Beau and Mike's Research Paper

Gene Mutations of Colorectal Cancer
Beau Kildow & Michael J. Buckley
Aka 8-Ball & MJB

Colorectal cancer is a type of cancer characterized by formations of tumors referred to as adenomatous polyps in the rectum and/or the colon (7). Colon cancer is the third most frequent type and second leading cause of cancer related deaths in the United States (4,8,14). In addition, 50% of the western world population will develop colorectal cancer by the age of 70 (7) and the overall survival rate for 5 years is 40% (2). Colorectal cancer, like all cancers, is a disease that can be described as cells that proliferate and invade surrounding tissue, potentially metastasizing throughout the body. Cells can become cancerous when mutations occur specifically in two classes of genes: proto-oncogenes and tumor suppressor genes. Proto-oncogenes control cell division and differentiation. When these genes become mutated, they can turn into oncogenes, which allow the cell to divide quickly and wildly (15). Tumor suppressor genes slow down cell division, repair DNA damage, and activate apoptosis. When these genes are mutated, they can be deactivated, allowing the cell to divide uncontrollably without undergoing apoptosis (20). Discovery and identification of tumor suppressor, oncogenes, and pathways that control the expression of these types of genes could lead to major advances in cancer therapy (2).
Studies suggest that 35% of all colorectal cancers are hereditary with 11% of colorectal cancer patients having at least one first degree relative with the same disease. The remaining 65% of the cases occur sporadically (14). The two hereditary neoplastic syndromes that are linked to colorectal cancer are familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) also known as Lynch Syndrome (14). Progression in colorectal cancer involves the genetic mechanisms of these two syndromes. In addition, the genes and mechanisms involved with sporadic colorectal cancer is unknown at this point; however, research suggests sporadic CRC is caused by low penetrance mutations, while hereditary colorectal cancer is caused by high penetrance mutations. Mutations to many oncogenes and tumor suppressor genes have been identified through studying hereditary and sporadic causes of colorectal cancer, suggesting possible targets for therapy.
FAP is a dominantly inherited autosomal disease that is caused by a mutation in the adenomatous polyposis coli (APC) gene. Patients who inherit this disease develop many benign polyps (adenomas) in their colon/rectum usually beginning at age 20 as a result of a germline mutation in the APC gene (7). Polyps do not form unless the APC allele from the unaffected parent mutates. These polyps do not turn invasive until mutations occur in the KRAS and p53 genes, along a sequence deletion on chromosome 18q (14) or 5q (7). The APC gene is known for its “gatekeeping” abilities in colorectal epithelial cell growth and proliferation. The APC gene is turned on to regulate and maintain equilibrium between cell death and cell proliferation by regulating the frequency of apoptosis. When the APC gene is mutated, an imbalance occurs and cell proliferation dominates. As a result, a little lesion forms which eventually turns into an adenoma (7). APC gene mutation is detected by the presence of a truncated APC protein as a result of a point, nonsense or frameshift mutation. After the APC gene mutates, a mutation in the p53 gene and KRAS genes causes cancerous colonic cells. Studies have shown that development of adenomas increases the chance of mutations in the p53 and KRAS genes. The p53 gene is a known tumor suppressor gene that once inactivated, leads to many types of cancer. However, a mutation to p53 alone does nothing to increase the chances of developing colorectal cancer (7). Additionally, the KRAS gene is an oncogene that leads to uncontrollable cell proliferation once activated. Studies have shown, like the p53 gene, that if KRAS is activated alone without a mutation to the APC gene, little or no tumors result (7). Consequently, colorectal cancerous tumor formation is a result of an accumulation of mutations with order specificity. These observations support the Knudson two-hit hypothesis, stating that cancer is a result of more than one mutation of different genes (12). Although FAP only accounts .2% of all colorectal cancers (14), the genes involved in FAP have also been identified as a possible cause of some sporadic cases. In addition tests have been developed to detect truncated APC proteins which may help in slowing the process of colorectal cancer formation through preventative measures.
In addition to FAP, HNPCC is the other autosomal dominantly inherited disease linked to colorectal cancer. HNPCC is caused by a germline mutation to DNA mismatch repair genes (MMR) such as MLH1, MSH2, MSH6, PMS1, and PMS2 (1). Mismatch repair genes code for proteins used for correcting missing or mismatched nucleotides during DNA replication (3). For example, MSH2 is specific for repairing G-T nucleotide mispairs (3). When these genes are mutated, DNA replication in that particular cell cannot correct DNA mistakes, thus leading to an increase frequency of other mutations. These mutations that occur as a result of mutations in MMR genes are usually at sites called microsatellites in the DNA. Microsatellites are regions in the DNA with repeated 1-6 nucleotide sequences (5). When microsatellites regions are mutated it is referred to as microsatellite instability (MI). MI is seen in most cases of HNPCC (13) and other forms of cancer (13). Studies have shown that colorectal cancers that have MI do not show signs of high mutation frequencies, indicating that mutations to MMR genes lead to high mutation rates. As a result, these high penetrance tumor susceptibility MMR genes lead to an accumulation of many mutations to oncogenes and tumor suppressor genes. Additionally, changes in lengths of microsatellite regions due to mutation causes truncation of many proteins, which enhances the progression of malignant cancer (5). HNPCC is known for high mutation rates that lead to rapid development to malignancy (7). Although HNPCC only accounts for 2-4% of colorectal cancers, the mechanisms of gene mutation involved is seen in many cases of sporadic cancers (14). Studies of HNPCC suggest how colorectal cancer is caused by mutations among many genes.
In contrast to hereditary causes, sporadic causes make up most cases of colorectal cancer. Although most cases of sporadic causes are unknown, some cases have been linked to genes involved in both FAP and HNCPP syndromes (6). Both mutations to APC and MMR genes in somatic cells cover the majority of sporadic colon cancer cases (7, 9). Kim et al. concluded that there are many biological similarities that indicate germline mutation and somatic mutations to MMR as causes to sporadic colorectal cancer (6). They also noted that in many cases there were deletions of polyadenine repeats and alterations of C-G dinucleotide repeats in microsatellite regions (6). In one study, 13% of sporadic colon cancer cases involved MI (7). In another study, 15-20% of sporadic colorectal cancer cancers had somatic inactivation of MMR genes due to hypermethylation (14). Furthermore, Birkenkamp-Demtroder et al. found one pathway involving chromosomal instability and MI for progression of sporadic colorectal cancer. They found a particular mitochondrial gene that lost expression in many cases of sporadic colorectal cancer. This gene coded for the enzyme SCAD. SCAD is responsible for β oxidation of Butyryl-CoA into Acetyl-CoA. Butyryl acid is the main source of energy for coloncytes. When there is a depletion of pyruvate, butyryl acid stimulates growth and cell division. However, when pyruvate is present, butyryl acid promotes apoptosis. When SCAD is mutated it becomes deactivated, resulting in a build up of butyryl acid, while depleting the concentration of pyruvate. As a result, growth and cell proliferation is promoted leading to a possible cause to carcinogenesis (2). Mitochondria are great locations for mutations to occur due to little DNA repair machinery. As well, mitochondria play a vital role in metabolism and thus are good locations to identify carcinogenic pathways (2).
One major discovery through research on HNPCC was the significance of the TGF-β pathway in colorectal cancer. This signaling pathway controls many cell processes, most importantly, cell proliferation and cell death. This pathway seems to be disrupted in many cases of cancer, especially colorectal cancer (14). In colorectal cells, TGF- β binds to the membrane protein receptor TGFBR2. This complex then binds to another membrane bound protein receptor TGFBR. This kicks on a phosphorylation cascade, phosphorylating the transcription factors SMAD2 and SMAD3 which then binds to SMAD4. The phosphorylated SMAD2/3-4 complex binds to DNA to regulate the transcription of growth suppressor genes in colonic cells (14). Some specific growth suppressor (tumor suppressor) genes are CDKN1A, CDKN1B, and CDKN2B. When these genes are activated via TGF- β signaling pathway, cell growth terminates (14). As a result, when mutations occur to any proteins involved in this pathway, cells continue to proliferate, leading to colorectal tumors. However, during late stages of colorectal cancer, studies reveal an increased expression of TGF- β. These results indicate high levels of TGF- β promote tumor growth, while a possible mutation to the TGF- β gene may lead to tumor initiation (14). Because TGF- β is involved with other cellular pathways, it is hypothesized that increased TGF- β expression leads to activation of tumor promoter effects. For example, TGF- β was shown to activate oncogenic pathways such as Ras/MAPK, JNK, and PI3/Akt. In addition, TFG- β is involved with the promotion of angiogenesis and immunosuppression (14).
Many recent studies on colorectal cancer involve the TGF- β signaling pathway. First, mutations to TGFBR2 are common in many colorectal cancer cases. TGFBR2 contains microsatellite sequences of adenine in exon 3 and guanine-thymine in exons 5 and 7. When MMR genes, like those noted in HNPCC, are inactivated, the microsatellite regions of TGFBR2 are highly prone to mutations. In fact, more than 80% of colorectal cancer cases that contain MI reveal frameshift mutations to TGFBR2 (14). Moreover, 30% of all colorectal cancer, including sporadic and hereditary, include TGFBR2 mutations. Biswas et al. showed that mice that lacked TGFBR2 had increased neoplastic proliferation in comparison to mice with TGFBR2 (16). However, some studies have shown that homozygous mutations to TGFBR2 allow TGF- β to bypass TGFBR2 protein and bind directly to TGFBR1, allowing the continuation of tumor suppressor gene activation(17). More studies are needed to directly relate mutated TGFBR2 to colorectal cancer.
In addition, mutations to TGFBR1 may relate to colorectal cancer. Although mutations to TGFBR1 have not been seen in many colorectal cancer cases, Pasche et al. found a polymorphic allele for this receptor that had a deletion of 3 alanines in a 9 alanine microsatellite region of exon 1 (18). This allele was termed TGFBR1*6A. In a study with 2438 cancer patients and 1846 healthy control subjects, the allelic frequency of TGFBR1*6A was 28.8% higher in the cancer patients. Also, additional studies showed that the TGFBR1*6A allelic frequency was 44% higher in cancer patients. Moreover, these studies suggest carriers of TGFBR1*6A are at high risk of developing colorectal cancer (14). Findings by Kemp et al., suggest that TGFBR1*6A is not a major colon cancer susceptibility gene, but in acts as a modifier with other unknown genes to induce cancer (19).
Lastly, mutations of SMAD genes can also disrupt the TGF-β signaling pathway. SMAD 4 gene was found to be germline mutated in 16-25% of colorectal cancer cases (14). These mutations result in adenomas and the gene acts as a tumor suppressor gene (14). As well, mutations to the SMAD 2 gene were seen in 6% of colorectal cancer cases (14). Both SMAD 2 and SMAD 4 genes are located on the 18q chromosome which is commonly mutated in colorectal cancer (14). Mutations to SMAD 3 are usually not found in colorectal cancer, but through studying this gene, it was found that gram-negative bacteria trigger gene mutations in the TGF-β signaling pathway. Currently, research studies are directed at the TGF-β pathway as a result of its prevalent involvement in colorectal cancer in order to discover a plausible treatment.
Furthermore, recent studies have shown an increased expression of cyclooxygenase-2 (COX2) in colorectal cancer (4, 10, 11). COX2 is an enzyme that catalyzes the formation of prostaglandin E2 (PGE2), which is known for inducing the expression of anti-apoptotic Bcl-2 protein (11) and vascular endothelial growth factor (VEGF) in colonic cells (4). Bcl-2 promotes growth and suppresses apoptosis in colorectal cancer. VEGF is also highly expressed in colorectal cancer. Also, PGE2 has been found to maintain vessel structure in colorectal cancer (13). The significance of these findings yield a possible treatment to colorectal cancer via commonly known non-steroidal anti-inflammatory drugs (NSAIDS). Many studies have shown an average 40-50% decrease in the risk of colorectal cancer through regular dosage of NSAIDS (11). NSAIDS work by deactivating COX enzymes, and thus halting the production of PGE2. Although this mechanism seems suitable for reducing colon cancer, the mechanism by which NSAIDS inhibit carcinogenesis is unknown (10). Moreover, this mechanism seems skeptical in that VEGF is also induced by the HIF-1 protein which is expressed during cellular hypoxia (4). Under low oxygen conditions, HIF-1α (a monomeric subunit of HIF-1) gets hydroxylated at proline residues 402 and 564. These proline residues act as binding surfaces for degradation proteins, however when they are blocked by hydroxylation, HIF-1α does not get degraded leading to the activation of VEGF (4). This mechanism is more well-known for VEGF expression, thus making evidence supporting NSAIDS blockage of VEGF production inconclusive as of now.
Lastly, similar mutated genes to that of FAP were identified as a result of red meat consumption. Animal fat, haem iron content, and meat preparation have all been proposed explanations of why meat consumption raises the risk of colorectal cancer. Cooking meat at high temperatures creates heterocyclic aromatic amines (HCAs). 2-amino-1-methyl-6-phenylimidazo [4, 5-b]pyridine (PhiP), is the most abundant of the different HCAs (21). Studies have shown that after the induction of colon tumors with PhiP in rats, the PhiP causes site specific mutations in the APC gene in these rats. This observation was accounted for by truncated APC proteins indicating a nonsense mutation like that seen in FAP. Likewise, red meat and processed meat can contain nitrosamines and their precursors which are formed endogenously. These compounds can become carcinogenic through alkylation (21). These N-nitroso compounds have been observed to activate G-A transitions in human colonic tissue on the 12th and 13th codons of the K-ras gene. According to the studies red meat may or may not cause these transitions. Moreover, the colorectal tumors had multiple mutations and could not be narrowed down to one specific mutation.
Although a small percentage of colorectal cancer is hereditary, the genes involved with corresponding syndromes are present in many sporadic cases. The identification of the APC and MMR genes through the studies in hereditary colorectal cancer have led to the identification of pathways like the TGF-β signaling pathway that have been linked to the majority of colorectal cancer progression. Although strong evidence supports the hypothesis that mutations to the TGF-β pathway lead to colorectal cancer, there are still many unsolved mysteries especially in roles of unknown genes. However, due to increase knowledge about the genes and pathways in colorectal cancer, mechanisms and environmental factors leading to mutations can be identified, and thus be prevented.

References
1. Akiyama, Yoshimitsu, Hisayoshi Sato, Toshio Yamada, Hiromi Nagasaki, Atsuo Tsuchiya, Rikiya Abe, and Yasushito Yuasa. “Germ-Line Mutation of the hMSH6/GTBP Gene in an Atypical Hereditary Nonpolyposis Colorectal Cancer Kindred.” Cancer Research 57 (1997): 3920-3923.
2. Birkenkanmp-Demtroder, Karin, Lise Lotte Christensen, Sanne Harder Olesen, Casper M. Frederiksen, Paivi Laiho, Lauri A. Aaltonen, Soren Laurberg, Flemming B. Sorensen, Rikke Hagemann, and Torben F. Orntoft. “Gene Expression in Colorectal Cancer.” Cancer Research 62 (2002): 4352-4363.
3. Fishel, Richard, Mary Kay Lescoe, M. R. S. Rao, Neal G. Copeland, Nancy A. Jenkins, Judy Garber, Michael Kane, and Richard Kolodner. “The Human Mutator Gene Homolog MSH2 and Its Association with Hereditary Nonpolyposis Colon Cancer.” Cell 75 (1993): 1027-1038.
4. Fukuda, Ryo, Brian Kelly, and Gregg L. Semenza. “Vascular Endothelial Growth Factor Gene Expression in Colon Cancer Cells Exposed to Prostaglandin E2 Is Mediated by Hypoxia-inducible Factor.” Cancer Research 63 (2003): 2330-2334.
5. Jackson, Aimee L., and Lawrence A. Loeb. “The Mutation Rate and Cancer.” Genetics Society of America 148 (1998): 1483-1490.
6. Kim, Hoguen, Jin Jen, Bert Vogelstein, and Stanley R. Hamilton. “Clinical and Pathological Characteristics of sporadic Colorectal Carcinomas with DNA Replication Errors in Microsatellite Sequences.” American Journal of Pathology 145 (1994): 148-154.
7. Kinzler, Kenneth W., and Bert Vogelstein. “Lessons from Hereditary Colorectal Cancer.” Cell 87 (1996): 159-170.
8. Kitahara, Osamu, Yoichi Furukawa, Toshihiro Tanaka, Chikashi Kihara, Kenji Ono, Renpei Yanagawa, Marcelo E. Nita, Toshihisa Takagi, Yusuke Nakamura, and Tatsuhiko Tsunoda. “Alteration of Gene Expression during Colorectal Carcinogenesis Revealed by cDNA Microarrays after Laser-Capture Microdissection of Tumor Tissues and Normal Epithelia.” Cancer Research 61 (2001): 3544-3549.
9. Parsons, Ramon, Lois L. Myeroff, Bo Liu, James K. V. Willson, Sanford D. Markowitz, Kenneth W. Kinzler, and Bert Vogelstein. “Microsatellite Instability and Mutations of the Transforming Growth Factor β Type II Receptor Gene in Colorectal Cancer.” Cancer Research 55 (1995): 5548-5550
10. Sano, Hajime, Yutaka Kawahito, Ronald L. Wilder, Akira Hashiramoto, Shigehiko Mukai, Kiyoshi Asai, Shigeru Kimura, Haruki Kato, Motoharu Kondo, and Timothy Hla. “Expression of Cyclooxygenase-1 and -2 in Human Colorectal Cancer.” Cancer Research 55 (1995): 3785-3789.
11. Sheng, Hongmiao, Jinyi Shao, Jason D. Morrow, R. Daniel Beauchamp, and Raymond N. DuBois. “Modulation of Apoptosis and Bcl-2 Expression by Prostaglandin E2 in Human Colon Cancer Cells.” Cancer Research 58 (1998): 362-366.
12. Valle, Laura, Tarsicio Serena-Acedo, Sandy Liyanarachchi, Heather Hampel, Ilene Comeras, Zhongyuan Li, Qinghua Zeng, Hong-Tao Zhang, Michael J. Pennison, Maureen Sadim, Boris Pasche, Stephan M. Tanner, Albert de la Chapelle. “Germline Allele-Specific Expression of TGFBR1 Confers an Increased Risk of Colorectal Cancer.” Science 321 (2008): 1361-1365.
13. Wang, Tian-Li, Carlo Rago, Natalie Silliman, Janine Ptak, Sanford Markowitz, James K. V. Willson, Giovanni, Parmigiani, Kenneth W. Kinzler, Bert Vogelstein, and Victor E. Velculescu. “Prevalence of somatic alterations in the colorectal cancer cell genome.” PNAS 99 (2002): 3076-3080.
14. Xu, Yanfei, and Boris Pasche. “TGF-β signaling alteration and susceptibility to colorectal cancer.” Human Molecular Genetics 16 (2007): R14-R20.
15. Yang, Ziheng, Simon Ro and Bruce Rannala. “Likelihood Models of Somatic Mutation and Codon Substitution in Cancer Genes.” Genetics Society of America 165 (2003): 695-705.
16. Biswas,S., Chytil, A., Washington, K., romero-Gallo, J., Munoz, N., Olechnowicz, J., Ferguson, K., Gauam, S., and Grady, W.M. “Transforming Growth Factor Beta Receptor Type II Inactivation promotes the Establishment and Progression of Colon Cancer. Cancer Research 64 (2004): 4687-4692.
17. Ilayas, M., Efstathiou, J.A., Straub, J., Kim, H.C. and Bodmer, W.F. “Transforming Growth Factor Beta Stimulation of Colorectal Cancer Cell Lines: Typer II Receptor Bypass and Changes in Adhesion Molecule Expression. PNAS 96 (1999): 3087-3091
18. Pasche, B., Luo, Y., Rao, P.H., Nimer, S.D., Dmitrovsky, E., Caron, P., Luzzatto, L., Offit, K., Cordon-Cardo, C., Renault, B. et al. “Type I Transforming Growth Factor Beta Receptor Maps to 9q22 and Exhibits a Polymorphism and a Rare Variant Within a Polyalanine Tract. Cancer Research 58 (1998): 2727-2732.
19. Kemp, Z.E., Carvajal-Carmona, L.G., Barclay, E., Gorman, M., Martin, L., Wood, W., Rowan, A., Donahue, C., Spain, S., Jaeger, E. et al. Evidence of Linkage to Chromosome 9q22.33 in Colorectal Cancer Kindreds from the United Kingdom. Cancer Research 66 (2006): 5003-5006.
20. The American Cancer Society. “Learn about cancer”. 2008. http://www.cancer.org/docroot/LRN/LRN_0.asp.

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