Aim: To investigate the therapeutic effect of combined integrin α6β4-targeted radioimmunotherapy (RIT) and PI3K/mTOR inhibitor BEZ235 in a pancreatic cancer model.
Methods: Phosphorylation of Akt, mTOR, the downstream effectors eukaryotic initiation factor 4E binding protein 1 (4EBP1) and S6 ribosomal protein (S6) were evaluated in BxPC-3 human pancreatic cancer cells treated with Yttrium-90 (90Y) labeled anti-integrin α6β4 antibody (ITGA6B4) and BEZ235 by western blotting. The cytotoxic effect of BEZ235 was investigated using a colony formation assay. Therapeutic efficacy enhancement by oral BEZ235 administration was assessed using mice bearing BxPC-3 xenograft tumors. Tumor volume measurements and immunohistochemical analyses (cell proliferation marker Ki-67, DNA damage marker p-H2AX and p-4EBP1 staining) of tumors were performed for evaluation of combined treatment with 90Y-ITGA6B4 plus BEZ235, or each arm alone.
Results: We found that phosphorylation of Akt (p-Akt), 4EBP1 (p-4EBP1) and S6 (p-S6) was inhibited by BEZ235. Colony formation in BxPC-3 cells was additively suppressed by the combination of 90Y-ITGA6B4 and BEZ235. Pretreatment with BEZ235 before 90Y-ITGA6B4 exposure resulted in significant reduction of cells plating efficiency (PE) (0.54 ± 0.11 vs 2.81 ± 0.14 with 185 kBq/mL 90Y-ITGA6B4 exposure, P < 0.01; 0.39 ± 0.08 vs 1.88 ± 0.09 with 370 kBq/mL 90Y-ITGA6B4 exposure, P < 0.01) when 5 × 103 cells per dish were plated. In vivo, the combined treatment with 90Y-ITGA6B4 plus BEZ235 enhanced the inhibition of tumor growth and statistically significant differences of relative tumor volume were observed for 27 d after the treatment start date when compared with the 90Y-ITGA6B4 single injection treatment (1.03 ± 0.38 vs 1.5 ± 0.15 at Day 27, P < 0.05), and for 41 d when compared with the BEZ235 treatment alone (1.8 ± 0.7 vs 3.14 ± 1.19 at Day 41, P < 0.05). Tumors from treatment groups showed reduction in volumes, decreased Ki-67-positive cells, increased p-H2AX-positive cells and decreased p-4EBP1 expression.
Conclusion: The therapeutic efficacy of 90Y-ITGA6B4-RIT can be improved by combining with dual PI3K and mTOR inhibitor, BEZ235, in a pancreatic cancer model suggesting potential clinical application.
Keywords: Anti-integrin α6β4 antibody; NVP-BEZ235; Pancreatic cancer; Radioimmunotherapy; Yttrium-90.