1. Introduction
Colorectal cancer (CRC) is one of the major causes of morbidity and mortality, representing the second major cause of cancer incidence among females and the third among males. Epidemiologists reported that in 2008 the annual worldwide incidence of CRC cases was 1.2 million, almost equally split between males and females [
1]. Worldwide incidence appears to be highly variable with increasing trends in countries historically considered at lower risk.
The pathogenesis of CRC is very complex and diverse and is also influenced by multiple factors, some of which are related to diet and lifestyle, while others are related to genetic predisposition. Another risk factor is the presence of long-standing inflammatory bowel diseases (IBD), either Crohn’s or ulcerative colitis [
2]. Several epidemiological studies have confirmed the involvement of numerous environmental and dietary factors, such as cigarette smoking, alcohol abuse, a diet high in fat and low in fiber, a sedentary lifestyle and obesity [
3]. Physical activity [
4], long-term therapy with low-dose aspirin [
5], and the Mediterranean diet [
6] have proved to have possible preventive effects.
The pathogenesis of CRC varies according to genetic or epigenetic changes, which are related to each other in varying degrees. They follow the multiple stages pattern theorized by Fearon and Vogelstein [
7]. Such genetic and epigenetic alterations are directly responsible for a specific event within the sequence that leads to CRC, by contributing to the “initiation” of neoplastic transformation of healthy epithelium and/or determining the “progression” towards more malignant stages of the illness.
The different pathways are characterized by distinctive models of genetic instability, subsequent clinical manifestations, and pathological behavior characteristics. Most CRC follows the chromosomal instability (CIN) pathway, characterized by widespread loss of heterozygosis (LOH) and gross chromosomal abnormalities [
8,
9]. The second involves approximately 15% of CRC and is due to derangement of the DNA Mismatch Repair (MMR) system and consequential microsatellite instability (MSI). The MMR system is responsible for the production of proteins that recognize and direct repair of single nucleotide mismatches at microsatellite sequences that escape the proofreading system of DNA polymerase.
In recent years, it has been established that other systems and pathways are involved in the pathogenesis of colorectal cancer, including abnormal DNA methylation, inflammation and, more recently the discovery that microRNA (miRNA) can actively contribute to the carcinogenic process. These, along with the aforementioned CIN, MSI and DNA methylation will be discussed.
3. MSI Pathway
The MSI pathway represents a form of genomic instability involved in the genesis of approximately 15% of sporadic colorectal cancer and >95% of Hereditary Non Polyposis Colorectal Cancer (HNPCC) syndrome. MSI is caused by the inactivity of the DNA Mismatch Repair (MMR) system. Disabled DNA MMR causes a 100-fold increase in the mutation rate in colorectal mucosa cells [
58]. The MMR system is a multi-protein system, which acts like a proofing machine to increase the fidelity of DNA replications by identifications and direct repair of mismatched nucleotides [
59,
60]. The MMR system acts only when an error eludes the intrinsic error checking system of DNA polymerase [
59]. In human cells, the functioning MMR system is composed of multiple interacting proteins including the human MutS homologue (MSH) 2, and human MutL homologue (MLH) 1.
CRC that develops through the MSI pathway presents peculiar clinical features: more often located in the proximal colon, with a poorly differentiated and a mucinous or medullary histotype, and often presents intense peritumoral and intratumoral lymphocytic infiltrations [
61]. In general, the prognosis and survival of patients affected by MSI-high CRC is better and longer than that of patients with CIN positive CRC [
61]. Importantly, MSI-high CRC does not respond to 5-fluorouracyl-based chemotherapies [
62].
In the HNPCC syndrome, CRC development is determined by germline mutation in one of the MMR components. HNPCC is an autosomal dominant genetic disorder characterized by a young age of onset (<50 years old) of colorectal cancer as well as other malignant tumors, including endometrial and ovarian cancers. In 95% of HNPCCs, mutations are present in
hMLH1 and
hMSH2 [
63]. The clinical manifestations can be diverse, depending upon which gene is involved and where the mutations occur [
64]. Defective
hMSH2 is associated with a 40%–60% increased risk of developing endometrial cancer, while defective
hMLH1 with a 50%–80% increased risk of developing CRC [
65,
66]. Furthermore mutations in
hMSH6 are associated with 11%–19% increased risk of developing gastric cancer [
67] while mutations in
hPMS2 with a 9%–12% increased risk of develop ovarian cancer [
68]. Recently, a subclass of the MMR deficient HNPCC families have been found to carry germline deletions of the Epithelial Cell Adhesion Molecule (EpCAM) resulting in
hMSH2 gene silencing [
69]. EPCAM carriers show a lower risk of developing endometrial cancers. HNPCC is a good example of a genotype-phenotype association, and the identification of mutation carriers is critical for implementing optimum screening and follow-up procedures [
63]. Also, in families with high suspected HNPCC, clinical parameters can help direct new suspected cases toward targeted genetic testing [
70].
In sporadic settings, MSI-high CRCs are mostly due to epigenetic silencing of the
hMLH1 gene promoter [
71–
74]. The resulting mutant phenotype, as in HNPCC settings, leads to inactivation of target genes, in particular tumor suppressors having a microsatellite sequence in their coding region. Importantly, sporadic MSI-high CRC cases harbor the
V600E mutation of the
BRAF oncogene, a member of the
RAF family involved in the mediation of cellular response to the growth signal through the RAS-RAF-MAP kinase [
75]. MSI-high sporadic CRCs display CIMP features (a combination of two pathways), and will be described further in the CIMP pathway section.
More than 80% of MSI-CRC harbor mutations of the TGF-β Receptor II (TGF-βRII) [
76]. TGF-βRII mutations are found in adenomas either featuring high-grade dysplasia or progressing to adenocarcinoma, and represent a common cause of neoplastic progression in the late and metastatic steps of MSI-High CRCs [
77]. Additionally, mutations in the Smad2 and Smad4 genes, part of the TGF-β pathway, are common in MSI-high CRCs [
78]. Smad4 mutations facilitate the switch to the tumor-promoting role of TGF-β signaling [
79]. Eppert and colleagues demonstrated that the loss of function of Smad2 contributes, independently of Smad4, to deactivated TGF-β signaling [
80]. Another mutational target in the genesis of MSI-high CRCs is the alteration of the 2 polyadenine (A8) tracts in exon 10 of the activin type 2 receptor (ACVR2). The ACVR2 gene encodes for a transmembrane receptor, whose activation causes differentiation and growth suppression signaling through the phosphorylation of Smad2 and Smad3 proteins. Jung and colleagues identified these mutations only in MSI-high CRCs—further demonstrating that the
ACVR2 mutation occurred frequently with
TGF-βR2 mutations [
81].
Another target gene in the MSI-high CRC pathway is the pro-apoptotic tumor suppressor gene
BAX. Homozygous frameshift mutations of
BAX occur in 50% of CRCs cases and promote the cell’s escape from intrinsic apoptosis mechanisms [
82,
83].
BAX gene mutations, like
TGF-βRII mutations, can be present in neoplastic progressions despite early adenoma mutations [
84]. However, Shima and colleagues studied the co-occurring mutations of
TGF-βRII and
BAX in a large cohort of patients, and demonstrated that MSI-high CRCs were associated with a better prognosis than MS stable CRCs, regardless of the presence of mutations of
TGF-βRII and
BAX [
85].
In addition to the above-mentioned genes frequently present in MSI-high CRC, other genes are present at a lower frequency (around 20%) including mutations in the MMR genes
hMSH3 (36.5%) and
hMSH6 (17.5%), Insulin Growth Factor Type 2 Receptor (IGFIIR) (22%),
BLM gene (16%), PIK3CA (15%), G protein-coupled receptor of Prostaglandin-endoperoxide synthase 2 (PTGS2) (33%) and
Cyclin D1 gene (28%) [
86–
90].
Recently, Baba and colleagues found that G protein-coupled receptor
PTGER2 overexpression, the downstream target of
PGE2, which is involved in inflammation and cancer, is strongly associated with MSI [
88].
Finally, Ogino demonstrated that the presence of
cyclin D1 in the colon neoplastic mucosa was found not only in patients with the altered CIN pathway, but also in those with the altered MSI pathway. Its overexpression was associated with lower colon cancer–specific, and overall, mortality [
91].
4. CIMP and the “Serrated” Pathway
A third pathway through which CRC progresses is the CpG island methylator phenotype (CIMP) [
92,
93]. It consists of the aberrant hypermethylation of CpG dinucleotide sequences localized in the promoter regions of genes involved in cell cycle regulation, apoptosis, angiogenesis, DNA repair, invasion and adhesion. The promoter hypermethylations cause the loss of gene expression. CIMP is found in approximately 20%–30% of CRC and it was reported that clinical features of CIMP CRCs are similar to those associated with MSI [
94]. An early event that is correlated with the progression of histologic grades is the silencing of the
p16INK4a tumor suppressor gene, whose loss of function causes uncontrolled cell proliferation, leading to neoplastic transformation [
95–
98].
Based on the number of methylated markers, the CIMP phenotype can be also divided into CIMP-high and CIMP-low. The
BRAF oncogene mutation is often identified in CIMP-high CRC and is associated with increased cell growth, progression of carcinogenesis, and high colon cancer specific mortality [
99]. However CIMP-high tumors, regardless of BRAF mutation, are associated with reduced colon cancer mortality [
99].
Importantly BRAF V600E mutations were found in 90% of CRC cases with sessile serrated adenoma (SSA) lesions and never in the conventional adenomas. The BRAF mutation is an early event in the serrated pathway and its forced expression will lead to a state of dormancy known as senescence. In SSA, BRAF mutations were found either in early hyperplastic polyps (the serrated precursors) or in the advanced dysplastic serrated polyps, confirming its role in neoplastic progression [
100–
102]. The SSA polyps and the BRAF mutation frequently have CIMP-high and MSI-high features; thus, researchers established that, in sporadic settings, CIMP-high microsatellite unstable CRCs derive from the serrated pathway [
101].
BRAF and KRAS mutations are mutually exclusive [
103]. Recently, researchers discovered that when KRAS mutation was found in CIMP CRCs, it is associated with lower markers of methylation, called CIMP-low. This is also frequently associated with mutations in the DNA repair gene Methylguanine Methyltransferase (MGMT) and with the loss of function of the PIK3CA [
98,
104,
105]. CIMP-low, in contrast with CIMP-high, appears to have different phenotype, with a low-level of DNA methylation [
106]. An alternative serrated pathway was extensively studied by Jass and colleagues, who described polyps in an “alternative serrated pathway”, as a hybrid of adenomatous and serrated polyps. They hypothesized that these polyps, carrying
K-ras mutation, represent only 2% of CRC, but present an extremely aggressive malignant potential, through inactivations of MGMT [
103,
107,
108].
The progression out of the senescence state can also be determined by the loss of
p53 function and by the silencing of insulin-like growth factor binding protein 7 (IGFBP7), an important mediator of the
p53-induced senescence [
109]. Ogino S. and colleagues also found that the silencing, and subsequently the downregulation, of cyclin-dependent kinase inhibitor-1B (CDKM1B or
p27) was associated with CIMP-high CRC and, like IGFBP7, was associated with dysfunctions in
p53 [
110]. DNA methyltransferase-3B (DMT3B) overexpression seems to play a role in establishing and maintaining the aformentioned methylation patterns [
111].
6. Conclusions
The findings that different molecular pathways are involved in colorectal cancer development have helped researchers build different models and understand how colorectal cancer initiates and progresses. However, the application of molecular markers on large-scale populations is now facilitating the understanding of the peculiar role of these alterations on disease behavior, prognosis and response to treatments. Among them, the CIMP pathway and the contribution from miRNA require further examination and investigation by researchers for a better and more complete understanding.
The results from the cited studies (
Table 1) will be useful for developing strategies, possibly with the use of multiple molecular markers, to predict future disease behavior in newly cancer-diagnosed patients. Importantly, this will help define therapeutic strategies, even with anti-inflammatory drugs, for each individual patient based on their molecular tumor profile. Interestingly, several molecular markers (BRAF and PI3KCA, to cite some) have been found to be predictors of colon cancer risk and mortality in relation to aspirin and anti-inflammatory drugs consumption. As stated above, inflammation is a key contributor to colorectal carcinogenesis and anti-inflammatory drugs have been extensively explored also as chemopreventive agents. The recent findings that long-term use of low-dose Aspirin is protective against colorectal cancer development, clearly indicates that anti-inflammatory drugs could be effectively used to prevent colorectal cancer [
5]. However, results from the CAPP-1 [
129] and CAPP-2 [
130] trials have yielded negative results on the use of aspirin in FAP and HNPCC populations, and the reasons are unclear. Thus selection of patients suitable for chemoprevention should be performed, and possibly baseline inflammatory markers could be of help for the selection process.