Matsumoto Ryuji, Tsuda Masumi, Wang Lei, Maishi Nako, Abe Takashige, Kimura Taichi, Tanino Mishie, Nishihara Hiroshi, Hida Kyoko, Ohba Yusuke, Shinohara Nobuo, Nonomura Katsuya, Tanaka Shinya
Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
Department of Urology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
Cancer Sci. 2015 Jun;106(6):709-717. doi: 10.1111/cas.12662. Epub 2015 Apr 22.
We have previously reported that an adaptor protein CRK, including CRK-I and CRK-II, plays essential roles in the malignant potential of various aggressive human cancers, suggesting the validity of targeting CRK in molecular targeted therapy of a wide range of cancers. Nevertheless, the role of CRK in human bladder cancer with marked invasion, characterized by distant metastasis and poor prognosis, remains obscure. In the present study, immunohistochemistry indicated a striking enhancement of CRK-I/-II, but not CRK-like, in human bladder cancer tissues compared to normal urothelium. We established CRK-knockdown bladder cancer cells using 5637 and UM-UC-3, which showed a significant decline in cell migration, invasion, and proliferation. It is noteworthy that an elimination of CRK conferred suppressed phosphorylation of c-Met and the downstream scaffold protein Gab1 in a hepatocyte growth factor-dependent and -independent manner. In epithelial-mesenchymal transition-related molecules, E-cadherin was upregulated by CRK elimination, whereas N-cadherin, vimentin, and Zeb1 were downregulated. A similar effect was observed following treatment with c-Met inhibitor SU11274. Depletion of CRK significantly decreased cell proliferation of 5637 and UM-UC-3, consistent with reduced activity of ERK. An orthotopic xenograft model with bioluminescent imaging revealed that CRK knockdown significantly attenuated not only tumor volume but also the number of circulating tumor cells, resulted in a complete abrogation of metastasis. Taken together, this evidence uncovered essential roles of CRK in invasive bladder cancer through the hepatocyte growth factor/c-Met/CRK feedback loop for epithelial-mesenchymal transition induction. Thus, CRK might be a potent molecular target in bladder cancer, particularly for preventing metastasis, leading to the resolution of clinically longstanding critical issues.
我们之前曾报道，一种衔接蛋白CRK，包括CRK-I和CRK-II，在多种侵袭性人类癌症的恶性潜能中发挥着重要作用，这表明在多种癌症的分子靶向治疗中靶向CRK具有有效性。然而，CRK在具有明显侵袭性、以远处转移和预后不良为特征的人类膀胱癌中的作用仍不清楚。在本研究中，免疫组织化学表明，与正常尿路上皮相比，人类膀胱癌组织中CRK-I/-II显著增强，而CRK样蛋白则未增强。我们使用5637和UM-UC-3细胞系建立了CRK敲低的膀胱癌细胞系，这些细胞系的细胞迁移、侵袭和增殖均显著下降。值得注意的是，消除CRK会以肝细胞生长因子依赖性和非依赖性方式抑制c-Met和下游支架蛋白Gab1的磷酸化。在上皮-间质转化相关分子中，消除CRK会上调E-钙黏蛋白，而下调N-钙黏蛋白、波形蛋白和锌指蛋白1（Zeb1）。用c-Met抑制剂SU11274处理后也观察到了类似的效果。CRK的缺失显著降低了5637和UM-UC-3细胞的增殖，这与细胞外信号调节激酶（ERK）活性降低一致。一个采用生物发光成像的原位异种移植模型显示，CRK敲低不仅显著减小了肿瘤体积，还减少了循环肿瘤细胞的数量，从而完全消除了转移。综上所述这些证据揭示了CRK在侵袭性膀胱癌中通过肝细胞生长因子/c-Met/CRK反馈环诱导上皮-间质转化所起的重要作用。因此，CRK可能是膀胱癌中一个有效的分子靶点，特别是对于预防转移，这将解决临床上长期存在的关键问题。
