CRC is the third most major type of cancer diagnosed in both sexes globally. Approximately 1.8 million new cases are reported annually that account for approximately 10% of all common cancers investigated globally, leading to approximately 9 million fatalities in 2018 itself that is 9.2% of all the cases investigated globally (31), as per the International Agency for Research on Cancer report 2018. Reports suggest a large topographical variation in the incidence and mortality of CRC among several countries worldwide (Fig. 1) (7). CRC is more prevalent in developed countries than economically transitioning countries (Brazil, Slovakia and China) where incidence rates have increased; however, the overall risk of CRC remains low. Incidence rates are decreasing with a higher human development index (HDI; North America and Europe), trailing a peak (USA, New Zealand and France), or increasing (Spain, Italy and Norway). In Saudi Arabia, CRC is the most common type of cancer in males (19.6%), while the third most common cancer in females (9.5%), causing highest cancer-related mortality (32). Several countries have taken major initiatives, such as screening, resulting in the early detection of CRC along with better treatment management that has decreased mortality rates. However, some countries still need to improve the screening process with a more effective medical set up, so that CRC can be detected at an early stage and thus treatment can be improved. Statistics suggest a spike in CRC growth and mortality rates after 50 years of age. An estimated 90% of worldwide cases and deaths have been observed after this age. It is also noteworthy that the incidence rate in males is 30% higher compared to females, with wider variations for rectal cancer (60% higher) than for colon cancer (30% higher). Females also exhibit a lower susceptibility to malignancy overall. Older females, however, (≥50 years) are more prone to developing adenomas in the proximal colon than males. Sex inequalities and lifestyle habits follow differences in exposures to risk factors, such as smoking and sex hormones, as well as complex interactions between these factors.

The majority of CRC cases are sporadic with no family history or a predisposition to illness in individuals, although almost 1 out of 3 individuals with a family history of CRC develop the disease. The average lifetime risk of developing CRC in most western populations is in the range of 3-5%. However, individuals with a family history of CRC in first-degree family members diagnosed at 50-70 years of age are at a double risk; the risk triples if the relative is <50 years of age at the time of diagnosis. The risk of developing CRC increases with ≥2 diagnosed first-degree family members at any stage. There may be several reasons for the increased risk, such as genetics, environmental factors, or a combination of both. Approximately 5-10% of the specific subgroup of individuals who develop CRC symptoms have inherited gene mutations that cause hereditary cancer syndrome, rendering them prone to developing the disease. It has been observed that FAP and HNPCC also known as Lynch syndrome, are the most prevailing inherited syndromes linked to CRC, accounting for approximately 2-4% of all CRC cases (38).

HNPCC is the most widespread inherited colorectal syndrome with an estimated 1 in 3,000 affected individuals in western populations (39). Germline mutations in any of the mismatch repair genes, namely MLH1, MSH2, MSH6, PMS2 and epithelial cell adhesion molecule (EPCAM) have been reported to be associated with HNPCC. Generally, tumors harbor MSI (40), which primarily occurs due to the inability to rectify strand slippage within repetitive DNA sequences, leading to alterations in the mononucleotide or dinucleotide repeats, thus also altering the size and arrangement of microsatellites, which are strewn throughout the genome (12). This can be identified by a PCR test and immunohistochemical analysis, which can pinpoint the loss of expression of the mismatch repair protein factors (40-43). Individuals affected with 'non-polyposis' tend to have polyps from the defined spectrum, leading to cancer progression within 2-3 years, as compared to the 8-10 years of the 'normal' community.

FAP is the second major type of hereditary CRC syndrome, which accounts for >1% of all CRC cases (44). This syndrome affects almost 1 in 11,300-37,600 individuals in the European Union (45) and is caused by hereditary germline mutation in the APC gene, which regulates the activity of the Wnt signaling pathway (46). Unlike patients with HNPCC who develop a few adenomas, patients with FAP tend to develop numerous adenomas, primarily in the distal colon at a younger age (44). Among the numerous adenomas in FAP, one or more adenomas undergo malignant transformation, virtually increasing the risk of developing CRC to 100% by 40 years of age, unless the colon, or sometimes the rectum, is not removed (47).

The incidence of EOCRC is increasing in countries, such as the USA and Canada at an alarming rate, becoming the second most common type of cancer, and the third most common cause of cancer-related mortality in individuals <50 years of age (48). Over the past 4 decades, the incidence of EOCRC has increased rapidly and by the year 2030, it is further expected to increase by >140% (49,50). The incidence rates are inversely associated with age i.e., increasing significantly in younger individuals and decreasing in older individuals. Up to 13% of cases of EOCRC have been reported to be linked to a germline mutation in mismatch repair genes mentioned above, underlying hereditary CRC syndromes (51). Although early-onset CRC patients have a higher risk of the prevalence of hereditary syndrome, approximately half the patients with early-onset CRC do not have any family history of the disease (52). Studies have indicated a prominent difference in pathological characteristics between the elderly and younger groups of CRC patients (53), demonstrating an increase in EOCRC at a younger age, but often being detected at a higher stage (54,55). CRC diagnosed in younger patients is commonly symptomatic, at a later stage, mucinous and involves poorly differentiated tumors (56-58). The cancer-specific survival of younger individuals is markedly higher than older individuals (53). No statistically significant difference in the disease-free survival between these 2 groups has been demonstrated over the past 5 years (63.2% in both the young and elderly group) (53). Between 1992 and 2014, there was a significant increase in the number of young male patients with CRC than older patients (53.3% in the young and 49.7% in the older group). Compared to patients of a screening age (≥50), younger patients with CRC were mostly of African origin (16.8 vs. 10.4%), Asian/Pacific Islander (API) (5.6 vs. 2.2%) and Hispanic (13.1 vs. 6.0%). In younger patients with CRC, there are high proportions of rectal (39.5 vs. 27.7%) and distal colon (43.9 vs. 33.8%) cancers as compared to elderly patients with a higher percentage of proximal colon cancers (58.2 vs. 48.2%) (54). However, the reason for EOCRC remains unclear, although certain risk factors, such as prolonged exposure to carcinogens and early childhood exposure serve as critical determinants of risk (31).

Several other physical factors, such as body weight, age, sex, body mass index (BMI) and lifestyle behaviors, such as smoking, etc. have also been associated with EOCRC. Weight loss may be an early symptom of EOCRC (59). Furthermore, females with a BMI of >30 have a higher risk of developing EOCRC (95% CI, 1.15, 3.25) compared to those with a normal BMI (60). Low et al suggested that smoking is not associated with the risk of EOCRC; neither current nor former smokers are at a risk of developing CRC as compared to non-smokers (59). Rectal cancer has a higher chance of developing into EOCRC than colon cancer, whereas, in late-onset cases, obesity is the major risk factor for colon cancer (61,62). These differences indicate several factors associated with EOCRC and further studies are required to identify the major associated risk factors.

A number of studies have concluded that the majority of human tumors are heterogeneous in nature forming a 'mutator-phenotype' due to continuous accumulation of multiple mutations that occur during cell division in cancer cells that generally function to maintain genetic stability (70). Both CIN and MSI are well defined and detected by karyotype and PCR-based analysis, respectively; however, due to difficulty in predicting random mutations, particularly point mutations that limit the search for evidence of a mutator phenotype at single base-levels (70), the 'mutator phenotype' may exhibit various manifestations, including increased mutation rates and genetic evolution of cancer cells that propel tumor progression (71). The mutator-phenotype may be an attractive target for cancer therapy due to common features in the majority of cancers (72).

CIN appears in 70% of sporadic CRC cases. CIN causes an alteration in chromosome number, involving gain or losses of the whole or a large part of chromosomal aneuploidies, resulting in a rearrangement of chromosomes (karyotyping) from cell to cell (73). LOH and an imbalance in chromosome number (aneuploidy) are the most common characteristics of CIN. There are several techniques to measure CIN, such as cytometry, LOH analysis, karyotyping, fluorescent in situ hybridization (FISH) and a recently developed technique known as comparative genomic hybridization (CGH). CGH utilizes DNA microarray or 'chips' that are commonly used to detect copy number variations (74). In this advanced CGH microarray technique, cloned DNA fragments with precise genomic positions are used against the metaphase chromosomal arrangement in conventional CGH (75). It is not always upfront to categorize tumors as CIN-positive vs. CIN-negative based on these different methods and criteria rather different sub-categories of CIN-high and CIN-low for CIN-positive tumors have been proposed in several studies (76-78). Moreover, CIN contributing to CRC tumorigenesis through the aggregation of mutations in specific oncogenes, including B-Raf proto-oncogene serine/threonine kinase (BRAF), KRAS proto-oncogene GTPase (KRAS), tumor protein p53 (TP53) and tumor-suppressor APC gene (12,75). The multistep genetic model proposal by Fearon and Vogelstein, which is now widely accepted, examines the different stages of tumor development i.e., from small adenomas to large adenocarcinomas (18). The model demonstrates the various events occurring at different stages of tumor development i.e., from normal colorectal epithelium to metastatic carcinomas (79). The first event is the mutation of APC, transforming normal colorectal epithelium to adenoma, followed by oncogenic KRAS mutation at early adenomatous stage and ultimately inactivation of the tumor-suppressor gene TP53 on chromosome 17p and the deletion of chromosome 18q occurring during the progression to malignancy (80-82). APC is a tumor suppressor gene located at chromosome 5, which is responsible for familial adenomatous polyposis constituting approximately 85% of colorectal cancer cases without a hereditary relationship (83,84). APC and CTNNB1 (β-catenin) are the most frequently mutated genes in CRC. APC mutation breaks the association between APC and β-catenin, resulting in a large amount of β-catenin in the cytoplasm and the overactivation of the Wnt signaling pathway. Followed by tumor-formation promoting gene translocation to the nucleus and interacting with other transcription factors involved in tumorigenesis and invasion (85). K-RAS is located on chromosome 12 and is one of the most bulging proto-oncogenes in colon carcinogenesis. RAS family proteins are involved in signal transduction. K-RAS activates the mitogen-activated protein kinase (MAPK) pathway eliciting the nuclear expression of early response genes. RAF proteins are activated by the GTPase activity of RAS (86). Thus, K-RAS mutations result in colon cancer formation in the early adenomatous stage and contribute to its formation by 37-41% (87,88). The allelic loss in chromosome 18q is observed in 70% of primary CRC cases in the late-stage adenomas (89) and exhibits a strong association with a poor prognosis (90). The inactivation of tumor-suppressor genes, including deleted in colon cancer (DCC), SMAD2 and SMAD4 present on the q arm of chromosome 18 due to LOH plays a significant role in CRC (18,82,91). The SMAD proteins are involved in TGF-β signaling and in the regulation of genes involved in cell cycle programming (92). Since SMAD2 and SMAD4 are located on chromosome 18q, the loss of chromosome 18q leads to the deregulation of the TGF-β signaling pathway and contributes to colorectal carcinogenesis (93,94). DCC is localized in the chromosome band 18q21.2 and is deleted in approximately 70% of the cases (95). However, no evidence supports the role of DCC in colorectal tumorigenesis (96). Patients with 18q LOH (70%) have an increased 5-year survival rate in stage II than those without 18q LOH (43%), which leads to the analysis of the impact of adjuvant therapy in stage II (97). There is an increase in the 5-year survival rate of patients with 18q LOH receiving adjuvant therapy compared to those without 18q LOH (90 vs. 37%; P=0.01) (97). The TP53 gene located on the short arm of chromosome 17p13.1 consists of 11 exons and 10 introns and is commonly lost in colorectal carcinoma (20,98). The TP53 mutation is most common in human cancers with 43.28% in CRC resulting in the loss of tumor suppressor activity or (gain of function) to support tumor progression (99). The majority of mutations occur in exon 5 to 8 (DNA binding domain) (100,101). To date, the majority of TP53 mutations detected in CRC are missense mutations with AT for GC substitution (102). p53 is known as the 'guard of the genome' due to its ability to respond to mutagenic stress, such as DNA-damage and repair, cell cycle arrest and apoptosis (103). It also inhibits the development of new blood vessels (angiogenesis) through the induction of TSP1 (104). However, a mutation in p53 leads to oligomerization of the wild-type and mutant p53, which can block the function of TP53, resulting in the loss of DNA binding specificity (105).

The second most common genomic instability is the hyper-mutable phenotype (MSI) (106). MSI generally occurs due to damaged mismatch repair (MMR) along with the slippage of DNA polymerase which creates a short-term insertion-deletion loop (IDL) (106). These defects result in the alteration of the size of the allele as compared to those detected in the normal cells of the same individual. The DNA MMR system has several proteins (such as MLH1, MSH2, MSH6 and PMS2) that repair single base pair mismatch, incorporated into micro-satellites during DNA synthesis to maintain genomic stability. MSI CRC mostly occurs in the proximal colon (107). Several studies have examined MSI a prognostic biomarker for CRC. The Bethesda guidelines proposed the first panel of MSI markers consisting of 5 microsatellite markers viz. mononucleotides (BAT25 and BAT26), and dinucleotides (D2S123, D5S346, and D17S250) to access the status of CRC (108). CRC can be classified based on the percentage of loci with MSI. In particular, >30% of unstable markers are classified as CRC with MSI-high (MSI-H), those with <30% markers exhibiting instability are termed MSI-low (MSI-L), and markers with no instability are termed microsatellite stable (MSS) (109). Defects in MMR genes occur either by mutational inactivation or by epigenetic silencing of CpG island hyper-methylation of the MLH1 gene promoter (9).

The results of immunohistochemistry of CRC reveal the interaction between MMR proteins PMS2 and MSH6 with other repair factors, such as MLH1 and MSH2, respectively. Therefore, the inactivation of MSH2 is frequently associated with the loss of the expression of MSH6, which is highly acceptable in MSH2 germline mutation. Similarly, MLH1 inactivation is frequently associated with the loss of expression of PMS2, which may result either from MLH1 germline mutation or by the epigenetic silencing of CpG island hyper-methylation of the MLH1 gene promoter. Germ-line mutations of MSH6 and PMS2 are generally associated with the individual loss of expression of MSH6 and PMS2 proteins, respectively (110).

CIMP represents a subset of CRC that contains a high density of hyper-methylated genes, causing transcriptional silencing within the promoter region, resulting in the loss of gene expression (111). CIMP contributes to approximately 30-35% cases of colorectal adenomas, occurring at an early stage and as a precursor to the serrated pathway of colorectal tumorigenesis (112-114). CpG island hypermethylation present in the tumor suppressor gene promoter region results in gene silencing. The hypermethylation of MLH1 leads to its silencing and dysregulates MMR (mismatch repair) function (114). CIMP constitutes a prominent molecular characteristic of the serrated neoplasia in 20-30% of CRC cases (115,116). CIMP has also been found histologically in patients with hyperplastic polyposis syndrome, suggesting that it is an important early event of the serrated neoplasia pathway (117). The inactivation of MLH1 by hypermethylation leads to the induction of MSI-H followed by additional mutations in MSH3, MSH6, Bax, insulin-like growth factor 2 receptor (IGF2R) and phosphatase and tensin homolog (PTEN), resulting in the development of dysplasia and cellular transformation (118,119). Studies suggest that the serrated pathway is responsible for the rapid development of CRC, as compared to patients with CRC with Lynch syndrome (120,121).

The Wnt/β-catenin pathway is highly conserved as it is essential to embryogenesis. Wnt proteins are growth stimulatory factors (the attached palmitoleic acid assisting in protein-binding). These proteins exhibit an abnormal cellular expression in patients with CRC. There are 19 Wnt genes present in mammals and all play regulatory roles in several biological and developmental processes, such as cell fate determination, cell cycle, proliferation and migration. Membrane surface cell receptors comprise frizzled (Fz) and low-density lipoprotein (LDL) receptor-related protein (LRP) complexes at the cell surface. Along with this, there exists an intracellular complex comprising of several proteins, such as β-catenin, dishevelled (Dsh), axin, glycogen synthase kinase-3β (GSK-3) and APC. The protein complex regulates the level of β-catenin in the cell by proteasomal degradation. Following phosphorylation and ubiquitination (by β-trcp) of β-catenin, the transcriptional regulator is degraded by the cellular proteasome. Upon ligand-binding, the degradation process is inhibited, leading to the accumulation of active phosphorylated β-catenin in the cell. The β-catenin then translocates into the nucleus and induces transcription. Mutations in the APC gene can lead to colon cancer (reported in 90% of cases). The overexpression of Wnt is associated with tumorigenic activity and encourages tumor growth. Mutations in the Wnt/β-catenin pathway lead to CRC development (170). This pathway also plays an essential role in tissue regeneration of hair, skin, intestine, etc. (171). The dysregulation of the Wnt pathway has been reported in a number of tumors, including CRC. The hyperactivation of this pathway is imperative for oncogenesis, leading to CRC development. Targeting Wnt/β-catenin can be effectively used for the development of small molecules (172-175).

A catalytic receptor tyrosine kinase (RTK), EGFR, is present on the cell surface, having an extracellular ligand-binding domain. EGF acts as a ligand and binds to EGFR, resulting in the autophosphorylation of the tyrosine residues on the intercellular side of the transmembrane protein. This offsets a chain of cellular events; an adaptor molecule Grb-2 interacts with the phosphorylated tyrosine through its SH2 domain, followed by interaction with the son of seven-less protein (SOS) through the SH3 domain of Grb-2. SOS, a guanine nucleotide exchange factor, enables the conversion of GTP from GDP on the RAS molecule, thereby activating it. Activation initiation results in a kinase cascade, activating mitogen-activated protein kinase kinase kinase-Raf (MAPKKK), mitogen-activated protein kinase kinase-MEK (MAPKK) and MAPK or extracellular signal-regulated kinase (ERK) in turn through phosphorylation. ERK regulates cellular events, such as the proliferation and survival of cells by targeting cytoplasmic or nuclear substrates. Cytoplasmic substrates include c-fos and c-Jun (dimerized by MAPK) which enter the nucleus and interact with the AP-1 motif of the DNA, initiating transcription. ERK also phosphorylates cytoplasmic substrate, ribosomal S6 kinase (RSK). The S6 protein can perform one of two functions including, negative regulation of the SOS molecule (effectively turning 'off' the signaling pathway by inhibiting the conversion of GTP from GDP) or entering the nucleus and regulating the CREB transcription factor. MAPK may also directly regulate the nuclear substrate MYC. Inactivation of the pathway can also occur through the hydrolysis of GTP through the GAP protein.

The MAPK pathway engages in various cellular processes such as growth, proliferation and survival of cells. The deregulation of the pathway results in the stimulation of growth, survival, angiogenesis and metastasis of neoplastic cells. The mutation of the K-Ras gene has been reported in early cancer stages in almost 40% of CRC cases. Abnormal regulation, amplification, increased copy number and the overexpression of EGFR promoting MAPK activation has been reported in cases of CRC and is being studied as a possible and promising target for treatment (173-175).

The PI3K pathway is associated with cell growth, proliferation and differentiation. The enzymatic receptor tyrosine kinase upon ligand-binding, autophosphorylates and activates phosphatidylinositol 3-kinase (PI3K) that has two subunits: p85 and p110. PI3K then, in turn, phosphorylates lipid protein, phosphatidylinositol-4, 5-biphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3). PIP3 signals proteins, such as 3-phosphoinositide-dependent protein kinase 1 (PDK1) that activates protein kinase B (AKT/PKB) by acting upon its serine and threonine residues. AKT may be of 3 subtypes (AKT-1, AKT-2 and AKT-3) depending upon whether it has been encoded by PKBα, PKBβ, or PKBγ, respectively. AKT targets downstream proteins, such as mammalian target of rapamycin (mTOR), which is responsible for cell cycle progression, proliferation, delayed apoptosis, growth and survival. Phosphatase and tensin homolog protein (PTEN) downregulate the pathway by dephosphorylating PIP3. PTEN is also a tumor-suppressing molecule. The aberrant expression of the pathway (inability to switch-off) results in continuous and unchecked growth and survival of cells leading to cancer. PI3K consists of 3 classes, of which type class 1A is the most prevalent. The abnormal expression of PI3K accounts for 30% of human cancers. Overall, it is shown to serve as an oncogenic factor in the growth and development of CRC. The overexpression of phosphorylated AKT has been linked with cell division and the suppression of apoptosis in 70% of patients with CRC, along with the abnormal expression of PTEN. Akt also targets downstream protein mTOR that has been shown to favor angiogenesis and growth; research into the use of aspirin (mTOR inhibitor) has demonstrated that it inhibits CRC progression (173,175).

Angiogenesis is an essential process for the formation of blood vessels contributing crucially to cancer initiation, cell proliferation, and growth, metastasis, and invasion. Identification of vascular endothelial growth factor (VEGF-A) and the generation of monoclonal antibodies inhibitor against VEGF-A led to the direct relationship between new blood vessel formation and carcinogenesis (176). Various pro-angiogenic and anti-angiogenic factors regulate angiogenesis like VEGF, FGF, TGF-α, TGF-β, PDGF, and angiopoietins which are released from the tumor microenvironment (177-179). The VEGF family of proteins is comprised of 5 proteins namely, VEGF-A, B, C, D and placental growth factor (PIGF). These proteins bind to VEGFR: VEGFR1, VEGFR2 and VEGFR3, a type of receptor tyrosine kinases on endothelial cells. There are 2 non-tyrosine kinase co-receptors, neuropilin-1 (NP-1) and NP-2. The diverse network between VEGF and VEGFR, VEGF-A, VEGF-B, and PIGF mainly contribute to angiogenesis. However, VEGF-C and VEGF-D predominantly contribute to lymph angiogenesis. VEGF-A and VEGF-B prominently bind to endothelial cells and on some non-endothelial cells via VEGFR-1 and -2 (180). VEGFR-3 is expressed on endothelial lymphatic cells and bind to VEGF-C and D with increased affinity (181).

VEGFR-1 belongs to receptor tyrosine kinase family protein known to be expressed on endothelial cells, inflammatory cells and tumor cells. VEGFR-1 regulates mainly differentiation and cell migration of endothelial cells and promotes epithelial cell differentiation during the early angiogenic event; however, it has an insignificant role in cell proliferation (182,183). Furthermore, VEGFR-1 activation mediates the activation of several downstream pathways, such as PI3K/AKT/MAPK/ERK in inflammatory cells, resulting in the upregulation of inflammatory cytokine and interleukin (IL) production, such as TNFα, IL-1β, IL-6 and IL-8, leading to cell migration. VEGFR-1 function is still unknown and mainly plays a regulatory role in the angiogenesis process. VEGFR-2 is a 200-230 kDa protein reported in its involvement in vascular formation. VEGFR-2 is mainly expressed in blood and lymphatic epithelial cells (183). VEGF-A binds to VEGFR-2 leads to activation of VEGFR-2 resulting in the activation of several downstream pathways, such as RAS/RAF/ERK/MAPK and PLCγ which promotes cell growth. The activation of VEGFR-2 also activates PI3K-AKT signaling, leading to the regulation of cell death (177-180,184). The binding of VEGF-C and -D to VEGFR-3 results in lymphatic vessel formation (185,186). Activated VEGFR-3 activates RAS-MAPK-ERK and PI3K-AKT/PKB pathways leading to differentiation, proliferation, survival, and migration of lymphatic endothelial cells (185-187). There is sufficient evidence to indicate that VEGF levels and VEGFR activity are elevated and considered to be associated with a poor prognosis in CRC (188). Elevated levels of VEGF are reported in the early and late advanced stages of CRC (189,190). The interaction between VEGF-VEGFR is regulated by K-RAS mutation, p53, Cox2 and hypoxia resulting in cell growth and migration in CRC (190-193). The pro-angiogenic function of this VEGF/VEGFR complex is critical at the primary site of tumor enhancing progression and migration and at the metastatic site for new vessel formation to promote cancer growth and survival. Targeting this complex with anti-VEGF or anti-VEGFR therapy may result in the depletion of tumor formation and metastasis.

HGF and cMET play an essential role in proliferation, survival, drug resistance and metastasis (194-198). HGF is the only ligand known for MET receptor tyrosine kinase and is secreted from mesenchymal tissues. An increased expression of HGF in tissue and serum is related to a poor prognosis in various solid tumors of the breast and gastrointestinal tumors (199-201). Patients with CRC with advanced disease symptoms are reported to have higher levels of serum HGF (202,203). MET belongs to the transmembrane receptor family known to express in hepatocytes, normal and malignant epithelial and endothelial cells, neural cells and hematopoietic cells (204-206). MET has been reported to be overexpressed in various malignant tumors, such as hepatocellular carcinoma, lung, breast, thyroid, kidney, gastric cancer and CRC (207-213). Several studies have demonstrated elevated levels of MET mRNA and protein in CRC during tumor progression and metastasis (214-216). HGF binding to MET receptor leads to the activation of MET signaling, which initiates various downstream signaling pathways, such as MAPK-ERK, PI3K-AKT, JAK-STAT and NF-κB, resulting in the regulation of hematopoiesis, wound healing and organ regeneration (195-200). Aberrant HGF-MET axes are comprised of gene amplification, overexpression, mutation, and ligand-mediated auto and paracrine signaling during oncogenesis (217). Other factors also modulate the HGF/MET pathway. Recently, it has been reported that a novel gene metastasis-associated in colon cancer 1 (MACC1) is a crucial player of HGF-MET signaling and regulates cancer progression and CRC metastasis (218). Increased levels of MACC1 have been observed in primary and metastatic CRC tissues. HGF induces the translocation of MACC1 from the cell membrane into the nucleus and binds to MET promoter, leading to an increased MET expression. The MET signaling pathway is also regulated by crosstalk with receptor tyrosine kinases mainly EGFR. MET and EGFR are both known to be overexpressed in CRC (219). The individual blocking of MET or EGFR has little effect on downstream ERK/PI3K activation due to the compensatory mechanism. Targeting both receptors by combined therapy results in the abrogation of the downstream pathway (220-222).

Recent data suggest that targeting immune-recognition and response may be effective in eradicating cancer cells. This strategy includes malignant tumors having different genetic and epigenetic signatures that may be identified and attacked by the host immune system expressing unique antigens. This process consists of many steps, such as T cell binding to MHC molecules presented by antigen-presenting cells (APCs). The next step involves signals mediated by co-stimulatory or inhibitory receptors that play a critical role in the T cells activation and tolerance (223,224). This dual-check mechanism is essential for avoiding excessive immune response in a normal scenario and attack diseased cells (225). The process of tumor cells evading host immune recognition and response is referred to as the immune escape and has been mentioned in cancer (226). Immune escape results from immunosuppressive factors, such as TGF-β of T_reg cells and IL-6 regulatory cells, or the loss of immunogenicity by the inhibition of MHC-1 (227). The activation of co-inhibitory receptors, also known as immune checkpoint receptors present on the surface of T cells, leads to cancer-mediated T cell inactivation. The immune checkpoint receptors expressed on the surface of T cells comprise of programmed death-1 (PD-1) and cytotoxic T lymphocyte antigen 4 (CTLA-4). Ligands for these receptors are known as PD-L1 and PD-L2 expressed on cancer, stromal and immune cells (228). Wang et al reported elevated levels of PD-L1 in metastatic CRC as compared to primary CRC, allowing its targeting with an immune response (229). High levels of T_reg cells have been found in CRC tissue as compared to adjacent normal tissue. These T_reg cells are known to express PD-1 and are crucial to the immune response to CRC (230).

The 4 types of Janus kinase proteins (JAKs) include JAK1-3 and TYK2 that interact with cytokine receptors present in the colon. Although they are associated with different cytokine receptors, they have a common mechanism of the intracellular pathway, including signal transducer and activator of transcription (STAT) protein. Upon ligand-binding and physiological transformation, the associated JAKs of the cytokine receptor autophosphorylate and proceed to phosphorylate specific residues on the receptors that act as docking sites for STAT proteins. Associated JAK proteins further phosphorylate these STAT proteins, causing their dissociation and dimer formation. These dimers translocate to the nucleus, identifying and binding to gamma activated sequence (GAS) elements, causing pro-inflammatory gene expression and transcription and playing a role in the pathogenesis of inflammatory bowel disease (IBD). Drugs developed to inhibit the functioning of JAK proteins bind and prevent their phosphorylation, effectively blocking the pathway. JAK protein can also signal PI3K protein (Akt pathway) and Ras protein (MAPK pathway) (172).

The TGF-β pathway plays a role in cell growth, division and adhesion. It also stimulates apoptosis and cellular differentiation. Its downstream targets include important cell cycle checkpoint genes (p21, p27 and p15) that trigger growth. TGF-β receptors occur as transmembrane heterodimers (type 1 and 2) with kinase domains (KDs) present in the intracellular part. Upon ligand binding, 2 of these heterodimers come together to form a complex through receptor dimerization. The KDs are activated through phosphorylation and further activate SMAD proteins present in the cytosol. SMAD2 and SMAD3 form phosphorylated heterodimers. This complex, joined by co-factor SMAD4, forms a heterotrimer. The heterotrimers then translocate into the nucleus and bind to TGF-β target genes and initiate transcription. TGF-β is known to function as a tumor suppressor, normally controlling cell division and the death of epithelial cells of the colon. CRC cells lose TGF-β, thereby resisting growth inhibition. However, in the later stages of CRC, TGF-β expression is increased and influences epithelia-to-mesenchymal transition (EMT), and as a result, increases invasion and cell migration thus subduing the normal cellular immune response. Mutations in SMAD4 have also been reported in cases of juvenile polyposis (172-175).

The Notch signaling pathway occurs intercellularly and is highly conserved. Mammals possess 4 types of notch receptors (Notch 1-4). Ligands are of 2 types, the Jagged protein family (JAG 1 and 2) and the Delta-like protein family (DLL 1, 3, and 4). A Notch receptor has 3 components, namely the notch extracellular domain (binds to the ligand), Notch intracellular domain (NICD) and the transmembrane component. Ligand activation of DLL or JAG proteins on the 'sending' cell occurs through ubiquitination by a mind bomb protein (MIB). The activated ligand then binds to the extracellular component of the notch receptor. A disintegrin and metalloproteinase (ADAM protease) cleaves the extracellular domain of the notch receptor (S2 cleavage). Subsequently, another protease, secretase gamma, cleaves NICD causing it to dissociate from the transmembrane domain of the receptor (S3 cleavage). NICD, now free to move in the cytosol, binds to and activates the CSL transcription factor (suppressor of the hairless) that forms a complex with co-activators MAML (mastermind-like proteins), and p300. The complex translocates into the nucleus where the p300 acts as a histone acetylase, causing the activation of transcription factors (ex-HES1) and the transcription of notch-target genes (example-MYC, p21). The downregulation of the pathway may occur by a ubiquitin ligase, f-box/WD-40 repeat-containing protein 7 (FBW7) that ubiquitinates NICD causing its proteasomal degradation. The overexpression of Notch-associated proteins and ligands (JAG, HES1, NICD, etc.) has been reported in CRC. Cell cycle and apoptotic regulation of target genes (p21 and PUMA genes) enhance the severity of CRC through Notch signaling. The pathway also influences CRC resistance to chemotherapeutic drugs (172-175,231).

The SHH signaling pathway is essential for the regeneration and differentiation of epithelial cells present in adult colons. Hedgehog (Hh) ligands produced in the endoplasmic reticulum of secretory cells are released through a membrane protein known as Dispatch. In a paracrine-type signaling event, these Hh molecules bind to Patched (Ptc) protein present in neighboring cell(s) thereby, inhibiting its function. Upon the inhibitory action of the Ptc molecule, the smoothened (Smo) protein molecule (previously inhibited by Ptc) is activated. The Smo protein present in the primary cilia of the intestine regulates the action of the 3 intracellular Gli proteins (Gli-1, Gli-2 and Gli-3). It releases Gli-2 (transcriptional activator) from the suppressor complex composed of Costal-2, Fused kinase (Fu) and SuFu (suppressor of fused), thereby, activating it. This Gli-2 then acts upon and phosphorylates the transcription factor, Gli-1, that translocates to the nucleus and acts upon SHH-target genes. Smo protein also acts upon and inhibits the function of Gli-3 (a transcriptional inhibitor). In CRC tissues, it is noted that the levels of the proteins SHH, Smo and Gli1 are uncharacteristically high (175). A previous study also reported that the subcutaneous transplantation of speckle-type POZ protein (SPOP) reduced the rate of tumor growth (in BALB/c nude mice) and increased apoptosis (in HCT116 cells) (232). In CRC, SPOP is shown to degrade Gli2 by ubiquitinating it (172). The arbitrary regulation of the signaling pathway, either by a mutation in Ptc (loss of function) or a mutation in Smo (gain of function), can lead to colon cancer development (233).

The Hippo signaling pathway controls cell proliferation, homeostasis and regeneration (Fig. 4). The main transcriptional regulator of the pathway is Yes-associated protein 1 (YAP). YAP and its homolog, PDZ-binding domain taffazin (TAZ) regulate the Hippo pathway. Upon the initiation of the pathway, the activation and phosphorylation of first, mammalian Ste20-like kinases 1/2 (MST1/2), and subsequently large tumor suppressor 1/2 (LATS1/2) occurs. LATS1/2, in turn, phosphorylates YAP/TAZ, resulting in its removal from the nucleus into the cytoplasm where it undergoes ubiquitin-mediated protein degradation. An abnormally high level of YAP/TAZ protein has been reported in solid tumors and amplifies the frequency of tumors (174,234).

Reactive oxygen species (ROS) cause oxidative damage to cells and are linked to CRC (Fig. 4). In response to the fatal effects of oxidative stress, the cell releases antioxidant and detoxification genes, such as NF-E2-related factor 2 (Nrf2). A Cap-n-collar transcription factor, Nrf2 identifies antioxidant response elements on target gene promoters and combats free-radical damage (by carcinogens, inflammation, etc.). Normally, Nrf2 is confined to the cytosol by Kelch-like ECH associated protein 1 (Keap1). Keap1 acts as a linker protein between Nrf2 and Cul3-based E3-ubiquitin ligase complex, causing ubiquitination of Nrf2 and its subsequent proteasomal degradation. Under conditions of oxidative stress, antioxidant response elements induce separation of Nrf2 and Keap1, leading to its nuclear transportation. There, Nrf2 undergoes dimerization and interacts with small musculoaponeurotic fibrosarcoma (Maf) proteins initiating the attachment of Nrf2 with antioxidant response elements, initiating transcriptional activation of target genes (174). The Nrf2/Keap1 pathway can help regulate the chemopreventive effects of various drugs for the treatment of CRC (174).

p53 plays an important role in maintaining the integrity of cellular processes and genetic material. It is a well-known tumor suppressor and spearheads repair and cellular apoptosis depending upon the extent of DNA damage (Fig. 4). Secreted in response to external stress such as UV rays, and hypoxia, p53 has a short half-life in the cell and is directed by a fool-proof regulatory system. Nicks or mutations caused due to external stress stimuli result in activation of the ataxia-telangiectasia mutated (ATM) protein kinase CHK2, whose absence can delay the action of p53. The activation of the p53 gene also results in the production of mouse double minute 2 homolog (MDM-2), an E-3 ubiquitin ligase. MDM-2 is a negative regulator that degrades the p53 gene by ubiquitinating it, thus setting up a self-governing 'loop' to maintain the p53 level in the cell. Oncogenic events can lead to the transcription of the p19 alternate reading frame (ARF) protein that inhibits the functioning of MDM-2, thus, failing to regulate p53 levels in the cell. Mutations in the p53 gene and aberrant function lead to a loss of cellular checkpoints and programmed cell death, thereby compromising genetic integrity (174). Mutations also encourage EMT, and the formation of adenomatous polyps, eventually conferring malignancy, i.e., CRC. It has also been noted that 80% of p53 mutations in CRC cases stemmed from missense mutations, largely in exons 4-8 (172).

The TRAIL-mediated signaling pathway is a candidate for anticancer therapy by selectively targeting cancer cells (Fig. 4). TRAIL receptors span the cellular membrane and act as a conduit for the extrinsic apoptotic pathway. TRAIL ligand binds to specific death domain receptors DR4/DR5 which augments the association of Fas-associated protein with death domain (FADD), an adaptor protein. FADD activates pro-caspase 8, initiating the apoptotic pathway (through recruitment of various caspases initiating the extrinsic, and cytochrome-c initiating the intrinsic apoptotic pathway) (172).