Mutational Analysis of Hedgehog Signaling Pathway Genes in Human Malignant Mesothelioma

Background
The Hedgehog (HH) signaling pathway is critical for embryonic development and adult homeostasis. Recent studies have identified regulatory roles for this pathway in certain cancers with mutations in the HH pathway genes. The extent to which mutations of the HH pathway genes are involved in the pathogenesis of malignant mesothelioma (MMe) is unknown.
Methodology/Principal Findings
Real-time PCR analysis of HH pathway genes PTCH1, GLI1 and GLI2 were performed on 7 human MMe cell lines. Exon sequencing of 13 HH pathway genes was also performed in cell lines and human MMe tumors. In silico programs were used to predict the likelihood that an amino-acid substitution would have a functional effect. GLI1, GLI2 and PTCH1 were highly expressed in MMe cells, indicative of active HH signaling. PTCH1, SMO and SUFU mutations were found in 2 of 11 MMe cell lines examined. A non-synonymous missense SUFU mutation (p.T411M) was identified in LO68 cells. In silico characterization of the SUFU mutant suggested that the p.T411M mutation might alter protein function. However, we were unable to demonstrate any functional effect of this mutation on Gli activity. Deletion of exons of the PTCH1 gene was found in JU77 cells, resulting in loss of one of two extracellular loops implicated in HH ligand binding and the intracellular C-terminal domain. A 3-bp insertion (69_70insCTG) in SMO, predicting an additional leucine residue in the signal peptide segment of SMO protein was also identified in LO68 cells and a MMe tumour.
Conclusions/Significance
We identified the first novel mutations in PTCH1, SUFU and SMO associated with MMe. Although HH pathway mutations are relatively rare in MMe, these data suggest a possible role for dysfunctional HH pathway in the pathogenesis of a subgroup of MMe and help rationalize the exploration of HH pathway inhibitors for MMe therapy.

背景
刺猬（Hedgehog, HH）信号通路对胚胎发育及成人稳态维持至关重要。近期研究证实，该通路在部分携带其自身基因突变的癌症中发挥调控作用。目前尚不明确刺猬信号通路基因突变在恶性胸膜间皮瘤（malignant mesothelioma, MMe）发病机制中的参与程度。

材料与方法/主要结果
针对7株人恶性胸膜间皮瘤细胞系，开展了刺猬信号通路基因PTCH1、GLI1及GLI2的实时定量聚合酶链反应（Real-time PCR）分析。同时还对细胞系及人恶性胸膜间皮瘤组织中的13个刺猬信号通路基因进行了外显子测序。采用计算机模拟（in silico）程序预测氨基酸替换产生功能影响的概率。结果显示，GLI1、GLI2及PTCH1在恶性胸膜间皮瘤细胞中呈高表达，提示刺猬信号通路处于激活状态。在所检测的11株恶性胸膜间皮瘤细胞系中，2株检出PTCH1、SMO及SUFU基因突变。LO68细胞中检出SUFU基因错义突变（p.T411M）。对该SUFU突变体的计算机模拟分析表明，p.T411M突变可能改变蛋白功能，但本研究未能证实该突变对Gli蛋白活性存在任何功能影响。JU77细胞中检出PTCH1基因外显子缺失，导致其编码蛋白的两个参与刺猬配体结合的胞外环之一及胞内C端结构域丢失。LO68细胞及1例恶性胸膜间皮瘤组织中还检出SMO基因存在3碱基插入（69_70insCTG），该突变预计会使SMO蛋白信号肽段额外增加1个亮氨酸残基。

结论与意义
本研究首次鉴定出与恶性胸膜间皮瘤相关的PTCH1、SUFU及SMO基因新型突变。尽管刺猬信号通路基因突变在恶性胸膜间皮瘤中相对少见，但本研究数据提示，功能异常的刺猬信号通路可能在部分恶性胸膜间皮瘤亚型的发病机制中发挥作用，同时也为探索刺猬信号通路抑制剂用于恶性胸膜间皮瘤治疗提供了理论依据。