Hansen Stine Ninel, Westergaard David, Thomsen Mathilde Borg Houlberg, Vistesen Mette, Do Khoa Nguyen, Fogh Louise, Belling Kirstine C, Wang Jun, Yang Huanming, Gupta Ramneek, Ditzel Henrik J, Moreira José, Brünner Nils, Stenvang Jan, Schrohl Anne-Sofie
Sino-Danish Breast Cancer Research Center, University of Copenhagen, Strandboulevarden 49, DK-2100, Copenhagen, Denmark.
Tumour Biol. 2015 Jun;36(6):4327-38. doi: 10.1007/s13277-015-3072-4. Epub 2015 Jan 18.
The microtubule-targeting taxanes are important in breast cancer therapy, but no predictive biomarkers have yet been identified with sufficient scientific evidence to allow clinical routine use. The purposes of the present study were to develop a cell-culture-based discovery platform for docetaxel resistance and thereby identify key molecular mechanisms and predictive molecular characteristics to docetaxel resistance. Two docetaxel-resistant cell lines, MCF7RES and MDARES, were generated from their respective parental cell lines MCF-7 and MDA-MB-231 by stepwise selection in docetaxel dose increments over 15 months. The cell lines were characterized regarding sensitivity to docetaxel and other chemotherapeutics and subjected to transcriptome-wide mRNA microarray profiling. MCF7RES and MDARES exhibited a biphasic growth inhibition pattern at increasing docetaxel concentrations. Gene expression analysis singled out ABCB1, which encodes permeability glycoprotein (Pgp), as the top upregulated gene in both MCF7RES and MDARES. Functional validation revealed Pgp as a key resistance mediator at low docetaxel concentrations (first-phase response), whereas additional resistance mechanisms appeared to be prominent at higher docetaxel concentrations (second-phase response). Additional resistance mechanisms were indicated by gene expression profiling, including genes in the interferon-inducible protein family in MCF7RES and cancer testis antigen family in MDARES. Also, upregulated expression of various ABC transporters, ECM-associated proteins, and lysosomal proteins was identified in both resistant cell lines. Finally, MCF7RES and MDARES presented with cross-resistance to epirubicin, but only MDARES showed cross-resistance to oxaliplatin. In conclusion, Pgp was identified as a key mediator of resistance to low docetaxel concentrations with other resistance mechanisms prominent at higher docetaxel concentrations. Supporting Pgp upregulation as one major mechanism of taxane resistance and cell-line-specific alterations as another, both MCF7RES and MDARES were cross-resistant to epirubicin (Pgp substrate), but only MDARES was cross-resistant to oxaliplatin (non-Pgp substrate).
微管靶向紫杉烷类药物在乳腺癌治疗中具有重要作用，但尚未鉴定出具有充分科学证据可用于临床常规使用的预测性生物标志物。本研究的目的是开发一个基于细胞培养的多西他赛耐药发现平台，从而确定多西他赛耐药的关键分子机制和预测分子特征。通过在15个月内逐步增加多西他赛剂量进行筛选，从各自的亲本细胞系MCF-7和MDA-MB-231中产生了两个多西他赛耐药细胞系MCF7RES和MDARES。对这些细胞系进行了多西他赛和其他化疗药物敏感性的表征，并进行了全转录组mRNA微阵列分析。随着多西他赛浓度的增加，MCF7RES和MDARES呈现出双相生长抑制模式。基因表达分析选出编码通透糖蛋白（Pgp）的ABCB1作为MCF7RES和MDARES中上调最明显的基因。功能验证表明，在低多西他赛浓度下（第一阶段反应），Pgp是关键的耐药介质，而在较高多西他赛浓度下（第二阶段反应），其他耐药机制似乎更为突出。基因表达谱显示了其他耐药机制，包括MCF7RES中干扰素诱导蛋白家族的基因和MDARES中癌胚抗原家族的基因。此外，在两个耐药细胞系中均鉴定出各种ABC转运蛋白、细胞外基质相关蛋白和溶酶体蛋白的表达上调。最后，MCF7RES和MDARES对表柔比星呈现交叉耐药，但只有MDARES对奥沙利铂呈现交叉耐药。总之，Pgp被确定为低多西他赛浓度耐药的关键介质，在较高多西他赛浓度下其他耐药机制更为突出。支持Pgp上调是紫杉烷耐药的一个主要机制，细胞系特异性改变是另一个机制，MCF7RES和MDARES对表柔比星（Pgp底物）均呈现交叉耐药，但只有MDARES对奥沙利铂（非Pgp底物）呈现交叉耐药。
