ABSTRACT
BACKGROUND: Approximately 45% of individuals diagnosed with Colorectal Cancer (CRC) also possess KRAS mutations. One developing therapeutic method for this disease is reovirus treatment. It is theorized that reovirus treatment on patients with KRAS mutated CRC cells would be successful due to the virus' innate oncolytic properties [1]. Reovirus, a stable form of nonenveloped double-stranded RNA, causes minor infections in humans under normal circumstances. However, when the virus encounters KRAS mutated cells, it has the potential to lyse them [2]. While this method of treatment to CRC has shown signs of success, we are still some ways from universal administration of reovirus as a treatment. This review seeks to utilize various studies, as well as our original research data, to investigate reovirus as an efficient method of treatment, with a focus on select growth, apoptotic and RAS-related genes, and their effectiveness of mitigating KRAS mutated CRC post reovirus treatment. Furthermore, the review highlights transcriptome analysis as an effective tool to examine these genes and their activity. It has been shown that reovirus treatment induces apoptosis and mitigates growth related gene activity. CONCLUSIONS: This review confirms the novelty of our findings on the efficacy of reovirus in CRC treatment. The study that this review article discusses concluded that 10 apoptotic and lymphocyte-related genes were found to be upregulated and 6 angiogenesis and Ras-related genes were found to be downregulated post reovirus treatment. These findings enforce the notion that reovirus could be used as a novel treatment for KRAS mutated CRC.
ABSTRACT
PURPOSE: Reovirus propagates with high efficiency in KRAS mutated colorectal cancer (CRC). About 45-50% of CRC patients possess a KRAS mutation. Oncolytic reovirus treatment in combination with chemotherapy was tested in patients possessing KRAS mutated metastatic CRC. This study evaluates the biological responses to reovirus treatment by determining the gene expression patterns in RAS-related signaling pathways. METHODS: Reovirus was administered as a 60-min intravenous infusion for 5 consecutive days every 28 days, at a tissue culture infective dose (TCID50) of 3×1010. Peripheral blood mononuclear cells (PBMCs) were isolated from whole-blood pre- and post-reovirus administration at 48 hr, day-8, and day-15. Clariom_D_Human_Assay was used to determine the expression of vital genes compared to pre-reovirus treatment by RNA sequencing. Using exported sample signals, ΔΔCt method was used to analyze the fold changes of genes within seven gene pathways. Significance was calculated by students-two-tail-t-test. Hierarchical clustering dendrogram was constructed by calculating Pearson's correlation coefficients. RESULTS: As compared to the control, SOS1[48 hr; 2.49X], RRAS [48 hr; 2.24X], PIK3CB [D8, D15; 2.27X, 3.16X], MIR 16-2 [D15; 1.70X], CHORDC1 [48 hr, D15; 1.89X, 4.54X], RTN4 [48 hr; 4.66X], FAM96A [48 hr; 4.54X], NFKB [D8, D15; 19.0X, 1.42X], CASP8 [D8, D15; 2.11X, 1.77X], and CASP9 [D8; 1.45X] are upregulated post-reovirus. NOS3 [D15; 0.61X], SYNE1 [D8, D15; 0.78X, 0.71X], ANGPT1 [D8; 0.62X], VEGFB [48 hr, D8, D15; 0.44X, 0.28X, 0.28X], JUN [D15; 0.69X], and IGF2 [D8; 0.73X] are downregulated post-reovirus. Fold change values were significant [p<0.05]. CONCLUSION: This study highlights reovirus as a novel treatment option for KRAS mutated CRC and showcases its effect on the expression of crucial genes.
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-ΔΔC t 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.
