Inhibition of autophagy exerts anti-colon cancer effects via apoptosis induced by p53 activation and ER stress.
BACKGROUND
Although some molecularly targeted drugs for colorectal cancer are used clinically and contribute to a better prognosis, the current median survival of advanced colorectal cancer patients is not sufficient. Autophagy, a basic cell survival mechanism mediated by recycling of cellular amino acids, plays an important role in cancer. Recently, autophagy has been highlighted as a promising new molecular target. The unfolded protein response (UPR) reportedly act in complementary fashion with autophagy in intestinal homeostasis. However, the roles of UPR in colon cancer under autophagic inhibition remain to be elucidated. We aim to clarify the inhibitory effect of autophagy on colon cancer.


METHODS
We crossed K19 (CreERT) and Atg5 (flox/flox) mice to generate Atg5 (flox/flox)/K19 (CreERT) mice. Atg5 (flox/flox)/K19 (CreERT) mice were first treated with azoxymethane/dextran sodium sulfate and then injected with tamoxifen to inhibit autophagy in CK19-positive epithelial cells. To examine the anti-cancer mechanisms of autophagic inhibition, we used colon cancer cell lines harboring different p53 gene statuses, as well as small interfering RNAs (siRNAs) targeting Atg5 and immunoglobulin heavy-chain binding protein (BiP), a chaperone to aid folding of unfolded proteins.


RESULTS
Colon tumors in Atg5 (flox/flox)/K19 (CreERT) mice showed loss of autophagic activity and decreased tumor size (the total tumor diameter was 28.1 mm in the control and 20.7 mm in Atg5 (flox/flox)/K19 (CreERT) mice, p = 0.036). We found that p53 and UPR/endoplasmic reticulum (ER) stress-related proteins, such as cleaved caspase 3, and CAAT/enhancer-binding protein homologous protein, are up-regulated in colon tumors of Atg5 (flox/flox)/K19 (CreERT) mice. Although Atg5 and BiP silencing, respectively, increased apoptosis in p53 wild type cells, Atg5 silencing alone did not show the same effect on apoptosis in p53 mutant cells. However, co-transfection of Atg5 and BiP siRNAs led to increased apoptosis in p53 mutant cells.


CONCLUSIONS
Blocking autophagy has potential in the treatment of colon cancer by inducing apoptosis via p53 and ER stress, and suppressing the UPR pathway is a valid strategy to overcome resistance to autophagic inhibition.
The Institute for Adult Diseases, Asahi Life Foundation, 2-2-6 Bakuro-cho, Nihon-Bashi, Chuo-ku, Tokyo, 113-8655, Japan. sakitani-tky@umin.ac.jp.|Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. sakitani-tky@umin.ac.jp.
Kosuke
K
Sakitani
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. hiratay-int@h.u-tokyo.ac.jp.
Yoshihiro
Y
Hirata
The Institute for Adult Diseases, Asahi Life Foundation, 2-2-6 Bakuro-cho, Nihon-Bashi, Chuo-ku, Tokyo, 113-8655, Japan. y.hikiba@gmail.com.
Yohko
Y
Hikiba
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. hayakawayoku@gmail.com.
Yoku
Y
Hayakawa
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. sihara-tky@umin.ac.jp.
Sozaburo
S
Ihara
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. suzuhiro817@yahoo.co.jp.
Hirobumi
H
Suzuki
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. s_nobu3wall@yahoo.co.jp.
Nobumi
N
Suzuki
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. st8039-fki@umin.ac.jp.
Takako
T
Serizawa
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. kinosita.hiroto@gmail.com.
Hiroto
H
Kinoshita
The Institute for Adult Diseases, Asahi Life Foundation, 2-2-6 Bakuro-cho, Nihon-Bashi, Chuo-ku, Tokyo, 113-8655, Japan. ksakamoto.spvir@gmail.com.
Kei
K
Sakamoto
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. hayaton0120@gmail.com.
Hayato
H
Nakagawa
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. ktate-tky@umin.ac.jp.
Keisuke
K
Tateishi
Gastroenterology Division, Yokohama City University Graduate School of Medicine, Yokohama, Japan. smaeda@yokohama-cu.ac.jp.
Shin
S
Maeda
Division of Clinical Genome Research, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. tikenoue@hotmail.com.
Tsuneo
T
Ikenoue
The Institute for Adult Diseases, Asahi Life Foundation, 2-2-6 Bakuro-cho, Nihon-Bashi, Chuo-ku, Tokyo, 113-8655, Japan. skawa@asahi-life.or.jp.
Shoji
S
Kawazu
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. kkoike-tky@umin.ac.jp.
Kazuhiko
K
Koike
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001343:Autophagy / Q000502:physiology*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000517:prevention & control*; D059865:Endoplasmic Reticulum Stress / Q000502:physiology*; D016158:Genes, p53 / Q000502:physiology; D045325:HCT116 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
D016159:Tumor Suppressor Protein p53