LOXL2新剪接亚型促进食管癌细胞分裂增殖分子作用机制研究

Our previous researches have observed a significantly abnormal overexpression of a remarkable functional gene of esophageal cancer termed Lysyl Oxidase-Like 2 protein (LOXL2). The function of this very protein was closely related to invasion, migration while was proliferation-independent in esophageal cancer cells. Recently, we discovered a novel splicing isoform of LOXL2 named L2Δ13 for its lack of the thirteenth exon. Through further experimental analysis, L2Δ13 was observed to be detained in cytoplasm and its enzymatic activity was found to reduce extremely comparing to the wild-type as well. Unexpectedly, we uncovered that besides promoting invasion and migration, L2Δ13 also gave the cancer cells stronger abilities to proliferate of which the molecular mechanism still remained a fascinating puzzle. Therefore, in this very project, a series of experimental approaches were designed including stable isotope labeling with amino acids in cell culture (SILAC), immune co-precipitation, protein mass spectrometry analysis, site-directed mutagenesis, etc. to comprehensively identify crucial L2Δ13-interacting proteins involved in cell proliferation promotion. Ultimately, from three aspects namely molecular biology, cytology and animal model, combined with specific inhibition of individual cell signal pathway and systematic bioinformatics analysis, our work profoundly revealed the molecular mechanism of L2Δ13 promoting the cell proliferation in esophageal cancer.

我们以往的实验结果表明，LOXL2（赖氨酰氧化酶样蛋白2）在食管癌中显著异常过表达，是一重要食管癌功能基因，与癌细胞的侵袭移动、淋巴结转移相关，与癌细胞的分裂增殖无关。最近，我们在食管癌中发现了LOXL2的一个新剪接亚型，因外显子13剪接缺失，被命名为L2Δ13。进一步的实验结果表明，与LOXL2原型相比，L2Δ13的酶活性极显著降低，且滞留于细胞浆；令人意外的是，除了促食管癌细胞侵袭移动能力变得更强外，L2Δ13还新增了十分明显的促食管癌细胞分裂增殖新功能，但分子作用机制如何这一重要科学问题尚不清楚。为此，本项目拟通过联合运用SILAC标记、免疫共沉淀、蛋白质谱和特异位点突变等实验方法，首先鉴定出L2Δ13促食管癌细胞分裂增殖关键相互作用蛋白，然后在分子、细胞和动物实验三个水平上，结合细胞信号通路特异性抑制实验和生物信息学系统分析，深刻揭示L2Δ13促进食管癌细胞分裂增殖分子作用机制。

赖氨酰氧化酶家族成员之一LOXL2（赖氨酰氧化酶样蛋白2）是一重要癌功能基因，在传统意义上发挥依赖于其酶活性的胞外功能。我们在以往研究中发现，食管癌中存在LOXL2的一个新剪接亚型L2Δ13，其酶活性极低且滞留于胞浆，但仍发挥促癌功能。然而，L2Δ13的临床意义、酶活性外功能以及分子调控的分子机制尚不清楚。基于本项目研究，我们发现：（1）LOXL2及其L2Δ13表达与食管癌患者预后密切相关，是独立预后预警因素。（2）体外和体内实验证明，LOXL2与L2Δ13均促进食管癌细胞分裂增殖、移动和侵袭，而L2Δ13在促食管癌细胞分裂增殖中展现出比野生型更强的能力，且LOXL2不同结构域缺失体在食管癌细胞中的酶活性、定位分布和生物学功能各异。（3）我们构建了一个基于蛋白互作网络的新型食管癌不良预后的三分子预后预警模型（由LOXL2、FN1和CDH1组成），并发现LOXL2促进食管癌细胞伪足形成，参与细胞骨架重排。（4）胞浆LOXL2与L2Δ13通过与细胞骨架结合蛋白互作，进而影响细胞骨架重排，最终促进食管癌细胞侵袭移动；L2Δ13蛋白通过与代谢相关蛋白互作，影响关键代谢酶的活性，进而引起细胞代谢流变化，最终促进食管癌细胞分裂增殖。（5）L2Δ13高表达的转基因小鼠糖脂代谢紊乱，此基因高表达联合NMBA致癌剂显著加重小鼠食管上皮损伤。总之，上述成果表明，LOXL2新剪切亚型L2Δ13在食管癌发生发展中发挥重要作用，将成为食管癌防治的新靶点；对于无酶活性且不分泌的L2Δ13的深入研究，为揭示LOXL2在胞浆中的新功能和新机制提供了充分的理论和实验依据。