Identification of LEFTY as a molecular marker for ovarian clear cell carcinoma

// Masashi Akiya 1 , Masaaki Yamazaki 1 , Toshihide Matsumoto 1 , Yusuke Kawashima 2 , Yasuko Oguri 1 , Sabine Kajita 1 , Daiki Kijima 1 , Risako Chiba 1 , Ako Yokoi 1 , Hiroyuki Takahashi 1 , Yoshio Kodera 2 and Makoto Saegusa 1 1 Department of Pathology, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan 2 Center for Disease Proteomics, School of Science, Kitasato University, Sagamihara, Kanagawa 252-0374, Japan Correspondence to: Makoto Saegusa, email: msaegusa@med.kitasato-u.ac.jp Keywords: LEFTY, ovarian clear cell carcinoma, cell proliferation, apoptosis, TGF-beta Received: November 22, 2016 Accepted: June 11, 2017 Published: June 29, 2017 ABSTRACT To identify proteins involved in ovarian clear cell carcinoma (OCCCa), shotgun proteomics analysis was applied using formalin-fixed and paraffin-embedded samples of ovarian carcinoma. Analysis of 1521 proteins revealed that 52 were differentially expressed between four OCCCa and 12 non-OCCCa samples. Of the highly expressed proteins in OCCCa, we focused on left-right determination factor (LEFTY), a novel member of the transforming growth factor-β superfamily. In 143 cases of ovarian epithelial carcinoma including 99 OCCCas and 44 non-OCCCas, LEFTY expression at both mRNA and protein levels was significantly higher in OCCCas compared with non-OCCCas, with the mRNA expression of LEFTY1 being predominant compared to that of LEFTY2 . OCCCa cells stably overexpressing LEFTY1 showed reduced cell proliferation, along with decreased pSmad2 expression, and also either displayed an activated p53/p21 waf1 pathway or increased p27 kip1 expression, directly or indirectly. Moreover, the treatment of stable cell lines with cisplatin led to increased apoptotic cells, together with the inhibition of protein expression of a pSmad2-mediated X-linked inhibitor of apoptosis and a decreased bcl2/bax ratio. Blocking LEFTY1 expression with a specific short hairpin RNA inhibited cisplatin-induced apoptosis, probably through the increased expression of both XIAP and bcl2, but not bax. In clinical samples, a significantly higher number of apoptotic cells and lower Ki-67 labeling indices were observed in OCCCas with a high LEFTY score relative to those with a low score. These findings suggest that LEFTY may be an excellent OCCCa-specific molecular marker, which has anti-tumor effects in altering cell proliferation and cellular susceptibility to apoptosis.