CXCL16/CXCR6轴通过诱导MSC-肺癌杂交瘤细胞EMT促进肺癌转移的作用及分子机制研究

Supported by grants from the National Natural Science Foundation of China (30871123), we observed that a variety of mesenchymal stem cells (MSCs) from the bone marrow are recruited to tumor sites, and play a role in facilitating epithelial mesenchymal transition(EMT). Supported by grants from the National Natural Science Foundation of China (81071728), we found that MSCs could facilitate the ability of tumor cells to acquire mesenchymal properties during a physical fusion event with lung cancer cells. However, the undrelying mechanism remains unclear. Our previous research showed, hybrids formed by fusion between human lung cancer cells and bone marrow-derived MSCs overproduce both CXCR6 and CXCL16 and correlated with EMT. JAK2 inhibitor can effectively inhibit EMT. Based on our previous reserch, we hypothesized that MSCs might promote EMT through fusion with lung cancer cells by CXCL16/CXCR6 axis mediated JAK2/STAT3 pathway. In order to verify the hypothesis, we plan to use a murine model of lung cancer and culture MSC-lung cancer hybrids to observe whether CXCL16/CXCR6 axis are used by hybrids to acquire mesenchymal properties and test JAK2/STAT3 pathway、PI3K and expression of EMT related transcription factors to elucidate the underlying mechanism. Finally verify through clinical research. These findings may explain how lug cancer cells acquire a motile phenotype so early and represent viable entities for the design of targeting therapies to prevent the establishment of distant metastasis of lung cancers cells.

在面上项目30871123支助下，我们发现骨髓来源间充质干细胞（MSC）能向肺癌病灶归巢，且与肺癌EMT相关。在面上项目81071728支助下，我们检测到MSC可与肺癌细胞在体内、外自发融合，产生的杂交瘤细胞具有EMT特性，然而其调控机制不明。本课题组前期研究发现，杂交瘤细胞同时高表达CXCR6和CXCL16，且与EMT密切相关，而抑制JAK2/STAT3通路可明显抑制杂交瘤细胞EMT发生。据此推测CRCL16/CXCR6轴可能通过JAK2/STAT3通路调控杂交瘤细胞发生EMT的假说。本课题拟构建肺癌动物模型及体外培养杂交瘤细胞，通过增强和干扰CXCL16/CXCR6轴观察对EMT的影响，并检测JAK2/STAT3通路、下游PI3K以及twist、snail等EMT关键调控因子的基因及蛋白的变化。最后通过临床标本验证。为阐明肺癌早期转移机制及制定新的靶向治疗措施抑制肺癌转移提供实验依据。

背景：据前期研究证实MSCs能向肺癌病灶归巢。并且MSCs可与肺癌细胞在体内、外自发融合成杂交瘤细胞具有EMT特性，但调控机制不明。研究表明CXCL16/CXCR6可以引导部分免疫细胞迁移至病灶，血管突破等过程。这与肿瘤发生迁移、侵袭过程类似。CXCL16/CXCR6在多种进展期的肿瘤表达水平增高。有文献报道，RANKL可以通过降低CXCL16的生成致使JAK2/STAT3信号通路失活。JAK2/STAT3是通过细胞因子刺激使其磷酸化而被激活。CXCL16/CXCR6是常见的趋化因子及受体。两者产生的生理学效应较为接近。另外，STAT3具有SH2的结构域，CXCR6具有SH2的结合位点。在多项肿瘤的研究中，CXCL16/CXCR6增高通常伴有JAK2/STAT3表达异常。综上可推测出两点假说：1、A549细胞产生外分泌型的CXCL16，能促进表达CXCR6的MSCs向肿瘤细胞迁移并与之融合从而激活CXCL16/CXCR6轴。2、CXCL6/CXCR6通过自分泌调控JAK2/STAT3，活化下游PI3K/AKT/mTOR通路，促使杂交瘤细胞发生EMT进而促进肺癌转移。.主要研究类容：1、揭示CXCL16/CXCR6轴在骨髓来源MSCs与肺癌细胞自发融合形成的在交流细胞EMT发生中的作用。2、揭示CXCL16/CXCR6轴在在就留细胞EMT中的调控机制，为肺癌转移八项治疗提供科学依据。.重要结果：.1、体外共培养的MSCs与肺癌细胞能够发生自发融合。且融合细胞呈EMT特性。2、杂交瘤细胞的CXCL16及CXCR6表达量较亲代细胞均有所上调。3、CXCL16的过表达或敲低能促进和抑制骨髓来源的MSCs与肺癌细胞自发融合的比例。4、揭示JAK2/STAT3信号传导分子通路是CXCL16/CXCR6轴在MSCs-肺腺癌杂交瘤细胞中的下游通路。.关键数据：1、MSC可与A5498发生自发融合。1、CXCL16能够趋化MSC及促进MSCs与A549细胞融合率。2、融合细胞后CXCL16、CXCR6表达均增高。3、Western Blot测定JAK2、STAT3表达量及磷酸化水平与CXCL16、CXCR6呈正相。.科学意义：.1、揭示了CXCR6/CXCL16在MSC-肺癌融合中的重要作用。2、证实了CXCL16/CXCR6与JAK2/STAT的相互关系。