Magistri Paolo, Battistelli Cecilia, Strippoli Raffaele, Petrucciani Niccolò, Pellinen Teijo, Rossi Lucia, Mangogna Livia, Aurello Paolo, D'Angelo Francesco, Tripodi Marco, Ramacciato Giovanni, Nigri Giuseppe
Department of Medical and Surgical Sciences and Translational Medicine, Sapienza-University of Rome, Rome, Italy.
Molecular Genetics Section, Department of Cellular Biotechnology and Hematology, Sapienza-University of Rome, Rome, Italy.
Front Pharmacol. 2018 Feb 2;8:956. doi: 10.3389/fphar.2017.00956. eCollection 2017.
Preliminary results of this work were presented at the 2016 Academic Surgical Congress, Jacksonville (FL), February 2-4 2016 (Original title: Selective Smo-Inhibition Interferes With Cellular Energetic Metabolism In Colorectal Cancer)This study was funded by "Sapienza-University of Rome" (Funds for young researchers) and "AIRC" (Italian Association for Cancer Research)Hedgehog inhibitor was kindly provided by Genentech, Inc.®. Colon Cancer (CC) is the fourth most frequently diagnosed tumor and the second leading cause of death in the USA. Abnormalities of Hedgehog pathway have been demonstrated in several types of human cancers, however the role of Hedgehog (Hh) in CC remain controversial. In this study, we analyzed the association between increased mRNA expression of and , two Hh target genes, and CC survival and recurrence by gene expression microarray from a cohort of 382 CC patients. We found that patients with increased expression of showed a statistically significant reduction in survival. In order to demonstrate a causal role of Hh pathway activation in the pathogenesis of CC, we treated HCT 116, SW480 and SW620 CC cells lines with GDC-0449, a pharmacological inhibitor of Smoothened (SMO). Treatment with GDC-0449 markedly reduced expression of Hh target genes , thus indicating that this pathway is constitutively active in CC cell lines. Moreover, GDC-0449 partially reduced cell proliferation, which was associated with upregulation of p21 and downregulation of CycD1. Finally, treatment with the same drug reduced migration and three-dimensional invasion, which were associated with downregulation of Snail1, the EMT master gene, and with induction of the epithelial markers Cytokeratin-18 and E-cadherin. These results were confirmed by SMO genetic silencing. Notably, treatment with 5E1, a Sonic Hedgehog-specific mAb, markedly reduced the expression of Hedgehog target genes, as well as inhibited cell proliferation and mediated reversion toward an epithelial phenotype. This suggests the existence of a Hedgehog autocrine signaling loop affecting cell plasticity and fostering cell proliferation and migration/invasion in CC cell lines. These discoveries encourage future investigations to better characterize the role of Hedgehog in cellular plasticity and invasion during the different steps of CC pathogenesis.
这项工作的初步结果于2016年2月2日至4日在佛罗里达州杰克逊维尔举行的2016年学术外科大会上公布（原标题：选择性Smo抑制干扰结直肠癌的细胞能量代谢）。本研究由“罗马第一大学”（青年研究人员基金）和“AIRC”（意大利癌症研究协会）资助。Hedgehog抑制剂由基因泰克公司慷慨提供。结肠癌（CC）是美国第四大最常被诊断出的肿瘤，也是第二大主要死因。Hedgehog信号通路的异常已在多种人类癌症中得到证实，然而Hedgehog（Hh）在CC中的作用仍存在争议。在本研究中，我们通过对382例CC患者队列的基因表达微阵列分析，研究了Hh靶基因 和 的mRNA表达增加与CC生存及复发之间的关联。我们发现 表达增加的患者生存率有统计学意义的降低。为了证明Hh信号通路激活在CC发病机制中的因果作用，我们用GDC - 0449（一种Smoothened（SMO）的药理抑制剂）处理HCT 116、SW480和SW620 CC细胞系。用GDC - 0449处理显著降低了Hh靶基因 的表达，从而表明该信号通路在CC细胞系中持续激活。此外，GDC - 0449部分降低了细胞增殖，这与p21上调和CycD1下调有关。最后，用同一药物处理降低了迁移和三维侵袭，这与EMT主基因Snail1下调以及上皮标志物细胞角蛋白 - 18和E - 钙黏蛋白的诱导有关。这些结果通过SMO基因沉默得到证实。值得注意的是，用Sonic Hedgehog特异性单克隆抗体5E1处理显著降低了Hedgehog靶基因的表达，同时抑制了细胞增殖并介导了向上皮表型的逆转。这表明存在一个影响细胞可塑性并促进CC细胞系中细胞增殖和迁移/侵袭的Hedgehog自分泌信号环。这些发现鼓励未来进行进一步研究，以更好地阐明Hedgehog在CC发病机制不同阶段的细胞可塑性和侵袭中的作用。
