角蛋白18在BCPR介导的乳腺癌细胞多药耐药中的作用和机制研究

Breast cancer resistant protein (BCRP) acts as one of markers of cancer stem cells (CSCs) and mediates the multidrug resistance (MDR) in a number of malignancies, but the mechanism of regualation on it is not clear. Epithelial-mesenchymal transition (EMT) is one of the important mechanisms of tumor invasion and metastasis, and NF-κB/snail pathway plays an important role in the regulation of EMT. The newest studies have demonstrated that EMT correlates with BCRP -mediated MDR and promotes the production of CSCs. Our laboratory previous studies found that cytokeratin 18 (CK18) was involved in MDR in breast cancer, inhibition of CK18 affected the expression of snail and induced EMT. Therefore, we speculate that CK18 induces EMT and regulates BCRP-mediated MDR in breast cancer through NF-κB/snail pathway. To test this hypothesis, the differential expression of CK18, EMT- associated proteins and pathway and CSCs markers were detected in human breast cancer cells, including drug-sensitive and -resistant cells with the MDR phenotype known as overexpression of BCRP. To explore the mechanism involved of CK18 in BCRP-mediated MDR, induction EMT via inhibition of CK18 or blockade of NF-κB/snail is to be executed to observe the effect on the BCRP expression and function, the ratio of CSCs. This study will provide new insights into prevention and treatment of malignant breast cancer.

乳腺癌耐药蛋白(BCRP)是肿瘤干细胞(CSCs)标志物之一，介导多种肿瘤的多药耐药(MDR)，但其调控机制不清。上皮间质转化(EMT)是肿瘤侵袭转移的重要机制，NF-κB/snail是其调节通路之一。最新研究发现，EMT与CSCs、BCRP介导的MDR均相关。我课题组前期结果提示，角蛋白(CK18)与BCRP介导的乳腺癌MDR有关，且抑制CK18可上调snail诱导EMT。据此，我们推测“CK18可能经NF-κB/snail通路诱导EMT调控BCRP介导的乳腺癌MDR”。本课题先观察CK18表达、EMT相关蛋白及NF-κB/snail通路、CSCs比例在人乳腺癌敏感株和BCRP高表达耐药株的差异，并进一步抑制CK18或阻断NF-κB/snail通路诱导EMT，观察对BCRP、CSCs的影响，阐明CK18调控BCRP介导的乳腺癌MDR的机制。本课题的完成将为恶性乳腺癌的有效防治提供新思路。

多药耐药(MDR)是导致化疗失败的重要原因。乳腺癌耐药蛋白(BCRP)是新近发现的膜转运蛋白，参与多种肿瘤包括乳腺癌的MDR，也被证实是干细胞(CSC)标记物之一，因此抑制BCRP介导的耐药显得尤为必要。角蛋白18(CK18)是构成细胞骨架的主要中间丝蛋白之一，CK18下调是上皮间质转化(EMT)的特征之一。我课题组前期结果发现，CK18与BCRP介导的乳腺癌MDR有关。因此，本研究着重探索了两个科学问题：(1)明确CK18在BCRP介导的MDR和CSC中的作用。(2)阐明EMT是否参与了CK18在耐药中的作用，并探讨其分子机制。通过本课题的实施，课题组首次发现了CK18在BCRP高表达的乳腺癌耐药株MCF-7/MX明显低于敏感株MCF-7, 敲低MCF-7中CK18促进BCRP表达、增加对多种化疗药物的耐药性、干性标记物Klf4和Oct4表达增加，提示CK18参与BCRP介导的乳腺癌MDR和干性；相对于MCF-7，耐药株MCF-7/MX呈间质表型、侵袭迁移能力增强，上皮标志物E-cadherin下调，间质标志物N-cadherin和vimentin上调，提示EMT与BCRP介导的耐药有关；作为EMT的重要调节通路，NF-κB/Snail在耐药株MCF-7/MX相较于MCF-7被明显激活；利用shRNA法敲低MCF-7细胞CK18表达诱导其EMT发生(细胞形态向间质转变、且侵袭迁移能力增强、上皮标志物下调，间质标志物上调)，提示CK18下调促进EMT发生；利用NF-κB抑制剂PDTC处理MCF-7/MX，可减少Snail表达、逆转其EMT进程。综合这些结果，我们得出结论：CK18 通过NF-κB/snail通路诱导 EMT调控 BCRP。我们还收集了60例乳腺浸润性导管癌患者的临床资料，用其石蜡切块行免疫组化分析，从临床水平证实CK18是乳腺癌预后的潜在预测指标。这些研究结果为耐药性乳腺癌的治疗提供了重要的理论依据，并为恶性乳腺癌的治疗提供了新的潜在治疗靶点。