Analysis of molecular aberrations of Wnt pathway gladiators in colorectal cancer in the Kashmiri population

Colorectal cancer (CRC) is a major cause of mortality and morbidity, and the third most common malignancy in the world [1]. The incidence of this malignancy shows considerable variation among racially or ethnically defined populations in multiracial/ethnic countries. It is the fourth most common cancer in men and the third most common in women worldwide [1]. Kashmir has been reported as being a high-incidence area for gastrointestinal (GIT) cancers [2, 3]. In the Kashmir valley, CRC represents the third most common GIT cancer after oesophageal and gastric cancer [4, 5].

It has been suggested that CRC is a multi-step process which arises from cumulative aberrations of a number of different genes (including tumour suppressor genes, proto-oncogenes, DNA repair genes, the genes encoding growth factors and their receptors, cell cycle checkpoint genes and apoptosis-related genes) or from epigenetic changes in DNA at different stages of development and progression [6, 7]. It is believed that mutations in the gene encoding adenomatous polyposis coli (APC) or that encoding β-catenin set the stage for the initiation and transformation of normal colonic epithelial cells. Further accumulation of mutations in other genes then contributes to the progression of cancer through adenoma - carcinoma - metastasis stages. The generally accepted model of CRC tumorigenesis for the majority of tumours has been a stepwise progression, in which mutations in APC are followed by several other mutations, including alterations in the genes encoding Kirsten ras (K-ras) and tumour protein 53 (TP53) [6–8]. During the accumulation of genetic changes, a complex signalling network is established among inactivated and activated cellular pathways [9].

The Wnt pathway regulates cell adhesion, morphology, proliferation, migration and structural remodelling [9, 10] and plays an important role in a variety of cellular processes, including proliferation, differentiation, survival, apoptosis and cell motility [11]. Loss of regulation of the Wnt pathway has been implicated in the development of several types of cancers, including colon, lung, breast, thyroid and prostate cancers and leukemia [12–15]. Two of the most important gladiator molecules of the Wnt pathway are APC and β-catenin.

APC is a classical tumour suppressor gene, located on 5q21, containing 21 exons. The APC transcript is 9.0 kilobases (kb) in length and the most common isoform of the APC protein contains 2,843 amino acids, with a molecular weight of 310 kD. Exon 15 of APC is most important, as it comprises > 75 per cent of the coding sequence of APC and hence is the common target for both germline and somatic mutations, which usually span codons 1286-1513 of this exon [16, 17]. This region represents the mutation cluster region (MCR), and 68-77 per cent of somatic mutations in APC occur in this region [18]. Mutations in APC are considered to be the earliest genetic aberrations in the initiation and progression of CRC,[7, 9, 19] and have also been found in ~60-80 per cent of sporadic carcinomas and adenomas [20, 21]. Using mutant mouse models, various genetic studies have demonstrated that mutations in APC are responsible for intestinal tumorigenesis [22–24]. Homozygous APC mutations in mice lead to embryonic lethality,[23, 25, 26] and conditional deletion of the gene in the adult mouse disrupts homeostasis, not only in the intestines but also in other tissues [27–29].

In addition to the mutational inactivation, hypermethylation of the gene promoter is another important mechanism associated with gene silencing [30]. In many tumours the hypermethylation of CpG islands in gene promoters has been found to be a frequent epigenetic change in cancers, and is usually associated with the loss of transcription of APC [31–38]. Hypermethylation of the APC gene promoters has been reported in about 20-48 per cent of human CRCs [32, 37, 39, 40].

The β-catenin gene is located at 3p22-p21.3 and encompasses 23.2 kb of DNA. It contains 16 exons, with a mRNA transcript of about 2343 base pairs (bp), encoding a 781-amino-acid-residue protein with a molecular weight of 92 kD [41]. This gene is mutated in up to 10 per cent of all sporadic CRC by point mutations or in frame deletions of the serine and threonine residues that are phosphorylated by glycogen synthase kinase 3-beta (GSK3β) [42]. These mutations result in the stabilisation of β-catenin and the activation of Wnt signalling. Mutations in the β-catenin gene occur in exclusivity to APC aberrations, as both molecules are components of the same pathway [19].

Based on the hypothesis that CRC carcinogenesis is a multi-step and multi-gene event, we designed this study to elucidate the role of APC and β-catenin in the development and progression of CRC in the Kashmiri population, and to correlate the gene aberrations and hypermethylation with the clinicopathological parameters of CRC cases.

The Kashmir valley, located in the northern division of India, has a unique ethnic population, living in temperate environmental conditions and with distinctive food habits, which, along with genetic factors, play a large role in the development of GIT cancers [3–5, 45]. As previously reported, the aetiology and incidence of various GIT cancers in this population has been attributed to a probable exposure to nitroso compounds, amines and nitrates reported to be present in local foodstuffs such as hoakhe suen (sun-dried vegetables), pharei and hoggade (sundried and/or smoked fish and meat), hakh (a leafy vegetable of the Brassica family), hot noon chai (salted tea), dried and pickled vegetables and red chilli, and also through smoking hukka (a water pipe) [2–5, 46].

According to the multi-step model of colorectal tumorigenesis,[7] the most common and principal causes of APC inactivation are gene aberrations. A somatic mutation in APC leads to a truncated protein in most sporadic CRCs [15, 36]. Hypermethylation of APC at the promoter region constitutes an alternative mechanism for APC inactivation in breast, lung and GIT cancers, especially CRCs [30, 33, 34, 38, 40, 47]. Combined with these two mechanisms of APC inactivation and the aberrations in the β-catenin gene, the Wnt pathway molecules play an important role in CRC development and progression [48, 49].

The present study involved the mutational analysis of exon 15 (MCR) of APC and exon 3 of the β-catenin gene and also the hypermethylation analysis of two promoters of APC. Although being the important genetic molecule of the Wnt pathway, and implicated in almost 60 per cent of sporadic CRCs, we found APC gene to be aberrant in only 12.79 per cent of CRCs, which was considerably lower than the previously reported frequencies [9, 19, 35, 50–53]. This low frequency suggests that APC may not be the foremost gene to be implicated in the development of CRCs in this population.

Furthermore, we found an SNP (G > A) at codon 1492 in 72 (83.7 per cent) CRC cases. Out of 72 cases, 53 were homozygous variants. This was a novel finding, as it has not been reported previously.

We also found a low frequency (8.1 per cent; seven of 86) of β-catenin mutations in CRC. These results were in line with those in the published literature [54–57]. Exon 3 of β-catenin contains a regulatory domain which is the hotspot for genetic aberrations. Mutations in this exon have been reported in various tumours, resulting in its nuclear accumulation and leading to progression of the tumour [9, 43, 58]. The mutations in the hotspot codons - 32, 33, 41, 45 and 49 - in exon 3 of β-catenin result in an amino acid change at the GSK-3β phosphorylation sites, which in turn affect the phosphorylation mechanism and result in the decreased sequestration of β-catenin by APC [58]. Furthermore, the mutation affecting codon 45 (TCT > TTT; Ser > Phe) was present in a Lynch syndrome patient, as has been reported previously [55, 56]. Also, six of seven tumour samples which harboured β-catenin gene mutations were wild-type for APC (MCR only), which further corroborated findings in the literature that mutations of the genes encoding these two Wnt pathway molecules are mutually exclusive [9, 54, 57, 59, 60]. Only one case (A9) had mutations in both genes (Tables 4 and 6). Overall, mutations in Wnt pathway molecule genes were found to be present in 20.9 per cent (18 of 86) of CRC cases. Thus, our observation identifies this pathway as being important in determining the development and progression of CRC but is less important than in other populations, where the mutational frequency of these Wnt gladiators is higher.

CpG island hypermethylation is one of the important mechanisms of gene inactivation. Cancer cell lines have in general demonstrated an increased frequency of hypermethylation by comparison with primary tumours [61]. Inactivation of tumour suppressor genes by promoter hypermethylation has been recognised to be as common as gene disruption by mutation in tumorigenesis [36, 37, 62, 63]. A number of studies on CRC around the globe have demonstrated the role of promoter hypermethylation of a number of different genes in the development and progression of CRC [32, 64, 65]. Promoter hypermethylation of APC, similarly to that of other genes, plays a pivotal role in the inactivation of APC, which in turn enhances tumour development [30, 33].

In the present study, we found hypermethylation in 54.65 per cent (47/86) of CRC cases, which is consistent with the results found in some other major studies, although markedly higher than reported in others [30, 32, 33]. However, only 26.7 per cent (23/86) of the tumours were hypermethylated at both the 1A and 1B promoters. This may be due to the fact that there is less mutational inactivation of APC in this population and also because this population is exposed to a special set of environmental challenges, such as extreme temperature, high altitude, special food habits and exposure to agricultural by-products such as pesticides and nitrosamines [5, 46]. As has been revealed in previous studies, promoter hypermethylation constitutes an alternative hit in the inactivation of APC in cancers,[39, 66] and we have identified the same phenomenon as the major cause of APC inactivation in our population. Various studies have shown transcriptional repression of APC by hypermethylation in tumours as well as cell lines [34, 67].

Arnold et al. demonstrated the loss of protein expression due to the promoter hypermethylation of APC [39]. In addition, 42.8 per cent (three of seven) of patients with a mutation in β-catenin were also found to have hypermethylation of APC. We found the methylation status of the APC promoter to be associated with age (> 60), tumour location (colon) and nodal status/tumour grade (C + D) in CRC.

We conclude that, in the Kashmir valley population, although mutational aberration of the genes encoding two pivotal molecules of the Wnt pathway - APC and β-catenin - occurs at a low frequency in CRC cases, the high level of epigenetic silencing of APC plays a pivotal role in the initial tumorigenesis and also enhances the chances of tumour development and progression to advanced stages.