AP1S1在乳腺癌中的表达及其对化疗耐药的影响

Role of AP1S1 in inducing chemotherapy resistance in breast cancer

目的 探究接头相关蛋白复合物1亚基σ1(AP1S1)基因表达水平与乳腺癌化疗患者预后之间的相关性,分析AP1S1在乳腺癌化疗耐药中的作用。方法 采用生物信息学方法,对癌症基因组图谱(TCGA)数据库中的乳腺癌患者和乳腺正常组织的AP1S1表达水平差异进行分析。运用Kaplan-Meier生存分析法,探究AP1S1表达水平与乳腺癌化疗患者无复发生存预后的关联。通过细胞培养、RNA提取、实时荧光定量PCR、蛋白质免疫印迹(Western blot)分析及药物敏感性实验等多种实验手段,基于AP1S1高表达的乳腺癌细胞模型,研究AP1S1基因高表达对乳腺癌细胞化疗耐药的影响,并考察AP1S1过表达对肿瘤细胞干性及上皮-间充质转化(EMT)特性标志物表达的作用。结果 乳腺癌组织中AP1S1基因的表达水平显著高于正常组织,差异有统计学意义(P<0.05)。在肿瘤不同分期,AP1S1基因的表达水平均表现出显著高于正常组织,差异均有统计学意义(P均<0.05);其中4期肿瘤的过表达水平更高,差异有统计学意义(P<0.05)。管腔(Luminal)型、人类表皮生长因子2(HER2)阳性型和三阴型乳腺癌组织较正常组织均表现出AP1S1基因高表达,差异均有统计学意义(P均<0.05);其中在HER2阳性型和三阴型乳腺癌组织中AP1S1表达水平更高,差异均有统计学意义(P均<0.05)。Kaplan-Meier生存分析发现,AP1S1高表达的乳腺癌患者在化疗后无复发生存期显著短于低表达组,差异有统计学意义(P=0.038);在三阴型、HER2阳性乳腺癌患者中,AP1S1高表达与化疗后预后不良显著相关(P=0.037、0.017);Luminal B型患者中,AP1S1高表达与较长的无复发生存期显著相关(P=0.007)。实时荧光定量PCR显示,与对照组比较,在AP1S1过表达的细胞系CAL-51-oe-AP1S1、Hs-578T-oe-AP1S1中AP1S1转录水平显著上调,差异均有统计学意义(t=23.51、6.22,P均<0.05);Western blot结果显示,与对照组比较,在AP1S1过表达的细胞系CAL-51-oe-AP1S1、Hs-578T-oe-AP1S1中AP1S1蛋白水平显著上调,差异均有统计学意义(P均<0.05);肿瘤药物敏感性基因组项目(GDSC)数据库中分析结果显示,乳腺癌细胞CAL-51、Hs-578T的半抑制浓度(IC50)分别为0.008 398、0.000 935μmol/L;药物毒性实验结果显示,过表达AP1S1显著增强了乳腺癌细胞CAL-51、Hs-578T对化疗药物多西紫杉醇的耐受性,差异均有统计学意义(t=8.74、9.90,P均<0.05);三维培养实验结果显示,过表达AP1S1的CAL-51细胞形成更多的球体细胞团,而对照组的结构在药物作用下显著恶化,差异有统计学意义(t=11.54,P<0.05)。与对照组比较,在CAL-51-oeAP1S1、Hs-578T-oe-AP1S1乳腺癌细胞系中,AP1S1过表达在转录水平上显著上调了干细胞标志基因纳诺克同源盒(NANOG)、辛烷结合转录因子4 (OCT4)、性别决定区相关HMG盒基因2 (SOX2)、B细胞特异性分化基因1(BMI1)以及多药耐药相关基因ATP结合盒转运蛋白G2(ABCG2)的表达,差异均有统计学意义(t=20.75、39.05、28.55、29.76、94.18,24.78、17.79、19.60、14.10、16.79,P均<0.05);同时显著降低了上皮钙黏蛋白(E-cadherin)(CDH1)和紧密连接蛋白1(TJP1)的mRNA表达,并增加了与EMT相关的标志物神经钙黏蛋白(N-cadherin)(CDH2)和波形蛋白(VIM)的表达,差异均有统计学意义(t=6.26、4.99、9.79、5.23,13.25、19.87、13.83、11.42,P均<0.05)。在CAL51细胞系中,Western blot结果显示,AP1S1的过表达导致干细胞标志基因SOX2的表达水平上升和多药耐药基因ABCG2的表达增加,同时显著抑制了细胞黏附蛋白E-cadherin的表达,并促进了N-cadherin的过表达,差异均有统计学意义(P均<0.05)。结论 AP1S1在乳腺癌中高表达,不仅促进化疗药物多西紫杉醇的耐药性,还促进乳腺癌细胞的干性增强并诱发EMT过程。

Objective To explore the correlation between the expression level of adaptor related protein complex 1 subunit sigma 1(AP1S1) gene and the prognosis of breast cancer patients undergoing chemotherapy, and analyze the role of AP1S1in chemotherapy resistance of breast cancer. Methods The expression of AP1S1 between breast cancer patients and breast normal tissues in The Cancer Genome Atlas(TCGA) database was analyzed by bioinformatics. Kaplan-Meier survival analysis was used to explore the association between AP1S1 expression level and relapse-free survival prognosis of breast cancer patients undergoing chemotherapy. Through cell culture, RNA extraction, real-time fluorescent quantitative PCR, Western blot analysis and drug sensitivity test, and other experimental methods, based on the breast cancer cell model with high expression of AP1S1, the effect of high expression of AP1S1 gene on chemotherapy resistance of breast cancer cells was revealed. The effect of overexpression of AP1S1 on the expression of stem and epithelial-mesenchymal transformation(EMT) markers in tumor cells was investigated. Results significantly higher than that in normal tissue;the difference was statistically significant(P<0.05). At different tumor stages, the expression level of AP1S1 gene was significantly higher than that of normal tissue, with statistical significance(all P<0.05). Among them, the overexpression level of AP1S1 gene of stage 4 tumors was higher, and the difference was statistically significant(P<0.05). Luminal,human epidermal growth factor receptor 2(HER2)-positive and triple-negative breast cancer tissues showed high expression level of AP1S1 gene compared with normal tissues, with statistical significance(all P<0.05). The expression levels of AP1S1 were higher in HER2-positive and triple-negative breast cancer tissues, and the differences were statistically significant(both P<0.05). Kaplan-Meier survival analysis showed that the relapse-free survival of breast cancer patients with high AP1S1 expression after chemotherapy was significantly shorter than that of patients with low AP1S1 expression,the difference was statistically significant(P=0.038). High AP1S1 expression was significantly associated with poor prognosis after chemotherapy in triple-negative and HER2-positive breast cancer patients(P=0.037, 0.017). In Luminal B patients, high AP1S1 expression was significantly associated with longer relapse-free survival(P=0.007). Real-time fluorescence quantitative PCR showed that compared with the control group, the transcription level of AP1S1 in the overexpressed cell lines CAL-51-oe-AP1S1 and Hs-578T-oe-AP1S1 was significantly up-regulated, with statistical significance(t=23.51,6.22;both P<0.05). Western blot results showed that compared with the control group, the AP1S1 protein levels in the AP1S1 overexpression cell lines CAL-51-oe-AP1S1 and Hs-578T-oe-AP1S1 were significantly upregulated,with statistical significance(all P<0.05). The results of The Genomis of Drug Sensitivity in Cancer Project(GDSC) database analysis showed that the inhibitory concentration 50%(IC50) of CAL-51 and Hs-578T were 0.008 398 and 0.000 935 μmol/L, respectively. Drug toxicity test results showed that overexpression of AP1S1 significantly enhanced the tolerance of breast cancer cells CAL-51 and Hs-578T to the chemotherapy drug docetaxel, with statistical significance(t=8.74, 9.90;both P<0.05). The results of three-dimensional culture experiment showed that CAL-51 cells overexpressing AP1S1 formed more spherical cell clusters, while the structure of the control group was significantly deteriorated under the action of drugs(t=11.54,P<0.05). Compared with the control group, in CAL-51-oe-AP1S1 and Hs-578T-oe-AP1S1 breast cancer cell lines, overexpression of AP1S1 significantly upregulated stem cell marker gene Nanog homeobox(NANOG), octamer-binding transcription factor 4(OCT4), SRY associated HMG box gene 2(SOX2), BMI1 polycomb ring finger oncogene(BMI1), and multi-drug resistance associated gene ATP-binding cassette subfamily G member 2(ABCG2) expression at the transcription level;the differences were statistically significant(t=20.75, 39.05, 28.55, 29.76, 94.18;24.78, 17.79, 19.60, 14.10, 16.79;all P<0.05). Meanwhile, the mRNA expression of epithelial cadherin(E-cadherin)(CDH1) and tight junction protein 1(TJP1) was significantly decreased;The expression of EMT-related markers neural cadherin(N-cadherin)(CDH2) and vimentin(VIM) were increased, and the differences were statistically significant(t=6.26, 4.99, 9.79, 5.23;13.25, 19.87, 13.83, 11.42;all P<0.05). In CAL51 cell line, Western blot results showed that overexpression of AP1S1 led to increased expression level of stem cell marker gene SOX2 and multi-drug resistance gene ABCG2, and significantly inhibited the expression of cell adhesion protein E-cadherin, it also promoted the overexpression of N-cadherin, and the differences were statistically significant(all P<0.05). Conclusion The high expression of AP1S1in breast cancer not only could promote the resistance of chemotherapy drug docetaxel, but also promote the dry enhancement of breast cancer cells and induces EMT process.