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Colorectal Cancer (CRC) presents in two varieties: hereditary and sporadic, and is a frequent cause of morbidity and mortality amid the Western society (1). It is well characterized by a series of diverse genetic cellular modifications, some of which involve two very important pathways in the process of CRC development. Colorectal Cancer carcinogenesis sees the modification of various genes, subsequently leading to progression through the stages of CRC and also the formation of metastases.
This paper will outline the key genes & pathways involved in the development of sporadic and hereditary Familial Adenomatous Polyposis (FAP) and discuss the effect of genetic modifications on cellular functionality, leading onto how this information will continue to benefit the evolution of new treatments, diagnosis, and patient prognosis.
"An abnormal growth of cells which tend to proliferate in an uncontrolled way and in some cases, to metastasize. (2)"
Tumors can present in a benign or malignant form, however only the malignant tumors are incorporated into the definition of cancer. They have the ability to invade, spread and colonize regions of the body as metastases; however, they arise from various changes in regulatory processes correlating to cellular proliferation and apoptosis; consequently a series of clonal cancerous growths may develop.
With multiple genetic mutations required to stimulate normal cells to become cancerous, a single cell line will require a lengthy period of time to amass the numerous mutations. Due to this, cancer is conceived to be a disease that strikes with older age rather than during a more youthful age. A prime example is in the development of colorectal cancer with which incidence distinctly increases with age (3, 4); supporting The Multiple Hit Hypothesis (5 ).
Furthermore, cancer is a multifactorial disease associated not only with obesity & smoking but also links viruses like hepatitis B & C, Papilloma & retro viruses to carcinogenesis (6).
Fundamentally, cancer is a disease with a genetic foundation underlying pathological changes in DNA. It does however differ from other genetic diseases because mutations occur in somatic cells.
Cancer can be identified by a number of mutations which result in the instability of the human genome. The instability hast the effect of vastly amplifying the net reproductive rate of cells. The mutations can occur in numerous genes and two key genes involved are proto-oncogenes & tumor suppressor genes (TSG) and these will give rise to two types of mutation:
Gain-of-function mutations leading to over activity e.g. the activation of K-ras in colorectal cancer. This requires a single mutational event in a proto-oncogene which will then stimulate the conversion to an oncogene. The effect of this on cells is the fuelling of excessive proliferation and survival.
Loss-of-function mutations which lead to under activity. This occurs in oncogenic Tumor Suppressor (TS) genes, e.g. Adenomatosis Polyposis Coli and Deleted in Colorectal Cancer genes in CRC, where two inactivating mutations are necessary for elimination of the gene's function rather than one because the gene could still be functional with one remaining allele. The functional elimination will allow cellular survival and proliferation to continue but it will do so in an unregulated manner.
Confirmation came from experimental isolation of Tumor Suppressor genes, verifying Knudson's "two-hit" hypothesis (4,7,8). Oncogenic mutation must occur in actively dividing cells, allowing mutations to present in daughter cells & induce cancer (6).
Colorectal Cancer is the 3rd dominating cancer in the UK with most cases manifesting from the epithelium lining the colon and rectum and affects both sexes equally. Clinical symptoms include loose stools & rectal bleeding, weight loss and anemia. Studies from EPIC also implicate diets heavy in fats, red and processed meat along with age & excess alcohol consumption, providing a positive correlation with western lifestyle and the epidemiology relating to CRC (9). After clinical diagnosis, the use of staging systems, including Dukes' & Tumour, Node, Metastasis (TNM), aid the decision to treat with adjuvant chemotherapy, radiotherapy or surgery (10).
Duke's Staging System (11)
The staging systems used for colorectal cancer, such as Duke's, indicate how far the cancer has spread. It plays an essential role in the treatment of cancer because the treatment options are often related to the stage of the cancer.
Duke's makes use of four categories: Dukes A, B, C or D.
Dukes A indicates that the cancer is limited to the innermost lining of the colon or rectum or intruding marginally into the muscle layer
Dukes B indicates the cancer has breached the muscle layer of the colon or rectum
Dukes C indicates the cancer has metastasized to at least one lymph node in the area
Dukes D indicates the spread of the cancer to a more distant region of the body such as the liver or lung. If it reaches this stage it is usually diagnosed as an advanced bowel cancer.
TNM staging is a newer concept beginning to be used across the globe and represents Tumour, Node, Metastasis.
The T stages reflect the size of the tumour:
T1 - The cancer has not gone past inner layer of the bowel.
T2 - Growth has progressed into the muscle layer of wall of the bowel.
T3 - It continues grow and it has reached the outer lining of the bowel wall or organs & body structures next to the bowel.
T4 - Growth has reached other parts of the bowel, organs or body structures which lay in close proximity to the bowel. Alternatively the tumour has penetrated through the membrane covering the outer wall of the bowel.
The N stages reflect the presence of cancer cells in the lymph nodes.
N0 - indicates no cancer cells in any nodes.
N1 - indicates approximately 1 to 3 lymph nodes within close range of the bowel contain cancerous cells.
N2 indicates the presence of cancer cells in 4 or more lymph nodes located further than 3cm away from the primary tumour in the bowel. Alternatively, there are cancer cells in lymph nodes which are connected to the main blood vessels on the perimeter of the bowel.
The two M stages tell if the cancer has metastasized or not.
M0 - The cancer has not spread to other organs
M1- The cancer has metastasized to other parts of the body
The principle difference between hereditary & sporadic CRC is the earlier presentation of the inherited form in combination with an increased frequency of adenomatous polyps (7). There are many different pathways which present in the carcinogenesis of the different varieties of CRC, examples include The Chromosomal Instability pathway (CIN) & The Microsatellite Instability Pathway (MSI); They are associated with two hereditary syndromes, Familial Adenomatous Polyposis (FAP) & Hereditary Non Polyposis Colorectal Cancer (HNPCC) respectively. The two syndromes increase the chances of developing colorectal cancer (CRC).
Hereditary Non Polyposis Colorectal Cancer - An autosomal dominant syndrome arising from possible changes in 5 genes. Occurrence in limited to an estimated 2 - 5% of CRC's. The related microsatellite instability develops from improperly repaired replication errors in microsatellites, which will stem from defects in mismatch genes with 1-7bp repeating units. As a result of the improper repair, repeats at specific loci vary, making the microsatellites unstable. MSI usually presents in HNPCC cases, which implicates the presence of a mutated mismatch repair enzyme, hence faulty DNA repair. It is from faulty DNA repair which produces mutations in Tumour Suppressor (TS) or oncogenes that cancer will arise (12,13).
The presence of masses of colonic polyps distributed throughout the large bowel is characteristic of Familial Adenomatous Polyposis (14). It is a trait with autosomal dominant inheritance associated with a germline mutation of the APC gene on chromosome 5q (15,16,17) and reflects the CIN pathway. This involves Loss Of Heterozygosity (LOH) at TS loci in APC, p53, and the Deleted in CRC (DCC) genes combined with mutational activation of proto-oncogenes, for example K-ras.
Both syndromes overlap in terms of the Tumour Suppressor & proto - oncogenes involved, but the frequency of LOH is a lot lower in the HNPCC syndrome (13). About 70% of sporadic cases follow the CIN pathway, relating to the adenoma-carcinoma sequence, while 10-15% of sporadic CRC demonstrate features typical of MSI (10). Nethertheless most CRC cases occur in individuals that lack a strong family history (18). From this we can deduce that most sporadic and the FAP Colorectal Cancers occur from mutations in the following genes:
1. APC 2. K-ras 3. DCC 4. p53
E-cadherin & cyclin D1 also implicated in colorectal carcinoma & mestastic tumours (10).
[Figure 1] The two major pathways associated with CRC development. 1) blue arrows ïƒ is initiated by the mutations in the mismatch repair genes and microsatellite instability followed by various other mutations
2) red arrows ïƒ initiaties with mutations in the adenomatous polyposis gene and chromosomal instability followed by mutations in K-ras, DCC and p53 genes (19)
The chromosomal instability Pathway (CIN)
Chromosomal Instability occurs during cell division and sees an increase in the rates of loss or gain of whole or large parts chromosomes. Consequently, aneuploidy and an increased rate of loss of heterozygosity (LOH) arise. If the LOH occurs at a higher rate, the rate of inactivation of Tumour Suppressor Genes will also accelerate; which is one of the key properties of the CIN pathway (20).
Chromosomal Instability occurs due mutations in CIN genes. These genes are responsible for maintaining the security of the genes of the cell. The pathway sees the various mutations of different regulatory genes which have a role in cell growth. These changes include: mutational activation of K-ras & allelic losses on chromosome 5q21 (APC), 7p (p53) and 18q (DCC). (21)
Adenomatous Polyposis Coli (APC) is a Tumour Suppressor gene located on chromosome 5q21. Evidence implicates APC as the most frequently mutated gene by means of inactivation, in virtually all FAP Colorectal Cancer germline and somatic tumour mutations. It can be found to be mutated in 70-80% of sporadic CRC patients (18, 23 ). A germline mutation was identified to be the causative gene for Familial Adenomatous Polyposis (FAP) by means of positional cloning & linkage analysis (22).
The APC gene consists of 15 exons, the largest being the 15th and disturbance of the APC gene typically occurs from mutations which create Premature Stop Codons resulting in a truncated expressed protein (10,23). In sporadic varieties of CRC, two somatic mutations are required for both alleles of the gene in comparison to Familial Adenomatous Polyposis where there is a Loss of heterozygosity, followed by a point mutation before clinical presentation of Colorectal Cancer. Mutations in the APC gene thus follow the Knudson's "two hit" hypothesis model of tumour suppressor inactivation.
The APC gene encodes a large protein suggested to regulate cell adhesion, cell migration, and apoptosis in cells which dwell within the colon ( 24).The protein has been found to bind to a number of proteins with a regulatory function. These proteins include: Î²-catenin, Î³-catenin & glycogen synthase kinase 3Î² (GSK3Î²). In colorectal cancers where the APC gene is mutated, it is unable to bind and, or coordinate regulation of Î²-catenin efficiently. As a result, Î²-catenin accumulates, forms complexes with transcription factors such as Tcf-4, & translocates to the nucleus. Here, Î²-catenin functions as a transcriptional co-activator, activating expression of Tcf-regulated genes. These mutations leave the defective Î²-catenin proteins oncogenic as APC & GSK3Î² are unable to regulate the mutated Î²-catenin (25).
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[Figure 2] A) In normal cells, proteins glycogen synthase kinase 3Î² (GSK3Î²), APC, & axin promote degradation of free cytosolic Î²- catenin. GSK3Î² activity and Î²-cat degradation are inhibited by Wnt pathway inactivation from action of the Frizzled receptor and dishevelled signaling protein.
(B) Mutation of APC in Colorectal cancer cells results in accumulation of Î²-catenin, binding to Tcf-4, leading to transcriptional activation of Tcf-4
target genes (26).
Mutational analysis techniques are in existence and each confers its advantages but also have their shortfall.
The denaturing gradient gel electrophoresis (DGGE) analysis. This involves amplification of exons using the Polymerase Chain Reaction and then application of a sample of an exon to an electrophoresis gel containing a denaturing agent. The gel will induce DNA to melt and consequently the DNA will spread throughout the gel making is possible to analyse single strands (27). DGGE analysis of exons 1-14 saw the identification of germline mutations, illustrating DGGE to the extent that it can be used for presymptomatic diagnosis of FAP. However the sensitivity to mutation detection is limited (28).
[Figure 3] DGGE analysis of exon 4 of APC in one family. The Numbers above the lanes relate to 1 individual within the family. In those affected a characteristic four-banded pattern, consisting of 2 homoduplex bands and 2 heteroduplex bands is illustrated (1, 2, 4, 6, 9, and 10). Unaffected individuals form a single homoduplex band (3, 5, 7, and 8) (28).
The protein truncation test (PTT). The protein truncation test aims to detect mutations which lead to early translation termination. The gene required for analysis is again amplified via the Polymerase Chain Reaction and the protein synthesized in vitro and analysed electrophoretically. It is a very sensitive method illustrated by the detection of APC mutations in tiny abnormal colonic polyps. However, it has limited use in identifying non-truncating genetic mutations (29).
[Figure 4} PTT analysis of exon 15. Sizes of the synthesized proteins are shown on the left. The normal control shows only the wild type polypeptide, whereas all the patients show a FAP characteristic additional truncated polypeptide (28).
These tests are however, contraindicated in sporadic Colorectal Cancer because most cases will arise in patients without a significant family history (18). Consequently, alternative investigations have been produced such as colonoscopy and faecal occult blood tests. These tests screen the colon and rectum but go a step further in the way that they also obtain specimens from biopsies and polypectomies for examination. They are ''gold standard'' methods of investigation.
Kirsten-ras (K-ras) is a GTPase protein encoded for by the K-RAS gene in humans. It links extracellular signals by means of membrane receptors to a cascade of intracellular signals, activated by the binding of GTP or inactivated with GDP-bound. Which is bound depends on the extracellular stimulus. Activational conversion to an oncogene can also occur from a point mutation as is the case during FAP or Sporadic carcinogenesis.
K-ras activation normally starts with the stimulation of a range of receptors like Tyrosine Kinase and cytokine receptors. Once bound to Guanine Exchange Factors (GEFs) such as Son of sevenless homolog 1 (SOS-1) and CDC25, a conformational change in the K-ras is induced and see the exchange of GDP for GTP.
GTPase activity is stimulated by numerous GTPase Activating Proteins which will prohibit an extended K-ras stimulated signal. Activated K-ras has the ability to can bind and activate numerous effector enzymes via various pathways, which give K-ras control of a multitude cell functions such as survival, growth and angiogenesis; all of which are attributes of the cancer phenotype (16, 30). Inactivation of K-ras will occur from the hydrolysis of GTP to GDP.
[Figure 5] Ras signalling pathways involved in the control of cellular growth, proliferation and survival. Activation of the signalling cascade can occur from mutation of the components shown in blue or deletion of those in yellow (31)
The major pathways activated by K-ras are RAF and PI3-K.
The RAF pathway - The RAF protein stimulates the activation of a series of cytoplasmic kinases and nuclear transcription factors. These will subsequently go onto induce gene expression for cell differentiation, proliferation, survival and apoptosis depending on the quality and quantity of the stimuli affecting the cell and the cell which the stimuli is for (30).
The PI3-K pathway functions within the control of apoptosis. It inactivates of pro-apoptotic factors, such as BAD and caspase 9 (30) but also stimulates the protein RAC. RAC has a role which is involved in the regulation of the actin cytoskeleton & sime of the pathways for transcription-factors (16, 32, 33). Furthermore, K-ras activates gene targets such as Cyclin D1 and vascular endothelial growth factor (VEGF) for proliferation and angiogenesis (10. 33).
Point mutations in K-ras lead to the K-ras protein being insensitive to 'GAP' induced hydrolysis of GTP. This will mean K-ras is held in the active state and the pathways it regulates are continuously active , extending cellular growth, survival and the effects of the rest of the unregulated pathways. Thus progressing Colorectal Cancer.
As a prognostic tool, the use of K-ras is yet to be definitive as studies conflict as illustrated by Anwar et al (34). Nethertheless, there are techniques such as antisense oligonucleotides and short interfering RNAs which possess potential K-ras inhibiting abilities. The mutated K-ras protein is also a possible target for cancer gene therapy as demonstrated by Dvory-Sobol et al (33). The methodology relies on the constant active Ras signalling pathway to over express pro-apoptotic genes using a Ras-responsive promoter. Limitations include difficulty in tumour cell specific expression of these therapeutically modified genes to a normal but therapeutic level of expression (3).
Unfortunately there is a lack of significant evidence on the K-ras oncogene to verify how important a role it plays in tumour genesis and thus gene therapy.
Deleted in Colorectal Cancer (DCC)
The product of DCC is a single transmembrane receptor. Located on chromosome 18q, inactivation occurs fairly late in the development of CRC and is related to metastasis. Its function is implicated in cell adhesion, differentiation & apoptosis. Point mutations will usually lead to a chromosomal deletion, insertion, Loss Of Heterozygosity and rearrangements, consequently there is inactivation or a loss of the DCC (35,36).
The DCC gene encodes a receptor for netrin-1, which has a role in axon guidance. Current knowledge of the gene implicates it as a member of emerging family 'the dependence receptors'. These receptors cause a cellular state of reliance on the binding ligand, and where the ligand is unavailable the dependence receptor will stimulate apoptosis. To date however, knowledge of DCC's role in CRC is lacking and its role as a prognostic tool is uncertain as other tumour suppressor genes, including SMAD4/2, are also said to be located on 18q. (37)
p53 is located on chromosome 17p, and its main role lies as a transcription factor,seeing the expression of a number of genes through tetrametric binding. It functions to suppress cancer through prevention of mutations and does this by arresting the cell cycle to repair the damage or undergo apoptosis if the damage is too severe and irreparable by transcription activation of p21 (38).
p53 genetic mutations include most commonly; deletions, insertions, truncation, or a point mutation in a single allele and Loss Of Heterozygosity in the other, following Knudson's two hit hypothesis. But approximately 80% of mutations are mis-sense mutations exchanging CG for AT and occurs mainly in five codons: 175, 245, 248, 273, and 282 (19). Mutations in p53 can result in a loss of-function and will therefore see DNA damage being copied to the progeny of the cell. Thus the mutations will accumulate and further the progression of CRC.
A standard immunohistochemical procedure (IHC) can be used for investigation into the over expression of p53. The process makes use of antigens such as proteins, in cells of a tissue and exploits the principle of specific antibody - antigen binding in tissues. With specific molecular markers associated with cellular processes, including apoptosis, Immunohistochemical staining would highlight the distribution and localization of molecular markers including over expressed proteins such as p53. This has clinical use as it has the potential to identify CRC patients likely to benefit from the standard chemotherapy treatment (39).
p53 appears essential for metastases to occur and its presence within a tumour acts as a strong prognostic attribute relating to poor survival due to an advanced stage in tumour genesis.
As illustrated by clinical trials performed with adenovirus-p53 enhancing the anti-tumour effects of tumour suppressor genes in relation to p53 has been suggested as a method for cancer treatment (40).
Genetic mutations which occur in colorectal cancer work as a collective through various steps to deregulate cellular processes and further the progression through the stages of colorectal cancer. The most significant genetic mutations occur in APC, K-ras, DCC and p53 with one if not more of these genes found to be defective in cases of Familial Adenomatous Polyposis and the majority of sporadic Varities.
The future not only lies in obtaining the intricate details pertaining to the genetics of Familial Adenomatous Polyposis and Sporadic Colorectal Cancer but also finding various means of using this knowledge to prevent, diagnose and produce therapeutics capable of targeting the specific cause of the Cancer.
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