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Open Access Highly Accessed Research article

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

Hiroaki Mita1, Minoru Toyota12, Fumio Aoki35, Hirofumi Akashi3, Reo Maruyama12, Yasushi Sasaki1, Hiromu Suzuki2, Masashi Idogawa1, Lisa Kashima1, Kazuyoshi Yanagihara6, Masahiro Fujita7, Masao Hosokawa7, Masanobu Kusano8, Sorin Vasile Sabau9, Haruyuki Tatsumi34, Kohzoh Imai2, Yasuhisa Shinomura2 and Takashi Tokino1*

Author Affiliations

1 Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Sapporo, Japan

2 First Department of Internal Medicine, Sapporo Medical University, Sapporo, Japan

3 Scholarly Communication Center, Sapporo Medical University, Sapporo, Japan

4 First Department of Anatomy, Sapporo Medical University, Sapporo, Japan

5 Department of Ecology and Evolution, State University of New York at Stony Brook, New York, USA

6 Central Animal Laboratory, National Cancer Center Research Institute, Tokyo, Japan

7 Keiyukai Sapporo Hospital, Sapporo, Japan

8 Otaru Kyokai Hospital, Otaru, Japan

9 Department of Human Science and Informatics, Tokai University, Sapporo, Japan

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BMC Cancer 2009, 9:198  doi:10.1186/1471-2407-9-198

Published: 23 June 2009

Abstract

Background

Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells.

Methods

DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively.

Results

DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity.

Conclusion

Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression.