Akiya Masashi, Yamazaki Masaaki, Matsumoto Toshihide, Kawashima Yusuke, Oguri Yasuko, Kajita Sabine, Kijima Daiki, Chiba Risako, Yokoi Ako, Takahashi Hiroyuki, Kodera Yoshio, Saegusa Makoto
Department of Pathology, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan.
Center for Disease Proteomics, School of Science, Kitasato University, Sagamihara, Kanagawa 252-0374, Japan.
Oncotarget. 2017 Jun 29;8(38):63646-63664. doi: 10.18632/oncotarget.18882. eCollection 2017 Sep 8.
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 being predominant compared to that of . OCCCa cells stably overexpressing LEFTY1 showed reduced cell proliferation, along with decreased pSmad2 expression, and also either displayed an activated p53/p21 pathway or increased p27 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.
为了鉴定参与卵巢透明细胞癌（OCCCa）的蛋白质，我们使用福尔马林固定石蜡包埋的卵巢癌样本进行了鸟枪法蛋白质组学分析。对1521种蛋白质的分析显示，在4例OCCCa样本和12例非OCCCa样本之间，有52种蛋白质表达存在差异。在OCCCa中高表达的蛋白质中，我们聚焦于左右决定因子（LEFTY），它是转化生长因子-β超家族的一个新成员。在143例卵巢上皮癌病例中，包括99例OCCCa和44例非OCCCa，与非OCCCa相比，OCCCa中LEFTY在mRNA和蛋白质水平的表达均显著更高，其中 的mRNA表达比 的更占优势。稳定过表达LEFTY1的OCCCa细胞显示细胞增殖减少，同时pSmad2表达降低，并且直接或间接表现出p53/p21途径激活或p27表达增加。此外，用顺铂处理稳定细胞系导致凋亡细胞增加，同时抑制pSmad2介导的X连锁凋亡抑制蛋白的蛋白质表达，并降低bcl2/bax比值。用特异性短发夹RNA阻断LEFTY1表达可抑制顺铂诱导的凋亡，可能是通过增加XIAP和bcl2的表达，但不影响bax。在临床样本中，相对于LEFTY评分低的OCCCa，LEFTY评分高的OCCCa中观察到凋亡细胞数量显著更多，Ki-67标记指数更低。这些发现表明，LEFTY可能是一种出色的OCCCa特异性分子标志物，在改变细胞增殖和细胞对凋亡的易感性方面具有抗肿瘤作用。
