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

Abstract

Purpose: To explore the effects of pelareorep on autophagy in multiple models of colorectal cancer, including patient-derived peripheral blood mononuclear cells (PBMCs). Experimental Design: HCT116 [KRAS mutant (mut)] and Hke3 [KRAS 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 (KRAS mut) and MC38/C57B6 (KRAS 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−ΔΔCt method. Results: 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 P < 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 P < 0.05). Induction of 13 transcripts in cell lines (>4 ×; 6 and 9 hours; P < 0.05), 12 transcripts in CT26 (qRT-PCR), and 14 transcripts in human PBMCs (P < 0.05) was observed. LC3 A/B, RICTOR, and RASD1 expression was upregulated in all three model systems. Conclusions: Pelareorep hijacks host autophagic machinery in KRAS-mut conditions to augment its propagation and preferential oncolysis of the cancer cells.

AMPK phosphorylates PPARδ to mediate its stabilization, inhibit glucose and glutamine uptake and colon tumor growth

“…AMPK induced misfolded PPARδ-S50 phosphorylation leading to inhibition of p62-mediated misfolded PPARδ autophagic degradation. Other reports suggest that KRAS could regulate autophagy (47,48), and SW480 and HCT116 are KRAS mutant colonic cancer cells, whereas the increased PPARδ protein levels were similar to KRAS wild type (CW-2, HT-29) colonic cancer cells (SFigure. 2).…”

PIK3C3 Inhibition Promotes Sensitivity to Colon Cancer Therapy by Inhibiting Cancer Stem Cells

“…However, an oncogenic function for autophagy has generally been aligned with specific oncogenic status of the cancer cells and/or genetic mutations [ 49 ]. In this regard, studies have suggested that cancer cells harboring K-ras mutation are autophagy addicted [ 49 , 50 ]. While our findings support the role of the autophagy in promoting the resistance to chemotherapy, they also suggest that autophagy is an essential property of colon cancer cells irrespective of the cancer stage or genetic mutations.…”

Transcriptome Signature of Immune Cells Post Reovirus Treatment in KRAS Mutated Colorectal Cancer

“…This study confirms that reovirus (commercially known as pelareorep) has a profound effect on gene expression in patients with KRAS mutated CRC. We have previously shown using the same transcriptome data set that immune related genes 8 , 46 and autophagy-related genes 47 are significantly upregulated upon reovirus treatment. Herein, we have further analyzed and reported the genes that are altered in seven important cellular pathways, including the RAS signaling pathway.…”