In Vitro and In Vivo Analysis of RTK Inhibitor Efficacy and Identification of Its Novel Targets in Glioblastomas

Abstract

Treatment for glioblastoma consists of radiotherapy and temozolomide-based chemotherapy. However, virtually all patients recur, leading to a fatal outcome. Receptor tyrosine kinase (RTK)-targeted therapy has been the focus of attention in novel treatment options for these patients. Here, we compared the efficacy of imatinib, sunitinib, and cediranib in glioblastoma models. In the present work, the biologic effect of the drugs was screened by viability, cell cycle, apoptosis, migration, and invasion in vitro assays or in vivo by chick chorioallantoic membrane assay. Intracellular signaling was assessed by Western blot and the RTK targets were identified using phospho-RTK arrays. The amplified status of KIT, PDGFRA, and VEGFR2 genes was assessed by quantitative polymerase chain reaction. In a panel of 10 glioblastoma cell lines, we showed that cediranib was the most potent. In addition, cediranib and sunitinib synergistically sensitize the cells to temozolomide. Cediranib efficacy was shown to associate with higher cytostatic and unique cytotoxic effects in vitro and both antitumoral and antiangiogenic activity in vivo, which could associate with its great capacity to inhibit mitogen-activated protein kinase (MAPK) and AKT pathways. The molecular status of KIT, PDGFRA, and VEGFR2 did not predict glioblastoma cell responsiveness to any of the RTK inhibitors. Importantly, phospho-RTK arrays revealed novel targets for cediranib and sunitinib therapy. In conclusion, the novel targets found may be of value as future biomarkers for therapy response in glioblastoma and lead to the rational selection of patients for effective molecular targeted treatment.

Molecular profiling, including TERT promoter mutations, of acral lentiginous melanomas

“…Some authors have identified KIT gene amplification as an alternative mechanism for KIT upregulation in these tumors [18][19][20]. However, we did not find any case with KIT gene amplification using quantitative real-time PCR, a very sensitive and specific methodology, previously optimized by us and reported by our group and other groups [34,36,67,68]. We observed an association between KIT mutation and higher Clark staging, indicating that KIT could be associated with a more aggressive disease.…”

“…In all assays, appropriate positive and negative controls of 4q12 loci amplification were included [36]. These controls had also been previously assessed by other methodologies, such as array-CGH and CISH [34,36,37].…”

Coordination of signalling networks and tumorigenic properties by ABL in glioblastoma cells

“…Such down-regulation of RTKs could be per se incompatible with the tumorigenic competence of GBM cells [45, 65–67]. Consistently, drugs targeting several RTKs at the same time elicit efficient responses on a range of GBM cells [45, 50, 54]. Besides RTKs, we show a severe alteration also of MAPKs, STAT3, and p53 intracellular pathways, each of them being able to contribute to GBM tumorigenicity [68, 69, 70].…”

Comparison of effects of anti-angiogenic agents in the zebrafish efficacy–toxicity model for translational anti-angiogenic drug discovery

“…This could be attributed to differences in mechanisms of angiogenesis inhibition associated with sorafenib, regorafenib, and other multikinase inhibitors and endothelial cell proliferation inhibitors. Akin to VEGFR inhibitors, sorafenib and regorafenib are associated with potent VEGFR inhibition in biochemical assays (sorafenib: IC 50 ∼ 26–100 nM; regorafenib: IC 50 ∼ 13–46 nM)29,34 and exhibit significant angiogenesis inhibition in the in vivo CAM assay at 1–3 μM 35,36…”