TβRⅡ对EML4-ALK阳性非小细胞肺癌生物学行为的调控和分子机制研究

It has been confirmed that EML4-ALK fusion gene is one of the driven genes in non-small cell lung cancer (NSCLC). We found the survival of NSCLC patients in the early stage with EML4-ALK positive was significantly better than the negative patients, however the survival of the patients in the local advanced stage with EML4-ALK positive was significantly poorer than the negative patients. Therefore, we assume that in the early stage the EML4-ALK-driven lung cancer not express invasively, and the biological changes in behavior in the late stage might be associated with other genetic abnormalities. Tumor cells could decrease the transforming growth factor receptor 2 (TβR Ⅱ) expression on the surface in the development of cancer, causing TGFβ from inhibiting the tumor growth early to lately promoting tumor invasion and metastasis. TβR Ⅱ plays an important role in the development of malignancy. In the term of this, we carried out the preliminary experiments which found that TβR Ⅱ expression of NSCLC with EML4-ALK fusion in the advanced stage was significantly lower than that in the early stage. Based on this evidences, we will construct the stable expression of TβR Ⅱ shRNA of the EML4-ALK-positive lung adenocarcinoma cell lines. First constructing lung cancer mouse model, to study TβR Ⅱhow to regulate the metastatic behavior of EML4-ALK-positive lung cancer and the related inflammation factor signal pathway, then to verify the mechanisms of TβR Ⅱin vitro which regulate the invasive behaviors of EML4-ALK positive lung cells founded in vivo. This study would help to reveal a possible molecular mechanisms of aggressive behavior in the EML4-ALK-positive NSCLC by the regulation of TβR Ⅱ, which provides a potential new target for the development of new drugs.

研究证实EML4-ALK融合基因是非小细胞肺癌（NSCLC）发生的驱动基因之一。我们发现早期ALK阳性患者预后明显好于阴性患者；然而在相对晚期ALK阳性者预后却明显差于阴性者。因此我们假设EML4-ALK驱动的肺癌早期侵袭性不强，但在后期可能合并其他遗传学异常而导致生物学行为的改变。肿瘤在发展过程中可以通过降低肿瘤细胞表面转化生长因子受体2（TβRⅡ）的表达，使TGFβ完成从早期抑制到晚期促进肿瘤进展角色的转化。鉴于此我们进行了预实验，结果发现晚期EML4-ALK阳性肺癌组织的TβRⅡ表达明显低于早期。本课题拟在此基础上构建稳定表达TβRⅡshRNA的EML4-ALK阳性肺癌细胞株，首先建立肺癌小鼠模型，研究TβRⅡ对EML4-ALK阳性肺癌转移行为的影响及相关炎症因子信号通路；然后体外细胞系中验证TβⅡ调控EML4-ALK阳性肺癌侵袭性行为的可能分子机制，为新药开发提供了可能的新靶点。

研究证实EML4-ALK融合基因是非小细胞肺癌（NSCLC）发生的驱动基因之一。我们发现早期ALK阳性患者预后明显好于阴性患者；然而在相对晚期ALK阳性者预后却明显差于阴性者。因此我们假设EML4-ALK驱动的肺癌早期侵袭性不强，但在后期可能合并其他遗传学异常而导致生物学行为的改变。肿瘤在发展过程中可以通过降低肿瘤细胞表面转化生长因子受体2（TβRⅡ）的表达，使TGFβ完成从早期抑制到晚期促进肿瘤进展角色的转化。鉴于此我们进行了预实验，结果发现晚期EML4-ALK阳性肺癌组织的TβRⅡ表达明显低于早期。本课题在前期研究基础上：首先构建TβRⅡshRNA慢病毒载体，构建EML4-ALK（variant 1）表达阳性H3122/ TβRⅡshRNA肺癌细胞株，研究TβRⅡ对H3122肺癌细胞增殖、侵袭等生物学行为的影响；其次建立H3122/ TβRⅡshRNA肺癌小鼠皮下成瘤和原位肺癌移植模型，研究TβRⅡ对EML4-ALK阳性肺癌生长转移生物学行为的影响。研究结果显示：H3122细胞沉默TβRⅡ表达后，体外肺癌细胞增殖能力无明显变化，但侵袭能力较对照组明显增强，体外ALK抑制剂药物抑制能力减弱；动物模型上显示BALC-nu/nu裸鼠H3122/ TβRⅡshRNA细胞株皮下成瘤能力明显增强，H3122/ TβRⅡshRNA原位裸鼠肺癌移植模型转移能力明显增强。本研究首次发现EML4-ALK阳性NSCLC患者肿瘤组织晚期TβRⅡ表达明显低于早期ALK阳性患者肿瘤组织，同时在体外EML4-ALK阳性肺癌细胞株上证实TβRⅡ低表达促进肿瘤细胞侵袭性增强，在体内动物模型上验证TβRⅡ低表达的ALK阳性肺癌生长转移能力明显增强，体外药物抑制实验结果显示TβRⅡ低表达是ALK抑制剂耐药的可能机制之一。本研究结果揭示了晚期EML4-ALK阳性非小细胞肺癌生物学行为复杂性的可能原因之一；晚期EML4-ALK阳性非小细胞肺癌靶向药耐药可能机制之一；同时为新药开发提供了可能的新靶点。