Development Of Colorectal Cancer Biology Essay

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Colorectal Cancer (CRC) is a frequent cause of morbidity and mortality among Western industrialized countries i.e. UK, USA, and New Zealand (1). It is seen as both a hereditary and sporadic disease, which is well characterized by its genetic alterations at the cellular level, which leads to its development. CRC is complex and genetically its carcinogenesis is made up of a variety of pathways, the major two being; Chromosomal Instability (CIN) (adenoma - carcinoma sequence) and Microsatellite Instability (MSI); with these pathways there are countless more gene alterations, leading to formation of metastases thus the progression of CRC.

Cancer is defined as 'any malignant tumour, including carcinoma and sarcoma which arises from the abnormal and uncontrolled division of cells that then invade and destroy the surrounding tissue' (2). Tumours can be differentiated in to two types, benign and malignant, where only the latter is classified as a cancer. Both of these types of tumours form due to breakdown of tightly regulated cellular processes of proliferation and apoptosis, resulting in aberrant cells which proliferate forming a large cluster of cells, recognized as a clonal cancerous growth. The major characteristic which differentiate them is that malignant tumours have the capacity to invade, spread and colonize other regions of the body forming metastases, an ability which benign tumours lack.

Cancer is a multifactorial disease with various risk factors such as obesity and tobacco-smoking. Some cancers have been linked to viruses as their causal agents i.e. Hepatitis B virus which causes liver cancer (3). However cancer is fundamentally a genetic disease, arising as a consequence of pathological changes in information carried by DNA. Mutations in cancer occur in somatic cells, unlike other genetic diseases, where mutations often occur in germ-line cells.

Cancer is primarily seen as a disease of old age due to the well known elucidation, that it takes more than one genetic mutation to turn a normal cell; cancerous and it takes a long time for a single cell line to accumulate these large number of mutations. This pattern is observed in many human cancers e.g. stomach and rectal cancers, where their incidences increase markedly with age (4); this supports the idea that cancers develop by an accumulation of mutations in a clone of cells; known as the Multiple Hit Hypothesis.

Cancer cells are mainly defined by their containment of many mutations and their genetically instability and it is this genomic instability that results in the amassing of mutations in genes critical for cancer which are proto-oncogenes and tumour suppressor genes (TS). Both these genes experience different types of mutations, as firstly gain-of-function mutations (overactivity mutation) convert proto-oncogenes into oncogenes, resulting in a single mutation event in the proto-oncogene leadings to its conversion into an oncogene and the excessive stimulation of cell survival and proliferation; an example of oncogene activation is that of K-ras in colorectal cancer.

Secondly loss-of-function mutations (underactivity mutation) in TS genes are oncogenic, here two inactivating mutations are required which functionally eliminate the gene (as the gene could still be functional with one allele), thus stimulating cellular survival and proliferation; i.e. APC and DCC gene in colorectal cancer, this was confirmed by experimental isolation of various tumour suppressor genes which substantiated Knudson's "two-hit" hypothesis for Rb (3). However a requirement for oncogenic mutation to induce cancer is that they must occur in actively dividing cells so that mutations are passed on to many progeny cells (4).

Colorectal Cancer

Colorectal cancer (CRC), estimated as the third most common cancer worldwide (5) and second leading cause of cancer-related deaths in the western world (6), affects both sexes equally, with approximately â…“ of a million new cases diagnosed in each gender group each year (5). Generally, those suspected of having CRC present clinically with episodes of rectal bleeding, changes in bowel habit, weight loss, anorexia, faecal incontinence and tenesmus (7).

Studies show diets high in fat and meat consumption (with special reference to barbequed meat) are associated with increased cancer rates (8), though this topic remains controversial, but there is a positive definitive correlation of western diet with CRC and cigarette smoking though only having weak positive association with CRC (9). Other risk factors include a strong family history of CRC, age, alcohol and physical inactivity; where as consumption of vegetables, fruits and non-steroidal anti-inflammatory drugs (due to a casual association of COX/PGE-2 to CRC (10)) have a negative association with CRC (9). Tumour staging systems of CRC such as TNM and Dukes' is an important prognostic tool but is dependant on time of diagnosis. They help in determining whether to give adjuvant radio- or chemotherapy; or surgery, with the latter being the most effective form of treatment (10).

CRC arises from the epithelium lining the colon and rectum. The inherited form is similar to sporadic form which occurs later in life. The major difference is that the inherited form strikes much earlier in life and usually with an increased frequency of adenomatous polyps (3). As previously stated there are many pathways associated with the carcinogenesis of CRC, one being the epigenetic pathway whose role in CRC initiation and progression is embedded in the basis of hypermethylation of specific promoter islands and histone modification leading to the transcriptional silencing of genes (11).

The two major pathways of CIN and MSI (fig 1) are associated with two common hereditary syndromes, FAP and hereditary non-polyposis colorectal cancer (HNPCC) respectively where FAP, is said to accounts for less than 1% of all CRC' (12) and HNPCC, with frequency of occurrence shown to be estimated between 1 and 5% of all CRCs (13). Other rarer syndromes occur such as hereditary flat adenoma and hereditary mixed polyposis.

HNPCC is an autosomal dominant trait, characterized by its early onset of pre-dominantly right-sided colorectal cancer and the tendency to develop multiple primary cancers at an early age (9). HNPCC is caused by MSI which is due to defects in DNA mismatch genes (e.g. MLH1, PMS2 etc) and the mutations in DNA mismatch repair enzymes lead to genetic instability reflected in errors in replication of the repetitive nucleotide repeats scattered throughout the genome. Although these replication errors produce mutations in many of the same TS genes and proto-oncogenes as the FAP pathway, though there is a significantly lower frequency of LOH in the HNPCC pathway (14).

The trademark of the FAP syndrome is the appearance of at least 100 colonic polyps distributed throughout the large bowel (9). This syndrome follows the CIN pathway as molecular alterations that occur in this pathway involve deletions of alleles of TS genes (LOH) in APC, p53, and the Deleted in CRC (DCC) gene (14) combined with mutational activation of proto-oncogenes, especially K-ras (20, 14) and is an autosomal dominant trait, associated with a germline mutation of the APC gene on chromosome 5q (15, 16).

Sporadic cases of CRC follow the same pathways as that of hereditary syndromes, but the majority of cases; about 70% of cases, follow the CIN pathway involving the adenoma-carcinoma sequence, while 10-15% of sporadic CRC (9) show the features characteristic of MSI. But most CRC are, however, sporadic and occur in individuals without a strong family history (7). So from these studies we can gather that most sporadic and the majority, if not all FAP CRCs occur due to the mutations in the key genes; APC, K-ras, DCC and p53 and the rationale to how this occurs will be discussed. In addition there is evidence of countless other genes associated with colorectal carcinoma and mestastic tumours such as E-cadherin and cyclin D1 (fig 1) (6, 17, 18).

Figure 1. Shows two major pathways associated with CRC development. One pathway (indicated with red arrows) initiates with mutations in the adenomatous polyposis (APC) gene and chromosomal instability (CIN) followed by mutations in K-ras, deleted in colorectal cancer (DCC) and p53 genes. The second pathway (indicated with blue arrows) is initiated by the mutations in the mismatch repair (MMR) genes and microsatellite instability (MSI) followed by additional mutations (Taken from 17)


Chromosomal Instability Pathway

The model of colorectal carcinogenesis proposed by Fearon and Vogelstein (20) describes the mutation of different cell growth regulatory genes during the transition from adenoma to carcinoma which includes the early mutation of the APC gene and then the following mutations in the K-ras, DCC and p53 genes. This model and its core structure remains a paradigm for mutational event regarded as essential to progression of CRC i.e. in FAP (19). This idea and its defining steps is characterized by its allelic losses on chromosome 5q (APC), 7p (p53) and 18q (DCC) and so is therefore called the chromosomal instability (CIN) pathway.

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 e.g. APC (17).


Adenomatous Polyposis Coli (APC) is a TS gene on chromosome 5q21 whose germline mutation was found to be the causative agent for FAP via molecular cloning (15, 16), is considered the initiating gate keeper gene in virtually all FAP CRCs and also in sporadic CRC where it's mutated in 60-80% of patients (17). The breakthrough leading to the identification of the APC gene came with the recognition of a patient with Gardner's syndrome, who had a deletion of chromosome 5q (9). In 1991, seven years after the advancement in the discovery of the gene by investigation of FAP families, a link was found to this same region via linkage analysis (21) and subsequently the APC gene was isolated, cloned and sequenced (9).

The APC gene consists of 21 exons and is contained within a 98-kilobase locus (22), but with the conventional form encoded by 15 exons. The largest exon, 15, comprises more than 75% of the coded sequence and seems to be the targeted region in the majority of germline mutations in FAP patients and somatic mutations in tumours (22). APC experiences a deviation from its customary functions via mutations normally created by frameshifts (small insertions or deletions), which in turn generate premature stop codons (PSCs), non-sense point mutations or splice site alteration that results in a truncated protein product which has reduced or absent tumour suppressor activity (9, 23). With PSCs being the mutation commonly experienced (15). APC mutations also follows the "two hit" hypothesis model of tumour suppressor inactivation as two somatic mutations are required in both alleles in sporadically acquired CRC and loss of heterozygosity (LOH) needed for those with FAP.

Figure 2. (A) In the presence of APC β-catenin is localized to the adherens junction. GSK-3β phosphorylates β-catenin in a complex and then is readily degraded by ubiquination at the proteosome. (B) When APC is mutated, β-catenin accumulates in the cytoplasm and the nucleus which results in constitutive transcription by the β-catenin/T cell factor-4 (Tcf-4) complex2,3. (Taken from 22)

The APC gene encodes a multifunctional protein which is involved in several cellular processes such as migration, cell adhesion and apoptosis. But its main function lays in its regulatory role within the Wnt signalling cascade (24) and its capacity to control the intracellular levels of β-catenin (25). The control of the transcription factor β-catenin by degradation allows the inhibition of cellular growth (15). This process occurs by the APC protein binding to β-catenin to form a complex with axin and GSK-3β, which is then degraded through ubiquination in the proteosome (15, 23, 26, 27). Inactivation of APC allows a free-pool of stabilized β-catenin to accumulate in the cytoplasm, which eventually traverses into the nucleus, where it drives the transcription of multiple genes implicated in tumour growth and invasion such as c-myc and cyclin D1, by heterodimerizing with Tcf-Lef transcription factor (fig 2).

APC is involved in both cell adhesion and migration due to its association with β-catenin which localizes in adherens junctions and the microtubule cytoskeleton (22, 25). Evidence for APC association with microtubules was reported by Nathke et al (28), as they showed by immunolocalization the accumulation of APC at the leading edges of integral microtubules in actively migrating epithelial cells (22, 61). Additional support for β-catenin comes from the analysis by Mahmoud NN et al (29) of intestinal tissue from mice with genetically altered levels of the APC gene, which showed altered migration, is facilitated by β-catenin dysregulation (15, 39). So therefore inactivation of APC would also lead to detachment of cells from primary tumours due to disruption of the cell-cell interactions and aberrant cell migration leading to further cancer progression and the formation of metastases.

The cloning of the APC gene was a major achievement which has facilitated the presymptomatic diagnosis in patients with family history of FAP by direct detection of the responsible mutation in at risk individuals. Many mutational analysis techniques are available each with there limitations and advantages. Examples of this are the denaturing gradient gel electrophoresis (DGGE) analysis and the protein truncation test (PTT). These tests where used by Van der Luijt et al (30), in an experiment that involved the molecular screening of the APC gene in 105 Dutch Kindreds with FAP for mutations, where DGGE analysed exons 1-14 in the APC and PTT analysed exon 15.

In the DGGE analysis firstly the exons are amplified by PCR and then a small sample of the exon 14 for example is applied to an electrophoresis gel that contains a denaturing agent. Certain denaturing gels are capable of inducing DNA to melt at various stages. As a result of this melting, the DNA spreads through the gel and can be analyzed for single components, even those as small as 200-700 base pairs (31) (fig 3). DGGE analysis of exons 1-14 resulted in the identification of germline mutations in 27 patients in Van der Luijt et al (30) studies which shows DGGE to an extent is an efficient strategy for presymptomatic diagnosis of FAP but there are limitations in the sensitivity of mutation detection.

Figure 3. DGGE analysis of APC exon 4 in family 38. Numbers above the lanes correspond to the individuals in the family. All the affected individuals (38-1, -2, -4, -6, -9, and -10) show a characteristic four-banded pattern of the variant, consisting of 2 homoduplex and 2 heteroduplex bands. Unaffected individuals (38-3, -5, -7, and -8) form a single homoduplex band. (Taken from 30)

In PTT, the gene required for analysis is amplified by PCR or in this case exon 15. Then the protein synthesized in vitro are then analyzed electrophoretically (fig 4). This method of analysis is very sensitive as it was demonstrated in the detection of APC mutations in tiny dysplastic colonic polyps (32). But has limitations in identifying non-truncating genetic mutations.

These two tests are adequate for patients with family history of FAP, but as most causes of CRC are sporadic without a family history (7) these test are contraindicated in those with sporadic CRC. So alternative investigative tests are carried out such as faecal occult blood tests and colonoscopy (gold standard for investigation), which allows screening of the colon and rectum and can then allow biopsies and polypectomies to obtain specimens for histological examination.

Figure 4. PTT analysis of exon 15. Sizes of the in vitro synthesized proteins are indicated on the left. The normal control shows only the wild type polypeptide, whereas all the patients show an additional truncated polypeptide characteristic of FAP. (Taken from 30)


Kirsten-ras (K-ras) is a proto-oncogene part of a 'ras family of monomeric G proteins which act as "molecular switches" linking extracellular signals through membrane receptors to intracellular signals; others within this subfamily include H-ras and N-ras' (33). It was discovered as a transforming protein in human tumours that was equivalent to the transforming protein encoded by retroviral oncogenes of the Kirsten murine sarcoma virus 30 years ago (33, 34, 35) . K-ras is essentially a 21 kDa membrane localized GTPase which may be in an activated state (GTP-bound status) or inactivated state (GDP-bound status) depending on extracellular physiological stimulus. And when constitutively activated by mutations leads to its conversion into an oncogene and this scenario normally occurs early in the multistage process of FAP and sporadic CRC tumourigenesis. K-ras is 188 proteins in length and its alteration into an oncogene occurs due to activating point mutations in codons 12, 13, 61 and 64, with most occurring in the latter (15, 33). These mutations occur in about 40-50% of colon carcinomas and approximately 50% of all sporadic CRC (36, 39).

K-ras activation is complex due to the variety of stimuli able to cause its initiation. But generally K-ras activation begins with stimulation of a vast array of upstream receptors such as tyrosine kinase and cytokine receptors and then due to various mechanisms, bound to Guanine Exchange Factors (GEFs) such as SOS-1 and CDC25 which are able to induce a conformational change in the K-ras leading to the exchange of the GDP for GTP (33, 37). Its inactivation occurs due to hydrolysis of GTP to GDP, but K-ras has an intrinsically low GTPase activity. So its GTPase activity is stimulated by numerous GTPase Activating Protein (GAPs) such as p120-GAP, leading to its inactivation and so prohibiting a protracted K-ras stimulated signal (37).

GTP-bound K-ras is able to bind and activate countless effector enzymes and its through these pathways such as RAF, JNK and phosphatidylinositol 3-kinase (PI3-K), that K-ras controls myriad cell functions such as growth, survival, angiogenesis and other aspects of cell behaviour that can contribute to the transformed phenotype (fig 5) (15, 34).

Figure 5. Simplified Overview of Ras activation and signalling cascade. This shows activated Ras interaction with multiple signalling pathways such as PI3-K, PLC, MKKI, RalGEF and RAF. (Taken from 33)

The major pathways activated by K-ras are that of RAF and PI3-K. In the resulting activation of RAF pathway by K-ras, the RAF protein induces a cascade activation of a series of cytoplasmic kinases and nuclear transcription factors (i.e., ETS) which may induce specific gene expression toward cell proliferation, differentiation, survival and apoptosis depending on cell type and quality and quantity of the stimuli (37). The PI3-K pathway coexists with several other pathways and some of its main functions lies in its control of apoptosis by the inactivation of pro-apoptotic factors, such as BAD and caspase 9 (37, 49) and its stimulation of the RHO family protein, RAC, that is involved in the regulation not only of the actin cytoskeleton but also of transcription-factor pathways - for example, by activating nuclear factor-κB which is significant in cellular survival (15, 34, 39). K-ras also activates; through these varied pathways, various gene targets such as cyclin D1 and vascular endothelial growth factor (VEGF) which both are well know for the roles in proliferation and angiogenesis respectively (15. 39).

In the result of point mutations to K-ras this leads to aberrant signalling of the G-protein, as these mutations renders the K-ras protein insensitive to GAP induced hydrolysis of GTP to GDP, which results in sustaining K-ras in a constitutively active state and so resulting in the pathways discussed above to be continuously active and deregulated, leading to the progression of CRC via enhanced cellular growth, survival etc.

Mutational Analysis of K-ras has been pin-pointed as a clinically relevant protein due to its early activation in CRC, so it can be used as a diagnostic marker and prognostic indicator (40). Some of the methods used is enriched PCR-colorimetric assay; believed to be a useful tool in diagnosis and prognosis by Santiago et al (40) and short oligonucleotide mass analysis which is a technique by which small sequences of mutated and wild-type DNA, produced by PCR amplification and restriction digestion, are characterized by HPLC-electrospray ionization tandem mass spectrometry. This method was adapted by Lleonart et al (41) to detect low levels of K-ras mutations in codon 12; which proved a success and was capable with multiplex PCR to detect multiple codon mutations and so could be used in routine K-ras analysis for diagnosis in stool or circulating DNA in the blood as a marker for early detection of CRC in both FAP and sporadic cases, when treatment may be curative.

The usage of K-ras as a prognostic indicator is still a debatable field as studies have conflicting evidences which are critically analysed by Anwar et al (62) and they concluded that the inconsistent evidenced could be due to a number of circumstances such as differing genomic analysis, population heterogeneity etc. But the mutated K-ras protein has been implicated as a possible route for cancer gene therapy by a number of methods. One promising method carried out by Dvory-Sobol et al (39) which focused on utilizing the constantly active Ras signalling pathway rather than inhibiting it, to selectively target transformed but not normal cells by over expressing pro-apoptotic genes using a Ras-responsive promoter. But this approach of reintroducing functional wild type genes such as p53 and pro-apoptotic genes has its limitations one being to solely introduce these therapeutically modified genes solely into the tumour cells and not normal cells, where it might inhibit cell division. Other constraints include getting the genes into all tumour cells, as even a tiny number of uncorrected cell are still lethal and the levels of proteins expressed from these introduced genes must be near normal to be effectual (4). But other techniques also exist such as antisense oligonucleotides and short interfering RNAs (siRNAs) which are potential K-ras inhibiting drugs but their drug delivery is limited (33, 34).

But most of these methods could be of little help to the elderly due to reports showing that the individuals 65 years of age and older with sporadic and FAP CRC have a significantly decreased probability of K-ras mutations in their adenomas (43). This could be overcome by primarily identifying the K-ras mutation if any and then administrating the appropriate treatment. But generally there has been a failure of available therapeutics to the K-ras oncogene to confirm its known importance in tumourigenesis and validity in targeted gene therapy.


Deleted in Colon Cancer (DCC) was initially identified as a tumour suppressor gene (44, 45), located on chromosome 18q and its inactivation is a relatively late event in the carcinogenesis of CRC and is closely correlated with metastases (15, 46). Mutation of the DCC gene is observed in approximately 50% of late adenomas and 70% of sporadic colorectal carcinomas (9). The DCC gene is a member of the Ig-superfamily, with 29 or more exons that spans a very large genomic region, which encodes several different protein products due to differential splicing (47, 48), which are transmembrane proteins, similar in structure to certain types of cell adhesion molecules such as neural-cell adhesion molecule (N-CAM). The DCC genes proposed functions are cell adhesion and differentiation, apoptosis and axon guidance. Point mutations in this gene are seen in 6% of sporadic CRC (48), chromosomal deletion; insertions, LOH and rearrangements, with all leading to the inactivation/loss of DCC which significantly correlates with hepatic metastases (46).

DCC gene encodes a receptor for netrin-1, a molecule involved in axon guidance (49) and there are suggestions that the DCC/netrin-1 interaction may contribute to regulation of proliferation or differentiation. This idea is consistent with studies which show how forced DCC expression causes an arrest in the G2/M cell cycle and so a mutation in DCC will thus abolish this function and therefore allowing cellular proliferation to occur (47). But one of the main functions attributed to the relationship between DCC/netrin-1 is that of cell survival. This was demonstrated by Mehlen et al (49) by showing that the expression of DCC in the absence of netrin-1 induces apoptosis, while the presence of netrin-1 blocks DCC-induced cell death and this seems to be viable in CRC as the loss of DCC will then allow transformed colonic epithelial cells to not be dependent on the local concentration of netrin-1 for its survival. Hence, DCC appears to be a member of the emerging family of the so-called dependence receptor. Which are receptors proposed to generate a cellular state of dependence on the ligand, and in settings in which the ligand is not available; the dependence receptor promotes apoptosis (47). But not much detail is know about the role of DCC in CRC so further research is needed.

DCC has been indicated as a good prognostic marker, but this role is still controversial and is discussed in a report by Anwar et al (62). This role can be further debated as the candidacy of this gene as a tumour suppressor has been called in to question, as mice heterozygous for DCC have been reported to lack the tumour predisposition phenotype (47). And moreover, other tumour suppressor genes, including SMAD4/2, have been reported on 18q (47). In particular, SMAD4 is currently a candidate gene because the inactivation of SMAD4 has been causally associated with progression of cancers (50). But nevertheless LOH of chromosome 18q is very common in CRC and its assessment to determine its status is accomplished by using microsatellite markers. But the detailed role of DCC in CRC has to be further studied as a not much is known.


p53 located on chromosome 17p is the most frequently mutated tumour suppressor gene in various kinds of human cancers (51). And from the Fearon and Vogelstein (20) model of colorectal carcinogenesis, p53 mutations generally occur late at the transition from adenoma to carcinoma and this genetic malformation occurs in about 40-50% of sporadic CRC (15). The p53 gene was originally discovered because its protein product complexes with the SV40 large T antigen (52) and was initially thought to be a proto-oncogene. But this was disproved when the importance of p53 was shown in inhibiting cellular transformations and its role was shown to be parallel to that of a tumour suppressor (53).

The p53 gene (shown below) is very important in cells due to its role in suppressing cancer and so it has been described as the 'the guardian of the genome' and 'master watchman' (54), due to its part in conserving stability by preventing genomic mutations by inducing cell cycle arrest to repair the damage or apoptosis, if the damage is too severe and irreparable (55). Mutations to the p53 gene will lead to an aberration of these functions and most of these mutations occur in highly conserved areas of exon 5 to 8 (56). The most common mutations seen are deletions, insertions, truncations, or point mutation in one allele and LOH in the other, in which the wild-type allele is, deleted (which matches Knudson's two hit hypothesis). But the majority of mutations (approx. 80%) are mis-sense mutations (GC to AT) which occur mainly in five hotspot codons (175, 245, 248, 273, and 282) (15).

p53 gene has other family members including p63 and p73 with all possessing similar properties (57). But the p53 gene encodes a p53 protein which is a 393 amino acid nuclear localized phosphoprotein and carries out its role primarily as a transcription factor, inducing the expression of a number of downstream genes by tetrameric binding. This role and others such as apoptosis are controlled by domains in this protein which include the transcription-activation domain (TAD) and homo-oligomerisation domain (OD). The regulation of p53 is controlled by processes such as phosphorylation by kinases, acetylation and proto-oncogenes like Mdm2 (53).

p53 works in preventing genomic instability and tumourigenesis by its functions such as cell cycle arrest and the main way in which this protein negatively controls the cell cycle is by its transcriptional activation of p21 (53). This was proven in studies carried out by Deng et al (58) in which mice homozygous negative for p21 could not arrest in G1 in response to DNA damage. p53 also induces other proteins to aid in cell arrest and potentially DNA repair an example being Gadd45α (53). But as stated above, if the damage is irreparable the cell undergoes apoptosis and the way p53 controls this program is not well known; but a number of downstream effector genes which are known pro-apoptotic inducers are upregulated by p53 this includes Bax and KILLER/DFR (53). Another function of p53 is to repress cellular promoters such c-myc and bcl-2 which expression of both, leads to cell cycle progression and cell survival respectively. Mutations in p53 will result in a loss-of-function, which will result in DNA damage being copied and propagated to the progeny of the cell, leading to an accumulation of mutations and therefore CRC progression.

The overexpression of p53 can be evaluated by a standard immunohistochemical procedure (IHC) which can be of clinical use in the identification of CRC patients likely to benefit from the standard chemotherapy treatment (56). The overexpression of p53 in the nucleus is seen in CRC due to the clonal proliferation of the cancer cells passing on the mutated p53 gene on to their progeny and thus resulting in the loss of functions described above in all their descendants; which facilitates tumourigenesis in CRC. Detection of p53 mutations in CRC is detected via procedures such as DGGE and single strand conformation polymorphism (SSCP). These procedures could be useful in both sporadic and FAP CRC to judge patient prognosis as patients with cancers involving a p53 mutation have a worse outcome and shorter survival time than patients whose cancers do not have a mutation in this gene (15). But this still seems to be controversial (62), but studies in favour of p53 prognostic factor seem to be of greater strength as the p53 is such a critical gene in colorectal carcinogenesis and it seems to be essential for metastases to occur and its presence in the tumour of the patient must present as a strong prognostic factor for poor survival due to a progressed tumour stage.

Enhancing anti-tumour affects of tumour suppressor genes has been suggested as a way for cancer treatment and gene therapy concerning the p53 gene seems to be quite promising owing to results seen with clinical trials performed with adenovirus-p53 (60).


In summary, the genetic alterations described here all work in tandem to cause a dysregulation in cellular activities and their regulation, so therefore causing the potential development of colorectal cancer. The genetic malformations in APC, K-ras, DCC and p53 are the major causes of carcinogenesis in virtually all those with the hereditary syndrome FAP and most sporadically acquired cancers which are depicted by the adenoma-carcinoma sequence model. Current studies seem to follow the general idea that all these genes function in the control and regulation of cellular functions thus suppressing tumourigenesis. So their mutations results in the deviation from cellular normality due to aberrant regulation of cellular mechanisms and regulation of critical effector enzymes and genes which lead to uncontrolled proliferation, survival and decreased cell adhesion and apoptosis etc and thus a selective growth advantage against normal cells.

But this report has placed the gene into different sections in the development of CRC, but we need to stress that all the genes and control pathways all interact in various ways such as the induction of APC functions by p53 (17) and that genetic mutations at different genes and sites can cause these specified genes to also malfunction and even their regulated control pathways such as Wnt pathway (43, 61).

Though the model proposed by Fearon and Vogelstein (20) is a good paradigm for FAP and most sporadic CRC we must also consider the factor other genetic mutations and pathways contribute to this disease; thus making CRC complex and heterogeneous (15).

However, not all is known about these major genetic alterations and recent data seem to believe that the simultaneous alterations of these genes is not a frequent event due to substantiations that claim p53 and K-ras mutations rarely co-exist in the same tumour (19). But however these studies can not take away the importance of these genes in colorectal carcinogenesis and so increased knowledge of these genes will allow for improved screening methods, curative treatments and maybe even gene replacement therapy such as that proposed by Cho et al (48) to use yeast artificial chromosomes (YAC) as possible replacements for mutated DCC gene segments, but there still lies an immense challenge to overcome the limitations described above to enable the clinical usage of gene therapy both possible and viable. The main focus and direction of this field, is to acquire an even greater detailed genetic and biological analysis of FAP and sporadic CRC and to link this knowledge to effective prevention, diagnosis and therapeutics in the near future.