Molecular Pathways of Colorectal Carcinogenesis are Promising Mistery?

The specific etiologic factors and pathogenetic mechanisms, underlying the development of cancers of the colon and rectum appear to be complex and heterogeneous. Despite the continuous advancement in diagnostic and therapeutic methods, despite the global and national programs for prevention it is evidenced increase in colorectal cancer (CRC) incidence and mortality.Further progress is needed in the field of non-invasive diagnostic methods to enable early diagnosis, pre and postoperative staging, and to assist in selecting the most suitable neo-adjuvant and adjuvant therapeutic methods and post-treatment follow-up. There are attempts to "personalise and individualise" anticancer therapy based on presence or absence of specific biomarkers. In this review we will focus on the prevalence, the latest progress made within the genomic and proteomic fields and their significance as possible prognostic and predictive markers in CRC patients.


Introduction
Colorectal cancers (CRC) are the third most common human malignancy, and are also the leading cause of cancer-related deaths worldwide [1]. Recent advances in detection, chemotherapeutic and biological agent based therapies, combined with liver resection have dramatically increased the survival rate of CRC patients [1]. However, CRC still remains an uncontrollable disease [2]. Overall, the lifetime risk of developing CRC is about 1 in 20 (1 in 17 for men and 1 in 26 for women (5.1%), and the mortality rate from CRC is also alarming. Relative CRC risk is defined by genetic predisposition and environmental factors, with age being the most important risk factor for sporadic CRC (>90% of sporadic CRCs occurs in individuals over the age of 50). Other risk factors include family history of CRC, a diet low in fibres and folate and high in fat and red meat, alcohol, cigarette smoking, sedentary occupation, obesity and diabetes. Approximately 5% of all CRC are due to inherited genetic mutations. Of the remaining 95% of cases, about 20% have a positive family history but cannot be categorized to any hereditary CRC syndrome. These are probably caused by genetic alterations secondary to an inherited predisposition, or common dietary and environmental factors [1][2][3].

The Adenoma-Carcinoma Sequence
CRCs evolve through a stepwise accumulation of genetic and epigenetic alterations, leading to the transformation of normal colonic mucosa into invasive cancer. Most CRCs arise within pre-existing adenomas which harbour some of the genetic fingerprints of malignant lesions [4]. This transformation is continuing during 10-15 years.The specific factors and pathogenetic mechanisms underlying the development of CRCs are complex and heterogeneous. Etiologic factors include environmental and dietary exposures; still the major challenge is defining specific agents that influence risk of CRCs [5].
Some encouraging advances are visible:specific gene defects that underlie several inherited forms of CRCs are defined, new insights into the constellation in molecular alterations for initiation and progression of sporadic tumours have been done, and the prevalence and mutations in several oncogenes and tumour suppressor genes are discovered.
The adenomatous polyp or adenoma is important precursor lesion to cancer [6]. Only a fraction of adenomas progress to cancer and adenomas>1 cm in size are estimated to have a 15% chance of progression to carcinoma during a 10 year period [7]. Individuals affected by syndromes (i.e. familial adenomatous polyposis) that strongly predispose to the development of adenomas, develop CRCs by the third to fifth decades of life.

Sporadic Colorectal Cancer
Genetic model of colorectal tumorigenesis is based on the oncogene and tumour suppressor gene alterations. Development of CRC is accordance of series of events leading to the transformation of normal mucosa to adenoma and then to carcinoma. Genomic instability is an integral part in this transformation process.Three distinct molecular pathways are important: Chromosomal Instability (CIN) pathway, Microsatellite Instability (MSI) pathway, and the CpG Island Methylator Phenotype (CIMP) pathway [8,9]. Some tumours are exhibiting features of more than one pathway.

Chromosomal instability
The most common cause of genomic instability in CRC is chromosomal instability (65-70% of sporadic CRCs). Gain or loss of whole chromosomes or chromosomal regions harbouring genes integral for the process of CRC carcinogenesis are specific for CIN which is coming out from defects in chromosome segregation with subsequent aneuploidy, telomere dysfunction, and/or defects in the DNA damage response mechanisms [14]. Higher levels of CIN are constantly associated with poorer disease free survival (DFS) [15].

Microsatellite instability
20% mismatch repair function is activated either by somatic mutations or epigenetic inactivation leading to microsatellite instability (MSI) [16,17]. Microsatellites are short repeat nucleotide sequences that are spread out over the whole genome [16,17]. Because of their repetitive manner they are prone to errors during replication. These errors are recognised and repaired by the DNA mismatch repair (MMR) system [18,19]. MSI is a consequence of the inability of the MMR system to correct errors and is reflected by frame shift mutations in the microsatellite repeats. Members of the MMR system which have been identified are MSH2, MLH1, MSH6, PMS2, MLH3, MSH3, PMS1, and Exo1.73. In about 60% of all CRCs, another form of MSI (EMAST-elevated microsatellite alterations at selected tetranucleotide repeats) is present [20].
There have been fewer mutations in K-ras and p53 [21]. In sporadic MSI-high CRCs, B-Raf V600E mutations are often founded [21]. Another one frequent mutation (in 90% of CRC with MSI) can be found; it is mutation in the polyadenine tract of transforming growth factor b type II receptor (TGFbRII) which inactivates gene function.
In the presence of such defective MMR function many genes are susceptible to mutations (also genes which are containing coding repeats involved in DNA signal cell cycle, and the transcription factors ) [21].
In clinical setting, an improved survival has been reported with adjuvant fluorouracil chemotherapy in MSI CRCs of germline origin but not in sporadic cases.Less data are available on predictive value of MIS on response to oxaliplatin based adjuvant chemotherapy [22].
PIK3CA mutation is found in 14% of CRCs. These tumours are mostly found at proximal colon, they are frequently mucinous, and associated with K-Ras mutation. CRCs with K-Ras mutation have mucinous differentiation more frequently than CRCs with wild-type K-Ras [26].

Tumour suppressor gene defects
Loss of heterozygosis (LOH) is a principal mechanism for one allele inactivation of certain tumour suppressor gene in cancer. Most CRCs (75%) have allelic loss of chromosome 17p [27]. The 17pLOH is targeting p53 gene for inactivation. The mutation of LOH of p53 is arising most frequently during the transition of adenoma/carcinoma sequence and may facilitate continued growth and the acquisition of invasive properties in CRCs [28]. Loss of 17p is a late event in the process of colorectal tumorigenesis, not reported in adenomas but in 75% of CRCs [29]. The p53 is a transcription factor with tumour suppressor activity that binds to a specific DNA sequence and activates a number of genes involved in cell cycle arrest, apoptosis, senescence, autophagy, and cellular metabolism. The p53 facilitates the cellular adaptation in response to different cellular stresses including DNA damage by mutagens, oncogenic stimulation, hypoxia, and telomere erosion [30]. 20-50% of sporadic CRC has allelic loss of chromosome 5q [31]. APC and the Mutated in Colorectal Cancer (MCC) genes are located on the long arm of chromosome 5. Somatic APC mutations are found in 60-80% of CRCs as well as in a large percentage of adenomas, indicating that APC mutation is an early event in the process of colorectal tumorigenesis [32]. APC belongs to the canonical Wnt/ wingless pathway and APC protein forms a complex with b-catenin, axin, and glycogen synthase kinase 3 (GSK3) [33]. Activation of the Wnt signalling pathway is a common feature in cancers and leads to its development, progression and metastasis. Wnt/β-catenin signalling pathway is interconnected with the MicroRNA-mediated gene regulation, forming a Wnt/β-catenin-microRNA regulatory network, and this is important for oncotherapy which is targeting the Wnt/βcatenin pathway [34,35]. Although it is known that activation of βcatenin and PI3K pathways are crucial points in the oncogenesis of CRCs, it still remains unknown whether these two pathways functions independently or cooperatively [36]. No association between polymorphisms in regions of frequent mitotic recombination on 5q and CRC risk was found, suggesting that local influences over the rate of loss of heterozygosis at APC can't explain inter-individual differences in susceptibility to colorectal cancer [37]. About 50-70% of primary CRCs and nearly 100% liver metastases express LOH of chromosome 18q. Loss of this locus increases the potential for metastasis and therefore this is a marker of poor prognosis in stages II/III of CRCs. Inactivation of a chromosome 18q tumour suppressor gene has a role in the later stages of carcinogenesis, cancer progression and metastasis [38][39][40]. SMAD4 and SMAD2 genes are tumour suppressor genes on chromosome 18q. Loss of SMAD4 protein expression correlates with poor prognosis [40]. The SMAD4 protein mediates downstream TGF beta signalling events via its function as a transcription factor beta. The transforming growth factor β (TGF-β) pathway acts as a double-edged sword in tumorigenesis. TGF-β is a potent tumour suppressor by constraining epithelial cell growth but is also important in the induction of epithelial-tomesenchymal transition (EMT), thereby enhancing invasiveness and metastasis. Recent data suggest that TGF-β signalling can be correlated with resistance against both targeted and conventional anticancer agents [41].
Deleted in colorectal cancer, gene (DCC) at 18q21.2 encodes a 170-190 kDa protein of the immunoglobulin superfamily and havea role in the regulation of cell adhesion and migration [42]. Genetic and epigenetic alterations collaborate in transcriptional silencing of DCC. Therefore, DDC was suggested as a candidate gene for targeted inactivation by chromosome 18q LOH [43].

Epigenetic changes in colorectal carcinogenesis
Different mutational defects in colorectal tumours lead to alterations in cellular signalling cascades and transcriptional regulation and consequently to many changes in gene expression in the neoplastic cells [44]. But it's clear that non-mutational or epigenetic mechanisms have also very important roles in cancer development process. By the genomic view of colorectal carcinogenesis, CRC genomes has different types of genomic alterations (oncogenic drivers?) from small-scale changes (i.e., point mutations or small index) to large-scale chromosomal copy number changes or rearrangements [45].

DNA methylation
Concordant methylation of the CG di-nucleotides in the promoter region of multiple genes is called CpG Island Methylator Phenotype (CIMP). Patients with CIMP tumours have distinct clinical and pathological characteristics.CRCs can also be classified based on the presence of MSI and CIMP [10]. Such a classification has five molecular subtypes, each with a different molecular profile and clinicopathological features. These are: 1. CIMP high/MSI high (12% of CRC); originates in serrated adenomas and is characterized by BRAF mutation and MLH1 methylation. 2. CIMP high/MSI low or microsatellite stable (8%); originates in serrated adenomas and is characterized by BRAF mutation and methylation of multiple genes. 3. CIMP low/MSI low or microsatellite stable (20%); originates in tubular, tubulovillus, or serrated adenomas and is characterized by chromosomal instability (CIN), K-ras mutation, and MGMT methylation [46]. Next group are sessile serrated adenomas (SSA) which have distinct molecular and pathological changes than traditional adenomas. SSA are progressing in cancer trough different pathway-the serrated neoplasia pathway. Fifth group are CIMP negative/microsatellite stable (57%);originates in traditional adenoma and they are characterized by CIN [46,47].
Cancer genomes seemed to be overall under methylated, but some genomic loci have focal DNA hyper methylation [48,49]. Focal hyper methylation leads to transcriptional silencing, especially at the CpG islands of gene promoters.Between the putative inactivating mechanisms of tumour suppressor genes in cancer genomes it is often preferred over the inactivation by irreversible nucleotide substitutions [50]. Cancer-associated DNA methylation seems to be more dynamic than anticipated, andDNA methylation profiling has been proposed as a toolof early CRC diagnosis using non-invasive resources (i.e., bloodor stool-based) [51][52][53][54]. A unique cancer genome-associated phenomenon in which ten to hundreds of chromosomal rearrangements occur in a "one-off" cellular event is named chromothripsis [55].
Comparison between primary and metastatic CRC genomes proved that most genomic arrangements are shared both by primary and metastatic genomes, indicating that metastasis occurs quite rapidly with few additional mutational events [55,56]. Although the global trend in CRC cells is hypomethylation, a number of CpG rich promoters show increased methylation with associated transcriptional silencing of the downstream gene. A large proportion of CRCs show hyper methylation and transcriptional silencing of potential tumour suppressor genes such as H1C1 on chromosome 17p and Wnt signalling antagonists.Subset of CRCs shows coordinate hypermethylation of numerous promoters' genes suggesting that regulation of DNA methylation may be globally disrupted. Also, adenomatous precursor lesions of CIMP cancers show a distinct histologic phenotype, SSAa (sessile serrated adenomas). In SSAs B-Raf mutations are commonly observed as grow of cancers [57]. Global DNA hypo-methylation also plays an important role in CRC development, possibly through hypo methylation-induced genomic instability. A classic example of hypo methylation and CRC development is LINE-1. LINE-1 or L1 retro transposons account for about 17% of human genome; their methylation status is an important indicator of global DNA methylation level. CpG sites (located within the LINE-1 repetitive DNA elements) in association with tumour genesis tend to undergo dimethylation, [58]. LINE-1 methylation is associated with poor prognosis [58].
Epigenetic inactivation of MMR genes most commonly affects hypermethylation of the MLH1 promoter. These tumours often initially present as SSAs, show hypermethylation of numerous genes (CpG islan methylation phenotype) and B-Raf mutations. In both familial and sporadic MSI-H tumours inactivation of genes with repetitive elements (microsatellites) in their coding sequence might contribute to tumour progression (e.g., TGFbetaIIR, BAX, and HNPCC) [59].

Posttranslational modifications of histones
Among seven distinct types of histone modifications, acetylation and methylation are most extensively characterized ones that are involved in CRC pathogenesis [60][61][62].
Histone acetylation is reversible modifications of lysine residues on histone "tails" and is controlled by histone acetyltransferase (HATs) and histone deacetylases (HDACs) [62]. HDACs are involved in multiple signalling pathways and they are present in numerous repressive chromatin complexes; they also play key roles in CRC development [63,64]. Nuclear expression of HDAC2 was observed in 81.9% of CRCs, vs 53.1% of normal tissues, respectively [65]. A genomic-wide analysis found out thatmicroRNAs (miRNAs) ismethylated in human colon carcinoma cells and it is known that DNA methylation and histone acetylation often act in concert to mediate gene expression in CRC cells leading to 5-FU resistance in CRC cells [66][67][68]. We need more efforts to understand the various epigenetic mechanisms and their cross-talk in the context of 5-FU resistance in CRC cells, which is one of the frontlines in CRC research and drug development.
The BRAF pathway has become a target of interest for molecular therapy, and many B-Raf specific inhibitors display a cytostatic response inducing senescence and are susceptible to acquired resistance [69,70]. Therapy with anti-EGFR antibodies is desirable in patients with advanced CRC and absence of K-ras or B-Raf mutation. MSI status is associated with significantly better prognosis. Defining tumour phenotype MSI or MSS and testing for the presence or absence of 18q chromosome deletion is very much desirable in standard 5-FUbased therapy. Chemotherapy FOLFOX4 regiment did not alter survival of patients with MSI [71]. Studies on CRC biomarkers need to continue to closely examine the relationship between therapy and CRC curability. Since personalized medicine has become more clinically relevant in the last few years, genotyping of tumours is of high importance because anticancer therapy which will be used on specific targets appears to be the future of CRC therapy.

Conclusion
CRC is a genomic disorder in which various types of genomic alterations, such as point mutations, genomic rearrangements, gene fusions, or chromosomal copy number alterations, can contribute to the initiation and progression of the disease. CRC genomes harbour various types of genomic alterations which may contribute to colorectal carcinogenesis as oncogenic drivers, but the full spectrum of driver genomic alterations in CRC genomes is still incomplete. Several studies have suggested that LOH of specific chromosomes, such as chromosome 8q, 17p or 18q may be useful in assessing prognosis in patients with stage II and III colorectal cancer.Studies on CRC biomarkers need to continue to closely examine the relationship between therapy and CRC curability. Nowadays combination therapy is likely to be the most effective management plan for the treatment of B-raf-mutated tumours. Defining tumour phenotype MSI or MSS and testing for the presence or absence of 18q chromosome deletion is very much desirable in standard 5-FU-based therapy. Future of CRC therapy is connected to tumour genotyping because anticancer therapy will be used on specific targets.