Gene therapy for colorectal cancer by an oncolytic adenovirus that targets loss of the insulin-like growth factor 2 imprinting system

Abstract

Background: Colorectal cancer is one of the most common malignant tumors worldwide. Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality observed in human colorectal neoplasms. Our aim was to investigate the feasibility of using the IGF2 imprinting system for targeted gene therapy of colorectal cancer.

Results: We constructed a novel oncolytic adenovirus, Ad315-E1A, and a replication-deficient recombinant adenovirus, Ad315-EGFP, driven by the IGF2 imprinting system by inserting the H19 promoter, CCCTC binding factor, enhancer, human adenovirus early region 1A (E1A) and enhanced green fluorescent protein (EGFP) reporter gene into a pDC-315 shuttle plasmid. Cell lines with IGF2 LOI (HCT-8 and HT-29), which were infected with Ad315-EGFP, produced EGFP. However, no EGFP was produced in cell lines with maintenance of imprinting (HCT116 and GES-1). We found that Ad315-E1A significantly decreased cell viability and induced apoptosis only in LOI cell lines in vitro. In addition, mice bearing HCT-8-xenografted tumors, which received intratumoral administration of the oncolytic adenovirus, showed significantly reduced tumor growth and enhanced survival.

Conclusions: Our recombinant oncolytic virus targeting the IGF2 LOI system inhibits LOI cell growth in vitro and in vivo, and provides a novel approach for targeted gene therapy.

Figure 1

Construction and expression characterization of Ad315-E1A and Ad315-EGFP in cell lines with different IGF2 imprinting status. (a) In IGF2 MOI cell lines, CTCF binding factors are bound to DMD sites and the enhancer cannot control the H19 promoter, resulting in no expression of E1A or EGFP. (b) In IGF2 LOI cell lines, H19 has promoter activity that initiates expression of the downstream sequence due to CTCF complex regression. ITR: inverted terminal repeats.

Figure 2

Cell-specific expression of EGFP in four cell lines infected with recombinant adenoviral vectors. (a) Positive expression of EGFP in LOI cell lines. Ad315-EGFP infection (10 PFU/cell) induced EGFP expression in HCT-8 and HT-29 cells. (b) Negative expression of EGFP in MOI cell lines. Fluorescence images show no EGFP expression in GES-1 cells and only weak EGFP expression in HCT116 cells infected with Ad315-EGFP (10 PFU/cell).

Figure 3

E1A mRNA and protein expression in cells infected with adenoviral vectors carrying the E1A gene. (a) RT-PCR analysis of E1A mRNA expression in four cell lines at 24 h after infection with Ad315-E1A or H101 (10 PFU/cell). (b) Western blot analysis of E1A protein expression in four cell lines at 48 h after infection with Ad315-E1A or H101 (10 PFU/cell). In the Ad315-E1A group, E1A mRNA and protein were expressed in HCT-8 and HT-29 cells, but not in GES-1 and HCT116 cells. E1A mRNA and protein expression was only detected in the P53 mutant cell line HT-29 in the H101 group.

Figure 4

In vitro effect of adenoviral vectors carrying the E1A gene. (a) Cell viability was determined by an MTT assay at 72 h after infection with recombinant adenoviral vectors (10 PFU/cell). Graphs are representative of three separate experiments. (b) Apoptosis was investigated using flow cytometric analysis at 72 h after infection with Ad315-E1A, Ad315-EGFP or H101 (10 PFU/cell). The bottom right quadrants in dot plots show the apoptosis of various cell lines infected with Ad315-E1A. Data are representative of three separate experiments.

Figure 5

Anti-tumor activity of Ad315-E1A in HCT-8 xenograft models. (a) Average volume of subcutaneous tumors after receiving various treatments. Values represent the means ± SD for eight mice per group. Statistical differences were evaluated by one-way analysis of variance. **p < 0.01 vs. tumor volume of the Ad315-EGFP group. (b) Percentage of mouse survival analyzed by the Kaplan-Meier method.

Figure 6

Enhanced E1A expression and apoptosis in vivo by Ad315-E1A treatment. (a) Histopathological responses in xenografted tumors on day 7 after treatment (H&E staining; original magnification: ×100). (b) E1A protein expression as measured by immunohistochemical staining. (c) Cell apoptosis in tumors after receiving the last treatment, as measured by a TUNEL assay. Original magnification: ×400. (d) Analysis of the E1A expression rate and apoptosis percentage.