Abstract
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 αv activation of the FAK/paxillin/AKT signaling pathway and effects on expression of the proliferative marker Ki67 were investigated. To assess effects of integrin αv 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 αv-mediated FAK/paxillin/AKT signaling pathway. EGFR expression increased with integrin αv knockdown GBM cells and may represent a compensatory survival mechanism. These results indicate that ECM proteins confer CAMDR through integrin αv in GBM cells.
Highlights
Glioblastoma (GBM) is the most common primary brain tumor and the most aggressive in nature
We found that laminin, vitronectin, and fibronectin, three main components of extracellular matrix (ECM) proteins, could affect cell adhesion-mediated drug resistance (CAMDR) in GBM cells
An in vitro assay showed that fibronectin induced the highest degree of attachment for the U87 GBM cell line and two patient-derived GBM cell lines (GBM6 and HK308), followed by vitronectin and laminin, respectively (Figure 1B)
Summary
Glioblastoma (GBM) is the most common primary brain tumor and the most aggressive in nature. Prospective data regarding use of carmustine wafers included all high-grade gliomas, and some of the survival benefits were largely a result of grade 3 patients with long-term survival (Chowdhary et al, 2015). Additional concomitant chemotherapeutics for newly diagnosed GBM have not shown an incremental survival benefit. Concomitant bevacizumab with TMZ against newly diagnosed GBM in two large phase III trials showed prolonged progression-free survival but failed to show survival benefit (Chinot et al, 2014; Gilbert et al, 2014). It failed to show overall survival improvement in patients with methylated MGMT promoter in phase III trials (Stupp et al, 2014; Nabors et al, 2015). Single-arm phase II studies showed promising results, a randomized phase III trail (ACT IV) failed to show benefit over the control group (Weller et al, 2017)
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