Masuda Taka-aki, Inoue Hiroshi, Sonoda Hideto, Mine Shinji, Yoshikawa Yasuji, Nakayama Keiko, Nakayama Kei-Ichi, Mori Masaki
Department of Molecular and Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan.
Cancer Res. 2002 Jul 1;62(13):3819-25.
Reduced expression level of p27, a cyclin-dependent kinase inhibitor, is associated with high aggressiveness and poor prognosis of various malignant tumors, including gastric carcinoma. S-phase kinase-associated protein 2 (Skp2), a member of the F-box family of substrate-recognition subunits of Skp1-Cullin-F-box ubiquitin-protein ligase complexes, is necessary for p27 ubiquitination and degradation. In the present study, we examined the clinical and biological significance of Skp2 expression in human gastric carcinoma and the relationship between the expression of Skp2 and p27. Northern blot analysis showed that Skp2 mRNA was overexpressed in carcinoma tissues (P < 0.05), and the high Skp2 expression group showed significantly poorer prognosis in 98 patients with gastric carcinoma (P < 0.05). Immunohistochemical analysis showed that Skp2 protein was expressed predominantly in carcinoma cells. We also found an inverse correlation between the expression of Skp2 mRNA and p27 protein in vivo (P < 0.01). To analyze the biological behavior of Skp2, we established stably Skp2-transfected gastric carcinoma cell lines. Western blot analysis showed that Skp2-transfected cells expressed lower levels of p27 protein than the control cells. Skp2-transfected cells showed significantly higher levels of growth rate (P < 0.05), percentage of bromodeoxyuridine-positive cells after serum starvation (P < 0.01), resistance to apoptosis induction by actinomycin D treatment (P < 0.05), and invasion potential (P < 0.01) than the control cells. These findings indicate that Skp2 expression can modulate the malignant phenotype of gastric carcinoma, possibly via p27 proteolysis. Skp2 can play an important role in gastric carcinoma progression and would be a novel target for the treatment of gastric carcinoma as well as a strong prognostic marker.
细胞周期蛋白依赖性激酶抑制剂p27表达水平降低与包括胃癌在内的多种恶性肿瘤的高侵袭性和不良预后相关。S期激酶相关蛋白2（Skp2）是Skp1-Cullin-F-box泛素蛋白连接酶复合物底物识别亚基的F-box家族成员，是p27泛素化和降解所必需的。在本研究中，我们检测了Skp2在人胃癌中的表达的临床和生物学意义以及Skp2与p27表达之间的关系。Northern印迹分析显示，Skp2 mRNA在癌组织中过表达（P<0.05），在98例胃癌患者中，Skp2高表达组的预后明显较差（P<0.05）。免疫组织化学分析显示，Skp2蛋白主要在癌细胞中表达。我们还发现体内Skp2 mRNA与p27蛋白的表达呈负相关（P<0.01）。为了分析Skp2的生物学行为，我们建立了稳定转染Skp2的胃癌细胞系。蛋白质印迹分析显示，转染Skp2的细胞比对照细胞表达更低水平的p27蛋白。与对照细胞相比，转染Skp2的细胞显示出显著更高的生长率（P<0.05）、血清饥饿后溴脱氧尿苷阳性细胞百分比（P<0.01）、对放线菌素D诱导凋亡的抗性（P<0.05）和侵袭潜能（P<0.01）。这些发现表明，Skp2表达可能通过p27蛋白水解调节胃癌的恶性表型。Skp2在胃癌进展中可能起重要作用，将成为治疗胃癌的新靶点以及强有力的预后标志物。
