated associations between variants in this family of genes and colorectal cancer. Several small studies suggested that the homozygous MMP-1 1607G genotype is associated with colorectal cancer, but this was not replicated in larger studies. In one of these studies, a MMP-3 variant causing lower enzyme activity was associated with colorectal cancer (OR = 2.1; 95% CI = 1.2–3.8). No consistent pattern of association between MMP-2, -3, or -9 promoter variants and colorectal cancer has been found.

Genetic Variants Affecting Angiogenesis

Angiogenesis is a key process in the development and progression of cancer (107). Signaling by vascular endothelial growth factor (VEGF) is an important rate-limiting step in angiogenesis (107). Four members of the VEGF family have been identified—VEGF-A, VEGF-B, VEGF-C, and VEGF-D (now designated FIGF, c-fos induced growth factor). VEGF-A is the most abundant in colorectal tissues, where increased VEGF-A expression has been observed (108,109). Increased expression of VEGF-A and VEGF-C has also been reported in colorectal cancer (108).

No associations with colorectal cancer have been found for any of three VEGF-A variants: -2578C/A, -634G/C, and +936C/T).

Very limited evidence is available to assess the importance of other genes thought to be implicated in angiogenesis and related inflammatory pathways in the development of colorectal cancer. Two studies reported no association with colorectal cancer of the G801A variant (rs1801157) of CXCL12.

Associations of colorectal cancer with variation in two other angiogenesis-related genes (PTGS2/COX2, IL-8) are reviewed in Sections “PTGS2/COX2” and “Cytokine genes.”

Genetic Variants Affecting Inhibition of Cell Growth

TGF-beta signaling pathway

TGF-β is a cell growth inhibitor that acts by binding to type I (TGFBR1) and type II (TGFBR2) transmembrane receptors to form a heteromeric complex, TGFBR1/TGFBR2. TGFBR2 phosphorylates TGFBR1, which in turn activates TGFBR1 kinase. Defects in this mechanism can lead to unrestricted cell growth due to the loss of growth inhibitory activity.

TGFB1: Two studies reported no association of variants in the TGFB1 gene (transforming growth factor-β1) with colorectal cancer.

TGF-beta receptors (TGFBR1, TGFBR2): A meta-analysis comprising 12 case-control studies of colorectal cancer, with a combined 1,585 cases and 4,399 controls, reported an association of TGFBR1*6A with colorectal cancer (OR 1.20, 95% CI 1.01– 1.43). Germline mutations of TGFBR2 may predispose to the development of HNPCC.

Cell cycle regulatory genes (CCND1)

Cyclin D1, encoded by the CCND1 gene, has a key role in the cell cycle. A recent meta-analysis comprising 12 case-control studies and a total of 8,260 cases reported a small but significant positive association of the G870A variant (rs603965) with colorectal cancer (OR = 1.19, 95% CI 1.06–1.34) (110). In a subsequent study, risk of familial (but not sporadic) colorectal cancer was increased in persons with homozygous AA genotypes, compared with GG homozygotes.

Common Low-Penetrance Variants Identified from Genome-Wide Association Studies

Recently, the increasing availability of multigene chips and microarrays has prompted a move toward “scanning” large numbers of SNPs for possible associations with disease. In this section, we briefly summarize genome-wide association studies (GWAS) of colorectal cancer that had been reported at the time of writing.

Known rare, high-penetrance germline mutations account for less than 5% of cases of colorectal cancer. Recent findings from GWAS have identified common genetic variants at six loci, increasing the proportion of colorectal cancer that can be associated with specific genetic risk factors. ORs are typically in the range of 1.1–1.4 for heterozygous carriers of the risk allele and 1.6–1.7 for homozygotes. The associations of these six loci with colorectal cancer tend to be consistent among studied populations in different parts of the world.

The first of these six loci, on chromosome 8q24, is close to POU5F1P1, a known transcription factor, and 340,873 bp telomeric to the oncogene MYC (111–113). Variants from several regions at this locus, separated by sites of recombination, confer independent risk and have also been shown to be associated with prostate and breast cancer risk.

The second locus, on chromosome 15q13.3, is known as CRAC1 or HMPS (hereditary mixed polyposis syndrome) (114). One SNP is located in the 3′ UTR of GREM1, which codes a bone morphogenetic protein (BMP) involved in the TGFbeta/BMP pathway that is causally involved in juvenile polyposis.

The third locus, SMAD7, on chromosome 18q21, is involved in TGF-beta and Wnt signaling (115). SMAD7 acts as an intracellular antagonist of TGF-beta signaling and changes in its expression have been shown to influence progression of colorectal cancer.

A fourth locus, at 8q23, contains the gene EIF3H, for which amplification and overexpression have been described in breast, prostate, and hepatocellular cancers (116). No causal gene has been identified at the fifth locus at 10p14 (116). The sixth locus, at 11q23, contains four open reading frames and a polymorphic binding site for micro-RNAs (117).

The six loci identified so far by GWAS account for <5% of excess familial colorectal cancer risk. Given the limited power of these studies to detect the least common variants, it seems likely that many (perhaps 50–100) additional common variants remain to be discovered. Although their individual effects on risk are small, the combined effects of several variants could produce much larger risks and so could be clinically useful in directing prevention strategies. Further development of risk profiling using common variants will require the identification of additional variants in larger GWAS and through meta-analysis of GWAS. Large, multinational cohort studies will be needed to validate such genetic risk predictive models.

Gene–Environment Interaction in the Etiology of Colorectal Cancer

Investigation of potential gene–environment interactions has focused on candidate genes with a role in metabolism of dietary, drug, and environmental constituents associated with risk of colorectal cancer. Studies have investigated interactions of variants of GSTM1, GSTT1, and CYP1A1 with tobacco smoking; APC variants with diet (intake of total fat and specific fat types) and lifestyle factors (taking hormone replacement therapy [HRT]); HFE C282Y and H63D with age, gender, ethnic group, other genes, smoking, alcohol intake and dietary intake of iron; CYP1A1, NAT1, and NAT2 with meat intake; variants of GSTM1, GSTT1, and CYP1A1 with vegetable intake; variants of MTHFR, MTR, MTRR, and CBS with intake of folate and related nutrients; variants of COX2 (PTGS2) PPARD, UGT1A6, CYP2C8, CYP2C9, and genes encoding the interleukins with NSAID use; and genes thought to influence hormone metabolism with HRT use.

Overall, consistent evidence of gene–environment interaction has not been observed. Although investigating the complex interplay of genes and environment is widely considered to offer much promise for improving our understanding of the etiology of complex diseases, including colorectal cancer, research in this area is challenging (118). Many studies of gene–environment interaction and colorectal cancer have lacked statistical power to detect interaction. For example, of four studies that assessed interactions of CYP1A1 variants with different levels of smoking and consumption of meat, vegetables, and fruit, two studies included only about 200 cases. The methods used to test for interaction have varied among studies, making it difficult to integrate and summarize the evidence. For example, some studies have classified persons as “smokers” or “nonsmokers” (or “smokers,” “former smokers,” or “nonsmokers”); however, others have collected detailed information concerning the number of cigarettes smoked per day, age when individuals started smoking, and number of years smoked. Furthermore, although the metabolism of any exposure is likely to depend on the balance among the relative activities of all enzymes active within the metabolic pathway, few studies have investigated interactions of exposures with combinations of genes (or SNPs) operating in such pathways. New analytical approaches for exploring gene pathways in disease etiology are under development but their performance characteristics and properties are not yet well understood.

Conclusions

Inherited genetic factors play an important role in the etiology of colorectal cancer. Rare high-penetrance mutations account for a small proportion of disease but their identification plays an important role in the clinical management of the high-risk families in which these mutations segregate. The results of most candidate gene association studies of colorectal cancer have not been replicated consistently. Many results can be considered false positives; others may represent very small effects, which will require replication in larger studies before firm conclusions can be reached.

More recently, genome-wide association studies have discovered many common, low-penetrance genetic variants associated with risk of colorectal cancer. These studies have been conducted by large-scale, international collaborations (see Chapter 6). Further research is required to identify causal variants and to investigate pathophysiological pathways. Before tests for multiple common variants are used for risk profiling (e.g., to guide prevention and treatment strategies), prospective studies in several populations will be needed to validate risk estimates and to demonstrate improved health outcomes.

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Tables

• Table 13-1
• Table 13-2
• Table 13-3

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