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. 2015;16(3):383-91.
doi: 10.1080/15384047.2014.1002331.

Heterozygous deletion of ATG5 in Apc(Min/+) mice promotes intestinal adenoma growth and enhances the antitumor efficacy of interferon-gamma

Lu Wang  1 , Yan Wang, Yuyin Lu, Qianyun Zhang, Xianjun Qu
Affiliations

Heterozygous deletion of ATG5 in Apc(Min/+) mice promotes intestinal adenoma growth and enhances the antitumor efficacy of interferon-gamma

Lu Wang et al. Cancer Biol Ther. 2015.

Abstract

Autophagy related gene 5 (ATG5) was lost in 23% of the patients with colorectal cancer (CRC) and the role of loss of ATG5 in the pathogenesis of CRC remains unclear. Knockdown of ATG5 in cancer cells enhances the antitumor efficacy of lots of chemotherapeutic agents. However, there is still no animal model to validate these in vitro observations in vivo. In this study, we found that heterozygous deletion of ATG5 in Apc(Min/+) mice increased the number and size of adenomas as compared with those in Apc(Min/+)ATG5(+/+) mice. To investigate whether ATG5 deficiency could sensitize tumors to chemotherapies, we compared the antitumor effects of Interferon-gamma (IFN-γ) between Apc(Min/+)ATG5(+/+) and Apc(Min/+)ATG5(+/-) mice, as IFN-γ is a potential tumor suppressor for CRC and has been used clinically as an efficient adjuvant to chemotherapy of cancer. We revealed that heterozygous deletion of ATG5 significantly enhanced the antitumor efficacy of IFN-γ. Early treatment of Apc(Min/+)ATG5(+/-) mice with IFN-γ decreased tumor incidence rate to 16.7% and reduced the number of adenomas by 95.5% and late treatment led to regression of tumor. Moreover, IFN-γ treatment did not cause any evident toxic reaction. Mechanistic analysis revealed that heterozygous deletion of ATG5 activated EGFR/ERK1/2 and Wnt/β-catenin pathways in adenomas of Apc(Min/+) mice and enhanced the effects of IFN-γ-dependent inhibition of these 2 pathways. Our results demonstrate that ATG5 plays important roles in intestinal tumor growth and combination of IFN-γ and ATG5 deficiency or ATG5-_targeted inhibition is a promising strategy for prevention and treatment of CRC.

Keywords: 5-FU, 5-fluorouracil; ATG5; ATG5, autophagy related gene 5; Apc, adenomatous polyposis coli; ApcMin/+ mouse; CRC, colorectal cancer; EGFR, epidermal growth factor receptor; Erk, extracellular signal-regulated kinase; IFN-γ; IFN-γ, Interferon-gamma; LC3, microtubule-associated protein 1 light chain 3; PCNA, proliferating cell nuclear antigen; colorectal cancer; heterozygous deletion; intestinal adenoma; siRNAs, small interfering RNAs.

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Figures

Figure 1.
Figure 1.
Heterozygous deletion of ATG5 promotes tumor growth in ApcMin/+ mice. (A) ATG5 and LC-3 protein levels in adenomas were determined by Western blotting with β-actin as a loading control. (B) Representative images of intestinal tracts from ApcMin/+ATG5+/+ and ApcMin/+ATG5+/- mice at age 6 months. (C) Micrographs of hematoxylin and eosin stained colonic tumor sections. Histological analysis showed that dysplasia, loss of nuclear polarity and complex crypt outlines with cribriform glands in adenomas of both ApcMin/+ATG5+/+ mice (a and a‘) and ApcMin/+ATG5+/− mice (b and b’). Heterozygous deletion of ATG5 had no significant effect on the malignant progression of Apc-mediated intestinal tumor. Images a‘ and b’ (×200 magnification) are high magnification of insets in a and b (×40 magnification), respectively.
Figure 2.
Figure 2.
IFN-γ treatments effectively prevent intestinal adenomas in ApcMin/+ATG5+/− mice. (A) Representative images of intestinal tracts from ATG5 deficient ApcMin/+ mice after early treatment with IFN-γ. (B) Micrographs of hematoxylin and eosin stained colonic tumor sections. Histological analysis of intestinal adenomas in ApcMin/+ATG5+/− mice receiving vehicle revealed well-formed adenomas with severe dysplasia (a and a‘). Following early treatment with IFN-γ, adenomas of ApcMin/+ATG5+/− mice mostly exhibited hyperplastic morphology without obvious dysplasia in the polypoid area of mucosa of intestine (b and b’). Images a‘ and b’ (×200 magnification) are high magnification of insets in a and b (×40 magnification), respectively.
Figure 3.
Figure 3.
IFN-γ treatments do not cause any significant toxic reaction. Effects of early or late treatment of ApcMin/+ATG5+/+ mice with IFN-γ on body weight (A), average diet (B) and the number of WBCs (C) and platelets (D). *P < 0.05.
Figure 4.
Figure 4.
Heterozygous deletion of ATG5 promotes cell proliferation, activates Wnt/β-catenin and EGFR/ERK1/2 pathways and enhances the effects of IFN-γ-dependent suppression of these 2 signaling pathways. Western blotting assay showed the protein levels of PCNA (A), apoptosis-related protein including bax, bcl-2 and PARP (B), Wnt signaling-related protein including nuclear β-catenin, cyclin D1 and Survivin (C) and EGFR/Erk1/2 signaling-related protein including EGFR, phospho-EGFR and phospho-Erk1/2 (D). The protein β-actin served as a loading control and for the study involving phospho-Erk1/2, total Erk1/2 served as a control. The data presented is representative of 3 experiments. Significant differences are not shown.
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