EML4-ALK fusion protein in Lung cancer cells enhances venous thrombogenicity through the pERK1/2-AP-1-tissue factor axis,Journal of Thrombosis and Thrombolysis

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EML4-ALK fusion protein in Lung cancer cells enhances venous thrombogenicity through the pERK1/2-AP-1-tissue factor axis
Journal of Thrombosis and Thrombolysis ( IF 2.3 ) Pub Date : 2023-11-08 , DOI: 10.1007/s11239-023-02916-5
Yanping Su _{1,

2} , Jiawen Yi ₁ , Yuan Zhang ₁ , Dong Leng ₃ , Xiaoxi Huang ₄ , Xinyu Shi ₁ , Yuhui Zhang ₁

Affiliation

Background

Accumulating evidence links the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangement to venous thromboembolism (VTE) in non-small cell lung cancer (NSCLC) patients. However, the corresponding mechanisms remain unclear.

Method

High-throughput sequencing analysis of H3122 human ALK-positive NSCLC cells treated with ALK inhibitor/ dimethyl sulfoxide (DMSO) was performed to identify coagulation-associated differential genes between EML4-ALK fusion protein inhibited cells and control cells. Sequentially, we confirmed its expression in NSCLC patients’ tissues and in the plasma of a subcutaneous xenograft mouse model. An inferior vena cava (IVC) ligation model was used to assess clot formation potential. Additionally, pathways involved in tissue factor (TF) regulation were explored in ALK-positive cell lines H3122 and H2228. Statistical significance was determined by Student t-test and one-way ANOVA using SPSS.

Results

Sequencing analysis identified a significant downregulation of TF after inhibiting EML4-ALK fusion protein activity in H3122 cells. In clinical NSCLC cases, TF expression was increased especially in ALK-positive NSCLC tissues. Meanwhile, H3122 and H2228 with high TF expression exhibited shorter plasma clotting time and higher TF activity versus ALK-negative H1299 and A549 in cell culture supernatant. Mice bearing H2228 tumor showed a higher concentration of tumor-derived TF and TF activity in plasma and the highest adjusted IVC clot weights. Limiting EML4-ALK protein phosphorylation downregulated extracellular regulated protein kinases 1/2 (ERK1/2)-activating the protein-1(AP-1) signaling pathway and thus attenuated TF expression.

Conclusion

EML4-ALK fusion protein may enhance venous thrombogenicity by regulating coagulation factor TF expression. There was potential involvement of the pERK1/2-AP-1 pathway in this process.

中文翻译：

肺癌细胞中的 EML4-ALK 融合蛋白通过 pERK1/2-AP-1-组织因子轴增强静脉血栓形成性

背景

越来越多的证据表明，棘皮动物微管相关蛋白样 4 （EML4） -间变性淋巴瘤激酶（ALK）重排与非小细胞肺癌（NSCLC）患者的静脉血栓栓塞（VTE）有关。然而，相应的机制仍不清楚。

方法

对 ALK 抑制剂/二甲基亚砜（DMSO）处理的 H3122 人 ALK 阳性 NSCLC 细胞进行高通量测序分析，以鉴定 EML4-ALK 融合蛋白抑制细胞与对照细胞之间的凝血相关差异基因。随后，我们证实了它在 NSCLC 患者组织和皮下异种移植小鼠模型血浆中的表达。使用下腔静脉（IVC）连接模型评估凝块形成潜力。此外，在 ALK 阳性细胞系 H3122 和 H2228 中探索了参与组织因子（TF）调节的途径。通过 Student t 检验和使用 SPSS 的单因素方差分析确定统计显着性。

结果

测序分析发现 H3122 细胞中抑制 EML4-ALK 融合蛋白活性后 TF 显著下调。在临床 NSCLC 病例中，TF 表达增加，尤其是在 ALK 阳性 NSCLC 组织中。同时，与细胞培养上清液中 ALK 阴性的 H1299 和 A549 相比，具有高 TF 表达的 H3122 和 H2228 表现出更短的血浆凝血时间和更高的 TF 活性。携带 H2228 肿瘤的小鼠血浆中肿瘤来源的 TF 和 TF 活性浓度较高，调整后的 IVC 凝块重量最高。限制 EML4-ALK 蛋白磷酸化下调细胞外调节蛋白激酶 1/2 （ERK1/2） - 激活蛋白-1 （AP-1）信号通路，从而减弱 TF 表达。