J Korean Med Sci.  2010 Mar;25(3):353-360. 10.3346/jkms.2010.25.3.353.

Growth and Invasion of Sporadic Colorectal Adenocarcinomas in Terms of Genetic Change

• Roh SA
• Choi EY
• Cho DH
• Jang SJ
• Kim SY
• Kim YS
• Kim JC

• KMID: 1778028
• DOI: http://doi.org/10.3346/jkms.2010.25.3.353

Abstract

Integrative genetic changes were examined in relation to tumor growth and progression of sporadic colorectal cancers. Ninety-two sporadic colorectal cancer patients and 12 human colorectal cancer cell lines were evaluated. Genetic changes in representative steps of colorectal tumorigenesis were determined. Biological characteristics, i.e., clinicopathologic parameters, expression of invasion-associated molecules, and in vitro invasion and migration, in association with these changes were further analyzed. Adenomatous polyposis coli (APC) and/or Wnt-activated alterations occurred in 66% patients, whereas mismatch repair (MMR) defects and/or RAF-mediated alterations were identified in 47% patients. The crossover rate between these two alterations was 26%. Differential mRNA expression of ARK5 was closely associated with that of MMP2, MMP9, and S100A4 (P< or =0.044-0.001). Additionally, enhanced ARK5 mRNA expression was more frequent in tumors displaying RAF-mediated alterations and crossover pathways (P=0.01 and 0.03, respectively). Upregulation of CEA mRNA was more common in the advanced stages (P=0.034), while VEGF expression was greater in poorly differentiated or mucinous tumors (P=0.042). The high expressions of MMP2 and MMP9 were closely associated with invasion and migration of colorectal tumors and cell lines. Our results conclusively show that specific pathways of colorectal tumorigenesis are closely associated with characteristic tumor growth and invasion.

Keyword

Colorectal Neoplasms; Sporadic; Tumorigenesis; Molecular; Growth; Invasion

MeSH Terms

*Adenocarcinoma/genetics/metabolism/pathology
Animals
Carcinoembryonic Antigen/genetics/metabolism
Cell Line, Tumor
Cell Movement
*Colorectal Neoplasms/genetics/metabolism/pathology
*Gene Expression Regulation, Neoplastic
Humans
Matrix Metalloproteinase 2/genetics/metabolism
Matrix Metalloproteinase 9/genetics/metabolism
Neoplasm Invasiveness
Protein Kinases/genetics/metabolism
Repressor Proteins/genetics/metabolism
S100 Proteins/genetics/metabolism
Vascular Endothelial Growth Factor A/genetics/metabolism
Carcinoembryonic Antigen
Repressor Proteins
S100 Proteins
Vascular Endothelial Growth Factor A
Protein Kinases
Matrix Metalloproteinase 2
Matrix Metalloproteinase 9

Figure

• 

  Fig. 1 mRNA expressions (tumor/normal epithelium) of invasion-associated genes (ARK5, CEA, MMP2, MMP9, S100A4, and VEGFA) in 12 colorectal cell lines. Enhanced expressions were relatively evident in AMC5 (ARK5, MMP2, MMP9, and VEGFA) and SW48 (ARK5, CEA, MMP2, MMP9, and VEGFA) cell lines. Tumor cDNA quantities were normalized in terms of the respective GADPH content.

• 

  Fig. 2 Gelatinolytic matrix metalloproteinase (MMP) activity in colorectal cancer cell lines detected by quantitative zymography. Molecular markers indicate active MMP-2, proMMP-2, active MMP-9, and proMMP-9 as 62 kDa, 72 kDa, 82 kDa, and 92 kDa, respectively.

• 

  Fig. 3 Tumor cell invasion and migration were examined on a modified Boyden chamber (Transwell coated with Matrigel for invasion). Mean number (±SEM) of invading cells of five fields of triplicate wells from three independent experiments. AMC5 and SW48 vs. Caco2, RKO, and WiDr, P<0.001-0.004.

Reference

1. Conlin A, Smith G, Carey FA, Wolf CR, Steele RJ. The prognostic significance of K-ras, p53, and APC mutations in colorectal carcinoma. Gut. 2005. 54:1283–1286.

2. Senda T, Iizuka-Kogo A, Onouchi T, Shimomura A. Adenomatous polyposis coli (APC) plays multiple roles in the intestinal and colorectal epithelia. Med Mol Morphol. 2007. 40:68–81.

3. Dong X, Seelan RS, Qian C, Mai M, Liu W. Genomic structure, chromosome mapping and expression analysis of the human AXIN2 gene. Cytogenet Cell Genet. 2001. 93:26–28.

4. Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD, Barker MA, Arnold S, McGivern A, Matsubara N, Tanaka N, Higuchi T, Young J, Jass JR, Leggett BA. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut. 2004. 53:1137–1144.

5. Salahshor S, Kressner U, Pâhlman L, Glimelius B, Lindmark G, Lindblom A. Colorectal cancer with and without microsatellite instability involves different genes. Genes Chromosomes Cancer. 1999. 26:247–252.

6. Young J, Simms LA, Biden KG, Wynter C, Whitehall V, Karamatic R, George J, Goldblatt J, Walpole I, Robin SA, Borten MM, Stitz R, Searle J, McKeone D, Fraser L, Purdie DR, Podger K, Price R, Buttenshaw R, Walsh MD, Barker M, Leggett BA, Jass JR. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol. 2001. 159:2107–2116.

7. Vinodhkumar R, Song YS, Ravikumar V, Ramakrishnan G, Devaki T. Depsipeptide a histone deacetylase inhibitor down regulates levels of matrix metalloproteinases 2 and 9 mRNA and protein expressions in lung cancer cells (A549). Chem Biol Interact. 2007. 165:220–229.

8. Cheung LW, Leung PC, Wong AS. Gonadotropin-releasing hormone promotes ovarian cancer cell invasiveness through c-Jun NH₂-terminal kinase-mediated activation of matrix metalloproteinase (MMP)-2 and MMP-9. Cancer Res. 2006. 66:10902–10910.

9. Collins HM, Morris TM, Watson SA. Spectrum of matrix metalloproteinase expression in primary and metastatic colon cancer: relationship to the tissue inhibitors of metalloproteinases and membrane type-1-matrix metalloproteinase. Br J Cancer. 2001. 84:1664–1670.

10. Veikkola T, Karkkainen M, Claesson-Welsh L, Alitalo K. Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res. 2000. 60:203–212.

11. Hanrahan V, Currie MJ, Gunningham SP, Morrin HR, Scott PA, Robinson BA, Fox SB. The angiogenic switch for vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, and VEGF-D in the adenoma-carcinoma sequence during colorectal cancer progression. J Pathol. 2003. 200:183–194.

12. Kusakai G, Suzuki A, Ogura T, Miyamoto S, Ochiai A, Kaminishi M, Esumi H. ARK5 expression in colorectal cancer and its implications for tumor progression. Am J Pathol. 2004. 164:987–995.

13. Obrink B. CEA adhesion molecules: multifunctional proteins with signal regulatory properties. Curr Opin Cell Biol. 1997. 9:616–626.

14. Miyoshi Y, Ando H, Nagase H, Nishisho I, Horii A, Miki Y, Mori T, Utsunomiya J, Baba S, Petersen G, Hamilton SR, Kinzler KW, Vogelstein B, Nakamura Y. Germ-line mutations of the APC gene in 53 familial adenomatous polyposis patients. Proc Natl Acad Sci USA. 1992. 89:4452–4456.

15. Nagasaka T, Sasamoto H, Notohara K, Cullings HM, Takeda M, Kimura K, Kambara T, MacPhee DG, Young J, Leggett BA, Jass JR, Tanaka N, Matsubara N. Colorectal cancer with mutation in BRAF, KRAS, and wild-type with respect to both oncogenes showing different patterns of DNA methylation. J Clin Oncol. 2004. 22:4584–4594.

16. Einspahr JG, Martinez ME, Jiang R, Hsu CH, Rashid A, Bhattacharrya AK, Ahnen DJ, Jacobs ET, Houlihan PS, Webb CR, Alberts DS, Hamilton SR. Associations of ki-ras proto-oncogene mutation and p53 gene overexpression in sporadic colorectal adenomas with demographic and clinicopathologic characteristics. Cancer Epidemiol Biomarkers Prev. 2006. 15:1443–1450.

17. Jover R, Payá A, Alenda C, Poveda MJ, Peiró G, Aranda FI, Pérez-Mateo M. Defective mismatch-repair colorectal cancer: clinicopathologic characteristics and usefulness of immunohistochemical analysis for diagnosis. Am J Clin Pathol. 2004. 122:389–394.

18. Kim JC, Lee KH, Ka IH, Koo KH, Roh SA, Kim HC, Yu CS, Kim TW, Chang HM, Gong GY, Kim JS. Characterization of mutator phenotype in familial colorectal cancer patients not fulfilling Amsterdam criteria. Clin Cancer Res. 2004. 10:6159–6168.

19. Harada K, Hiraoka S, Kato J, Horii J, Fujita H, Sakaguchi K, Shiratori Y. Genetic and epigenetic alterations of Ras signalling pathway in colorectal neoplasia: analysis based on tumour clinicopathological features. Br J Cancer. 2007. 97:1425–1431.

20. Kim JC, Cho YK, Roh SA, Yu CS, Gong G, Jang SJ, Kim SY, Kim YS. Individual tumorigenesis pathways of sporadic colorectal adenocarcinomas are associated with the biological behavior of tumors. Cancer Sci. 2008. 99:1348–1354.

21. Illemann M, Bird N, Majeed A, Sehested M, Laerum OD, Lund LR, Dano K, Nielsen BS. MMP-9 is differentially expressed in primary human colorectal adenocarcinomas and their metastases. Mol Cancer Res. 2006. 4:293–302.

22. Théret N, Musso O, Campion JP, Turlin B, Loréal O, L'Helgoualc'h A, Clément B. Overexpression of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 in liver from patients with gastrointestinal adenocarcinoma and no detectable metastasis. Int J Cancer. 1997. 74:426–432.

23. Zeng ZS, Guillem JG. Unique activation of matrix metalloproteinase-9 within human liver metastasis from colorectal cancer. Br J Cancer. 1998. 78:349–353.

24. Chan CC, Menges M, Orzechowski HD, Orendain N, Pistorius G, Feifel G, Zeitz M, Stallmach A. Increased matrix metalloproteinase 2 concentration and transcript expression in advanced colorectal carcinomas. Int J Colorectal Dis. 2001. 16:133–140.

25. Giatromanolaki A, Koukourakis MI, Sivridis E, Chlouverakis G, Vourvouhaki E, Turley H, Harris AL, Gatter KC. Activated VEGFR2/KDR pathway in tumour cells and tumour associated vessels of colorectal cancer. Eur J Clin Invest. 2007. 37:878–886.

26. Mizukami Y, Kohgo Y, Chung DC. Hypoxia inducible factor-1 independent pathways in tumor angiogenesis. Clin Cancer Res. 2007. 13:5670–5674.

27. Kusakai G, Suzuki A, Ogura T, Kaminishi M, Esumi H. Strong association of ARK5 with tumor invasion and metastasis. J Exp Clin Cancer Res. 2004. 23:263–268.

28. Xing M. BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev. 2007. 28:742–762.

29. Stein U, Arlt F, Walther W, Smith J, Waldman T, Harris ED, Mertins SD, Heizmann CW, Allard D, Birchmeier W, Schlag PM, Shoemaker RH. The metastasis-associated gene S100A4 is a novel target of beta-catenin/T-cell factor signaling in colon cancer. Gastroenterology. 2006. 131:1486–1500.

30. Levy M, Visokai V, Lipska L, Topolcan O. Tumor markers in staging and prognosis of colorectal carcinoma. Neoplasma. 2008. 55:138–142.