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. 2013;8(3):e57724.
doi: 10.1371/journal.pone.0057724. Epub 2013 Mar 4.

Met is the most frequently amplified gene in endometriosis-associated ovarian clear cell adenocarcinoma and correlates with worsened prognosis

Yoriko Yamashita  1 , Shinya Akatsuka, Kanako Shinjo, Yasushi Yatabe, Hiroharu Kobayashi, Hiroshi Seko, Hiroaki Kajiyama, Fumitaka Kikkawa, Takashi Takahashi, Shinya Toyokuni
Affiliations

Met is the most frequently amplified gene in endometriosis-associated ovarian clear cell adenocarcinoma and correlates with worsened prognosis

Yoriko Yamashita et al. PLoS One. 2013.

Abstract

Clear cell adenocarcinoma of the ovary (OCC) is a chemo-resistant tumor with a relatively poor prognosis and is frequently associated with endometriosis. Although it is assumed that oxidative stress plays some role in the malignant transformation of this tumor, the characteristic molecular events leading to carcinogenesis remain unknown. In this study, an array-based comparative genomic hybridization (CGH) analysis revealed Met gene amplification in 4/13 OCC primary tumors and 2/8 OCC cell lines. Amplification of the AKT2 gene, which is a downstream component of the Met/PI3K signaling pathway, was also observed in 5/21 samples by array-based CGH analysis. In one patient, both the Met and AKT2 genes were amplified. These findings were confirmed using fluorescence in situ hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemistry. In total, 73 OCC cases were evaluated using real-time quantitative PCR; 37.0% demonstrated Met gene amplification (>4 copies), and 8.2% had AKT2 amplification. Furthermore, stage 1 and 2 patients with Met gene amplification had significantly worse survival than patients without Met gene amplification (p<0.05). Met knockdown by shRNA resulted in reduced viability of OCC cells with Met amplification due to increased apoptosis and cellular senescence, suggesting that the Met signaling pathway plays an important role in OCC carcinogenesis. Thus, we believe that targeted inhibition of the Met pathway may be a promising treatment for OCC.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Representative array-based CGH analysis data of chromosomes 1–22 and X.
The data of 4 ovarian clear cell adenocarcinoma samples (cc1, cc8, cc11, cc13), 1 endometrioid adenocarcinoma sample (em1), and 3 cell lines (RMG-II, JHOC-5, and JHOC-8) are shown. Met amplification is observed in cc1, cc8, and cc13 and JHOC-5 and JHOC-8 cells. AKT2 amplification is observed in cc11 and cc13 and JHOC-8 and RMG-II cells.
Figure 2
Figure 2. Gene and chromosome views of the amplified regions in array-based CGH analysis.
Genomic changes in the 21 ovarian clear cell adenocarcinoma samples are shown. High, clear peaks within 2 Mb are observed in 6/21 samples at the genomic region encoding Met (upper row). The amplified region including AKT2 is also shown (lower row).
Figure 3
Figure 3. FISH analysis for confirmation of Met amplification.
A representative nucleus of a Met-amplified cell (cc1) is shown in the upper figure (Green: Met probe, Orange: CEP 7; centromere 7 probe, Blue; DAPI). The lower graph shows the FISH signal number (MET/CEP 7 ratio) of the 4 Met-amplified ovarian clear cell adenocarcinoma samples (cc1, cc2, cc8, cc13) and an endometrioid adenocarcinoma case (em1) without Met amplification. A total of 60 cells were counted for each sample, average numbers (Met: CEP7) were as follows; cc1(18∶2.0), cc2(4.4∶2.1),cc8(10∶2.2), cc13(4.6/1.7), em1(1.7/1.9). All values were then normalized with that of em1 as 1.0.
Figure 4
Figure 4. QPCR confirmation of Met and AKT2 amplification.
A. Copy number change analysis of Met in 13 ovarian clear cell adenocarcinoma samples are shown. Four samples (cc1, cc2, cc8, cc13) had a Met/hTERT ratio greater than 1.5. B. Copy number change analysis of Met in 8 ovarian clear cell adenocarcinoma cell lines are shown. Two cell lines (JHOC-5 and JHOC-8) had a Met/hTERT ratio greater than 1.5. C. Copy number change analysis of AKT2 in 13 ovarian clear cell adenocarcinoma samples are shown. Three samples (cc5, cc11, cc13) had an AKT2/hTERT ratio greater than 1.5. D. The qPCR results for the copy number change analysis of AKT2 in 8 ovarian clear cell adenocarcinoma cell lines are shown. Two cell lines (RMG-II and JHOC-8) had a Met/hTERT ratio greater than 1.5.
Figure 5
Figure 5. Confirmation of Met amplification at the protein level.
A. The immunoblotting results for Met protein expression in ovarian clear cell adenocarcinoma cell lines. Two cell lines (JHOC-5 and JHOC-8) with Met gene amplification show stronger intensities. B. The immunostaining results of representative cases (cc2, Met-amplified ovarian clear cell adenocarcinoma (OCC) case; cc5, OCC case without Met amplification; em2, endometrioid adenocarcinoma case without Met amplification). Positive staining for c-Met antibody was further divided to 2 groups: 2+ and 1+ (see text for details).
Figure 6
Figure 6. AKT1 and AKT2 expression in ovarian clear cell adenocarcinoma.
A. Western blot analyses of protein expression using AKT antibodies in ovarian clear cell adenocarcinoma cell lines. Various intensities are observed by immunoblotting with AKT1, AKT2, and pan-AKT phosphorylated antibodies (serine 473 phosphorylated-AKT). B. A qRT-PCR analysis revealed relatively higher expression of AKT2 compared to AKT1 at the mRNA level.
Figure 7
Figure 7. The Kaplan-Meier curve of stage 1 and 2 ovarian clear cell adenocarcinoma patients with or without Met amplification.
Patients with Met amplification had a significantly worse prognosis (p<0.05).
Figure 8
Figure 8. Knockdown of Met in ovarian clear cell adenocarcinoma cell lines.
A. Confirmation of knockdown by Western blotting. Signal intensity ratio (Met/actin) are shown in numbers. B. Cell viability assay after transfection using retroviral vectors encoding short-hairpin RNAs targeting Met (Metsh2 and Metsh5) or control, shNC, in ovarian clear cell adenocarcinoma (OCC) cell lines. A decrease in cell viability in OCC cell lines with Met amplification (JHOC-5 and JHOC-8) is evident compared to ES-2 (OCC cell without Met amplification) or control HeLa cells. *p<0.05, ** p<0.01 C. Morphologically, Met-amplified cells (JHOC-5) expressing Metsh and enhanced green fluorescent protein (EGFP) became larger and were positively stained with SA-βGal (a senescence marker) compared with control. The number of apoptotic cells with nuclear staining in TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay also significantly increased as a result of Met knockdown in cells with Met amplification (JHOC-5 and JHOC-8) or without Met amplification (ES-2), compared with controls.
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This study was supported by a Grant-in-Aid from the Japan Society of the Promotion of Science, Tokyo, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.