Yu Qi, Xiao Weikun, Sun Songping, Sohrabi Alireza, Liang Jesse, Seidlits Stephanie K
Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA, United States.
Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang, China.
Front Cell Dev Biol. 2021 Mar 23;9:616580. doi: 10.3389/fcell.2021.616580. eCollection 2021.
Chemotherapy resistance to glioblastoma (GBM) remains an obstacle that is difficult to overcome, leading to poor prognosis of GBM patients. Many previous studies have focused on resistance mechanisms intrinsic to cancer cells; the microenvironment surrounding tumor cells has been found more recently to have significant impacts on the response to chemotherapeutic agents. Extracellular matrix (ECM) proteins may confer cell adhesion-mediated drug resistance (CAMDR). Here, expression of the ECM proteins laminin, vitronectin, and fibronectin was assessed in clinical GBM tumors using immunohistochemistry. Then, patient-derived GBM cells grown in monolayers on precoated laminin, vitronectin, or fibronectin substrates were treated with cilengitide, an integrin inhibitor, and/or carmustine, an alkylating chemotherapy. Cell adhesion and viability were quantified. Transcription factor (TF) activities were assessed over time using a bioluminescent assay in which GBM cells were transduced with lentiviruses containing consensus binding sites for specific TFs linked to expression a firefly luciferase reporter. Apoptosis, mediated by p53, was analyzed by Western blotting and immunocytofluorescence. Integrin α activation of the FAK/paxillin/AKT signaling pathway and effects on expression of the proliferative marker Ki67 were investigated. To assess effects of integrin α activation of AKT and ERK pathways, which are typically deregulated in GBM, and expression of epidermal growth factor receptor (EGFR), which is amplified and/or mutated in many GBM tumors, shRNA knockdown was used. Laminin, vitronectin, and fibronectin were abundant in clinical GBM tumors and promoted CAMDR in GBM cells cultured on precoated substrates. Cilengitide treatment induced cell detachment, which was most pronounced for cells cultured on vitronectin. Cilengitide treatment increased cytotoxicity of carmustine, reversing CAMDR. ECM adhesion increased activity of NFκB and decreased that of p53, leading to suppression of p53-mediated apoptosis and upregulation of multidrug resistance gene 1 (MDR1; also known as ABCB1 or P-glycoprotein). Expression of Ki67 was correlative with activation of the integrin α -mediated FAK/paxillin/AKT signaling pathway. EGFR expression increased with integrin α knockdown GBM cells and may represent a compensatory survival mechanism. These results indicate that ECM proteins confer CAMDR through integrin α in GBM cells.
胶质母细胞瘤（GBM）的化疗耐药性仍然是一个难以克服的障碍，导致GBM患者预后不良。许多先前的研究都集中在癌细胞内在的耐药机制上；最近发现肿瘤细胞周围的微环境对化疗药物的反应有重大影响。细胞外基质（ECM）蛋白可能赋予细胞黏附介导的耐药性（CAMDR）。在此，我们使用免疫组织化学方法评估了临床GBM肿瘤中ECM蛋白层粘连蛋白、玻连蛋白和纤连蛋白的表达。然后，将在预包被有层粘连蛋白、玻连蛋白或纤连蛋白的基质上单层培养的患者来源的GBM细胞用整合素抑制剂西仑吉肽和/或烷化化疗药物卡莫司汀进行处理。对细胞黏附和活力进行了定量分析。使用生物发光测定法随时间评估转录因子（TF）活性，在该测定中，GBM细胞用含有与萤火虫荧光素酶报告基因表达相关的特定TF的共有结合位点的慢病毒进行转导。通过蛋白质印迹和免疫细胞荧光分析由p53介导的细胞凋亡。研究了整合素α激活FAK/paxillin/AKT信号通路以及对增殖标志物Ki67表达的影响。为了评估整合素α激活通常在GBM中失调的AKT和ERK通路的作用以及在许多GBM肿瘤中扩增和/或突变的表皮生长因子受体（EGFR）的表达，使用了短发夹RNA（shRNA）敲低技术。层粘连蛋白、玻连蛋白和纤连蛋白在临床GBM肿瘤中含量丰富，并促进在预包被基质上培养的GBM细胞产生CAMDR。西仑吉肽处理诱导细胞脱离，这在玻连蛋白上培养的细胞中最为明显。西仑吉肽处理增加了卡莫司汀的细胞毒性，逆转了CAMDR。ECM黏附增加了NFκB的活性并降低了p53的活性，导致p53介导的细胞凋亡受到抑制以及多药耐药基因1（MDR1；也称为ABCB1或P-糖蛋白）上调。Ki67的表达与整合素α介导的FAK/paxillin/AKT信号通路的激活相关。整合素α敲低的GBM细胞中EGFR表达增加，这可能代表一种代偿性生存机制。这些结果表明，ECM蛋白通过整合素α在GBM细胞中赋予CAMDR。
