Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer

Accepted Manuscript Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer Xi Cheng, Haoran Feng, Haoxuan Wu, Zhijian Jin, Xiaonan Shen, Jie Kuang, Zhen Huo, Xianze Chen, Haoji Gao, Feng Ye, Xiaopin Ji, Xiaoqian Jing, Yaqi Zhang, Tao Zhang, Weihua Qiu, Ren Zhao PII:

S0304-3835(18)30385-9

DOI:

10.1016/j.canlet.2018.05.046

Reference:

CAN 13930

To appear in:

Cancer Letters

Received Date: 14 March 2018 Revised Date:

24 May 2018

Accepted Date: 25 May 2018

Please cite this article as: X. Cheng, H. Feng, H. Wu, Z. Jin, X. Shen, J. Kuang, Z. Huo, X. Chen, H. Gao, F. Ye, X. Ji, X. Jing, Y. Zhang, T. Zhang, W. Qiu, R. Zhao, Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer, Cancer Letters (2018), doi: 10.1016/j.canlet.2018.05.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer

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Xi Chenga,b,c1, Haoran Fenga,b1, Haoxuan Wua,b1, Zhijian Jina,b, Xiaonan Shend, Jie Kuanga,b, Zhen Huoa,b, Xianze Chena,b, Haoji Gaoa,b, Feng Yea,b, Xiaopin Jia,b, Xiaoqian Jinga,b, Yaqi Zhanga,b, Tao Zhanga,b***, Weihua Qiua,b**, Ren Zhaoa,b,*.

Medicine, Shanghai, China, 200025

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a. Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of

b. Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University

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School of Medicine, Shanghai, China, 200025

c. Department of Biological Sciences, Boler-Parseghian Center for Rare and Neglected Diseases, Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN 46556; d. Department of Gastroenterology, Renji Hospital, Shanghai Jiaotong University School of

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Medicine, Shanghai, China, 200001

These authors contributed equally to this work.

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*Corresponding author: Ren Zhao, MD&PHD, E-mail: [email protected]

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**Corresponding author: Weihua Qiu, MD&PHD, E-mail: [email protected] ***Corresponding author: Tao Zhang, MD, E-mail: [email protected] Financial support: Nature Science Foundation of China (NSFC: 81772558), Clinical Skill and Innovation 3-year program of Shanghai Hospital Development Center (16CR2064B), Ph.D. Innovation Fund of Shanghai Jiaotong University School of Medicine (BXJ201709) and “Visiting Programs for Graduate Students of Shanghai Jiaotong University School of Medicine”.

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Abstract Apatinib, a novel tyrosine kinase inhibitor (TKI), has been confirmed for its efficacy and safety

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in the treatment of advanced gastric carcinoma and some other solid tumors. However, the direct functional mechanisms of tumor lethality mediated by apatinib have not yet been fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, in this study, we demonstrated that apatinib could induce both apoptosis and autophagy in human

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colorectal cancer (CRC) via a mechanism that involved endoplasmic reticulum (ER) stress.

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Moreover, activation of the IRE1α pathway from apatinib-induced ER stress is responsible for the induction of autophagy; however, blocking autophagy could enhance the apoptosis in apatinib-treated human CRC cell lines. Furthermore, the combination of apatinib with autophagy inhibitor chloroquine (CQ) tends to have the most significant anti-tumor effect of CRC both in vitro and in vivo. Overall, our data show that because apatinib treatment could induce ER stress-

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related apoptosis and protective autophagy in human CRC cell lines, targeting autophagy is a promising therapeutic strategy to relieve apatinib drug resistance in CRC.

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Keywords:

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Tyrosine kinase inhibitor; Chloroquine; IRE1α; Drug resistance

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1. Introduction Colorectal cancer (CRC) is one of the most common cancers in the world, accounting for

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nearly 8.5% of total cancer related death annually [1-3]. Although most primary tumors can be surgically resected, late-stage CRC patients still suffer from poor 5-year survival, especially stage IV CRC patients, who have a less than 10% 5-year over survival rate[4]. Moleculetargeting agents inhibit the proliferation of tumor cells using the molecular biological differences

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between tumor cells and normal cells to kill tumor cells. Complete surgical treatment plus

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standard chemotherapy regimens and molecular-targeting drugs could significantly contribute to prolonging CRC patients’ overall survival (OS)[5, 6].

The endoplasmic reticulum (ER), composed of membranous tubules and vesicles, has many essential functions, such as being a Ca2+ reservoir, and facilitates the secretion of correctly

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folded proteins [7, 8]. Distressing normal ER processes would lead to an accumulation of unfolded proteins and trigger the unfolded protein response (UPR)[9]. To compensate for the damage induced by ER stress, UPR enhanced the protein-folding ability of the ER and facilitated

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proteasomal degradation of unfolded or misfolded proteins by reducing global protein synthesis with autophagy[10]. ER stress could also induce apoptosis by many chemotherapeutic drugs,

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contributing a prominent mode of cytotoxicity [11, 12]. Apatinib, a novel tyrosine kinase inhibitor (TKI), can inhibit multiple tumor-related kinases, such as vascular endothelial growth factor receptor-2 (VEGFR-2), rearranged during transfection (RET), platelet-derived growth factor- β (PDGF- β ) and other tumor-correlated kinase. Our previous study indicated that apatinib could inhibit solid tumor angiogenesis by suppressing Akt/GSK3β/ANG[13-15]. In this study, we discovered that direct stimulation of ER stress by

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apatinib in CRC induced autophagy through the upregulation of the IRE1 signaling pathway; meanwhile, inhibiting autophagy could stimulate ER stress-associated CRC cell apoptosis both

be a potential therapeutic implication for the treatment of CRC.

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2. Materials and Methods

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in vitro an in vivo. Furthermore, we hypothesized that targeting apatinib-induced autophagy may

2.1 Cell culture and reagents

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Human CRC cell lines HT29 and HCT116 were purchased from ATCC (Rockville, MD). All cells were cultured in high glucose PRMI, 10% fetal bovine serum (FBS), and 1% P/S (100 IU/ml penicillin and 100 IU/ml streptomycin) at 37°C and 5% CO2. To measure the autophagic flux of CRC cell lines, HT29 and HCT116 were transfected with RFP-GFP-hLC3 lentivirus (GM-

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1314L204H-S, Genomeditech, Shanghai), transfection details as described previously[16]. Apatinib was obtained from Hengrui Medicine Co. Ltd. (Jiangsu, China), dissolved in DMSO and then diluted with RMPI medium for vitro experiments, and it was dissolved in CMC for vivo

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experiments. Chloroquine (CQ) (Sigma, C6628, USA) was dissolved in PBS and diluted with RMPI for experiments. JNK inhibitor (SP600125) was purchased from Santa Cruz Biotechnology

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Inc (sc200635, Santa Cruz, USA). 2.2 Cell viability assays and colony formation assay The cytotoxicity of apatinib on CRC cells was estimated using CCk-8 assay (Dojindo, Japan). As we previously described [17], cells were plated in 96-well plates at 2,000 cells per well 24 h before the start of treatment. Then, cells were treated with apatinib at 0, 5, 10, 20, 40 and 60 µM for 24, 48 and 72 h. For the colony formation assay, 1,000 cells were plated in 6-well plates and

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cultured with different concentrations of apatinib at 37 C for 2 weeks. After staining with 0.1% crystal violet in methanol for 30 min, the number of colonies were visualized and quantified.

Transmission

electron

microscopy

was

used

to

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2.3 Electron microscopy identify

autophagosomes/autolysosomes as we previously described [18].

ultrastructure

of

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2.4 Confocal microscopy

the

Cells were fixed using 4% paraformaldehyde (158127, Sigma) for 20 min. Then, cells were

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permeabilized with 0.1% Triton X-100 (T8787, Sigma) for 15 min at room temperature, washed with PBS and blocked with PBS containing 0.5% bovine serum albumin (BSA) and 0.15% glycine (BSA buffer) for 1 h at room temperature. The slices were treated with DAPI (32670,

2.5 Western blot

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Sigma) and imaged using confocal microscopy (LSM510, Zeiss).

Western blot was performed as previously described [19]. Antibody LC3 (12741), IRE1

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(9956), PERK (5683), CHOP (9956), JNK (9252) and P62 (23214) were purchased from Cell Signaling, Caspase4 (M029-3) was purchased from MBL international corporation (Japan),

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GRP78 (ab21685) were purchased from Abcam (USA). GAPDH (sc-32233) was purchased from Santa Cruz Biotechnology Inc (Santa Cruz (USA). 2.6 Xenograft tumor model, immunochemistry and TUNEL assay CRC xenograft mice models were performed as previously described using human CRC cell line HT29[19]. Two weeks after inoculation, the mice were randomly divided into four groups: control group (PBS only), CQ single treatment group, apatinib single treatment group and CQ

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plus apatinib group (N=4 per group). Mice were administered a daily oral gavage with 50 mg/kg PBS, CQ, apatinib or CQ plus apatinib. Mice were killed 40 days after CRC cell inoculation, and xenograft tumors were weighed and fixed for immunohistochemistry (IHC) staining. The staining

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was conducted to the manufacture’s protocol (Immunostain SP kit, Dako Cytomation, USA) and the results of IHC were determined by the staining intensity and the number of positive cells. Antibodies used for IHC included antibodies against Ki67 (9027, Cell Signaling) and CD31 (3528,

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Cell Signaling). Three experienced pathologists who were blinded to patient’s characteristics independently determined an immunohistochemistry result. A TUNEL assay was performed to

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detect the apoptotic cells, through an In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science, USA), and a detailed operational procedure was performed as previously described [15].

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2.7 Flow cytometry assay

An Annexin V-FITC Apoptosis Detection kit (BD Pharmingen, Franklin Lakes, NJ, USA) was used to detect the apoptosis of cells. The detailed procedure was similar to that previously

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described [17].

2.8 Intracellular Ca2+ detection

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The levels of intracellular Ca2+ were detected using Fluo-3 AM (S1056, Beyotime, Shanghai, China). Human CRC cell HT29 and HCT116 were treated with different concentrations of apatinib (0 µM, 5 µM and 10 µM) for 24 h, and then, cells were treated and harvested according to manufacturer’s instruction. Flow cytometry were used to detect the fluorescence intensities of Fluo-3 combined with Ca2+. 2.9 RNA Interference

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Small interfering RNAs (siRNAs) targeting ATG5, IRE1-α were purchased from GenePharma， (Shanghai, China). The siRNA Sequence of ATG5 is (5'-GGAGTCACAGCTCTTCCTT-3'),

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IRE1- α (5'-CTACTGGATGATAAATTTGCTTCA-3'). Human CRC cell lines HT29 and HCT116 were both transfected with ATG5-siRNA and IRE1-α-siRNA using Lipo3000 (Thermo

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Fisher Scientific, USA), using the detailed procedure similar to that previously described [20]. 2.10 Statistical Analyses

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An analysis of variance (ANOVA) and Student’s t test were used for comparison among groups. The Mann-Whitney U test was used for comparison of tumor volume. Categorical data was evaluated with a chi-square test or Fisher exact test. A p-value less than 0.05 was significant.

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3. Results

3.1 Apatinib inhibits colorectal cancer cells proliferation To identify the effect of apatinib on the proliferation of CRC cell lines, human CRC cell lines

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HT29 and HCT116 were treated with a series of different concentrations of apatinib (0, 5, 10, 20, 40 and 60 µM) for 24 h, 48 h and 72 h. We discovered that apatinib at concentrations of 5, 10, 20,

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40, and 60µM could inhibit human CRC cell lines HT29 and HCT116 in a time- and dosedependent manner (Fig. 1A); the IC50 values of these 2 CRC cells were shown in (Table 1) and (Fig. 1B). A contact-dependent proliferation assay by plate cloning formation was performed to further confirm the effect of apatinib on inhibiting CRC cell proliferation, where fewer clones were observed in HT29 and HCT116 after apatinib treatment (Fig. 1C). Taken together, these data

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indicated that apatinib could suppress CRC cell in vitro proliferation in a time- and dosedependent manner.

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3.2 Apatinib induces apoptosis in human CRC cell lines Induction of apoptosis is a prominent mode of cytotoxicity for many chemotherapeutic drugs. A flow cytometry assay was performed to investigate whether apatinib could induce CRC cell

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apoptosis in vitro. The percentage of apoptotic HT29 and HCT116 cells was higher after apatinib treatment compared to the control groups (Fig. 1D). The rates of apoptosis were (3.49% and

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5.06%) in HT29 and HCT116 cells, respectively, after being incubated with 5 µM apatinib. These rates were significantly higher than those observed in the control group (0.28% and 0.37% for HT29 and HCT116 respectively, P<0.05). Furthermore, the apoptosis rates increased after higher apatinib treatment concentrations (7.46% and 9.15% for HT29 and HCT116). Thus, our data

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demonstrated that apatinib could induce apoptosis in human CRC cell lines. 3.3 Apatinib induces ER stress in human CRC cells We previously discovered that apatinib could inhibit human CRC cell growth, but the

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underlying mechanisms have not been studied in detail. Furthermore, we discovered that apatinib treatment could induce the cellular vacuolization of CRC cells via light microscopy (Fig. 2A). To

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further investigate this phenomenon, transmission electron microscopy was used to intensively observe the apatinib-treated CRC cells. More dilated cytoplasmic vacuoles were identified in apatinib-treated CRC cells HT 29 and HCT116 (Fig. 2B) than the control group; dilated cytoplasmic vacuoles were recognized as dilated ER lumens, indicating an increase in ER stress. Calcium homeostasis is an important function of the ER; meanwhile, calcium disorders result in ER stress. In this study, Fluo-3 AM was used to label cytoplasmic calcium in the CRC cell lines

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HT29 and HCT116, then through flow cytometry assay, a significantly dose-dependent elevated cytoplasmic calcium level was detected in apatinib treatment CRC cell groups (Fig. 2C). To

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further clarify this, the expression levels of UPR target marker GRP78, IRE1-α, PERK and CHOP were assessed, we found that apatinib treatment could significantly increase the expression of GRP78, IRE1ɑ and CHOP (Fig. 2D). Meanwhile, the expression of PERK is upregulated due to

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the elevated dose of apatinib, but this difference was not significant. These findings strongly demonstrated that apatinib could significantly induce ER stress in human CRC cell lines.

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3.4 Apatinib induces ER stress-related apoptosis in CRC

Furthermore, if ER stress is so excessive or prolonged that it exceeds the protective ability of UPR, the highly insensitive UPR eventually results in cell apoptosis. Human caspase-4 is one of the closest paralogues of rodent caspase-12 and may be associated with ER stress. As shown in

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(Fig. 2D), the expression level of caspase-4 was significantly elevated after being incubated with different concentrations of apatinib; these data aligned with flow cytometry results in Fig. 1D. Additionally, after knockdown of IRE1ɑ, the UPR sensor that has a protective effect in ER stress

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related apoptosis, we observed a significant increase of apatinib-induced CRC apoptosis (Fig. 4A). These findings demonstrated that apatinib could induce ER stress-related apoptosis in human

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CRC cell lines.

3.5 Apatinib treatment could increase the autophagy flux in the CRC cell lines ER stress is closely related to the activation of autophagy that is an important and evolutionarily conserved mechanism for maintaining cellular homeostasis; meanwhile, autophagy is a "double-edged sword" for cancer cells as it can either promote or suppress survival and proliferation in the tumor microenvironment. To clarify whether apatinib could induce autophagy

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in CRC cell lines, we evaluated the expression of LC3II, a good autophagosome formation indicator, the expression of LC3II was parallel to the increase of apatinib treatment concentration (Fig. 3A). Meanwhile, autophagy-related protein Atg5 and P62, the hallmark of autophagy

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showed a similar tendency towards expression after apatinib treatment (Fig. 3A). Autophagy is a dynamic process, and both upregulation of autophagy as well as high levels of late autophagy inhibition cause autophagosome accumulation, which could increase the expression level of

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LC3II. To distinguish autophagy induction or autophagy inhibition, HT29 and HCT116 were transfected with RFP-GFP-LC3 fusion gene lentivirus, the autophagosomes were labeled with

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yellow dots (a merge of green and red fluorescence), while autolysosomes were labeled with red dots only. After being incubated with 10 µM apatinib for 24 h, we detected a parallel increased number of both yellow dots and red dots to the concentrations of apatinib through fluorescence images in these RFP-GFP-LC3-lentivirus-transfected HT29 and HCT116 cells, (Fig. 3B). After

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further investigating the confocal microscopy view, more positive LC3 cells and more LC3 positive dots per transfected cells were observed after apatinib treatment (Fig. 3C). Additionally, via electron microscopy, we identified an increased number of autophagosomes/autolysosomes in

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apatinib-treated CRC cells HT29 and HCT116 (Fig. 3D), which was consistent with the results of western and confocal microscopy. Taken together, these findings demonstrated that autophagic

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flux in human CRC cells was induced by apatinib. 3.6 The IRE1 ɑ signaling pathway is responsible for activation of apatinib-induced autophagy in CRC

To further assess whether autophagy was induced by ER stress in apatinib-treated CRC cells, siRNA targeting IRE1ɑ was used to knockdown the expression level of IRE1ɑ in human CRC

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cell lines. After knockdown of IRE1ɑ in apatinib-treated CRC cells, the expression levels of JNK, CHOP, P62 and LC3II were significantly decreased (Fig. 4A, C); meanwhile, a higher apoptosis rate was observed after downregulating IRE1ɑ in apatinib-treated CRC cells (Fig. 4A, B).

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Furthermore, after knockdown of IRE1ɑ in apatinib-treated RFP-GFP labeled CRC cells, a significant decrease of yellow and red dots was observed in the siIRE1ɑ group than the control

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group (Fig. 4D). IRE1ɑ could facilitate JNK pathway-induced ER- stress, and to further clarify

this, a JNK inhibitor SP600125 was employed to block IRE1ɑ-JNK pathway. As shown in (Fig.

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4E), we confirm a synergistic effect of JNK inhibitor with apatinib treatment. These data indicated that IRE1ɑ is responsible for apatinib induced CRC cell autophagy. 3.7 Inhibition of autophagy could enhance apatinib-induced apoptosis in human CRC cell

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lines

Autophagy could promote cell survival, and it could also induce cell death. To investigate the role apatinib played in CRC cells, chloroquine (CQ), an autophagy inhibitor, was used to block

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autophagy in apatinib-treated CRC cells. After CQ blocked the last steps of autophagic degradation, an enhanced apatinib-induced accumulation of LC3II was observed (Fig. 5A).

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Moreover, after knockdown of ATG5 in CRC cells, decreased levels of P62 and LC3II were detected (Fig. 5B). Furthermore, confocal microscopy was used to evaluate the blocking effect of CQ in apatinib-treated CRC cells. After CQ plus apatinib double treatment, RFP-GFP-LC3lentivirus-transfected HT29 and HCT116 cells expressed fewer yellow and red dots than the apatinib single treatment cells (Fig. 5C, D, E). Furthermore, through flow cytometry we discovered that the apoptosis rates of CRC cells were significantly higher in CQ plus apatinib CRC cells than apatinib single treatment group (HT29: 9.24% vs 16.83%, P<0.05) and (HCT116:

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9.85% vs 18.63%, P<0.05), (Fig. 5F). This means that blocking apatinib-induced autophagy could increase CRC cell line apoptosis in vitro, these data suggested that apatinib might induce protective autophagy in CRC cell lines. These data supported the idea that block autophagy could

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significantly elevate cell apoptosis in apatinib-treated human CRC cell lines in vitro.

3.8 In vivo tumor suppression induced by apatinib is enhanced by chloroquine(CQ)

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CQ could suppress autophagosome-lysosomal fusion events. We evaluated whether treatment with CQ could potentiate the anti-tumor effect of apatinib in xenograft tumor modeling. CRC cell

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line HT29 was inoculated subcutaneously into the flank of nude mice and they were divided into four groups: (1) mice treated with PBS only, (2) mice treated with CQ only, (3) mice treated with apatinib only and (4) mice treated with apatinib plus CQ. The xenograft tumors were allowed to develop for 40 days after injection, and the treatment was started in day 14 after being

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subcutaneously inoculated.

No significant tumor suppression was investigated in the CQ only-treated group compared with the PBS-treated control group (Fig. 6A). Meanwhile, apatinib treatment with or without CQ

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could lead to a significant tumor suppression in all nude mice. Moreover, apatinib plus CQ treatment contributed a superior anti-tumor volume effect than either agent’s single treatment

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group (Fig. 6A, B, C). Immunohistochemistry was used to evaluate the proliferation marker Ki67 and angiogenesis marker CD31 of the xenograft tumor; we observed that apatinib plus CQ treatments most significantly decreased the Ki67 and CD31expression level than other groups (Fig. 6D). Furthermore, a significantly higher percentage of TUNEL-positive cells were detected in the apatinib plus CQ treatment group compared with other groups (Fig, 6D). Taken together, these findings strongly indicated that autophagy inhibitor CQ could enhance the anti-tumor effect of apatinib in vivo.

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4. Discussion CRC, despite numerous therapeutic and screening attempts, still remains a major life-

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threatening malignancy. The common treatment for CRC is standard chemotherapy and target therapy followed by complete surgical resection [21-23]. Apatinib is a highly selective tyrosine kinase inhibitor to VEGFR2, which exerts promising anti-tumor effect in various tumors[21, 24, 25]. Our previous study had indicated that apatinib could suppress anaplastic thyroid carcinoma

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(ATC) angiogenesis by blocking Akt/GSK3β/ANG pathway[15]. In this study, we demonstrated

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that apatinib could induce apoptosis in human CRC cell lines via ER stress. Furthermore, blocking ER stress-involved autophagy could significantly enhance apatinib-induced CRC cell line apoptosis both in vitro and in vivo (Fig. 7).

It has been reported that apatinib could promote autophagy and apoptosis through

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VEGFR2/STAT3/BCL-2 signaling in osteosarcoma[26]; meanwhile, Lu et al [27] described the phenomenon that apatinib could induce autophagy in CRC cell lines in vitro, but no further mechanism was discovered. Here in this study, we first discovered that apatinib could block CRC

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cell proliferation in vitro while inducing apoptosis in a time- and dose-dependent manner. Then, in further research on these apatinib-treated CRC cell lines, dilated cytoplasmic vacuoles were

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detected via electric light microscopy in apatinib-treated CRC cell lines and dilated cytoplasmic vacuoles were recognized as dilated ER lumens, which indicates an increase in ER stress. Furthermore, the western blot results also indicated an increasing trend of ER stress markers in apatinib-treated CRC cell lines. ER stress could activate autophagy in many cancer types; meanwhile, autophagy activation can facilitate survival of cancer cells (protective autophagy) or contribute to cancer cell death (cytotoxic/nonprotective autophagy) [28-30]. A better understanding of the dichotomy roles of

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autophagy in cancer biology can help to identify or design new drugs able to induce/enhance (or block) autophagic flux[31]. Here, in our study, we identified that ER-stress could drive autophagy in CRC cell lines via IRE1α pathway after apatinib treatment. Furthermore, we discovered that

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apatinib treatment-induced autophagy activation tends to have a protective role in human CRC cell lines. Meanwhile, blocking autophagy using CQ and siRNA targeting Atg5 could both significantly induce apoptosis of CRC cell lines in vitro. Additionally, using the combination of

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CQ plus apatinib treatment tends to have the most significant tumor suppressive effect in xenograft subcutaneous tumor in nude mice compared with single apatinib treatment or single CQ

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treatment.

Drug resistance is a universal but important phenomenon in cancer treatment. Many chemotherapy or targeting drugs tend to have drug resistance, which could decrease therapy efficacy[32, 33]. Apatinib was found to induce autophagy in several cancer types, but what kinds

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of autophagy it produced had not been fully discovered. Furthermore, how these treatments induce autophagy and whether such autophagy contributes to tumor suppression or is a mechanism of therapy resistance remains uncertain[33, 34]. We demonstrated that apatinib could

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induce autophagy via ER stress through the IRE1α signaling pathway in CRC cell lines;

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meanwhile, apatinib-induced protective autophagy in CRC cell lines might be a novel mechanism of drug resistance, and blocking autophagy could enhance the anti-tumor effect of apatinib in CRC cell lines.

In conclusion, our data unravel a novel mechanism whereby apatinib inhibits human CRC cell line proliferation and promotes apoptosis in a dose- and time-dependent manner. Additionally, apatinib could induce protective autophagy via ER stress, and blocking protective autophagy could enhance the anti-tumor effect of apatinib in human CRC cell lines. These findings indicate

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that targeting autophagy is a promising therapeutic strategy to improve the apatinib treatment efficacy of CRC cancer while providing a basis for future clinical trials to explore whether to use

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CQ as a potential adjuvant with apatinib in the treatment of CRC. Acknowledgement

Nature Science Foundation of China (NSFC: 81772558), Clinical Skill and Innovation 3-year

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program of Shanghai Hospital Development Center (16CR2064B), Ph.D. Innovation Fund of Shanghai Jiaotong University School of Medicine (BXJ201709) and “Visiting Programs for

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Graduate Students of Shanghai Jiaotong University School of Medicine”. Conflicts of Interest

No potential conflicts of interest are disclosed.

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Figure 1. Apatinib could suppress CRC cell proliferation and induce apoptosis in vitro. A. CCK8 assay showed that apatinib could suppress CRC cells in vitro proliferation in a timeand dose-dependent manner. B. IC50 of different apatinib-treated time of HT29 and HCT116. C. Apatinib could decrease plant colony numbers of CRC cells. D. Flow cytometry indicated that apatinib could induce CRC cell apoptosis. *, P < 0.05; **, P <0.01; ***, P < 0.001.

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Figure 2. ER stress induced by apatinib in CRC cell lines. A. Cellular vacuolization under a light microscope after apatinib treatment (10 µM) for 24 h. B.

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CRC cell lines HT29 and HCT116 incubated with apatinib (10 µM) for 24 h were visualized by electron microscopy, arrows point to ER. C. CRC cells HT29 and HCT116 were treated with different concentration of apatinib (0, 5 and 10 µM) for 24 h, then cells were loader with Fluo3am for 30 min, cytosolic calcium was monitored by flow cytometry. D. The expression level of

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IRE1α, PERK, GRP78, CHOP and Caspase4 were detected by western blot after being incubated

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with different concentrations of apatinib (0, 5, 10 and 20 µM) for 24 h. *, P < 0.05; **, P <0.01; ***, P < 0.001.

Figure 3. Apatinib-induced autophagy in CRC cell lines.

A. The expression level of autophagy markers Atg5, P62 and LC3 were evaluated by western blot

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after being incubated with different concentrations of apatinib (0, 5, 10 and 20 µM) for 24 h. B&C. Representative images and quantification results of early autophagosomes shown in 24 h apatinib (10 µM) incubated CRC cells HT29 and HCT116 transfected with RFP-GFP-LC3

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lentivirus. D. Electron microscopy showed increased autophagosomes/autolysosomes in CRC cells HT29 and HCT116 after being incubated with apatinib (10 µM) for 24 h. *, P < 0.05; **, P

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<0.01; ***, P < 0.001.

Figure 4. Autophagy after ER stress is activated by the IRE1α pathway. A. The expression levels of JNK, CHOP and Caspase4 decreased after downregulating IRE1α in 24 h apatinib- (10 µM) treated CRC cells HT29 and HCT116. B. More apoptosis cells were detected by flow cytometry after knockdown of IRE1α in HT29 and HCT116. C. The expression levels of JNK, P62 and LC3 decreased after downregulating IRE1α in 24 h apatinib- (10 µM)

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treated CRC cell HT29 and HCT116. D. Representative images of early autophagosomes shown in 24 h apatinib (10 µM) incubated CRC cells HT29 and HCT116 transfected with RFP-GFP-LC3 lentivirus after downregulating IRE1α. E. After treatment with JNK inhibitor SP600125, more

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apoptosis and less autophagy were detected in HT29 and HCT116. *, P < 0.05; **, P <0.01; ***, P <

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Figure 5. Autophagy protects against apatinib-induced CRC cell apoptosis.

A. The expression levels of P62 and LC3 were detected after treatment with apatinib (10 µM) or

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apatinib plus CQ (both 10 µM) for 24 h. B. The expression levels of P62 and LC3 were detected after knockdown ATG5 in apatinib- (10 µM) treated CRC cells HT29 and HCT116 for 24 h. C, D&E. Representative images of early autophagosomes shown in CRC cells HT29 and HCT116 transfected with RFP-GFP-LC3 lentivirus after being treated with PBS, apatinib(10 µM), apatinib

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plus CQ (both 10 µM) for 24 h. F. Apatinib plus CQ could induce more apoptosis of CRC cells than single apatinib-treated or control groups in CRC cells. *, P < 0.05; **, P <0.01; ***, P < 0.001.

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Figure 6. CQ could enhance the in vivo anti-tumor effect of apatinib in CRC cell lines. A. Apatinib plus CQ showed more significant anti-tumor effects than apatinib only treatment; CQ

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single treatment showed no anti-tumor effect. B. Quantified results of tumor weight. C. Quantified results of tumor volume. D. Representative IHC staining images of Ki67, CD31 and TUNEL assay were performed in these 4 groups of tumor samples. P < 0.05; **, P <0.01; ***, P < 0.001. Figure 7. Schematic diagram of autophagy mediated by ER stress enhances the apatinibinduced apoptosis of CRC cells.

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Table 1. IC50 value of apatinib in various CRC cell lines.

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Table1. IC50Value of Apatinib in variuos CRC cell lines IC50(µM) CRC cell lines 24h 48h 72h HT29 56.6 51.14 32.86 HCT116 48.76 44.11 29.25

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vitro. A. CCK8 assay showed that apatinib could suppress CRC cells in vitro proliferation in a time- and dose-dependent manner. B. IC50 of different apatinib-treated time of HT29

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and HCT116. C. Apatinib could decrease plant colony numbers of CRC cells. D. Flow cytometry indicated that apatinib could induce CRC cell apoptosis. *, P < 0.05; **, P

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<0.01; ***, P < 0.001.

Figure 2. ER stress induced by apatinib in CRC cell lines.

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A. Cellular vacuolization under a light microscope after apatinib treatment (10 µM) for 24 h. B. CRC cell lines HT29 and HCT116 incubated with apatinib (10 µM) for 24 h

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were visualized by electron microscopy, arrows point to ER. C. CRC cells HT29 and HCT116 were treated with different concentration of apatinib (0, 5 and 10 µM) for 24

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h, then cells were loader with Fluo-3am for 30 min, cytosolic calcium was monitored by flow cytometry. D. The expression level of IRE1α, PERK, GRP78, CHOP and Caspase4 were detected by western blot after being incubated with different concentrations of apatinib (0, 5, 10 and 20 µM) for 24 h. *, P < 0.05; **, P <0.01; ***, P < 0.001. Figure 3. Apatinib-induced autophagy in CRC cell lines.

ACCEPTED MANUSCRIPT A. The expression level of autophagy markers Atg5, P62 and LC3 were evaluated by western blot after being incubated with different concentrations of apatinib (0, 5, 10 and 20 µM) for 24 h. B&C. Representative images and quantification results of early

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autophagosomes shown in 24 h apatinib (10 µM) incubated CRC cells HT29 and HCT116 transfected with RFP-GFP-LC3 lentivirus. D. Electron microscopy showed increased autophagosomes/autolysosomes in CRC cells HT29 and HCT116 after being

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incubated with apatinib (10 µM) for 24 h. *, P < 0.05; **, P <0.01; ***, P < 0.001.

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Figure 4. Autophagy after ER stress is activated by the IRE1α pathway. A. The expression levels of JNK, CHOP and Caspase4 decreased after downregulating IRE1α in 24 h apatinib- (10 µM) treated CRC cells HT29 and HCT116. B. More apoptosis cells were detected by flow cytometry after knockdown of IRE1α in HT29

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and HCT116. C. The expression levels of JNK, P62 and LC3 decreased after downregulating IRE1α in 24 h apatinib- (10 µM) treated CRC cell HT29 and HCT116.

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D. Representative images of early autophagosomes shown in 24 h apatinib (10 µM) incubated CRC cells HT29 and HCT116 transfected with RFP-GFP-LC3 lentivirus

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after downregulating IRE1α. E. After treatment with JNK inhibitor SP600125, more apoptosis and less autophagy were detected in HT29 and HCT116. *, P < 0.05; **, P <0.01;

***, P < 0.001.

Figure 5. Autophagy protects against apatinib-induced CRC cell apoptosis. A. The expression levels of P62 and LC3 were detected after treatment with apatinib (10 µM) or apatinib plus CQ (both 10 µM) for 24 h. B. The expression levels of P62

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after being treated with PBS, apatinib(10 µM), apatinib plus CQ (both 10 µM) for 24 h. F. Apatinib plus CQ could induce more apoptosis of CRC cells than single apatinib-treated or control groups in CRC cells. *, P < 0.05; **, P <0.01; ***, P <

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0.001.

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Figure 6. CQ could enhance the in vivo anti-tumor effect of apatinib in CRC cell lines.

A. Apatinib plus CQ showed more significant anti-tumor effects than apatinib only treatment; CQ single treatment showed no anti-tumor effect. B. Quantified results of

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tumor weight. C. Quantified results of tumor volume. D. Representative IHC staining images of Ki67, CD31 and TUNEL assay were performed in these 4 groups of tumor

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samples. P < 0.05; **, P <0.01; ***, P < 0.001.

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Figure 7. Schematic diagram of autophagy mediated by ER stress enhances the apatinib-induced apoptosis of CRC cells. Table 1. IC50 value of apatinib in various CRC cell lines.

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Highlights 1. We demonstrate that Apatinib could induce both apoptosis and autophagy in human

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colorectal cancer (CRC) via a mechanism that involved endoplasmic reticulum (ER)

2. We further discovered that activation of IRE1α pathway from Apatinib induced ER stress

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is responsible for the induction of autophagy,

3. We found that blocking autophagy could enhance the apoptosis in Apatinib treated human

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CRC cell lines, indicated that Apatinib could induce protective autophagy in CRC cells

4. We demonstrated that combination of Apatinib with autophagy inhibitor chloroquine (CQ) tend to have the most significant anti-tumor effect of CRC both in vitro and in vivo.

5. Our data identifies that Apatinib treatment could induce ER stress related apoptosis and

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protective autophagy in human CRC cell lines, targeting autophagy is a promising

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therapeutic strategy to relieve Apatinib drug resistance in CRC.

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No potential conflicts of interest are disclosed.