Oncolytic Reovirus (pelareorep) Induces Autophagy in KRAS-mutated Colorectal Cancer.

Abstract

To explore the effects of pelareorep on autophagy in multiple models of colorectal cancer, including patient-derived peripheral blood mononuclear cells (PBMCs). HCT116 [KRAS mutant (mut)] and Hke3 [<i>KRAS</i> wild-type (WT)] cells were treated with pelareorep (multiplicity of infection, 5) and harvested at 6 and 9 hours. LC3 A/B expression was determined by immunofluorescence and flow cytometry; five autophagic proteins were analyzed by Western blotting. The expression of 88 autophagy genes was determined by qRT-PCR. Syngeneic mouse models, CT26/Balb-C (<i>KRAS</i> mut) and MC38/C57B6 (<i>KRAS</i> WT), were developed and treated with pelareorep (10 × 10⁶ plaque-forming unit/day) intraperitoneally. Protein and RNA were extracted from harvested tumor tissues. PBMCs from five experimental and three control patients were sampled at 0 (pre) and 48 hours, and on days 8 and 15. The gene expression normalized to "pre" was determined using 2^{-ΔΔ<i>C</i> _t} method. Pelareorep induced significant upregulation of LC3 A/B in HCT116 as compared with Hke3 cells by immunofluorescence (3.24 × and 8.67 ×), flow cytometry (2.37 × and 2.58 ×), and autophagosome formation (2.02 × and 1.57 ×), at 6 and 9 hours, respectively; all <i>P</i> < 0.05. Western blot analysis showed an increase in LC3 A/B (2.38 × and 6.82 ×) and Beclin1 (1.17 × and 1.24 ×) at 6 and 9 hours, ATG5 (2.4 ×) and P-62 (1.52 ×) at 6 hours, and VPS-34 (1.39 ×) at 9 hours (all <i>P</i> < 0.05). Induction of 13 transcripts in cell lines (>4 ×; 6 and 9 hours; <i>P</i> < 0.05), 12 transcripts in CT26 (qRT-PCR), and 14 transcripts in human PBMCs (<i>P</i> < 0.05) was observed. <i>LC3 A/B, RICTOR</i>, and <i>RASD1</i> expression was upregulated in all three model systems. Pelareorep hijacks host autophagic machinery in <i>KRAS</i>-mut conditions to augment its propagation and preferential oncolysis of the cancer cells.