Background
Colorectal cancer is the second leading cause of cancer-related death in the United States [
1]. Although some molecularly targeted drugs, such as anti-vascular endothelial cell growth factor (VEGF) and anti-epidermal growth factor receptor (EGFR) antibodies, are used clinically and contribute to a better prognosis, the current median survival of stage IV colorectal cancer patients receiving chemotherapy is shorter than 3 years [
2]. Macroautophagy (hereafter referred to as autophagy), which is a highly conserved cell survival mechanism mediated by the recycling of cellular amino acids [
3], has been highlighted as a promising new molecular target [
3,
4]. For example, chloroquine (CQ), originally developed as an anti-malarial agent, prevents autophagic activity and has been used in several clinical trials of cancer treatment as an autophagic inhibitor [
5,
6].
Autophagy is typically activated under conditions of amino acid starvation, and autophagy marker proteins include light chain 3 (LC3) and p62 [
5]. p62 (also called SQSTM1) is a selective substrate of autophagy, and its accumulation is observed when autophagy is inhibited [
7]. This p62 dysregulation is reportedly associated with proliferation of cancer, including colorectal cancer [
8,
9]. Preclinical evidence has shown that the role of autophagy in cancer differs depending on the situation. For instance, although autophagic inhibition promotes cancer initiation, it can suppress growth of some malignancies, such as lung cancer and hepatocellular carcinoma [
10‐
12]. Quite recently, Rosenfeldt et al. showed that the effect of autophagic inhibition on pancreatic cancer depends on p53 status [
13].
The endoplasmic reticulum (ER) is an organelle involved in protein folding, and ER stress refers to the condition leading to accumulation of misfolded proteins. The unfolded protein response (UPR), which is the cellular response to increased ER stress, is a basic mechanism of maintaining cell homeostasis [
14,
15]. Under ER stress, immunoglobulin heavy-chain binding protein (BiP) and phosphorylated eukaryotic initiation factor 2α (eIF2α) are upregulated [
16,
17]. BiP acts as a chaperone to aid folding of unfolded proteins, and eIF2α suppresses general mRNA translation. Severe ER stress can induce apoptotic cell death, and CAAT/enhancer-binding protein homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) have been reported to play a critical role in the induction of apoptosis [
18]. Recently, Adolph et al. showed that autophagy and UPR act in complementary fashion in Paneth cells to maintain intestinal homeostasis [
19]. However, the roles of autophagy and UPR in colon cancer remain to be elucidated.
To investigate the involvement of autophagy in colon cancer
in vivo, we treated mice mutant for
Atg5, an indispensable gene for autophagy [
20], with azoxymethane/dextran sodium sulfate (AOM/DSS), which is an established animal model used to induce and analyze colon cancer [
21,
22]. Since systemic Atg5 deletion causes neonatal lethality [
23], we used
K19
CreERT
mice to inhibit autophagy specifically in CK19 positive-cell which is known as a marker of epithelial cell [
24]. In this report, by genetic inhibition of autophagy and CQ treatment, we showed that suppression of autophagy has an anti-colorectal cancer effect via apoptosis induced by p53 activation and ER stress
in vivo and
in vitro.
Methods
Mice
K19
CreERT
mice were kindly provided by Guoqiang Gu (Vanderbilt University, Nashville, TN, USA) [
24].
ROSA26-lox-stop-lox-YFP reporter (
ROSA-YFP) mice, obtained from the Jackson Laboratory, were crossed with
K19
CreERT
mice to generate
K19
CreERT
/
ROSA-YFP mice.
Atg5
flox/flox
mice have been described previously [
25] and were kindly provided by Dr. Noboru Mizushima (Tokyo University, Tokyo, Japan).
Atg5
flox/flox
mice were crossed with
K19
CreERT
mice to generate
Atg5
flox/flox
/K19
CreERT+
mice. C57BL/6 J (B6) mice were from CLEA Japan (Tokyo, Japan). All mice used were of the B6 background. For tamoxifen (TAM) treatment, mice were injected with 10 mg/kg TAM (Cayman Chemical, Ann Arbor, MI, USA) intraperitoneally (i.p.) three times (on days 1, 3, and 5). For CQ treatment, mice were injected with 50 mg/kg CQ (Sigma-Aldrich, St. Louis, MO, USA) i.p. at the times indicated. All animal studies were approved by the Animal Care and Use Ethics Committee at the Institute for Adult Diseases, Asahi Life Foundation.
Tumor induction
Atg5
flox/flox
/K19
CreERT+
(Atg5-deficient mice) and Atg5
flox/flox
mice (Cre-negative littermates, used as control mice) were injected i.p. with 12.5 mg/kg AOM (Sigma-Aldrich) on day 1. After 5 days, mice received water supplemented with 2.5 % DSS (MP Biomedicals, Irvine, CA, USA) for 5 days, after which the mice were maintained on regular water for 14 days and subjected to two further DSS treatment cycles. On days 60, 62, and 64, the mice were injected i.p. with 10 mg/kg TAM. On day 67, the mice were sacrificed to analyze colon tumors. Macroscopic colon tumors were counted, and the longest diameter of each tumor was measured using a caliper in a blinded fashion.
Cell lines
Four established colon cancer cell lines, HCT116, SW48, DLD1, and SW837, were used [
26,
27]. HCT116 and SW48 cells harbor the wild type p53 gene, while DLD1 and SW837 cells are mutated in the p53 gene [
26,
27]. HCT116 cells were maintained in McCoy’s 5A medium containing 10 % fetal bovine serum (FBS). SW48 and SW837 cells were maintained in Leibovitz’s L-15 medium containing 10 % FBS. DLD1 cells were maintained in RPMI 1640 medium containing 10 % FBS. Hank’s Buffered Salt Solution (HBSS) was used to induce amino acid starvation conditions. The cell lines were obtained from the American Type Culture Collection (Baltimore, MD, USA), and all media formulations were obtained from Sigma-Aldrich.
Antibodies and reagents
The following primary antibodies were used for immunoblotting and immunohistochemistry: anti-Atg5, anti-Atg7, anti-LC3, anti-p62, anti-PARP, anti-cleaved caspase 3, anti-BiP, anti-p53, anti-phospho-eIF2α, anti-phospho-JNK, anti-phospho-Chk1, anti-phospho-p53, anti-actin (all from Cell Signaling, Beverly, MA, USA), anti-CK19, anti-proliferating cell nuclear antigen (PCNA) (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Ki67 (Dako, Carpinteria, CA, USA), anti-p53 (Vector Laboratories, Birmingham, CA, USA), anti-CHOP (Thermo Fisher Scientific, Waltham, MA, USA), and anti-yellow fluorescent protein (YFP) (MBL, Tokyo, Japan). CQ diphosphate salt (Sigma-Aldrich) was dissolved in PBS at the indicated concentrations.
RNA interference
Small interfering RNAs (siRNAs) targeting Atg5 (MISSION siRNA, Sigma-Aldrich) and BiP (Dharmacon siGENOME SMART pool siRNA, GE Healthcare, Pittsburg, PA, USA) or the non-silencing control (5’-AATTCTCCGAACGTGTCACGT-3’) were transfected into cells using Lipofectamine RNAimax (Invitrogen, Waltham, MA, USA) for 72 h. Immunoblotting was used to verify that the siRNAs reduced cellular protein expression by more than 80 %.
Immunoblotting
Cells or mouse tissues were disrupted in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X [Sigma-Aldrich], 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin). The lysates were electrophoresed by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (GE Healthcare), and blocked for 1 h in Tris-buffered saline-Tween 20 with 5 % dry milk. The membrane was incubated overnight at 4 °C with the primary antibody and subsequently washed and incubated with a secondary horseradish peroxidase (HRP)-conjugated antibody. The immunocomplexes were detected using a chemiluminescence detection kit (Luminata Classico Western HRP; Merck Millipore, Darmstadt, Germany). Images were obtained using the LAS 4000 image analyzer (Fujifilm, Tokyo, Japan).
Immunohistochemistry
Formalin-fixed and paraffin-embedded mouse tissues were cut at a thickness of 3 μm, deparaffinized, and incubated in citrate buffer at 95 °C for 40 min for antigen retrieval. Endogenous peroxidase activity was blocked using 3 % H2O2. The tissue sections were incubated overnight with rabbit primary antibody, followed by a polyclonal goat anti-rabbit immunoglobulins/biotinylated secondary antibody (DAKO) for 30 min, and then exposed to Streptavidin/HRP (DAKO) for 10 min. The Mouse-on-Mouse Immunodetection kit (Vector Laboratories) was used as the mouse primary antibody for mouse tissues, according to the manufacturer’s instructions. The chromogenic reaction was performed using the Liquid DAB Substrate Chromogen System (Dako). YFP expression in the colons of mice was examined by immunofluorescence staining. The tissues were incubated with anti-YFP antibody followed by Alexa Flour 594-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR, USA) for 30 min, and the nuclei were visualized by DAPI staining (Takara, Tokyo, Japan) for 1 min. The proportion of Ki67-positive cells was determined by counting more than 500 cells in three Ki67-concentrated lesions, and the numbers of cleaved caspase 3-positive cells per field were counted.
Real-time RT-PCR
Total cellular RNA samples were isolated from mouse colon tissues and from HCT116 cells using NucleoSpin RNA II (Takara). The cDNAs were generated from 1-μg total RNA by reverse transcription using Transcriptor Universal cDNA Master (Roche, Branchburg, NJ, USA). The mRNA expression levels of mouse Atg5, interleukin (IL) 1-β, chemokine (C-X-C motif) ligand 1 (CXCL1), p53 upregulated modulator of apoptosis (PUMA), Noxa, Bax, CHOP, BiP, spliced X-box binding protein 1 (sXBP1) and of human Atg5, Ulk1, Atg7, C-X-C chemokine receptor type 4 (CXCR4), SOX9, CD44, CXCL1, IL8, cellular inhibitor of apoptosis protein 1 (cIAP1), PUMA, Noxa, Bax, CHOP, BiP, and spliced XBP1 were determined by quantitative real-time RT-PCR using the LightCycler 480 instrument II real-time PCR System (Roche). GAPDH mRNA was used as an internal control. The primer sequences used are available on request.
Flow cytometric analysis of apoptosis
Colon cancer cells (1.0 × 104/ml) were seeded into 24-well plates and 24 h later were treated with siRNAs for 72 h or with CQ for 24 h. Cells were detached by trypsinization and exposed to early and late apoptotic detection reagents (GFP Certified Apoptosis Detection Kit, Enzo life Sciences, Farmingdale, NY, USA) for 15 min. Samples were analyzed by flow cytometry using the FL2 channel for Annexin V detection (early apoptosis) and the FL3 channel for PI detection (late apoptosis) using a fluorescence-activated cell sorter (FACS) (accuriC6, BD Biosciences, Ann Arbor, MI, USA).
Cell growth assay
The extent of cell growth was assessed using the Cell Counting Kit-8 (CCK-8) from Dojindo Laboratories (Kumamoto, Japan). Cells (1.0 × 104/ml) were seeded into 96-well plates (day 0) and 24 h later were transfected with siRNAs (day 1) for 48 h. CCK-8 solution was added to each well for 2 h. The absorbance at 450 nm was determined using a multi-mode reader (SpectraMax, Molecular Devices, Sunnyvale, CA, USA).
Statistical analysis
Statistical analysis was performed using Welch’s t-test, Mann–Whitney U-test, and one-way analysis of variance with Dunnett’s multiple comparison test, where appropriate. Differences were considered statistically significant at p < 0.05.
Discussion
In this study, using
Atg5
flox/flox
/K19
CreERT
mice and the AOM/DSS procedure, we examined whether autophagic inhibition is effective in colon cancer treatment and showed that suppression of autophagy exerts anti-colon cancer effects
in vivo. Our results, schematically summarized in Fig.
5f, suggested that blocking autophagy has the potential to treat colon cancer through apoptosis induced by p53 activation and ER stress. In addition, we demonstrated in colon cancer cell lines that the anti-colon cancer effect of autophagic inhibition depends on p53 status, and that UPR inhibition is a prospective alternative treatment candidate for colon cancer.
Several authors have reported that autophagic inhibition has promising anti-colon cancer effects in human colon cancer cell lines [
37,
38]. The mouse model used in this experiment, exploiting Cre/loxP technology, enabled examination of not only molecule-specific but also CK19-expressing cell-specific therapies. Molecular functions, including those of autophagy related-proteins, sometimes differ according to the organ or cell type, such as somatic versus hematopoietic cells. In fact, many tissue-specific autophagy ablation models using the Cre/loxP system have been published. For example, hepatocyte-specific autophagic inhibition in Alb-Cre mice resulted in multiple liver tumors, while dendritic cell-specific autophagic inhibition in CD11c-Cre mice exhibited antigen presentation dysfunction [
10,
39]. Our CK19-Cre model has the advantage of greater CK19 expression in colon tumors than in normal colonic mucosa. Indeed, we showed that autophagic inhibition had a suppressive effect on tumor size but not on tumor number. Therefore, we believe that TAM-induced autophagic inhibition is a suitable model for examining colon cancer progression in a setting in which the influence on cancer initiation has been minimized. Regarding cancer initiation, knockout mouse models displaying constitutive autophagic inhibition appear to be useful. Since genome-wide association studies have implicated that autophagy related 16-like 1 (
ATG16L1) gene polymorphisms are associated with risk of inflammatory bowel disease [
40,
41], previous researchers have established autophagy-deficiency in an intestinal epithelial cell model using Villin-Cre mice [
19,
42‐
44]. These mice showed Paneth cell abnormalities and insufficient defense against bacteria. The existence of a cascade resulting from infection and leading to inflammation and cancer has been widely accepted [
34], and it is likely that inhibition of constitutive autophagy in the intestine ultimately leads to cancer initiation. However, further investigation is required to clarify the role of autophagy in cancer initiation.
With clinical use of molecular targeting agents, the development of strategies to overcome cases of drug resistance, such as patients unresponsive to anti-EGFR agents who possess specific gene mutations, such as K-RAS mutations, is a major challenge [
45,
46]. In this experiment, human colon cancer cell lines harboring mutant p53 genes showed resistance to autophagic inhibition, in accordance with a previous report [
38]. Although mutation of the p53 gene is a major genetic alteration in human colon cancer [
47,
48], the absence of this mutation has been reported in rodent colon tumors [
22,
49]. Therefore, our
in vivo results showing anti-tumor effects by autophagic inhibition might be due to the normal p53 gene expression in this rodent model. A combination of molecular-targeted agents seems to be a valid strategy to overcome drug resistance. We assume that anti-autophagic and anti-UPR drugs have a positive interaction, since UPR up-regulation under autophagic inhibition seems to be a compensatory pathway for colon tumor cell survival.
In fact, HCT116 cells showed an elevation in apoptosis after co-transfection with siRNAs targeting Atg5 and BiP compared with transfection of the individual siRNAs (Fig.
5c). There are three major ER stress sensors: protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF6), and inositol-requiring enzyme 1 (IRE1) [
50]. These sensors are in a complex that contains BiP in the unstressed state. ER stress correlation with the dissociation of BiP and activates the censors [
50]. Since our analysis of autophagic inhibition showed that sXBP1 mRNA and JNK phosphorylation were not up-regulated, we believe that the ATF6-XBP1 and IRE1-XBP1-JNK pathways are not critical for UPR activation induced by loss of autophagy. In contrast, CHOP and eIF2α phosphorylation were upregulated under autophagy-suppressed conditions, indicating activation of the PERK-eIF2α-CHOP axis.
In this study, using siRNA-mediated BiP silencing, we found that BiP plays roles in ER stress and subsequent apoptosis (Fig.
5a, d), as has also been reported in previous studies [
35,
51]. If the activated UPR fails to alleviate ER stress in the BiP-silenced state, pathways for apoptosis including the induction of pro-apoptotic transcriptional factor CHOP can become activated [
52]. We also showed that autophagic inhibition caused ER stress, leading to up-regulation of BiP protein
in vivo and
in vitro (Fig.
2d and
5a). On the other hand, BiP mRNA reportedly is not always up-regulated under ER stress, since BiP expression is also controlled at the translational level [
53]. This is a possible explanation for our finding that autophagic inhibition did not increase BiP mRNA
in vitro (Fig.
4a). Under amino acid-free conditions, the effect of autophagic inhibition on increased BiP protein expression was exaggerated (Fig.
5a) and is in line with previous reports showing the importance of autophagy and UPR for cell survival, especially under starvation conditions [
4,
18].
In the clinical setting, the effect of molecular-targeted therapy depends on many factors, such as genomic diversity among patients, the drug’s influence on somatic and hematopoietic cells, the drug’s interactions in cancer microenvironments, among other factors. The effect of CQ treatment may differ from that of genetic inhibition of autophagy. For example, CXCL1 mRNA expression in HCT116 cells was up-regulated by CQ treatment but not by siRNA targeting Atg5 (Fig.
5a). CQ treatment has the potential to affect hematopoietic cells directly or through stimulation of epithelial cells to induce chemokines. We also detect up-regulation of IL1-β mRNA by inhibition of autophagy
in vivo (Fig.
3a). It is in line with the previous report that shows the up-regulation of IL1-β and IL18 in autophagy-deficient mice [
54]. These cytokines might affect the immune response
in vivo and it might be a plausible explanation of the differences between up-regulated mRNAs in the analysis of mice (Fig.
3a) and cell-based study (Fig.
4b). We believe the rodent model in this experiment could be applied to various situations using multiple mutant mouse strains and the Cre/loxP technology, as well as for
in vivo administration of potential reagents to investigate colon cancer mechanisms.
Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
K Sakitani, Y Hikiba performed experiments. K Sakitani, Y Hirata wrote the paper. K Sakitani, Y Hirata, Y Hikiba, Y Hayakawa, SI, HS, NS, TS, HK, K Sakamoto, HN, KT, SM, TI, SK, KK analyzed data. All authors approved the final manuscript.