YC-1

Inhibition of autophagy by YC-1 promotes gefitinib induced apoptosis by targeting FOXO1 in gefitinib-resistant NSCLC cells
Hui Hu a, Xiao-Wei Zhang a, b, Lin Li a, b, Ming-Ning Hu a, b, Wen-Qian Hu a, b, Jing-Ying Zhang b, c,
Xiao-Kang Miao b, c, Wen-Le Yang b, c, Ling-Yun Mou a, c,*
a School of Life Science Lanzhou University, Lanzhou, 730000, PR China
b Basic Medical Sciences & Research Unit of Peptide Science, Chinese Academy of Medical Sciences, 2019RU066, Lanzhou University, Lanzhou, 730000, China
c Key Laboratory of Preclinical Study for New Drugs of Gansu Province, School of Basic Medical Science, Lanzhou University, Lanzhou, 730000, PR China

A R T I C L E I N F O

Keywords:
Autophagy Apoptosis Gefitinib FOXO
Lung cancer
Epidermal growth factor receptor

A B S T R A C T

Non-small cell lung cancer (NSCLC) is the most common cancer in the world. Gefitinib, an inhibitor of EGFR tyrosine kinase, is highly effective in treating NSCLC patients with activating EGFR mutations (L858R or EX19del). However, despite excellent disease control with gefitinib therapy, innate resistance and inevitable acquired resistance represent immense challenges in NSCLC therapy. Gefitinib potently induces cytoprotective autophagy, which has been implied to contribute to both innate and acquired resistance to gefitinib in NSCLC cells. Currently, abrogation of autophagy is considered a promising strategy for NSCLC therapy. In the present study, YC-1, an inhibitor of HIF-1α, was first found to significantly inhibit the autophagy induced by gefitinib by disrupting the fusion of autophagosomes and lysosomes and thereby enhancing the proapoptotic effect of gefi- tinib in gefitinib-resistant NSCLC cells. Furthermore, the combinational anti-autophagic and pro-apoptotic effect of gefitinib and YC-1 was demonstrated to be associated with an enhanced of forkhead boX protein O1 (FOXO1) transcriptional activity which resulted from an increase in the p-FOXO1 protein level in gefitinib-resistant NSCLC cells. Our data suggest that inhibition of autophagy by targeting FOXO1 may be a feasible therapeutic strategy to overcome both innate and acquired resistance to EGFR-TKIs.

1. Introduction
Lung cancer is the leading cause of cancer related deaths in China and worldwide and non-small cell lung cancer (NSCLC) accounts for approXimately 87% of lung cancer cases (Ferlay et al., 2013; Jemal et al., 2011). The first generation EGFR tyrosine kinase inhibitor, gefitinib, has exhibited good antitumor activity and survival benefit in NSCLC patients with activating EGFR mutations (L858R or EX19del). However, the ef- ficacy of gefitinib in NSCLC clinical therapy is challenged by innate and acquired resistance factors (Dancey, 2004; P´erez-Soler et al., 2004; Pao and Chmielecki, 2010).
Macroautophagy (autophagy hereafter) is an evolutionarily conserved transport pathway crucial for maintaining cellular homeo- stasis through a lysosome-dependent degradative pathway. A recent

autophagy can play a positive and negative role in the carcinogenesis and tumor progression of NSCLC. Several studies have shown that chronic EGFR-TKI treatment induces cellular stress and autophagy in a tyrosine kinase-independent manner, which leads to survival benefits and confers resistance to EGFR-TKI treatment in NSCLC cells (Drag- owska et al., 2013; Han et al., 2011; Jutten and Rouschop, 2014; Li et al., 2013; Sugita et al., 2015; Tang et al., 2015). Abrogation of autophagy, with pharmacological inhibitors has been shown to overcome EGFR-TKI resistance and is considered a promising strategy for NSCLC therapy (Zhang et al., 2015). Recent studies have demonstrated that the auto- phagy inhibitor chloroquine (CQ) and its derivative hydroXychloroquine (HCQ) restore sensitivity to gefitinib and erlotinib in NSCLC cells with innate and acquired resistance, respectively (Han et al., 2011; Li et al., 2013; Sugita et al., 2015; Tang et al., 2015). Furthermore, 3-MA and Baf

series of studies suggests that EGFR signaling is involved in autophagy

A1 were found to enhance the cytotoXic effect of cisplatin in

suppression (Wei et al., 2013) and EGFR-TKIs upregulate autophagy in many cancer cells (Dragowska et al., 2013; Li and Fan, 2010). Indeed,

cisplatin-resistant cells (Lin et al., 2017a; Zhang et al., 2015). In contrast, several lines of evidence have suggested that autophagy itself

* Corresponding author. Institute of Biochemistry and Molecular Biology, School of Life Science, Lanzhou University, 222 TianShui South Road, Lanzhou, 730000, PR China.
E-mail address: [email protected] (L.-Y. Mou).
https://doi.org/10.1016/j.ejphar.2021.174346
Received 6 April 2021; Received in revised form 7 July 2021; Accepted 12 July 2021
Available online 13 July 2021
0014-2999/© 2021 Elsevier B.V. All rights reserved.

may be a mechanism of caspase- and apoptosis-independent cell death (Brech et al., 2009; Nikoletopoulou et al., 2013). In NSCLC cells with an activating EGFR mutation, EGFR-TKIs activate autophagy by disrupting the interaction between Beclin-1 and EGFR and autophagy induction contributes to EGFR-TKI responses (Wei et al., 2013). Chen et al. observed that curcumin could overcome innate resistance in NSCLC cells by inducing autophagy-related cell death (Chen et al., 2019). The dual role of autophagy in both tumor suppression and promotion represents a major challenge when targeting this pathway in NSCLC cancer therapy. Therefore, determining the exact mechanisms linking autophagy and apoptosis in cancer will guide the potential use of autophagy inducers or inhibitors in the future treatment of NSCLC cancers.
3-(5-HydroXymethyl-2-furyl)-1-benzyl indazole (YC-1) was initially
described as an activator of soluble guanylyl cyclase (sGC), which pro- motes antiplatelet aggregation and vascular relaxation (Galle et al., 1999). Recent studies revealed that YC-1 could inhibit tumor growth, suppress angiogenesis and enhance the antitumor effects of chemo- therapy or radiation by downregulating hypoXia-inducible factor 1α (HIF-1α) under normal and hypoXic conditions (Chen et al., 2008; Chun et al., 2004; Liu et al., 2011). HIF-1α is critical for chemotherapy induced enrichment of cancer stem cells, making it an attractive thera- peutic target for solid tumor. Some studies have found hypoXia-inducible factors (HIFs) are associated with chemotherapy resistance in breast cancer and glioblastoma multiforme. Chemotherapy induces HIF-dependent expression of interleukin (IL)-6 and IL-8, which promote the breast cancer stem cells (BCSCs) and glioblastoma stem cells (GSCs) phenotype. Coadministration of HIF-1α inhibitors, which inhibits HIF-1α protein accumulation, blocked chemotherapy-induced expression of IL-6, IL-8, and MDR-1, and blocked BCSCs and GSCs enrichment (Likus et al., 2016; Lu et al., 2015; Sharifzad et al., 2019). We previously reported that gefitinib treatment induced EGFR arrest in the early endosome and YC-1 could potentiate the antitumor activity of gefitinib by promoting endocytic trafficking and degradation of EGFR in NSCLC cells with the wild-type (innate resistant) or L858R/T790M-mutated (acquired resistant) EGFRs (Hu et al., 2020). However, the endosomal arrest of EGFR induced by EGFR-TKIs is
associated with the initiation of autophagy (Tan et al., 2016), and whether YC-1 can affect the autophagy induced by gefitinib and thereby enhance the sensitivity of gefitinib to gefitinib-resistant NSCLC cells remains unknown. Here, we report for the first time that YC-1 could potentiate the proapoptotic effect of gefitinib by inhibiting gefitinib-induced autophagy by blocking the fusion of autophagosomes and lysosomes. Additionally, the data presented in this study demon- strated a new and unexpected function of FOXO1 in the combinational activity of gefitinib and YC-1 on autophagy and apoptosis, thus providing clues to explain why inhibition of autophagy overcomes both innate and acquired resistance to EGFR-TKIs.
2. Materials and methods
2.1. Chemicals, plasmids and cell lines
Gefitinib (Iressa), AS1842856 and chloroquine (CQ) were purchased from Selleck Chemicals (Houston, TX, USA). 3-Methyladenine (3-MA), bafilomycin A1 (Baf A1) and YC-1 were purchased from Sigma-Aldrich
(St. Louis, MO, USA). The compounds were dissolved in dimethyl sulf- oXide (DMSO) or PBS at 100 mM and aliquots were stored at 20 ◦C until use. The final working concentration of DMSO (plus compound) in
all working assays was lower than 0.5%. All the cell lines (NCI–H1975, NCI–H1944 and HeLa) were purchased from American Typical Cell Collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 medium containing 10% (vol/vol) fetal bovine serum at 37 ◦C in a hu-
midified incubator containing 5% CO2. The cell culture reagents, including RPMI-1640, FBS, trypsin-EDTA, sodium pyruvate and penicillin-streptomycin solutions, were all obtained from Life Technol- ogies (Paisley, UK).

2.2. GFP-LC3 dots quantitative analyses
HeLa cells stably expressing GFP-LC3 were generated as previously described (Lao et al., 2014). Briefly, HeLa cells were transfected with the pGFP-LC3 plasmid using Lipofectamine 2000 Transfection Reagent (Life Technologies, Carlsbad, CA, USA). The cells were then treated with 800 μg/ml G418 (Life Technologies, Grand Island, NY, USA) for 7 days. The surviving cell clones were continually cultured with 400 μg/ml G418 and named HeLa-GFP-LC3 cells. HeLa-GFP-LC3 cells were seeded overnight into a Collagen-I-coated Cell Carrier-96 Ultra Microplate (PerkinElmer, Waltham, MA, USA). After treatment with different con- centrations of compounds in triplicate for 24 h, the cells were fiXed with 4% paraformaldehyde (Boster, Shanghai, China) and washed 3 times with PBS. Nuclei were then stained with Hoechst 33342 (Life Technol- ogies, Waltham, MA, USA). Images were acquired with an LSM710 confocal microscope with a 63 oil immersion objective (Carl Zeiss, Oberkochen, Germany) and a High-Content Operetta imaging system with a 63 water immersion objective (Perkin Elmer, Waltham, MA, USA). The fluorescence intensity of LC3 puncta per cell was quantified with Harmony 4.8 software (Perkin Elmer, Waltham, MA, USA).
2.3. Stable HeLa cells overexpressing of RFP-GFP-LC3 (tfLC3) and confocal microscopy
cDNA encoding mRFP-GFP-LC3 (tfLC3) was excised from ptfLC3 (Addgene, 21074) and sub-cloned into the lentiviral vector, pCDH1- MCS1-EF1-puro (System Bioscience, CD510A-1). Stable HeLa cells overexpressing the mRFP-GFP-LC3 (tfLC3) gene were generated as previously described (Lin et al., 2017b). HeLa_tfLC3 cells were then seeded into a Collagen-I-coated Cell Carrier-96 Ultra Microplate over- night and then treated with different concentrations of compounds in triplicate. Images were acquired with an LSM710 confocal microscope with a 63 oil immersion objective and a High-Content Operetta im- aging system with a 63 water immersion objective. The mRFP-LC3 dots and yellow dots per cell were quantified with Harmony 4.8 software.
2.4. Real-time PCR analysis
Total RNA was extracted from cells using an RNeasy Mini Kit (Qia- gen, Hilden, Germany) after cells were harvested at the end of the cor- responding treatment. Total RNA (500 ng) was reverse transcribed in a volume of 20 μl by using the iScript cDNA Synthesis Kit (Bio-Rad, Her- cules, CA, USA). RT-PCR was subsequently performed using SYBR Green PCR Master MiX (Roche, Indianapolis, IN, USA) according to the man- ufacturer’s instructions. The PCR conditions were programmed as fol-
lows: initial denaturation at 95 ◦C for 10 min, followed by 45 cycles of
10 s at 95 ◦C, 60 ◦C for 10 s and an extension temperature of 72 ◦C for 15 s; 72 ◦C for then 10 min, and 55 ◦C increased to 95 ◦C for melt curves. The primers used in the quantitative RT-PCR are listed as follows:
SQSTM1 (5′-GCACCCCAATGTGATCTGC-3′, 5′-CGCTACA-
CAAGTCGTAGTCTGG-3′) GAPDH (5′-GGAGCGAGATCCCTCCAAAAT- 3′, 5′-GGCTGTTGTCATACTTCTCATGG-3′)
The reaction was performed on a Light Cycler 480 II Instrument (Roche Diagnostic, Inc.). EXpression levels were normalized against the
internal reference gene GAPDH, and the fold difference (relative abun-
dance) was calculated using the formula 2—ΔΔCT and plotted as the mean.
2.5. Immunofluorescence staining
HeLa-GFP-LC3 cells (as described in section 2.2) were fiXed in 4% paraformaldehyde and permeabilized with staining buffer containing 0.05% Triton X-100. Cells were then incubated with specific primary antibodies Mouse anti- LBPA (# MABT837 from Sigma) overnight at
4 ◦C and then washed with PBS and incubated for 1 h with the Alexa-594

donkey anti-mouse IgG (#A21203 from Life Technologies) at 20 μg/ml. Confocal imaging was performed using a Zeiss LSM710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) with a 63 oil immersion objective lens. To ensure that the images collected from all specimens were equivalent, hardware acquisition parameters were optimized for imaging cells under control conditions, and all acquisition parameters were then held constant for imaging cells under the remaining experimental treatment conditions. Channels were imaged sequentially to ensure that there was no bleed through of signals. No further adjustment of brightness, contrast, or threshold was performed. The Pearson correlation coefficient (PCC) was calculated to quantify the colocalization of LC3-GFP and LBPA. The PCC was quantified by Zen 2012 software (Carl Zeiss, Germany) between the stack of images from 2 channels.
2.6. Cell viability assay
The CellTiter-Glo Luminescent Cell Viability Assay (Promega, Mad- ison, WI, USA) was performed to determine the cell growth inhibition effect of the test compounds. Cells were exposed to the indicated con- centrations of test compounds for 24 h or 72 h. Luminescence was read on a microplate reader (Envision 2104, PerkinElmer). Luminescence data were converted to the growth fraction by comparison with the luminescence readout for the untreated control, and IC50 values were determined from the graphical data. Each cell line was tested in at least three independent experiments. The inhibitory rate of the drug combi- nation was calculated as (luminescence autophagy inhibitor – lumines-
cence combination)/luminescence autophagy inhibitor × 100%.
2.7. Caspase-3/7 activity assay
Cells were treated with test compounds at the indicated concentra- tions for 24 h. A caspase- 3/7-Glo assay (Promega, Madison, WI, USA) was performed according to the manufacturer’s instructions. Lumines- cence was read on a microplate reader (Envision 2104, PerkinElmer, Waltham, MA, USA). Caspase-3/7 activity was normalized to the num- ber of viable cells (as determined by the cell viability assay), and caspase-3/7-fold induction was determined as the caspase-3/7 activity ratio between treated and control cells.
2.8. Western blot analysis
All reagents and instruments were obtained from Life Technologies (Carlsbad, CA, USA), unless otherwise stated. Cells were collected by trypsin and washed with PBS. The cell pellet was lysed using RIPA buffer with protease and phosphatase inhibitors (Roche, Indianapolis, IN, USA). The total protein concentration was determined using a BCA Protein Assay Kit. Western blot and image analyses were conducted as described previously (Hu et al., 2020). Antibody catalog numbers and vendors were as follows: rabbit anti-FOXO1#2880, rabbit anti-phospho-FOXO1 (Ser256) #9461, rabbit anti-FOXO3α #12829, rabbit anti-caspase-3 #9662, rabbit anti-caspase-7 #9492, and rabbit anti-GAPDH #5174 (Cell Signaling Technology, Beverly, MA, USA); rabbit anti-LC3 #L8918 (Sigma, St. louis, MO, USA); and rabbit anti-p62/SQSTM1 #PM045 (MBL, Kyoto, Japan). The HRP-conjugated secondary antibodies used for western blot analysis were purchased from Cell Signaling Technology. Protein levels were quantified via ImageJ software and normalized to GAPDH. Levels in untreated cells were set equal to 1.
2.9. Quantification of lysosomal proteolytic activity
DQ-Red–BSA (Life Technologies, Carlsbad, CA, USA) was used to determine the lysosomal proteolytic activities according to the manu- facturer’s instructions. In brief, NCI–H1975 and NCI–H1944 cells were seeded into a Collagen-I coated Cell Carrier-96 Ultra Microplate and

allowed to grow overnight to yield approXimately 80% confluence. The cells were treated with DQ-Red–BSA (final concentration, 10 μg/ml) for 18 h. The cells were then washed 3 times with PBS before being treated with GEF, YC-1 or GEF/YC-1 for 6 h. The cells were then fiXed with 4% paraformaldehyde, and nuclei were then stained with DAPI. The 96-well plate was read by a High-Content Operetta CLS imaging system with a
40 water immersion objective. The fluorescence intensity of DQ-Red BSA was quantified with Harmony 4.8 software.

2.10. FOXO1 transcriptional activity reporter assay
Cells were transfected with a commercial FOXO reporter construct (BPS Bioscience, San Diego, CA, USA) using Lipofectamine 3000 Transfection Reagent (Life Technologies, Carlsbad, CA, USA). The construct contained a firefly luciferase gene under the control of mul- timers of the FOXO responsive element located upstream of a minimal promoter, and it was premiXed with a constitutively expressing Renilla luciferase vector, which serveed as an internal control for the trans- fection efficiency. After overnight treatment with compounds as indi- cated for 24 h, the cells were lysed and luciferase activity was analyzed using dual-luciferase assay kit according to the manufacturer in- structions (Promega, Madison, WI, USA). To obtain the normalized luciferase activity for the FOXO1 reporter, the ratio of firefly lumines- cence from the FOXO1 reporter to Renilla luminescence from the control Renilla luciferase vector was calculated.

2.11. RNA interference
To silence FOXO gene expression, FOXO1 and FOXO3α siRNAs were synthesized by Life Technologies. The FOXO1 target sequence was (5′- 3′) GAGCGTGCCCTACTTCAAG and corresponded to nucleotides 576–594 relative to the first nucleotide of the start codon of the human FOXO1 coding sequence. The human FoXo3α siRNA target sequence was (5′-3′) GAGCTCTTGGTGGATCATC, which corresponded to 690–708 bp (Potente et al., 2005). Control siRNA (AM4611) was purchased from Life Technologies. Cells were transfected with 100 nM siRNA at 80% confluence for 24 h. Lipofectamine RNAiMAX Transfection Reagent
(Life Technologies, Carlsbad, CA, USA) was used for transfections following the protocols provided by the manufacturer. Twenty-two hours after transfection, cells were seeded into 96-well plates or 6-well plates for different assays. Seventy-two hours posttransfection, a west- ern blot assay was performed with the specific antibody to check the efficiency of knockdown.

2.12. RT2 profiler PCR array
Pathway-focused gene expression profiling was performed using a 384-well human Apoptosis PCR array and human Autophagy PCR array (PAHS-012ZG and PAHS-084ZG, Qiagen, Valencia, CA, USA). Briefly, NCI–H1975 cells were seeded into 6-well plates and incubated with the control, 30 μM gefitinib, 25 μM YC-1 or gefitinib plus YC-1 for 24 h.
Total RNA was extracted and reverse transcribed as explained in the ‘RNA isolation and cDNA synthesis’ section. The cDNA template was miXed with the appropriate amount of RT2 SYBR-Green qPCR MastermiX (Qiagen GmbH, Hilden, Germany), 10 μl was aliquoted into each well of
the same plate, and then the real-time PCR cycling program was per- formed on Light Cycler 480 II Instrument (Roche Diagnostic, Inc.) following the manufacturer’s instructions. Qiagen’s online web analysis tool (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php) was used to analyze PCR array data, and fold change was calculated by determining the ratio of mRNA levels to control values by using the Δ
threshold cycle (Ct) method (2—ΔΔCt). All data were normalized to
GAPDH. Genes with a fold change 1.8 were considered to be genes of interest. Three biological replicates were investigated for each group.

Fig. 1. Effects of YC-1 on gefitinib (GEF) -mediated cell autophagy. (A) HeLa-GFP-LC3 cells were exposed to compounds as indicated for 24 h. Fluorescent images were obtained with a confocal microscope and typical images are presented. Scale bar represents 10 μm. (B) Cells were analyzed by a High-Content Operetta CLS imaging system with a 60 × water immersion objective. The fluorescence intensity of GFP-LC3 puncta/cell was quantified with Harmony 4.8 software. (C–D) Cells were treated with compounds as indicated for 24 h. Cell lysates and total mRNA were prepared for Western blotting assay (C) and real-time PCR analysis (D). Western blotting and real-time PCR assays were performed three independent times, and representative images are shown. Error bars indicate the mean S.D. from three
independent experiments. *P < 0.05, **P < 0.01, significant difference versus control cells. #P < 0.05, ##P < 0.01, significant difference versus gefitinib treated cells. The “n.s” indicates “no significant difference”. 2.13. Overall survival and pathway analysis of core genes The Kaplan-Meier plotter is a commonly used website tool for assessing the effect on survival of a great number of genes (Sza´sz et al., 2016). The log rank P value and hazard ratio (HR) with 95% confidence intervals were computed and are shown on the plot. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to visualize the core gene enrichment of pathways (P < 0.05). 2.14. Statistical analysis Statistical analyses were conducted with GraphPad Prism (GraphPad software Inc., CA, USA). Unless otherwise indicated, data were obtained from a minimum of three trials and are expressed as the mean ± stan- dard deviation (S.D.) (n = 3). Statistical significance was determined using two-tailed Student’s t-test and one-way ANOVA as specified. Dif- ferences were considered statistically significant at P < 0.05. 3. Results 3.1. YC-1 inhibits gefitinib-induced cell autophagy In our previous study, we observed that YC-1 could significantly potentiate the antitumor activity of gefitinib by promoting endocytic trafficking and degradation of EGFR in gefitinib-resistant NSCLC cells (Hu et al., 2020). The endosomal arrest of EGFR induced by EGFR TKIs is associated with the initiation of cytoprotective autophagy (Tan et al., 2016). Therefore, we studied whether YC-1 affected gefitinib-induced autophagy in gefitinib-resistant NSCLC cell lines. Here, we describe a protocol for the analysis of relevant parameters of autophagic fluX using HeLa cells stably expressing EGFP-LC3. Accumulated evidences have suggested that these cells are a convenient tool to determine the influ- ence of the downregulation or overexpression of specific proteins in the autophagic fluX as well as the analysis of autophagy-modulating com- pounds (Kaizuka et al., 2016; Sandra and Ricardo, 2016). The accu- mulation of GFP-LC3, a fluorescent autophagosomal marker, was detected with a fluorescence microscope. Gefitinib induced significant GFP-LC3 puncta accumulation in HeLa-GFP-LC3 cells after 24 h of (caption on next page) Fig. 2. YC-1 blocks the fusion of autophagosomes and lysosomes in gefitinib (GEF)-induced autophagy. (A) HeLa_tfLC3 stable cells treated with compounds as indicated for 24 h. Fluorescent images were obtained with a confocal microscope and typical images are presented. Scale bar represents 10 μm. (B) Cells were analyzed by a High-Content Operetta CLS imaging system with a 60 × water immersion objective. The fluorescence intensity of mRFP-LC3 and yellow dots/cell were quantified with Harmony 4.8 software. (C) Cells stained and analyzed by High-Content Operetta CLS imaging system with a 40 × water immersion objective. The fluorescence intensity of DQ-BSA/cell was quantified with Harmony 4.8 software. ((D) LC3-GFP/LBPA co-localization in cells after treated with GEF, YC-1 and GEF/ YC-1 for 24 h were analyzed by confocal microscope with 63 × oil immersion objective. (E) Colocalization was determined using the Zen 2012 software. Error bars indicate mean ± S.E.M. (n > 150 cells from three independent experiments). Scale bar represents 10 μm ##P < 0.01, significant difference versus gefitinib treated cells. (F) Caspase-3/7 activity was determined after GEF, YC-1, 3-MA or combination treatment as indicated for 24 h. Caspase readings were normalized to cell number determined by CellTiter-Glo Luminescent Cell Viability Assay. *P < 0.05, **P < 0.01, significant difference versus control cells. #P < 0.05, ##P < 0.01, significant difference versus gefitinib treated cells. δP < 0.05, δδP < 0.01, significantly different from the same treatment without 3-MA. Error bars indicate the mean ± S.D. from three independent experiments. treatment but YC-1 did not (Fig. 1, A-B and Fig. S2). However, the combination of YC-1 and gefitinib significantly enhanced the number of GFP-LC3 puncta, which was much greater than that induced by gefitinib single treatment. Additionally, in either NCI–H1944 cells with wild-type EGFR or NCI-1975 cells with L858R/T790M-mutated EGFR, immuno- blot analyses showed that gefitinib markedly increased the level of LC3-II protein, which was further enhanced by the combination of YC-1 and gefitinib (Fig. 1C). Increases in GFP-LC3 puncta and LC3-II protein can result from the induction or inhibition of autophagic fluX. SQSTM1, a receptor-binding protein mediating autophagy cargo degradation, serves as a link be- tween LC3 and ubiquitinated substrates (Heckmann et al., 2013; Yamamoto et al., 1998). To distinguish whether the enhanced LC3-II accumulation by YC-1 combined with gefitinib is due to the augmen- tation of autophagy or rather a block in downstream steps, we detected the protein level of SQSTM1. Immunoblot analysis showed a significant increase in SQSTM1 at 24 h after cotreatment with YC-1 and gefitinib (Fig. 1C), which reflects an inhibition of autophagic degradation. To determine whether the alteration of SQSTM1 protein was due to a change in mRNA level, we analyzed the expression of SQSTM1 mRNA levels in the presence of gefitinib and/or YC-1 for 24 h. As shown in Fig. 1D, in comparison with gefitinib single treatment, cotreatment of YC-1 and gefitinib did not significantly alter the SQSTM1 mRNA levels in the test cell lines. These results suggest that YC-1 enhances the total SQSTM1 protein level without interfering with mRNA transcription. Together, these data indicate that YC-1 inhibits gefitinib-induced autophagy. 3.2. YC-1 blocks the fusion of autophagosomes with lysosomes in gefitinib-induced autophagy A later step of autophagy is the fusion of autophagosomes with ly- sosomes and inhibition of this process damages the autophagic pathway. Tandem mRFP-GFP-LC3 fluorescence analysis was used to visualize the transition from neutral autophagosomes to acidic autolysosomes (Kimura et al., 2007). In this assay, RFP fluorescence was more stable in acidic compartments of lysosomes whereas GFP fluorescence was rapidly quenched. Accordingly, autophagy induction results in increased numbers of both yellow and red puncta while autophagy in- hibition at a later step (inhibition of autophagosome maturation or fusion with the lysosome) results in an increase in the number of yellow puncta and a concurrent decrease in red puncta (Yoshii and Mizushima, 2017). We found that gefitinib (30 μM) treatment significantly increased the number of yellow and red puncta (Fig. 2, A-B) in HeLa_tfLC3 stable cell line, suggesting the formation of autolysosomes. However, as shown Table 1 IC50 Value of compound in NCI–H1975 or NCI–H1944 Cells, 72 h. Compound NCI–H1975 NCI–H1944 GEF 11.56 ± 2.93 μM 16.82 ± 2.10 μM YC-1 23.04 ± 5.99 μM 7.74 ± 4.51 μM CQ 22.25 ± 1.78 μM 21.07 ± 5.38 μM Baf A1 5.24 ± 1.93 nM 4.19 ± 0.43 nM 3-MA 6.60 ± 1.36 mM 4.64 ± 1.09 mM in Fig. 2, A-B, cotreatment with YC-1 and gefitinib dramatically increased the number of yellow puncta and was accompanied by a decrease in red puncta, indicating a blockage of the late stage of autophagy. To determine whether increasing in the number of yellow puncta was due to suppression of lysosomal activity, we next examined lysosomal activity by measuring the fluorescence of DQ-Red BSA. DQ-Red BSA is a BSA derivative heavily labeled with BODIPY TR-X dye, resulting in self- quenching of the dye. Degradation of DQ-Red BSA in lysosomes formed smaller protein fragments with dequenching fluorophores that could be quantified by measuring the fluorescent brightness in cells. As shown in Fig. 2C, dequenching of DQ-red BSA occurred in gefitinib- or YC-1- treated cells, indicating an increase in lysosomal activity. Interest- ingly, cotreatment of YC-1 and gefitinib further enhanced the dequenching of DQ-BSA, indicating that the increase in yellow puncta was not due to suppression of lysosomal activity. Indeed, in eukaryotic cells, lysosomes are a major degradative organelle for both endocytosis and autophagy. Therefore, we speculated that the enhancement of lysosome activity may contribute to the combinational effect of YC-1 and gefitinib on endocytic degradation of EGFR (Hu et al., 2020). Together, these findings suggest that YC-1 inhibits gefitinib-induced autophagy by blocking the fusion of autophagosomes and lysosomes. In our previous study, YC-1 was observed to accelerate the endocytic trafficking and degradation of EGFR delayed by gefitinib, we considered that accumulation of LC3 also could be an effect produced by an enhanced fusion of autophagosomes with late endosomes. Lysobi- sphosphatidic acid (LBPA) is a phospholipid found at high concentra- tions on the internal membranes of late endosomes, where it may play an important role in the degradation of glycolipids and the transport of membrane proteins and lipids (Pillay et al., 2002). Therefore, a coloc- alization analysis was carried out in HeLa-GFP-LC3 cells, using GFP-LC3, as a marker of autophagosomes, and LBPA, as a marker of late endo- somes. As shown in Fig. 2, D-E, the combination treatment of gefitinib and YC-1 increases the colocalization of LBPA with LC3. In summary, our results indicated that the combination treatment of gefitinib and YC-1 facilitates the autophagic fluX, increasing the fusion of autopha- gosomes with late endosomes but not with lysosomes. Next, we hypothesized that if the combinatorial activity of gefitinib and YC-1 in NSCLC cells was due to the suppression of the fusion of autophagosomes and lysosomes, blockade of the early phase of auto- phagy may abolish their synergistic effect. The class III PI3K inhibitor 3- methyladenine (3-MA) is commonly used to selectively block the early phase of autophagy (Heckmann et al., 2013). Cells were pretreated with 3-MA (5 mM) for 30 min to block the autophagy pathway first and 3-MA treatment slightly increased the caspase-3/7 activity in both NCI–H1975 and NCI–H1944 cells (Fig. 2F). In the presence of 3-MA (5 mM), the further enhancement of caspase-3/7 activity was significantly attenu- ated, indicating that blockade of the early phase of autophagy may abolish their synergistic effect (Fig. 2F). Taken together, these data further support that YC-1 inhibits gefitinib-induced autophagy by blocking the fusion of autophagosomes and lysosomes, and this activity may serve as an underlying mechanism of the combinational effect of YC-1 and gefitinib to overcome insensitivity in NSCLC cells. Fig. 3. Synergistic effects of autophagy inhibitors and gefitinib (GEF) on caspase-3/7 activity (A–B) Caspase-3/7 activity assay was examined after 24 h of treatment with indicated compounds. Caspase readings normalized to cell number determined by CellTiter-Glo Luminescent Cell Viability Assay. Error bars indicate the mean S.D. from three independent experiments. *P < 0.05, **P < 0.01 significantly different from the control (no treatment). . #P < 0.05, # #P < 0.01, significant difference versus gefitinib treated cells. 3.3. YC-1 shows more potent combinatorial activity with gefitinib than other autophagy inhibitors in NSCLC cells Increasing research has demonstrated that drug resistance in cancer therapy can be abrogated by the inhibition of autophagy (Amaravadi et al., 2007). Therefore, we compared the effects of YC-1 and several autophagy inhibitors on improving the sensitivity of NSCLC cells to Table 2 List of the genes induced or repressed in NCI–H1975 cells (autophagy pathway). No. Gene Symbol Fold Change GEF plus YC-1 vs. GEF S.D 1 IFNG 2.83 0.75 2 IGF1 2.72 1.66 3 PIK3CG 2.32 0.86 4 MAP1LC3B 1.86 0.10 5 ATG4A 0.6 0.06 6 HTT 0.55 0.11 7 ATG7 0.54 0.01 8 APP 0.51 0.05 9 TGM2 0.49 0.06 10 TNFSF10 0.49 0.00 11 DAPK1 0.48 0.01 12 CTSS 0.36 0.10 13 ATG10 0.35 0.03 Table 3 List of the genes induced or repressed in NCI–H1975 cells (apoptosis pathway). No. Gene Symbol Fold Change GEF plus YC-1 vs. GEF S.D 1 TP73 0.54 0.27 2 CIDEA 0.53 0.53 3 TNFRSF9 0.52 0.37 4 DAPK1 0.51 0.32 5 BNIP3L 0.51 0.19 6 FASLG 0.49 0.20 7 BCL10 0.48 0.22 8 PYCARD 0.47 0.19 9 CFLAR 0.47 0.16 10 TNFRSF11B 0.47 0.23 11 BIRC6 0.47 0.16 12 RIPK2 0.47 0.22 13 IL10 0.45 0.05 14 CRADD 0.44 0.30 15 TNFSF8 0.43 0.39 16 CASP1 0.4 0.20 17 TNF 0.36 0.19 18 BRAF 0.36 0.20 19 CD70 0.34 0.18 20 BIRC3 0.33 0.17 21 NAIP 0.31 0.19 22 TNFSF10 0.31 0.20 23 CASP14 0.26 0.26 24 BCL2A1 0.16 0.14 gefitinib. Three commonly used autophagy inhibitors, 3-methyladenine (3-MA), chloroquine (CQ) and bafilomycin A1 (Baf A1), were examined. First, dose response curves were established to determine the IC50 values for gefitinib, YC-1 and three autophagy inhibitors in two gefitinib-resistant NSCLC cell lines (Table 1). We then evaluated the combined effects of gefitinib and IC50 concentrations of YC-1, CQ, Baf A1 or 3-MA on caspase3/7 activity of NCI–H1975 and NCI–H1944 cells. As shown in Fig. 3, A-B, YC-1, CQ, Baf A1 and 3-MA all significantly enhanced the caspase-3/7 activity of gefitinib. In addition, YC-1 plus gefitinib showed the strongest effects on caspase-3/7 activity compared to three commonly used autophagy inhibitors. Due to the potent syn- ergistic effect induced by YC-1 combined with gefitinib, it is worthwhile to further explore the underlying mechanisms that link autophagy and apoptosis to govern cell fate decisions. 3.4. Combination effects of gefitinib and YC-1 on apoptosis and autophagy-related gene expression The RT2 Profiler PCR Array allows for the simultaneous analysis of 84 genes relevant to a specific pathway or disease state. In this study, we employed human apoptosis and autophagy RT2 Profiler PCR Arrays, to determine the changes induced by gefitinib (30 μM) either alone or in combination with YC-1 (25 μM) on the gene expression profiles in NCI–H1975 cells. A change of at least 1.8-fold with gefitinib in Fig. 4. Prognostic value of the 11 core genes. Overall survival curves of 11 core genes are plotted for all lung cancer patients (n = 1927). Data analyzed using Kaplan- Meier Plotter (www.kmplot.com). (A) Three of 11 genes had a significantly good survival rate, and (B) 8 of 11 genes had a significantly worse survival rate (P < 0.05). Table 4 Analysis of 11 core genes via KEGG pathway enrichment. Pathway Name Count % P-value Genes for all lung cancer cases, while expression of FOXO4 and FOXO6 (Fig. S1) was not significantly associated with overall survival. Taken together, we speculated that FOXO1 or FOXO3 might be involved in the combinatorial activity of gefitinib and YC-1 on autophagy inhibition and FOXO signaling pathway 4 36.4 2.40E- 04 IL10, IGF1, FASLG, PI3KCG apoptosis induction. 3.5. FOXO1 (but not FOXO3) contributes to the efficacy of the combination with YC-1 (in comparison with expression of the gefitinib alone cells) was established as a criterion, and we found that treatment with gefitinib and YC-1 further upregulated the expression of 4 auto- phagy pathway related genes and downregulated the expression of 9 autophagy pathway and 24 apoptosis pathway related genes (Tables 2–3). Next, the Kaplan-Meier plotter (http://kmplot.com/analysis) was employed to predict the prognostic information of the 35 identified genes. Among the genes examined, 11 genes were found to be signifi- cantly associated with the overall survival of patients with lung cancer and were consistent with the expression trend (P < 0.05) (Fig. 4, A-B). To understand the possible pathways of these 11 genes, KEGG pathway enrichment was analyzed via DAVID (P < 0.05). The results showed that four genes (IL10, IGF1, FASLG and PI3KCG) were markedly enriched in the FOXO signaling pathway (P 2.4E-04, Table 4). The FOXO family consists of four submembers (FOXO1, FOXO3, FOXO4 and FOXO6). We investigated the prognostic value of FOXO family members in lung cancer patients through the Kaplan-Meier plotter database again. Of these, high expression of FOXO1 and FOXO3 mRNA was significantly associated with good overall survival combination of YC-1 and gefitinib in NSCLC cells To confirm the role of FOXO1 and FOXO3 in the combinatorial ac- tivity of gefitinib and YC-1 on autophagy and apoptosis, siRNAs specific targeting FOXO1 or FOXO3 were used to knock down the expression of FOXO1 and FOXO3, respectively. The effective knockdown of FOXO1 and FOXO3 was confirmed by western blotting. Interestingly, loss of FOXO1 in both cell lines strikingly reduced the expression of FOXO3, but not vice versa (Fig. 5A). Additionally, loss of FOXO1 expression mark- edly attenuated the synergistic growth inhibition and proapoptotic ef- fect of combined gefitinib and YC-1 treatments, although the loss of FOXO3 did not show these apparent effects (Fig. 5B–C). Compared to the control siRNA-treated samples, knockdown of FOXO1 expression consistently markedly decreased the synergistic effect of combined gefitinib and YC-1 on the protein levels of cleaved caspase 3, 7 and LC3- II. (Fig. 5D). Together, these findings suggests that FOXO1 plays the main role in the synergistic effect on autophagy inhibition and apoptosis induction by combining of YC-1 and gefitinib in NSCLC cells. Fig. 5. FOXO1 is involved in the combinatorial activity of gefitinib (GEF) and YC-1. (A) Protein level of FOXO1 or FOXO3 in cells transiently transfected with specific siRNAs for 72 h examined by western blot assay. (B–C) NCI–H1975 and NCI–H1944 Cells transiently transfected with FOXO1 or FOXO3 siRNAs for 24 h and then stimulated with compounds as indicated for another 24 or 72 h. (B) Caspase-3/7 activity was determined. (C) Cell viability was then analyzed using CellTiter-Glo Assay. For the GEF and YC-1 combination treatment group, the percentage of inhibition of cell growth was calculated relative to 10 μM YC-1 single treated cells (set to 100%). (D) Cell lysates collected for western blotting analysis. GAPDH served as the loading control. A representative blot of three separate experiments is shown. Values represent the mean ± S.D. (n = 3). #P < 0.05, ##P < 0.01, significantly different from the same treatment of the siRNA control group. 3.6. YC-1 enhances the FOXO1 transcriptional activity of gefitinib in gefitinib-resistant NSCLC cell lines Accumulating evidence highlights that the phosphorylation of Ser- 256 is critical for the nuclear exclusion of FOXO1, and FOXO1 nuclear-cytoplasmic shuttling regulates its transcriptional activity (Gopal et al., 2017; Van Der Heide et al., 2004). Here, we measured the change in FOXO1 phosphorylation at the protein level. As expected, after treatment with gefitinib and YC-1, the protein level of phosphor- ylated FOXO1 was significantly reduced (Fig. 6A). To determine whether downregulation of phosphorylated FOXO1 after combination treatment resulted in an increase in transcriptional activity, we next examined the transcriptional activity of FOXO1 with a FOXO reporter assay. Interestingly, the transcriptional activity of FOXO1 slightly increased after treatment with 30 μM gefitinib, and was significantly further elevated after combination treatment in both NCI–H1975 and NCI–H1944 cells (Fig. 6B). Collectively, our data suggested that the combination treatment of gefitinib and YC-1 promoted FOXO1 tran- scriptional activity in gefitinib-resistant NSCLC cells. 3.7. Inhibition of FOXO1 transcriptional activity with AS1842856 leads to a blockage of the combinatorial activity of gefitinib and YC-1 AS1842856 is a specific FOXO1 antagonist and can potently inhibit the DNA binding of FOXO1 and its transcriptional activity (Gopal et al., 2017; Nagashima et al., 2010). NCI–H1975 and NCI–H1944 cells were Fig. 6. Combination treatment with gefitinib (GEF) and YC-1 promotes FOXO1 nuclear translocation and transcriptional activity. (A) Cells treated with compounds as indicated for 24 h. Then, cell lysates were collected for western blotting analysis. GAPDH served as a loading control. A representative blot of three separate experiments is shown. (B) Cells transfected with a luciferase reporter plasmid under the control of the FOXO1 promoter and incubated with compounds as indicated for 24 h. Values represent the mean ± S.D. (n = 3). *P < 0.05, **P < 0.01, significantly different from the control (no treatment). #P < 0.05, ##P < 0.01, significant difference versus gefitinib-treated cells. incubated with gefitinib, YC-1 and gefitinib plus YC-1 in the presence and absence of 1 μM AS1842856 for 24 h. We observed the transcrip- tional activity of FOXO1 induced by gefitinib plus YC-1 was significantly reduced by AS1842856 (Fig. 7A). Additionally, compared with gefitinib and YC-1 alone, cotreatment with gefitinib and YC-1 significantly increased cell apoptosis. In the presence of AS1842856 (1 μM), the combinatorial activity of gefitinib and YC-1 was obviously reduced (Fig. 7B). Next, the major mediators of apoptosis and autophagy were examined by western blot analysis. The protein levels of cleaved caspase 3, 7 and LC3-II were significantly increased in NCI–H1975 and NCI–H1944 cells treated with YC-1 and gefitinib simultaneously compared to either gefitinib or YC-1 alone. Consistently, AS1842856 significantly blocked this combinatorial activity of gefitinib and YC-1 (Fig. 7C). These data suggested that the transcriptional activity of FOXO1 is critical for the combinatorial activity of gefitinib and YC-1 on autophagy inhibition and apoptosis induction. 4. Discussion In a previous study, we found that YC-1 exhibited an extraordinary ability to sensitize gefitinib-resistant NSCLC cells to gefitinib treatment and downregulate kinase-independent EGFR cellular signaling in the presence of gefitinib(Hu et al., 2020). Autophagy is known to be an important kinase-independent function of EGFR, and it is essential for cancer cell survival and the development of drug resistance (Tan et al., 2016). Therefore, following a previous study, we investigated whether YC-1 can affect the autophagy induced by gefitinib. In this study, we investigated the effects of gefitinib alone or in combination with YC-1 on the initial stage of autophagy, autophagosome formation, and late-stage autolysosome formation. Gefitinib was demonstrated to increase the numbers of GFP-LC3 puncta and the protein levels of LC3-II as previ- ously reported (Han et al., 2011). Tandem mRFP-GFP-LC3 fluorescence analysis showed an increase in the number of both yellow and red LC3 puncta in the presence of gefitinib, which indicated an induction of autophagy by gefitinib in NSCLC cells. However, YC-1 was demon- strated to further increase the levels of LC3-II protein and GFP-LC3 puncta, thus impairing the degradation of SQSTM1 protein. An in- crease in the number of yellow puncta and a concurrent decrease in red puncta were observed in the presence of gefitinib and YC-1 based on a mRFP-GFP-LC3 fluorescence analysis, which indicated a blockage of the late stage of autophagy. Meanwhile, gefitinib plus YC-1 significantly enhanced the lysosomal proteolytic activity, as shown by DQ-Red BSA fluorescence analysis, suggesting that the accumulation of yellow puncta and the decrease in red puncta were not due to the impairment of lysosomal activity but rather were caused by a blockage of autophagosome-lysosome fusion. In our previous study, YC-1 was observed to accelerate the endocytic trafficking and degradation of EGFR delayed by gefitinib, and the lysosomal protease inhibitor E64D attenuated the proapoptotic effects of gefitinib plus YC-1. The endocytosis and autophagy pathways have a Fig. 7. AS1842856 blocks the combinatorial activity of gefitinib (GEF) and YC-1. (A) Cells transfected with a luciferase reporter plasmid under the control of the FOXO1 promoter and incubated with compounds as indicated for 24 h. (B) Caspase-3/7 activity determined after compound treatment of NCI–H1975 and NCI–H1944 cells for 24 h. Caspase readings were normalized to cell number determined by CellTiter-Glo Luminescent Cell Viability Assay. (C) Cell extracts subjected to western blot analysis after 24 h of treatment, and the expression of LC3-II, total and cleaved caspase 3 and caspase 7 was detected. GAPDH served as loading control. A representative blot of three separate experiments is shown. Values represent the mean ± S.D. (n = 3). #P < 0.05, ##P < 0.01, significantly different from the same treatment without AS1842856. common endpoint at lysosomes, where their cargoes are degraded. Previously, Ghislat et al. proposed a double and opposite role of annexin A5 in regulating the endocytic and autophagic pathways. They found annexin A5 can promote delivery of autophagosomes to lysosomes and inhibit endocytosis (Ghislat et al., 2012). Here, our finding indicated that YC-1 can inhibit the fusion of autophagosome with lysosomes and promote endocytosis induced by gefitinib by an enhanced fusion of autophagosomes with late endosomes (Fig. 2). Taken together, it is reasonable to assume that the blockade of autophagosome-lysosome fusion and the facilitation of EGFR endocytic trafficking and degrada- tion may work synergistically to amplify the combinatorial activity of gefitinib and YC-1 on apoptosis induction, although further in- vestigations are needed to explain how these two pathways crosstalk to improve the sensitivity of NSCLC cells to gefitinib. Next, we investigated whether autophagy blockade was a potential mechanism underlying the synergistic enhancement of proapoptotic activity via gefitinib and YC-1 combination treatment. Here, we found that blocking the early phase of autophagy (3-MA) could reverse the synergistic proapoptotic effect of YC-1 and gefitinib. To our knowledge, our study is the first to reveal that YC-1 could potentiate the proapo- ptotic effect of gefitinib by inhibiting gefitinib-induced autophagy via the blocking of autophagosome and lysosome fusion in NSCLC cells. Accordingly, we examined the effect of three common autophagy inhibitors (Baf A1, CQ and 3-MA) on the anti-NSCLC activity of gefitinib (Fig. 3, A-B). Consistently, all the autophagy inhibitors significantly enhanced the caspase-3/7 activity of gefitinib. Surprisingly, compared with these three autophagy inhibitors, YC-1 showed a much stronger ability to potentiate the proapoptotic effects of gefitinib. Because of these promising results of YC-1 plus gefitinib treatment in NCI–H1944 and NCI–H1975 cells, it is worthwhile to further investigate the un- derlying mechanisms to design innovative therapeutic strategies to overcome both primary and acquired resistance to EGFR-TKIs. First, we evaluated the change in gene expression in autophagy- and apoptosis- related pathways with the RT2 Profiler PCR array after 24 h of treat- ment with gefitinib or gefitinib plus YC-1. Then, genes meeting the criteria were analyzed with the online Kaplan-Meier plotter (http://k mplot.com/analysis/) and DAVID (https://david.ncifcrf.gov/) data- bases. The FOXO1/3 signaling pathway was predicted as a potential mechanism involving the combinational effect of YC-1 and gefitinib on autophagy and apoptosis in NSCLC cells. Using siRNAs specifically tar- geting FOXO1 or FOXO3, we observed that knockdown of FOXO1 in both NCI–H1975 and NCI–H1944 cells strikingly reduced the expression of FOXO3, but not vice versa. Additionally, the concurrent loss of FOXO1 and FOXO3 attenuated the growth inhibition and proapoptotic effect of YC-1 and gefitinib but not the loss of FOXO3 alone (Fig. 5, A-D). Mammalian FOXO transcription factors, including FOXO1, FOXO3, FOXO4 and FOXO6, play an important role in glucose homeostasis, energy metabolism, cell cycle arrest, oXidative stress resistance, and apoptosis (Beretta et al., 2019; Farhan et al., 2017; Tran et al., 2002; van der Vos and Coffer, 2011). Among them, FOXO1 was suggested as a potential tumor suppressor gene in NSCLC development and progression (Gao et al., 2018; Maekawa et al., 2009). The possibility of interfering with FOXO1 localization has been proposed as a valuable approach to improving cell sensitivity to chemotherapy because nuclear retention of FOXO1 may favor the induction of proapoptotic genes (Beretta et al., 2019; Corno et al., 2018; Cossa et al., 2014). Analysis of the clinical NSCLC samples displayed a strong correlation between positive staining for phosphorylated FOXO1, detected in 68% of tumors, and Akt acti- vation (Balsara et al., 2004). Several studies have suggested that inac- tivation of the PI3K/Akt pathway resulted in an increase of FOXO1 phosphorylation at serine 256, which is essential for nuclear exclusion of FOXO1 (Brunet et al., 1999; Zhang et al., 2002). Consistently, Sangodkar et al. reported that addition of erlotinib to HCC827 cells decreased FOXO1 phosphorylation at serine 256 and led to FOXO1 accumulation in the nucleus. The increase in nuclear FOXO1 resulted in increased transcriptional activation of KLF6 and subsequent induction of apoptosis (Sangodkar et al., 2012). Additionally, the translocation of FOXO1 from the nucleus to the cytoplasm is essential for stress-induced autophagy in HCT-116 and HeLa cells, and FOXO1 depletion is sufficient to inhibit stress-induced autophagy in HCT-116 cells, even in the presence of FOXO3 and FOXO4, indicating that this effect is unique to FOXO1 (Zhao et al., 2010; Zhou et al., 2012). In a previous study, we found that compared with the single treatment, the combination treatment with gefitinib and YC-1 significantly reduced the protein level of phosphor- ylated AKT in NCI–H1944 and NCI–H1975 cells (Hu et al., 2020). In the present study, as expected, the p-FOXO1 (ser-256) level was signifi- cantly decreased and transcriptional activity of FOXO1 were signifi- cantly increased in NCI–H1975 and NCI–H1944 cells. We hypothesize that the enhanced transcriptional activity of FOXO1 contributes to the synergistic effect of gefitinib and YC-1 on autophagy and apoptosis. To address these questions, a FOXO1-specific antagonist (AS1842856) was used. AS1842856 can potently repress FOXO1-mediated promoter ac- tivity and shows no activity towards other forkhead proteins including FOXO3 (Nagashima et al., 2010). In the presence of AS1842856, we found that the combined effects of gefitinib and YC-1 on FOXO1 tran- scriptional activity were significantly abrogated. Accordingly, the combinatorial activity of gefitinib and YC-1 on autophagy and apoptosis was significantly attenuated (Fig. 7, A-C). Together, our data suggest that the combinational anti-autophagic and pro-apoptotic effects of gefitinib and YC-1 might be due to the enhancement of FOXO1 tran- scriptional activity by downregulating p-FOXO1(ser-256) levels and promoting FOXO1 nuclear translocation in gefitinib-resistant NSCLC cells. Collectively, our study provides a new therapeutic strategy for EGFR- TKI-resistant NSCLC patients and reveals that FOXO1 represents a novel molecular target that responds to cotreatment with gefitinib and YC-1, which might contribute to sequential anti-autophagic and pro- apoptotic effects. Although the detailed molecular mechanisms remain unclear, our study provides new insights into the crosstalk between autophagy and apoptosis. CRediT authorship contribution statement Hui Hu: Conceptualization, Investigation, Methodology, Analyzed the data, Contributed to the writing of the manuscript. Xiao-Wei Zhang: Investigation, Methodology, Analyzed the data. Lin Li: Investigation, Methodology. Ming-Ning Hu: Investigation, Methodology. Wen-Qian Hu: Investigation, Methodology. Jing-Ying Zhang: Investigation, Methodology. Xiao-Kang Miao: Investigation, Methodology. Wen-Le Yang: Analyzed the data. Ling-Yun Mou: Conceptualization, Analyzed the data, Contributed to the writing of the manuscript, Funding acquisition. Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant 81874315, 81302798); the CAMS Innovation Fund for Medical Sciences (Grant 2019-I2M-5–074); the Program for the Ministry of Education “Peptide Drugs” Innovation Team (Grant IRT_15R27) and the Science and Technology Project of Gansu Province (Grant 18JR2RA031). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ejphar.2021.174346. References Amaravadi, R.K., Yu, D., Lum, J.J., Bui, T., Christophorou, M.A., Evan, G.I., Thomas- Tikhonenko, A., Thompson, C.B., 2007. Autophagy inhibition enhances therapy- induced apoptosis in a Myc-induced model of lymphoma. J. Clin. Invest. 117, 326–336. Balsara, B.R., Pei, J., Mitsuuchi, Y., Page, R., Klein-Szanto, A., Wang, H., Unger, M., Testa, J.R., 2004. Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions. Carcinogenesis 25, 2053–2059. 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