Mutations in the APC gene on chromosome 5q21 locus are considered as one of the earliest events in the initiation and progression of colorectal cancer. In FAP patients, allelic mutation of the APC gene followed by a loss of heterozygosity (LOH) is a common feature. Notably, mutations in the APC gene also are found in 60 to 80% of sporadic colorectal cancers and adenomas. FAP patients with APC mutations are prone to hundreds to thousands of colorectal adenomas and early onset carcinoma. FAP patients also are prone to small intestinal adenomas (and carcinomas), intra-abdominal desmoids and osteomas tumors (Gardner's syndrome), congenital hypertrophy of retinal pigment epithelium (CHRPE), fundic gland polyps in the stomach, pancreas and thyroid, dental abnormalities, and epidermal cysts 67. Mutations in APC are also associated with malignant brain tumors known as Turcot's syndrome 8. The APC locus on chromosome 5q21 shows loss of heterozygosity (LOH) in approximately 25% of breast cancers 9. Approximately 18% of somatic breast cancers carry APC gene mutations 10. Furthermore, LOH at the APC gene locus is frequently seen in the early stages of non-small cell lung cancers 11. In an animal model for carcinogen-induced lung tumors, a decrease in APC gene expression, rather than an increase in APC mutations, has been shown to be associated with tumorigenesis 12. The diverse effects of mutations in APC gene indicates that this molecule plays a key role in the regulation of cell growth in a number of colonic and extra-colonic tissues. It has been suggested that the overexpression of APC causes a G₁/S phase checkpoint in serum-treated NIH-3T3 cells via components of the pRB pathway 13. A role for APC in the G₂/M phase transition is also expected, given that APC is hyperphosphorylated during M-phase and is a target of the M-phase kinase p34Cdc2 1415. In a recent study, a direct role of APC in S and G₂ phase arrest of cell cycle has been described in cell lines immortalized mouse epidermal keratinocytes, immortalized canine kidney epithelial, mouse squamous cell carcinoma and skin papilloma, and human colon carcimona 16.

APC is expressed constitutively within the normal colonic epithelium. The APC gene product is a 310 kDa protein localized in both the cytoplasm and the nucleus. Recently, it has been shown that the APC gene is inducible, which is transcriptionally up-regulated by p53 in response to DNA damage 171819. p53 is a negative regulator of normal cell growth and division. Although mutations in the p53 gene is also widely present in colorectal cancers, its consequence on the expression of the wild-type or mutant APC gene is not clear 2. In an earlier study, in the Apc^Min/+ (Min, multiple intestinal neoplasia) mice model, an increased multiplicity and invasiveness of intestinal adenomas was associated with deficiency for p53 20. Also, the occurrence of desmoid fibromas in these mice was strongly enhanced by p53 deficiency. The structure of the APC protein with different protein interaction domains are given in Figure 2. At the N-terminal site, the APC protein contains oligomerization and Armadillo-repeat binding domains; and at the C-terminal site there are EB1 and tumor suppressor protein DLG binding domains. The APC protein also contains three 15-amino acids and seven 20-amino acids repeat regions in which the latter is involved in the negative regulation of β-catenin protein levels in cells (Fig. 2). The first 20-amino acid-repeat in the APC gene is located at the 5'-end of the mutation cluster region (MCR). An association has been shown between severe polyposis phenotype and germ-line mutations in the MCR 21222324. Selective pressure for an MCR mutant has been proposed based on the germ-line mutation in FAP 25. Patients with mutations outside of the MCR region have a milder phenotype. A recent study has shown that animals with homozygous truncating mutations at codon 1638 of APC did not develop tumors and survived through adulthood 26.

It has been reported recently that APC functions as a nuclear-cytoplasmic shuttling protein and as a β-catenin chaperone 2728293031. There are at least five APC nuclear export signals (NESs). Among them, two are Rev-type NESs located at the N-terminal region and the other three are located in the 20-amino acid repeat region of β-catenin binding motif 2932. The nuclear export of APC is mediated by CRM1/Exportin receptor pathway 28293033. The APC protein has two nuclear localization signals, NLS1 (1767–1772; amino acids GKKKKP) and NLS2 (2048–2053; amino acids PKKKKP). The nuclear localization of APC is controlled by post-translational modification of NLS residues by protein kinase 2 and/or protein kinase A 323334.

APC may act as a negative regulator of β-catenin signaling in the transformation of colonic epithelial cells and in melanoma progression 353637. The role of the Wingless/Wnt signaling pathway has been described in Drosophila, Xenopus, and in vertebrates. This pathway is important in organ development, cellular proliferation, morphology, motility, and the fate of embryonic cells 383940. In a simple model shown in Figure 3, Wingless/Wnt signaling regulates the assembly of a complex consisting of: axin (and its homologs Axinl and conductin), APC, β-catenin, and glycogen synthase-3β kinase (GSK3β). Axin (Axil/conductin) binds to form a complex with APC, β-catenin, and GSK3β to promote β-catenin phosphorylation and subsequent binding with slim (β-TrCP) which mediates its ubiquitination and degradation in the proteasome 4142. GSK3β regulates this process by phosphorylating β-catenin, APC and Axin (Axil/conductin) complex 434445. Activation of the Wingless/Wnt signaling pathway inhibits GSK3β and stabilizes β-catenin 4647. Mutations of β-catenin or truncation of APC, which occur both in colon cancer and melanoma cells, increases the stability and transcriptional activity of β-catenin 3748. The stabilized pool of β-catenin associates with members of the T-cell factor (Tcf)-lymphoid enhancer factor (Lef) family of transcription factors. There are four known members of the Tcf and Lef family in mammals, one of which, the human Tcf4 gene, is expressed specifically in colon cancer cells 35. The β-catenin/Tcf4 complex regulates the proto-oncogene and cell cycle regulator c-myc 48, the G₁/S-regulating cyclin D1 49, the gene encoding the matrix-degrading metalloproteinase, matrysin 50, the AP-1 transcription factors c-jun and fra-1; and the urokinase-type plasminogen activator receptor gene 51. From this discussion, it is clear that the inactivation of APC causes activation of β-catenin, which results in the constitutive activation of Tcf/Lef response genes.

In many cases, it has been observed that colon tumors carrying mutations in the APC gene also carried increased levels of c-Myc, a known factor for cellular proliferation. Recently, a direct link has been established between APC gene mutation, β-catenin activation and c-myc gene up-regulation in colon cancer development. The increased expression of c-Myc through Wnt-signaling pathway up-regulates the expression of Cdk4 gene, whose product is responsible for cell cycle regulation in G₁ phase 52. The c-myc gene encodes a transcription factor of helix-loop-helix leucine zipper family that binds as a heterodimer with Max to E boxes (CACGTG) on target promoters, for example Cdk4 gene, and activates its expression. Max can also interact with Mad and Mxi1 and down-regulate c-Myc target gene expression. It has been suggested that the increased levels of Cdk4 protein can phosphorylate pRB. The E2F/DP transcription factor then dissociates from the hyperphosphorylated pRB, which actively transcribes genes involved in cell cycle progression through G₁ phase. It is also suggested that the increased levels of Cdk4 may sequester cell cycle kinase inhibitor p21, p27, and p16. This sequestration may account for the ability of c-Myc overexpression to substitute for p16 deficiency as noted in mouse fibroblast transformation 53. These studies thus establish a link among APC gene mutation, β-catenin stabilization, c-myc gene activation, and Cdk4/cyclin D1/pRB/p16 pathway in colorectal cancer development.

Actin cytoskeletal integrity is necessary to maintain the shape and adherence junctions of cells. The imbalance in actin cytoskeletal integrity can cause disturbance in cell-cell adhesion and cell migration. The role of APC in actin cytoskeletal maintenance is predicted through its interaction with β-catenin. β-catenin establishes a link between APC and actin by providing a bridge to α-catenin 53. In Drosophila, mutations in E-APC have shown to affect the organization of adherence junctions 545556. Another link of APC with actin is shown through its interaction with PDZ domain of DLG protein. Since APC co-localizes with DLG in the cytoplasm in rat colon epithelial cells, the APC-DLG complex may participate in regulation of cell cycle progression 57. Mutant APC lacking the S/TXV motif for DLG binding exhibits weaker cell cycle blocking activity at Go/G1 phase than the intact APC 58.

Interaction of APC with β-catenin and through β-catenin interaction with the members of the cadherin family of proteins have been implicated in cell-cell adhesion 2459. As shown in Figure 4, the C-terminal domain of E-cadherin interacts with β- and γ-catenin, which associate with α-catenin and form an E-cadherin complex with actin cytoskeleton. This complex maintains the stable cell-cell adhesion 59. APC becomes a part of the cell-cell adhesion complex linked with E-cadherin, since it directly binds with β-catenin, γ-catenin, and actin filament 2460. The tyrosine phosphorylation of β-catenin by epidermal growth factor (EGF), hepatocyte growth factor (HGF) and c-Met receptors is important in modulating cadherin-catenin complexes from membrane bound form to free cytosolic form 61. The phosphorylation of β-catenin at tyrosine residue, which is blocked by tyrosine phosphatase Pez, is involved in epithelial cell migration 62. If the Wnt-pathway and the EGFR or c-Met receptors pathway are activated at the same time, then the degradation of β-catenin can be inhibited and it may translocate to the nucleus, bind to the Lef-Tcf transcription factor, and down-regulate the transcription of E-cadherin gene, CDH1, expression 63. These complex interactions may finally result in the reduction in E-cadherin-mediated cell-cell adhesion and proliferation of cells 646566.

Another important role for APC is assigned in cell migration. Colonic epithelial cells, derived from a committed stem cell, divide in the lower two-third of the crypts and migrate rapidly to the surface to form a single layer. During migration, they are differentiated into absorptive, secretory, paneth and endocrine cells. The function of a wild-type APC is necessary in maintaining the direction of upward movement of these cells along the crypt-villus axis. Loss of wild-type APC functions due to loss of expression or mutations affect cell migration. These cells, instead of migrating upwards towards the gut lumen, migrate aberrantly or less efficiently towards the crypt base where they accumulate and form polyps 67. In due time, these cells become aneuploid due to defects in chromosome segregation as well as acquiring β-catenin stabilization and activation of genes for cell proliferation. The mechanisms by which APC might be involved in cell migration can be understood by its association with the kinesin superfamily-associated protein KAP3 that has been established in cell-cell adhesion and migration. It has been shown that APC, mediated by KAP3, interacts with kinesin motor proteins which transport it as well as β-catenin along the microtubules to the growing ends of the cytoskeletal protruding into motile cell membranes 6869. At the tip of the microtubule, APC interacts with the end-binding protein EB1 and protein tyrosine phosphatase PTP-BL. PTP-BL modulates the steady state levels of tyrosine phosphorylations of APC associated proteins such as β-catenin and GSK3β. In fact, GSK3β kinase activity has been implicated in microtubule dynamics 7071.

Recently, experimental evidence was presented describing the mechanisms by which the mutated APC might play a role in the migration of colorectal tumor cells 7273. In these studies, an interaction of APC has been shown with APC-stimulated guanine nucleotide exchange factor (ASEF) that may regulate the actin cytoskeletal network 72. APC binds with ASEF and controls its activity. ASEF is activated in colorectal cancer cells containing truncated APC. Active ASEF decreases E-cadherin-mediated cell-cell adhesion and promotes cell migration. Thus, the dynamic association of APC, EB1, ASEF, catenins, EGFR or c-Met receptor, PTP-BL and E-cadherin proteins at cell-cell adherence junctions and microtubule ends play an important role in cell-cell communication, cell migration and carcinogenesis.

Both sporadic and hereditary colorectal cancers exhibit a defined set of biological and genetic cell heterogeneity through a series of molecular events. Two specific pathologically distinct genetic pathways for colorectal cancer have been identified – the chromosomal instability (CIN) pathway and the microsatellite instability (MSI) pathway. The CIN and MSI are associated with two major inherited syndromes, familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC), respectively. The MSI leads to a 1,000-fold increase in the rate of subtle DNA changes, whereas CIN enhances the rate at which gross chromosomal changes occur during cell division such as chromosome breaks, duplication, rearrangements, and deletions. The latest findings describing these two pathways are discussed below.

Aneuploidy, the abnormal number of chromosomes both quantitatively and qualitatively, is proposed to be a common characteristic of colon cancer cells. It is believed that the aneuploidy in colon cancer cells arises during mitosis through a defective cell division leading to CIN. CIN is a common feature of about 85% colorectal cancers, and it has been detected in the smallest adenoma, suggesting that CIN may occur at very early stages of colorectal cancer development. The mechanism(s) by which CIN is generated in colon cancer cells is largely unclear. Since the main feature of CIN is aneuploidy, it has been predicted that it may arise due to structural changes to the chromosomes and abnormal mitosis. In the year 2001, two different research groups linked mutations in APC gene with CIN 7475. Their results suggested that the wild-type APC may be involved in maintaining the proper connection of microtubules with chromosomes. APC performs a bridging function between microtubules and chromosomes. APC binds at the plus end of the microtubule through EB1, stretches it to the chromosomes, and inserts them into kinetochores after binding with Bub1. APC co-localizes to kinetochores and form complexes with Bub1 and Bub3, the two mitotic checkpoint proteins 75. The successful complex formation may facilitate proper growth of spindle formation and helps in maintaining euploidy (Fig. 5A). Once the APC gene is mutated, the truncated APC protein may loose its ability to bind with Bub1, and it may become unable to properly maintain the attachment of microtubules at kinetochores, resulting in defect in segregation of chromosomes (Fig. 5B).

These studies also pointed out another interesting observation associated with CIN. It has been found that after blocking apoptosis in either Apc^Min/+ or Apc1638T ES cells, the number of aberrant chromosomes were much greater than in control ES cells that were unable to undergo apoptosis 74. From these results, a multiple-hit hypothesis of colorectal cancer development has been suggested. The chromosome segregation defect in colon cancer cells with mutated APC gene could lie dormant until an additional genetic-hit suppresses the mitotic checkpoint or the apoptosis of defective cells. In this regard, the proper functioning of the hSecurin, a protein which is necessary for the completion of the anaphase portion of mitosis, is critical to maintain euploidy, since the loss of hSecurin is often associated with the loss of chromosomes at a high frequency 76. Furthermore, telomere dysfunction has been shown to promote CIN that triggered early carcinogenesis in the Apc^Min/+ Terc^-/- mouse models, while telomerase activation restored genomic stability to a level permissive for tumor progression 77. These studies suggested that early and transient telomere dysfunction is a major mechanism underlying CIN of human cancer. Thus, multiple factors may play together in colorectal cancer development from normal epithelial cells – adenoma – carcinoma stages.

Microsatellite instability (MSI) is characterized by the size variation of microsatellites in tumor DNA as compared to matching normal DNA due to defects in the mismatch repair (MMR) system. MMR system is critical for the maintenance of genomic stability. MMR increases the fidelity of DNA replication by identifying and excising single-base mismatches and insertion-deletion loops that may arise during DNA replication. Thus, the MMR system serves a DNA damage surveillance function by preventing incorrect base pairing or avoiding insertion-deletion loops by slippage of DNA polymerase 78. Once cells compromise with these functions, then it may lead to accumulation of mutations resulting in the initiation of cancer. The MMR genes are involved in one of the most prevalent cancer syndromes in humans known as hereditary nonpolyposis colon cancer (HNPCC) or Lynch syndrome 79. HNPCC is characterized as an autosomal dominant inherited disease with 85–90% gene penetrance for early-onset of colorectal carcinoma. HNPCC accounts for about 5–7% of cases of colorectal carcinoma. The molecular diagnosis of HNPCC is based on determining MMR genes for germ-line mutations. There are at least six different proteins required for the complete MMR system. These proteins are hMSH2, hMLH1, hPMS1, hPMS2, hMSH3 and hMSH6 (GTBP). Recently, mutational germ-line analysis of hMSH2 and hMLH1 genes has been suggested to cause early onset in colorectal cancer patients 80. hMSH2 forms a heterodimer with either hMSH6 or hMSH3 (depending on the type of lesion to be repaired) and binds to the mismatch site. The complex of hMSH2 with hMSH6 is called hMutSα, and it is required for the correction of single-base mispairs. The complex of hMSH2 with hMSH3 is called hMutSβ, and it is required for the correction of insertion-deletion loops 81. Subsequently, a heterodimer of hMLH1 and hPMS2 proteins are recruited by hMutSα or hMutSβ proteins to the mismatch recognition complex, which along with other proteins are involved in excision, resynthesis and ligation of DNA. Mutations in the hMLH1 and hPMS2 have been found in about 90% of HNPCC cases. Mutations in other MMR genes have been less frequent in HNPCC patients 79. In many sporadic colon cancers, hypermethylation of the hMLH1 gene promoter resulting in its transcriptional silencing has been observed more than mutations. Both mutations and methylation of hMLH1 gene have been linked with MSI playing a causal role in the initiation of colorectal cancer 828384. Recently, the CpG island methylator phenotype (CIMP) pathway has been suggested as a novel mutator pathway that predisposes to colonic tumorigenesis. In many cases of the sporadic colon cancers, the MSI can be induced by CIMP, followed by hMLH1 gene's promoter methylation, loss of hMLH1 gene expression, and resultant MMR deficiency 85. Mutation rate in cells with MMR deficiency are 100–1,000-fold greater than in normal cells. There are many targets of MMR gene inactivation; however, the precise stage of tumorigenesis in which mutation of the wild-type MMR gene occurs is not clear. There are few well studied gene targets of MMR, whose mutations in the microsatellite region have been found in gastrointestinal tumors. These gene targets are the transforming growth factor-β receptor II (TGFβRII), hMSH3, hMSH6, insulin like growth factor II receptor (IGFIIR), phosphatase and tension homologue deleted on chromosome ten gene (PTEN), caspase 5, Bloom (BLM) syndrome helicase, T-cell factor 4 (Tcf-4), the cell cycle regulator E2F4, and antiapoptotic gene Bax 86878889909192939495.

In colon cancers, transforming growth factor-β (TGFβ) signaling potently inhibits the growth of normal epithelial cells, since the tumor cells are frequently resistant to TGFβ; they cause pre-neoplastic lesions, increase motility and spread cancer. The structural basis for TGFβ resistance in colon cancers is defined due to somatic mutations that inactivate TGFβ receptor II (TGFβRII) 86. In human colon cancer cell lines with high rates of MSI, the mutations in the TGFβRII gene was found 96. These are primarily frameshift mutations that add or delete one or two adenine bases within or from 10 base-pair poly(A) repeat in the cysteine-rich coding region (codons 125–128) of the TGFβRII gene. Mutations in this region consist of 1- or 2-base deletions or insertions. The other microsatellite region in the TGFβRII gene is a poly(GT)₃ that was found with insertion of an extra GT in this region 869697. These mutations produce truncated proteins that lack the cytoplasmic domain. As much as 90% of the colorectal cancers with MSI have the mutated TGFβRII gene. TGFβIIR responses are connected with Smads, tumor suppressor gene products, which help to initiate TGFβ-mediated gene transcription (Fig. 6). The transcriptional regulation of Type 1 plasminogen inhibitor (PAI-1) and cyclin dependent kinase inhibitor p15 genes are controlled by TGFβ signaling. PAI-1 is the primary inhibitor of tissue-type plasminogen activator (tPA) and urokinase-type 1 plasminogen activator (uPA) 98. TGFβ inhibits cell proliferation by inducing a G₁-phase cell cycle arrest acting through increased expression of p15 99. Thus, the loss of TGFβIIR or Smad4 can abolish TGFβ-signaling and advocate cell proliferation and development of colorectal cancer. In a mouse Apc^Min/+ mouse model, the combination of Smad4 and APC gene mutations have been shown to cause rapid tumor formation of the benign lesions arising only from the APC gene deficiency 100. These studies, reiterate the human data that put forward the hypothesis that mutations in the TGFIIR gene contribute to adenoma – carcinoma stage progression in colon.

Insulin like growth factor II receptor (IGFIIR) plays a critical role in cell growth, survival and differentiation; however, once mutated, these receptors play a major role in tumorigenesis 101102. The IGFIIR/mannose-6 phosphate receptor (M6PR) interacts with both IGF-II and latent TGFβ1 ligands and acts as a tumor growth suppressor 103. The IGFIIR performs its growth suppressive function by two different mechanisms. In one case, it binds and stimulates the plasmin-mediated cleavage and activation of the latent TGFβ1; in the second case, it participates in the internalization and degradation of its own ligand IGFII, a mitogen 104. The IGFIIR gene harbors several microsatellites within its coding region; one of them is poly(G)₈ repeat which is often mutated with 1- or 2-base pair deletions or insertions. These mutations produce frameshifts and premature stop codons 89. Thus, the loss of IGFIIR function due to mutations in MSI cells may enhance cell proliferation due to accumulation and activation of IGFII and down-regulation of TGFβ1.

The PTEN tumor suppressor gene, also known as mutated in multiple advanced cancers (MMAC) or TGFβ-regulated and epithelial cell-enriched phosphatase (TEP-1), is located on chromosome 10q23. A homologous deletion of this gene has been found in sporadic glioma, endometrial, melanoma, prostate, renal, meningioma and small cell lung carcinomas. Germ-line mutations in PTEN gene is linked with autosomal dominant inherited cancer syndromes such as Cowden's disease, Lhermitte-Duclos disease, and Bannqayan-Zonana syndrome, Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, and Proteus-like syndromes 105106107108109110. In endometrial and colorectal cancers, the mutations in PTEN gene occur in MMR function-deficient cells 109111. The coding region of the PTEN gene contains several microsallites, among which a repeat of poly(A)₆ tract in exon 7 and 8 are most susceptible to frameshift mutations 112. PTEN is involved in cell cycle arrest and apoptosis through negatively regulating the survival signaling mediated by phosphatidylinositol 3-phsophate (PIP3) kinase (PI3K) and its down-stream target, a serine/threonine kinase Akt (also called protein kinase B) 113114. Once cells receive signals through growth factors, hormones, or extracellular matrix components, the Akt is recruited by PIP3 to the plasma membrane, where it is phosphorylated at the Ser473/Thr308 115. The phosphorylated Akt is involved in cell survival, proliferation and migration. Thus, the loss in PTEN causes elevation of intracellular PIP3 level that stimulates Akt kianse activity. The active Akt leads to tumorigenesis due to loss of proliferative and apoptotic controls of cell growth. The genetic data of PTEN gene mutations found in human tumors were further supported by animal studies. In a mouse model study, the PTEN^-/- homozygous null mice was found to be embryonic lethal; however, the PTEN^-/+ mice developed neoplasm in various organs, including breast, prostate, endometrium, adrenal, lymphoid, skin, colon, thyroid, liver and thymus 116.

The ability of pRb to regulate cell cycle progression is dependent upon its interaction with E2F/DP family of transcription factors which are required for the regulation of a large number of genes involved in cell proliferation 117118119. There are at least six E2F proteins which can be categorized into distinct subgroups that act in direct opposition to one another to promote either cellular proliferation or cell-cycle exit and terminal differentiation. The E2F1, E2F2 and E2F3 proteins can be grouped in one subgroup since they all directly interact with pRb and are strong transcriptional activators. The E2F4 and E2F5 proteins are comparatively less strong transcriptional activators or sometimes act as repressors by recruiting the pocket proteins (BOX 2) and their associated histone-modifying enzymes. The E2F6 acts as a repressor through its interaction with Bmi1-containing Polycom group of proteins 120121122. The E2F4 and E2F5 transcription factors are also different than their other family members in that these factors lack the cyclin A binding domain. Furthermore, there is another critical feature in the E2F4 gene which is distinct from its family members. The coding region of the E2F4 gene contains a longer spacer segment of a repeated sequence of trinucleotide (CAG)₁₃ that encodes 13 consecutive serine residues; this region is highly vulnerable to frameshift mutations in MSI cells. In MMR defective colorectal tumors, mutations in the E2F4 gene have been observed 91. Thus, defective E2F4 gene expression in MSI colon cancer cells can possibly promote cell cycle progression through G_o/G₁ transition.

Approximately 50% of the tumors with MSI are found with mutations in the Bax gene. These mutations produce frameshift mutations within a coding region of eight nucleotide stretch of guanine residues (G₈) 93123. Bax heterodimerizes with anti-apoptotic protein Bcl2 and induces apoptosis. In the presence of apoptotic signals, BH3 domain proteins tBid are activated and bound with Bax. The interaction of tBid with Bax brings conformational changes in Bax, and this promotes its translocation to the mitochondria. Bax oligomerizes and inserts into the outer mitochondrial membrane where it forms channels to release cytochrome c (Cyt c) into the cytoplasm. The released Cyt c forms a complex called apoptosome with Apaf-1, dATP and pro-caspase 9 124. The apoptosome recruits and processes pro-caspase 9 to form a holoenzyme complex, which in turn recruits and activates the effector caspases leading to apoptosis. Thus, the expression of mutated Bax protein may fail to release Cyt c and increase the Bax-Bcl2 ratio resulting in the escape from apoptosis and inducing initiation of colorectal carcinogenesis.

Bloom syndrome (BS) is a rare autosomal recessive inherited cancer predisposition caused by inactivation of the RecQ family helicase, BLM 125126. The genetic disorder of the BLM gene is characterized by growth deficiency, impaired fertility, proportional dwarfism, sun-sensitive telangiectatic erythema, immunodeficiency and premature ageing. The function of the BLM helicase is implicated in reinitiating DNA replication forks that are blocked at lesions, thereby promoting chromosome stability, which is characterized by elevated sister chromatid exchanges (SCEs), as well as chromosomal breaks, deletions, and rearrangements. Also, BLM helicases perform essential roles in genetic recombination, transcription, and DNA repair 127128. In all the above systems, the helicase activities derive energy from hydrolysis of ATP. BLM is a part of BRCA1-associated genome surveillance complex (BASC) which includes MSH2, MHS6, MLH1, ATM, RAD50-MRE11-NBS1 complex, and replication factor C 129. BLM also interacts with heterotrimeric, single-stranded DNA binding protein, replication protein A (RPA), which after binding, stimulates BLM helicase activity 130. The genetic structure of the BLM gene coding region contains 4,437 base-pairs and encodes 1,417 amino acids. The BLM helicase has different motifs which are assigned to different activities. Motifs Ia, III and V contain residues that establish direct contact with ssDNA and motifs I, II-IV and VI form a pocket for binding ATP 131. It has been observed that many point mutations in BS patients are confined to these conserved helicase motifs, thus disrupting its ATPase and helicase activity. DNA sequencing from the sporadic gastrointestinal tumors showed abnormal bands caused by a deletion of one adenine residue in the poly-adenine tract (reduction from nine to eight adenines) in the coding region of the BLM gene 95132133. Since these tumors had defective MMR genes, mutations in the BLM gene is directly linked with MSI and chromosomal instability pathways. Mutations in BLM gene has been associated with frameshifts in Bax, hMSH6 and/or hMSH3 with multiple unstable mono- and trinucleotide repeats located in coding regions that were significantly higher than that observed in microsatellite mutator phenotype positive tumors without BLM frameshifts. From these studies, it has been suggested that BLM loss of function by MSI in sporadic gastrointestinal tumors might be an intermediate mutational event, which may increase the instability of a pre-existent unstable genotype.

In Figure 3, disruption of the APC/β-catenin pathway is shown as a critical step in the development of colorectal and other cancers. The oncogenic β-catenin translocates into nucleus, heterodimerizes with the Tcf/Lef family of proteins, and transcribes target genes. The transcription factor Tcf-4 is one of the Tcf-family proteins, which forms a transcription complex with β-catenin and plays an important role in maintenance of normal epithelial growth and development of colorectal tumors 35. The Tcf-4/β-catenin transcriptional activity is inhibited by protein Icat (inhibitor of β-catenin and Tcf-4), which blocks the interaction between β-catenin and Tcf-4 and thereby antagonizes Wnt-signaling 134. Since the APC/Tcf-Lef/β-catenin pathway plays an important role in colorectal cancer development, mutational analysis of these genes is critical to evaluate their functions. The mutations and their consequences in APC and CTTNB (β-catenin) genes are discussed earlier, which are characteristics of CIN. However, the mutations in the Tcf-4 gene were found in MSI cells 135136137. In MSI cells, an error-prone poly(A)₉ monorepeat in exon 17 of the Tcf-4 gene frequently shows frameshift mutations by deletion of one nucleotide producing a short and medium isoform of the Tcf-4 protein. The transactivating properties of these shorter Tcf-4 isoforms in the development of colorectal cancers are still not clear. Recently, it has been suggested that the frameshift mutations in the poly(A)₉ region of the Tcf-4 gene may in fact produce a gain in function of the shorter Tcf-4 isoform due to loss of binding sites of the transcriptional suppressor molecules, CtBP or Grg/TLE 138139140. These mutated Tcf-4 proteins also lose the binding of chromatin remodeling complexes containing Osa/Brahma 141. Thus, based on these studies, it is expected that mutated Tcf-4 protein may acquire increased transcriptional activity by increased binding affinity for β-catenin or by facilitating the structural organization of chromatin 142. Finally, it has been suggested that mutations in the Tcf-4 gene may not have significant effect and thus it may contribute marginally to the colorectal carcinogenesis or act as passenger mutations 140.