缝隙连接蛋白26调控EMT介导的非小细胞肺癌吉非替尼耐药及分子机制

Gefitinib, a molecular-targeted drug, is widely used in non-small-cell lung cancer (NSCLC) patients at advanced stages who failed on other anticancer drugs. Despite initial responses, the patients eventually progress by unknown mechanisms of chemoresistance. It has been reported that cytotoxicity of various chemotherapeutic agents are enhanced by the presence of functional gap junctions (GJ) composed by connexins (Cx) between the targeted cells. Loss of GJIC is well accepted to be correlated with tumorigenic phenotypes. Our preliminary study showed that overexpression of Cx26 in H460 cells (a gefitinib-resistant NSCLC cell line) enhanced the sensitivity to gefitinib, upregulated the expression of E-cadherin, inhibited epithelial-mesenchymal transition (EMT) and invasive ability. Moreover, overexpression of Cx26 could inhibit the Akt activity, and combination treatment of Cx26 overexpression with specific PI3K inhibitor LY294002 resulted in a synergistic increase in E-cadherin expression in H460 cells. Specifically, our study showed that inhibition of gap junctional intercellular communication (GJIC) by oleamide (a widely accepted GJIC inhibitor) did not affect the enhanced effect on E-cadherin expression by Cx26 overexpression. Thus, we propose that Cx26 itself, reverses the EMT-mediated gefitinib resistance of NSCLC, most likely through inactivation of PI3K/Akt pathways in a GJIC-independent manner. Based on these findings, the present study will further explore the effect of Cx26 on EMT and its mediated gefitinib resistance of NSCLC as well as the underline molecular mechanisms in cells, animals and specimens of patients. Our study will lay an important theoretical basis for subsequent clinical application.

吉非替尼是晚期非小细胞肺癌（NSCLC）化疗失败后常用的分子靶向药物，但最终仍出现耐药，机制不明。Connexin（Cx）组成的gap junction（GJ）可增强多种化疗药的细胞毒性，但肿瘤发展过程中常伴GJ缺失。我们前期研究表明，过表达Cx26可增加NSCLC耐药细胞对吉非替尼的敏感性，并上调E-cadherin表达、抑制上皮间质转化（EMT）及侵袭力。过表达Cx26还可抑制耐药细胞Akt活性，PI3K抑制剂可协同Cx26上调E-cadherin表达。用GJ功能抑制剂处理不影响Cx26表达诱导的E-cadherin上调。由此我们提出科学假设：Cx26可独立于GJ功能的形成，本身能抑制PI3K/Akt信号通路激活从而逆转EMT介导的NSCLC吉非替尼耐药。本课题将通过细胞、动物及临床标本明确Cx26对NSCLC的EMT及其介导的吉非替尼耐药的调控作用及分子机制，为临床应用奠定理论基础。

吉非替尼获得性耐药是晚期非小细胞肺癌（non-small-cell lung cancer，NSCLC）化疗失败的主要原因，然而其机制尚未明确。在本研究中，我们首次发现缝隙连接蛋白26（Connexin 26，Cx26）是NSCLC细胞中主要表达的Cx亚型。通过在NSCLC吉非替尼敏感细胞株HCC827、PC9上，逐步递增吉非替尼浓度诱导获得吉非替尼耐药株 HCC827 GR、PC9 GR；实验结果显示，在这些吉非替尼耐药株中，HCC827 GR、PC9 GR细胞的Cx26表达明显高于其亲本细胞，且GR细胞的耐药性与其上皮间质化（Epithelial-mesenchymal transition，EMT）有关；免疫荧光结果显示，Cx26主要表达在HCC827 GR、PC9 GR及其亲本细胞的细胞浆中，且这4株细胞均无细胞缝隙连接功能（gap-junctional intercellular communication，GJIC）；在体外及体内实验中，通过慢病毒载体感染技术上调HCC827、PC9细胞胞浆Cx26的表达，可诱导细胞EMT并增强细胞吉非替尼耐药性，而当下调Cx26表达时可逆转细胞EMT并下调细胞吉非替尼耐药性；除此之外，过表达Cx26可激活细胞PI3K/Akt信号通路，且Cx26诱导的细胞EMT及吉非替尼耐药可被PI3K/Akt抑制剂所逆转，抑制PI3K/Akt信号通路可下调Cx26表达。因此，Cx26可独立于其所组成的GJIC与PI3K/Akt之间存在相互调控的正反馈循环，且这个正反馈循环可通过诱导NSCLC细胞EMT从而促进其吉非替尼获得性耐药。

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