Islam S S, Mokhtari R B, Noman A S, Uddin M, Rahman M Z, Azadi M A, Zlotta A, van der Kwast T, Yeger H, Farhat W A
Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada.
Division of Urology, The Hospital for Sick Children, Toronto, ON, Canada.
Mol Carcinog. 2016 May;55(5):537-51. doi: 10.1002/mc.22300. Epub 2015 Mar 1.
Activation of the sonic hedgehog (Shh) signaling pathway controls tumorigenesis in a variety of cancers. Here, we show a role for Shh signaling in the promotion of epithelial-to-mesenchymal transition (EMT), tumorigenicity, and stemness in the bladder cancer. EMT induction was assessed by the decreased expression of E-cadherin and ZO-1 and increased expression of N-cadherin. The induced EMT was associated with increased cell motility, invasiveness, and clonogenicity. These progression relevant behaviors were attenuated by treatment with Hh inhibitors cyclopamine and GDC-0449, and after knockdown by Shh-siRNA, and led to reversal of the EMT phenotype. The results with HTB-9 were confirmed using a second bladder cancer cell line, BFTC905 (DM). In a xenograft mouse model TGF-β1 treated HTB-9 cells exhibited enhanced tumor growth. Although normal bladder epithelial cells could also undergo EMT and upregulate Shh with TGF-β1 they did not exhibit tumorigenicity. The TGF-β1 treated HTB-9 xenografts showed strong evidence for a switch to a more stem cell like phenotype, with functional activation of CD133, Sox2, Nanog, and Oct4. The bladder cancer specific stem cell markers CK5 and CK14 were upregulated in the TGF-β1 treated xenograft tumor samples, while CD44 remained unchanged in both treated and untreated tumors. Immunohistochemical analysis of 22 primary human bladder tumors indicated that Shh expression was positively correlated with tumor grade and stage. Elevated expression of Ki-67, Shh, Gli2, and N-cadherin were observed in the high grade and stage human bladder tumor samples, and conversely, the downregulation of these genes were observed in the low grade and stage tumor samples. Collectively, this study indicates that TGF-β1-induced Shh may regulate EMT and tumorigenicity in bladder cancer. Our studies reveal that the TGF-β1 induction of EMT and Shh is cell type context dependent. Thus, targeting the Shh pathway could be clinically beneficial in the ability to reverse the EMT phenotype of tumor cells and potentially inhibit bladder cancer progression and metastasis.
音猬因子（Shh）信号通路的激活在多种癌症的肿瘤发生过程中发挥着控制作用。在此，我们揭示了Shh信号在促进膀胱癌上皮-间质转化（EMT）、致瘤性和干性方面的作用。通过E-钙黏蛋白和紧密连接蛋白-1（ZO-1）表达降低以及N-钙黏蛋白表达增加来评估EMT诱导情况。诱导的EMT与细胞运动性、侵袭性和克隆形成能力增强相关。用Hh抑制剂环杷明和GDC-0449处理后，以及在敲低Shh-siRNA后，这些与进展相关的行为减弱，并导致EMT表型逆转。使用第二个膀胱癌细胞系BFTC905（DM）证实了HTB-9的结果。在异种移植小鼠模型中，经转化生长因子-β1（TGF-β1）处理的HTB-9细胞表现出增强的肿瘤生长。尽管正常膀胱上皮细胞在TGF-β1作用下也可发生EMT并上调Shh，但它们不具有致瘤性。经TGF-β1处理的HTB-9异种移植瘤显示出有力证据，表明其转变为更具干细胞样的表型，CD133、Sox2、Nanog和Oct4功能激活。在经TGF-β1处理的异种移植瘤样本中，膀胱癌特异性干细胞标志物细胞角蛋白5（CK5）和细胞角蛋白14（CK14）上调，而在处理和未处理的肿瘤中，CD44均保持不变。对22例原发性人膀胱肿瘤的免疫组织化学分析表明，Shh表达与肿瘤分级和分期呈正相关。在高级别和高分期的人膀胱肿瘤样本中观察到Ki-67、Shh、Gli2和N-钙黏蛋白表达升高，相反，在低级别和低分期肿瘤样本中观察到这些基因的下调。总体而言，本研究表明TGF-β1诱导的Shh可能调节膀胱癌中的EMT和致瘤性。我们的研究表明，TGF-β1诱导的EMT和Shh具有细胞类型背景依赖性。因此，靶向Shh信号通路在逆转肿瘤细胞的EMT表型以及潜在抑制膀胱癌进展和转移方面可能具有临床益处。
