J Breast Cancer.  2010 Mar;13(1):37-45. 10.4048/jbc.2010.13.1.37.

COL18A1 as the Candidate Gene for the Prognostic Marker of Breast Cancer According to the Analysis of the DNA Copy Number Variation by Array CGH

• Hwang KT
• Chung JK
• Jung IM
• Heo SC
• Ahn YJ
• Ahn HS
• Chang MS
• Kim JA
• Han W
• Noh DY

• KMID: 2286558
• DOI: http://doi.org/10.4048/jbc.2010.13.1.37

Abstract

PURPOSE
We tried to select and validate the candidate gene for the prognostic marker of breast cancer by comparing the analysis of copy number variation (CNV) between normal breast tissues and breast cancer tissues by performing array comparative genomic hybridization (CGH).
METHODS
Array CGH was performed with using the fresh frozen tissues of 77 breast cancer patients. We selected the clones with more than a 20% frequency of gain or loss, and the clones with gain or loss in more than 2 consecutive clones. We finally selected the clones that were statistically significant on the survival analysis. We searched for the candidate gene that belonged to the candidate clones and we selected the final candidate gene that is assumed to be most related to the carcinogenesis of breast cancer by searching for information of the individual gene. We performed RT-PCR to validate the RNA expression of the final candidate gene with using the breast tissues of another 20 breast cancer patients.
RESULTS
Eleven (10 in the gain group and 1 in the loss group) clones were finally selected as candidate clones. The significant CNVs with gain were found in the regions of 1q23.1, 1q41, 1q44, 5p15.33, 8q21.3, 15q26.3, 17q12 and 21q22.3 and the significant CNV with loss was found in 14q32.33. COL18A1 (21q22.3) was selected as the final candidate gene and the RT-PCR results revealed that the expression of COL18A1 was up-regulated in the cancer tissues of 18 of the other 20 (90%) breast cancer patients.
CONCLUSION
We selected COL18A1 (21q22.3) as the candidate gene for the prognostic marker of breast cancer by comparing the analysis of CNVs from the array CGH. The RNA of COL18A1 was over-expressed in breast cancer tissue, as determined by RT-PCR.

Keyword

Breast neoplasms; Collagen type XVIII; Gene amplification; Microarray analysis

MeSH Terms

Breast
Breast Neoplasms
Clone Cells
Coat Protein Complex I
Collagen Type XVIII
Comparative Genomic Hybridization
DNA
DNA Copy Number Variations
Gene Amplification
Humans
Microarray Analysis
RNA
Coat Protein Complex I
Collagen Type XVIII
DNA
RNA

Figure

• 

  Figure 1 Survival curve of systemic recurrence-free survival analysis for the clone which contains COL18A1 gene (c2806) by Kaplan-Meier test. The survival of the group with gain of c2806 clone was better than that without gain of c2806 clone and the difference of survival between two groups was statistically significant (p=0.008) by log rank test.

• 

  Figure 2 Results of validation test by RT-PCR for the RNA expression of COL18A1. The RNA expression of COL18A1 was up-regulated in breast cancer tissues compared to normal breast tissues in 18 of 20 (90%) breast cancer patients. Difference of the RNA expression was statistically significant (p<0.0001). RPLP0 was used as reference gene. OD ratio=(OD[COL18A1_Caner]/OD [RPLP0_Cancer])/(OD[COL18A1_Normal]/OD[RPLP0_Normal]. OD=optical density.

Reference

1. Sotiriou C, Piccart MJ. Taking gene-expression profiling to the clinic: when will molecular signatures become relevant to patient care? Nat Rev Cancer. 2007. 7:545–553.

2. Tan DS, Reis-Filho JS. Comparative genomic hybridisation arrays: high-throughput tools to determine targeted therapy in breast cancer. Pathobiology. 2008. 75:63–74.

3. Davies JJ, Wilson IM, Lam WL. Array CGH technologies and their applications to cancer genomes. Chromosome Res. 2005. 13:237–248.

4. van Beers EH, Nederlof PM. Array-CGH and breast cancer. Breast Cancer Res. 2006. 8:210.

5. Hwang KT, Han W, Cho J, Lee JW, Ko E, Kim EK, et al. Genomic copy number alterations as predictive markers of systemic recurrence in breast cancer. Int J Cancer. 2008. 123:1807–1815.

6. Chebil G, Bendahl PO, Fernö M. South Sweden Breast Cancer Group. North Sweden Breast Cancer Group. Estrogen and progesterone receptor assay in paraffin-embedded breast cancer--reproducibility of assessment. Acta Oncol. 2003. 42:43–47.

7. Kim HS, Jung JH, Park HY, Lee YH, Chung EJ, Kim MK, et al. Gene expression profile analysis of human breast cancer using cDNA microarrys. J Korean Breast Cancer Soc. 2003. 6:58–67.

8. Han W, Chung KW, Ahn SJ, Noh DY, Youn YK, Oh SK, et al. Gene expression profiles of primary breast cancer tissue using cDNA microarray. J Korean Breast Cancer Soc. 2002. 5:284–290.

9. Kim MY, Yim SH, Kwon MS, Kim TM, Shin SH, Kang HM, et al. Recurrent genomic alterations with impact on survival in colorectal cancer identified by genome-wide array comparative genomic hybridization. Gastroenterology. 2006. 131:1913–1924.

10. Thomassen M, Tan Q, Kruse TA. Gene expression meta-analysis identifies chromosomal regions and candidate genes involved in breast cancer metastasis. Breast Cancer Res Treat. 2009. 113:239–249.

11. Nakatani K, Thompson DA, Barthel A, Sakaue H, Liu W, Weigel RJ, et al. Up-regulation of Akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines. J Biol Chem. 1999. 274:21528–21532.

12. Liu D, Rudland PS, Sibson DR, Barraclough R. Identification of mRNAs differentially-expressed between benign and malignant breast tumour cells. Br J Cancer. 2002. 87:423–431.

13. Han W, Han MR, Kang JJ, Bae JY, Lee JH, Bae YJ, et al. Genomic alterations identified by array comparative genomic hybridization as prognostic markers in tamoxifen-treated estrogen receptor-positive breast cancer. BMC Cancer. 2006. 6:92.

14. Naylor TL, Greshock J, Wang Y, Colligon T, Yu QC, Clemmer V, et al. High resolution genomic analysis of sporadic breast cancer using array-based comparative genomic hybridization. Breast Cancer Res. 2005. 7:R1186–R1198.

15. Nessling M, Richter K, Schwaenen C, Roerig P, Wrobel G, Wessendorf S, et al. Candidate genes in breast cancer revealed by microarray-based comparative genomic hybridization of archived tissue. Cancer Res. 2005. 65:439–447.

16. Rennstam K, Ahlstedt-Soini M, Baldetorp B, Bendahl PO, Borg A, Karhu R, et al. Patterns of chromosomal imbalances defines subgroups of breast cancer with distinct clinical features and prognosis. A study of 305 tumors by comparative genomic hybridization. Cancer Res. 2003. 63:8861–8868.

17. Dang C, Zhang Y, Ma Q, Shimahara Y. Expression of nerve growth factor receptors is correlated with progression and prognosis of human pancreatic cancer. J Gastroenterol Hepatol. 2006. 21:850–858.

18. Brzeziańska E, Karbownik M, Migdalska-Sek M, Pastuszak-Lewandoska D, Wloch J, Lewiński A. Molecular analysis of the RET and NTRK1 gene rearrangements in papillary thyroid carcinoma in the Polish population. Molecular analysis of the RET and NTRK1 gene rearrangements in papillary thyroid carcinoma in the Polish population. Mutat Res. 2006. 599:26–35.

19. Tokusashi Y, Asai K, Tamakawa S, Yamamoto M, Yoshie M, Yaginuma Y, et al. Expression of NGF in hepatocellular carcinoma cells with its receptors in non-tumor cell components. Int J Cancer. 2005. 114:39–45.

20. Iizasa T, Chang H, Suzuki M, Otsuji M, Yokoi S, Chiyo M, et al. Overexpression of collagen XVIII is associated with poor outcome and elevated levels of circulating serum endostatin in non-small cell lung cancer. Clin Cancer Res. 2004. 10:5361–5366.

21. Swidzińska E, Ossolińska M, Naumnik W, Trojan S, Chyczewska E. Serum endostatin levels in patients with lung carcinoma. Rocz Akad Med Bialymst. 2005. 50:197–200.

22. Woo IS, Kim KA, Jeon HM, Hong SH, Rho SY, Koh SJ, et al. Pretreatment serum endostatin as a prognostic indicator in metastatic gastric carcinoma. Int J Cancer. 2006. 119:2901–2906.

23. Kikuchi E, Menendez S, Ohori M, Cordon-Cardo C, Kasahara N, Bochner BH. Inhibition of orthotopic human bladder tumor growth by lentiviral gene transfer of endostatin. Clin Cancer Res. 2004. 10:1835–1842.

24. Hu TH, Huang CC, Wu CL, Lin PR, Liu SY, Lin JW, et al. Increased endostatin/collagen XVIII expression correlates with elevated VEGF level and poor prognosis in hepatocellular carcinoma. Mod Pathol. 2005. 18:663–672.

25. Balasubramanian SP, Cross SS, Globe J, Cox A, Brown NJ, Reed MW. Endostatin gene variation and protein levels in breast cancer susceptibility and severity. BMC Cancer. 2007. 7:107.

26. Beck MT, Chen NY, Franek KJ, Chen WY. Prolactin antagonist-endostatin fusion protein as a targeted dual-functional therapeutic agent for breast cancer. Cancer Res. 2003. 63:3598–3604.

27. Lourenço GJ, Cardoso-Filho C, Gonçales NS, Shinzato JY, Zeferino LC, Nascimento H, et al. A high risk of occurrence of sporadic breast cancer in individuals with the 104NN polymorphism of the COL18A1 gene. Breast Cancer Res Treat. 2006. 100:335–338.

28. Chari R, Lockwood WW, Lam WL. Computational methods for the analysis of array comparative genomic hybridization. Cancer Inform. 2007. 2:48–58.