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Molecular genetic aspects of colorectal cancer development
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Colorectal cancer (CRC) is one of the most common cancer diseases in the world after lung and female breast cancer and approximately 945 000 new cases are diagnosed every year. CRC is caused by genetic alterations in the DNA, which results in cell cycle acceleration, escape from apoptosis, senescence, angiogenesis, invasion and metastasis. In this thesis, we have investigated molecular genetic alterations for the development of CRC and focused on the MAPK pathway, HIF-1 α and NOS2 genes.

Alterations in the MAPK pathway have been found in several different cancer forms, including CRe. In the present study, we found somatic mutations in the MAPK pathway in 50% of the CRCs; 40% of the tumors carried mutations in the KRAS gene and 10% carried BRAF mutations. No genetic alterations were found in the ARAF or RAF-1 genes. B&4F gene mutations were present only in exon 15 and were associated with micro satellite instability. Three mutation types were identified; V599E, D593G and K600N, whereof the latter has not previously been described.

The hypoxia inducible factor (HIF)-la protein is involved in the oxygen sensing mechanism and several tumor types show HIF-la overexpression due to hypoxia. At normoxia, HIF-la is degraded by interaction with the von Hippel-Lindau (VHL) tumor suppressor protein followed by an ubiquitin-proteasome dependent degradation mechanism, which prevents HIF-l a from nuclear translocation and transcription of downstream target genes. Fifteen percent of CRC patients and normal healthy population was found to carry the P582S polymorphism in the HIF-1 α gene, which previously has been associated to higher transactivating capacity. In the present study, the polymorphism was associated to ulcerative tumor development. In addition, loss of heterozygosity of the wild type P582 allele in heterozygotes may contribute to the development of ulcerative CRCs. However, the overall mechanism for ulcerative tumor development is still unclear.

Nitric oxide (NO) is involved in several physiological processes, such as apoptosis, neurotransmission, angiogenesis and immune defence and is produced by three nitric oxide synthases; NOSl-3. In the present study, NOS2 upregulation was identified in CRCs compared to normal intestinal mucosa. Moreover, the contribution of NOS2 in CRC development was investigated in APCMin/+ and APCMin/+ NOS2-/- mice. The APCMin/+ NOS-/- mice developed a higher polyp frequency compared to APCMin/+ mice, indicating a protective role for the presence of NOS2 in intestinal cancer development. The elevated polyp formation in the APCMin/+ NOS-/- mice was independent of the expression of Notch-l and p21. We also investigated whether polymorphisms in the NOS2 promoter affected the onset of CRC, but no differences in allele or genotype frequencies were observed in normal healthy population compared to CRC patients.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2005. , 83 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 878
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-31156Local ID: 16894ISBN: 91-7373-856-5 (print)OAI: oai:DiVA.org:liu-31156DiVA: diva2:251979
Public defence
2005-01-21, Berzeliussalen, Halsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-09-25Bibliographically approved
List of papers
1. Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas
Open this publication in new window or tab >>Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas
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2004 (English)In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 25, no 4, 527-533 p.Article in journal (Refereed) Published
Abstract [en]

Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1–2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-24437 (URN)10.1093/carcin/bgh049 (DOI)6544 (Local ID)6544 (Archive number)6544 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
2. Association between ulcerative growth and hypoxia inducible factor-1α polymorphisms in colorectal cancer patients
Open this publication in new window or tab >>Association between ulcerative growth and hypoxia inducible factor-1α polymorphisms in colorectal cancer patients
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2006 (English)In: Molecular Carcinogenesis, ISSN 0899-1987, E-ISSN 1098-2744, Vol. 45, no 11, 833-840 p.Article in journal (Refereed) Published
Abstract [en]

The hypoxia inducible factor-1α (HIF-1α) has been found to be involved in several different physiological mechanisms, such as blood-vessel formation, apoptosis, and erythropoiesis. HIF-1α is hydroxylated at normoxia and rapidly degraded via the von Hippel–Lindau (VHL)/ubiquitin-proteasome degradation system to prevent angiogenesis. In a previous study, the C1772T (P582S) and the G1790A (A588T) polymorphisms were identified in the human HIF-1α gene, which was shown to have a higher transactivating capability in vitro compared to the wild type allele. However, the role for these polymorphisms in vivo is still unclear. In the present investigation, we have therefore studied the role of the two polymorphic variants in the development of colorectal cancer (CRC) with PCR/RFLP (restriction fragment length polymorphism), single strand conformation analysis (SSCA), and immunohistochemistry (IHC). A significant higher-risk was identified between patients heterozygous for the C1772T polymorphism and the more severe ulcerative growth pattern compared to homozygous C1772C wild type tumors (RR = 5.2; 95% CI 1.26–21.6; P = 0.006). This was also verified on the allelic level (RR = 6.5; 95% CI 1.58–26.8; P = 0.001). In addition, patients carrying one or more polymorphic alleles in either the HIF-1α C1772T or the G1790A polymorphisms display significant higher risk for the development of ulcerative CRCs (RR = 4.17; 95% CI = 1.33–13.08; P = 0.004). These results suggest that the HIF-1α polymorpisms are an important factor for development of a subset of ulcerative intestinal tumors. Future screening of the polymorphic HIF-1α allele may therefore be of importance in the selection of treatment strategies of CRC.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-35755 (URN)10.1002/mc.20209 (DOI)28446 (Local ID)28446 (Archive number)28446 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13Bibliographically approved
3. Nitric oxide synthase 2 mRNA expression in relation to p53 and adenomatous polyposis coli mutations in primary colorectal adenocarcinomas
Open this publication in new window or tab >>Nitric oxide synthase 2 mRNA expression in relation to p53 and adenomatous polyposis coli mutations in primary colorectal adenocarcinomas
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2002 (English)In: Surgery, ISSN 0039-6060, E-ISSN 1532-7361, Vol. 131, no 4, 384-392 p.Article in journal (Refereed) Published
Abstract [en]

Background. The inducible nitric (NO) synthase 2 (NOS2) is upregulated in breast, brain, colon, and gynecological tumors, which indicate that NO may have a role in tumorigenesis. However, little is known about the role and regulation of NOS2 in colorectal carcinomas. Recent in vitro experiments have implicated that NOS2 is downregulated by p53 accumulation. Virtual analysis of the NOS2 promoter showed putative TCF-4/Lef-1 response elements, which indicate a potential regulation of NOS2 expression by activation of the adenomatous polyposis coli (APC)/β-catenin pathway.

Methods. NOS2 mRNA expression was investigated in 59 colorectal carcinomas by reverse transcriptase/real-time polymerase chain reaction and related to mutations in the p53, APC, and β-catenin genes. Presence of NOS2 protein was studied by Western blot, and the localization was studied by immunohistochemistry. Loss of heterozygosity was studied in the region of the NOS2 gene.

Results. The NOS2 mRNA and protein expression were significantly higher in tumors than in control tissue. Immunohistochemistry revealed extensive NOS2 staining in the epithelial cells and, to a minor degree, in leukocytes. Increased NOS2 mRNA expression was found in Dukes' stages A and B compared with the C and D stages. No relationship was found between elevated NOS2 expression and loss of heterozygosity in the later stages according to Dukes' classification or mutations in the p53, APC, or β-catenin genes.

Conclusions. Inactivating mutations in the p53 and APC pathways are not the main explanation for the increased NOS2 expression found in colorectal tumors.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25021 (URN)10.1067/msy.2002.121888 (DOI)9442 (Local ID)9442 (Archive number)9442 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Nitric oxide synthase 2 (NOS2) promoter polymorphisms in colorectal cancer
Open this publication in new window or tab >>Nitric oxide synthase 2 (NOS2) promoter polymorphisms in colorectal cancer
2005 (English)In: Cancer Letters, ISSN 0304-3835, E-ISSN 1872-7980, Vol. 225, no 1, 99-103 p.Article in journal (Refereed) Published
Abstract [en]

Previously, increased expression of nitric oxide synthase 2 (NOS2) in colorectal cancer (CRC) has been identified. The NOS2 gene is transcriptionally regulated, which suggests that polymorphisms in the NOS2 promoter may have a role for CRC development and progression. The genotyping was performed with PCR/RFLP, single strand conformation analysis or MegaBACE genotyping of normal blood DNA from CRC patients and normal healthy controls. However, no significant association between NOS2 polymorphisms and CRC onset or clinical outcome was evident. In conclusion, these results, therefore, suggest that NOS2 promoter polymorphisms have a limited effect on the onset or progression of CRC.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-30426 (URN)10.1016/j.canlet.2005.02.006 (DOI)15988 (Local ID)15988 (Archive number)15988 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically approved
5. Promotion of intestinal polyposis in nitric oxide synthase 2 (NOS2) deficient Min mice and expression of genes in the Notch-1 pathway
Open this publication in new window or tab >>Promotion of intestinal polyposis in nitric oxide synthase 2 (NOS2) deficient Min mice and expression of genes in the Notch-1 pathway
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Nitric oxide synthase 2 (NOS2) expression has been found in several different tumor types, including colorectal cancers, but the role of NOS2 expression for cancer development is not fully understood. In the present study, we have investigated the role of NOS2 for intestinal polyp development in the APC Min/+ mouse and studied the mRNA expression by real time PCR of Notch-1 and p21 in normal murine small intestinal tissue and polyps from APC Min/+ NOS2+/+ and APC Min/+ NOS2-/- mice. A significant higher polyp frequency was found in mice with APC Min/+ NOS2-/- genotype compared to APC Min/+ NOS2+/+ mice. The expression of Notch-1 was significantly increased in polyps from the APC Min/+ NOS2+/+ mice compared to wild type small intestinal mucosa, but no difference was evident between the APC Min/+ NOS2+/+ and APC Min/+ NOS2-/- mice, which indicates that NOS2 expression does not affect the Notch-1 expression. No significant difference was found between the different mouse groups regarding the expression of p21. Collectively, NOS2 expression is a protective factor in intestinal polyposis, but its role in polyp development is still unclear.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-81928 (URN)
Available from: 2012-09-25 Created: 2012-09-25 Last updated: 2012-09-25Bibliographically approved

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