Molecular Mechanism of Tumorigenesis of Colorectal Cancers

• Main Authors: Ching-Sheng Yeh, 葉景生
• Other Authors: Shiu-Ru Lin
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/66154132127440452726

博士 === 高雄醫學大學 === 醫學研究所博士班 === 96 === Colorectal cancer (CRC) is the third most fatal cancer in Taiwan, next to liver cancer and lung cancer. Every year more than 7,000 people are diagnosed with the disease. There are four stages in the course of the disease: the 5-year survival rate of stage I CRC is about 90%, stage II is about 60-80%, stage III is about 30-60 %. At stage IV CRC, where distant metastasis occurs, the 5-year survival rate is reduced to 5%. The prevalence of CRC has increased gradually in recent years, therefore, effectively diagnosing and treating CRC is critical to identifying the risk factors and the molecular mechanisms. Cancer studies have progressed considerably, for example, many oncogenes and tumor suppressor genes have been found to be involved in cancer development. However the understanding of the causes of CRC and the molecular mechanisms are yet to be established. CRC can be caused by individual genetic make-up and its interaction with environmental factors through multi-step development processes. A plausible scenario is that cells lose their normal functions, such as gene deletion and other mutations, when stimulated by environmental factors then become cancerous. To further understand the molecular pathogenesis and the related signaling pathways in CRC patients in Taiwan, especially the correlative relationship between high fat or high carbohydrate diet and CRC incidence, a series of experiments were designed and conducted with the emphasis on defining the roles of various molecules in CRC development. Microarray technology was employed, combined with bioinformatics and genomics methodologies to study the “molecular mechanisms of tumorigenesis of colorectal cancer” by analyzing 10 CRC patients in Taiwan that were selected by a matching process. Subsequently, a cDNA microarray chip (TGS-4K array) was used to analyze the gene expression difference between normal tissues and tissues from CRC affected areas. >From the results, gene groups, which are up-regulated in tumor tissues, were identified. The identifications were then verified by northern blot and direct sequencing techniques. cDNA microarray analysis revealed 157 up-regulated gene groups and 281 down-regulated gene groups. After in-depth analyses performed by bioinformatics software GOMiner, DAVID and KEGG, it was found that metabolism-related gene groups, among 14 categories of gene groups defined by genomics theory, are the most closely related (21%, p＝0.0046). Furthermore, it was shown that gene groups related to fatty acid metabolism (22.6%), bile acid metabolism (25.1%) and glycolysis (23.1%) are the most modulated, indicating their involvement in rectum/colon cancer development. The relationship between CRC and western diets (high fat and/or high carbohydrate) and the possible molecular mechanisms regulating metabolic pathways and gene expression were also discussed. Using linoleic acid-treated CRL-1790 cells, mimicking high-fat uptake through diet to study the gene regulation upon high fat ingestion eight genes related to fat metabolism were found up-regulated, including EHHADH (enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase), ECHS1 (enoyl Coenzyme A hydratase, short chain, 1, mitochondrial), GCDH (glutaryl-Coenzyme A dehydrogenase), ACOX2 (acyl-Coenzyme A oxidase 2, branched chain), ACADS (acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain precursor), CPT1B (carnitine palmitoyltransferase 1B), ACSL5 (acyl-CoA synthetase long-chain family member 5) and CYP4A11 (cytochrome P450, family 4, subfamily A, polypeptide 11). In addition, SW480 and HCT116 cells were treated with deoxycholic acid for 15, 30, 45, 60, 90 and 120 minutes. It was found that the deoxycholic acid treated cells showed an elevation of ATP activity by 12.35 %, 20.08 %, 29.69 %, 33.56 %, 45.85 %, 54.32 %, respectively. While the HCT116 treated cells, showed an elevation of ATP activity by 3.69 %, 5.59 %, 8.52 %, 12.26 %, 16.52 %, 19.71%, respectively. >From the experiments conducted using the cell lines described above, it was further confirmed that genes CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), AKR1D1 (aldo-keto reductase family 1, member D1), ALDH1A1 (aldehyde dehydrogenase 1 family, member A1), BAAT (bile acid Coenzyme A: amino acid N-acyltransferase), ABCA1 (ATP-binding cassette, sub-family A, member 1), APOC3 (apolipoprotein C-III), LIPA (lipase A), LIPE (lipase, hormone-sensitive), PTGER2 (prostaglandin E receptor 2) and PTGS2 (prostaglandin-endoperoxide synthase 2) were up-regulated. SW480 and SW620 cells were also used to study the gene regulation with high carbohydrate intake, where it was shown that during glycolysis eight genes, HK1 (nuclear gene encoding mitochondrial protein, transcript variant 1), GPI (glucose phosphate isomerase), GAPD (glyceraldehyde-3-phosphate dehydrogenase), PGK1 (phosphoglycerate kinase 1), PGK2 (phosphoglycerate kinase 2), ENO2 (enolase 2), PKM2 (pyruvate kinase, muscle, transcript variant 2) and GLUT1 (facilitated glucose transporter, member 1) are over-expressed. In our study, we also found that during hypoxia of cancer tissues the resulting up-regulation of HIF-2α (hypoxia-inducible factor 2, alpha subunit) would in turn up-regulate the GLU1 gene, further activating glycolysis. The correlative relationship between glycolysis and hypoxia-initiated processes was evaluated using SW480 and SW620 cells, where nine genes related to hypoxia were investigated under different environments. The result showed that HIF-1α and GLUT1 mRNA levels increased during hypoxia. Tests in cancer tissues also confirmed that GLUT1 (facilitated glucose transporter, member 1), HIF-1α (hypoxia-inducible factor 1, alpha subunit) and HIF-2α (endothelial PAS domain protein 1) were up-regulated at stage IV colorectal cancer. In summary, when cancer cells are replicated at a high rate, nearby tissues experience hypoxia, causing HIF up-regulating of the transcription of downstream target genes such as VEGF (vascular endothelial growth factor), GLUT1 (facilitated glucose transporter, member 1), PDGF (platelet-derived growth factor), EGFR (epidermal growth factor receptor) and TGF-α (transforming growth factor- α), further enhancing glycolysis and consequentially providing more energy. Additionally, the end products of fatty acid metabolism, secondary cholic salts, are confirmed carcinogens, which facilitate cancer development in epithelia cells. In conclusion, by inspecting the metabolic pathways and gene expression of related enzymes, we established an outline of CRC development initiated from polyp/cancer transition on the basis of stimuli-triggered molecular pathology hypothesis. This study demonstrates that high fat/carbohydrate diets may contribute to cancer development and that hypoxia is related to CRC development. Moreover, the correlation between specific molecular mechanisms and cancer development was also established. In the future, molecules, as identified in this study, could be potentially used as molecular markers in colorectal cancer diagnosis, radiotherapy, or as chemotherapy efficacy indicators to improve rectum/colon cancer diagnosis, treatment and drug development.