A novel inhibitor isoimperatorin targeting YWHAZ suppresses human lung adenocarcinoma activity by activating autophagy

doi:10.21203/rs.3.rs-4276470/v1

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A novel inhibitor isoimperatorin targeting YWHAZ suppresses human lung adenocarcinoma activity by activating autophagy

https://doi.org/10.21203/rs.3.rs-4276470/v1

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Background: Precise targeted therapy for lung cancer is a key strategy that benefits patients. Existing research indicates that Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) plays a role as an oncogene in the occurrence and development of lung cancer. However, there are currently few targeted inhibitors developed against YWHAZ. This study investigated a novel small-molecule drug, isoimperatorin, targeted inhibit YWHAZ and further indicated the anti-tumor effects in vitro and in vivo. We intend to provide a promising therapeutic strategy for lung adenocarcinoma with high YWHAZ gene expression.

Methods: The Cancer Genome Atlas (TCGA) database was used to assess YWHAZ expression in Non-small cell lung cancer and normal lung tissues. We detected YWHAZ expression in different types of lung cancer tissue. A high-throughput dual-luciferase reporter system was used to screen YWHAZ-targeting inhibitors. The fuction experiment of tumor cell and mice xenografts were used to investigate the roles of YWHAZ and the antitumor activity of the targeted inhibitors in lung adenocarcinoma in vitro and in vivo. Western blotting was conducted to preliminarily explore potential mechanisms.

Results: We revealed high expression of YWHAZ in clinical lung cancer tissues, which was associated with poor prognosis. Knockdown of YWHAZ in lung adenocarcinoma cells impaired the malignant functions, and inhibited the tumor growth in mice xenograft. Isoimperatorin exhibited significant inhibitory effects on the viability of lung adenocarcinoma cells, but its effect on the cells with YWHAZ knockdown was not significant. Treatment with isoimperatorin in lung adenocarcinoma cells inhibited the malignant functions and suppressed tumor growth in mice xenografts. More importantly, we found PI3K/AKT signaling pathway is suppressed after YWHAZ was targeting inhibited by isoimperatorin, while autophagy-related proteins and apoptosis-related proteins were activated.

Conclusion: Our study indicates that YWHAZ plays an oncogenic role as a core target molecule in lung adenocarcinoma. As a novelly targeted inhibitor, isoimperatorin effectively inhibited YWHAZ gene with potent antitumor activities in lung adenocarcinoma. The underlying mechanism may involve inhibition of PI3K/AKT signaling pathway, then activating cell autophagy, and subsequently promoting cell apoptosis.

isoimperatorin

YWHAZ

lung adenocarcinoma

inhibitor

autophagy

Lung cancer is a malignant tumor with high incidence and mortality rates globally^[1]. It thus serves as a substantial threat to human life and health. Non-small cell lung cancer (NSCLC) contributes to approximately 85% of all lung cancer cases. Lung adenocarcinoma (LUAD) was the predominant subtype of NSCLC, constituting approximately 55% of NSCLC cases^[2]. The exact pathogenic mechanisms underlying LUAD remain unclear. It results in limited therapeutic efficacy against LUAD. The diversity of pathogenetic molecular during LUAD development makes it a challenge in current treatment strategies^[³^-5]. Therefore, it is important to identify key molecular in the LUAD evolution for implementing precise molecular-targeted therapies to improve patient prognosis.

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ or 14-3-3ζ) serves as the fundamental protein in regulating multiple molecular signaling pathways during tumor development^{[6, 7]}. YWHAZ interacts with proteins related to biological processes, such as cell apoptosis, cell cycle, cell proliferation, and cell adhesion^[8-11]. YWHAZ is regulated by non-coding RNAs in tumors to control malignant biological behaviors by activating or inhibiting its downstream signaling pathways^[12-14]. We previously proved that YWHAZ is a downstream target of miR-451a and YWHAZ expression is negatively correlated with miR-451a in LUAD. The malignant functions of LUAD can be controlled when YWHAZ is targeted and inhibited by overexpressed miR-451a. Some studies have shown that as a core molecule in tumors, the decrease of YWHAZ expression can lead to the inhibition of tumor malignant functions. However, research and development of drugs or inhibitors for targeting YWHAZ is currently limited and molecular mechanisms of YWHAZ signaling pathway in tumors are also unclear.

Traditional Chinese Medicine serves as a crucial source of antitumor drugs and represents a treasure trove for modern small-molecule drugs^[15,16]. Therefore, screening of TCM drugs targeting YWHAZ inhibition may offer new therapeutic options for LUDA. We established a dual luciferase reporter system targeting the YWHAZ promoter in A549 cells. Then, a drug library containing 1,000 TCM small molecules was screened, identifying the small molecule isoimperatorin (ISO), which effectively reduces the fluorescence signal of the YWHAZ promoter. ISO is a natural coumarin exhibiting properties such as analgesic, antibacterial, vasodilatory, and anticancer activities. However, no current related research has reported on the specific action mechanisms of ISO in LUDA, as well as its drug safety profile.

In this study, our data showed that YWHAZ plays an oncogenic role in LUDA. Inhibition of YWHAZ expression can prevent the malignant functions of LUDA. ISO is a novel small-molecule drug that can selectively inhibit YWHAZ. We further investigated the anti-tumor effects of ISO in vitro and in vivo. A preliminary exploration of molecular mechanisms underlying the ISO-induced targeted inhibition of YWHAZ to prevent LUAD progression. This study aims to provide potential candidate drugs for the clinical precision targeted therapy of LUAD.

Cell culture

The human NSCLC cell line A549 and H1299 cells were purchased from the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at a controlled atmosphere with 37°C, 95% relative humidity, 5% CO2.

RNA preparation and quantitative real-time polymerase chain reaction (RT-qPCR)

The total RNA from cells were isolated with TRIzol reagent according to the manufacturer’s instructions. The total RNA was converted into cDNA according to the reverse transcription kit (TaKaRa, Japan). RT-qPCR analysis was performed using SYBR Green PCRmix 2 kit (Solarbio, Beijing, China) according to supplier instructions. YWHAZ：forward 5'- AGGGAGAGCGTCGTCTTAGT-3',

reverse 5'-AGGATTTGCTCAAGGAGCCC-3'.

β-actin：forward 5'-CCCATCTATGAGGGTTACGC-3',

reverse 5'- TTTAATGTCACGCACGATTTC-3'.

Western blotting

Cells or tissues were lysed with RIPA buffer supplemented with protease and phosphatase inhibitors. The lysates were denatured by heating for 10 min at 99°C and subjected to 4%–10% SDS-polyacrylamide gel electrophoresis. Then, the proteins were transferred to polyvinylidene fluoride membranes, which were blocked and probed with primary and secondary antibodies. The membranes were detected on the basis of chemiluminescence.

Plasmid construction and transfection

The lentiviral vector was constructed and packaged by Hanheng company (Wuhan,China), including the lentivirus of silencing YWHAZ gene HBLV-YWHAZ shRNA-ZsGreen-PURO and the negative control virus. The cells were transfected into two groups, sh-NC group and sh-YWHAZ group.

Hematoxylin and eosin(HE) staining and immunohistochemistry(IHC)

Tissue sections (thick: 4 μm) were deparaffinized through graded alcohols and subjected to microwave pretreatment for antigen retrieval. Each section was incubated with 0.3% H₂O₂for 10 mins. Nonspecific binding sites were blocked through incubation of the sections with 2% FBS for 15 mins. Each section was incubated with anti‐YWHAZ or other primary antibody overnight at 4°C, and then incubated with the secondary antibody biotinylated antimouse immunoglobulin for 30 mins, followed by incubation with the conjugated horseradish peroxidase streptavidin for 30 mins. Finally, sections were then counterstained with hematoxylin, dehydrated, and mounted.

High-throughput dual-luciferase reporter system

pGL4-pYWHAZ luciferase reporter plasmid was constructed in A549 cells. A library of 1000 small molecules of traditional Chinese medicine was screened. A549 cells carrying the fluorescence reporter system and dissolved small-molecule drugs were cultured in a 96-well plate for 24 hours. High-throughput screening of reduced fluorescence signals using a plate reader was performed to identify small-molecule drugs capable of inhibiting YWHAZ expression.

Cell counting kit-8 (CCK-8) assay

Cell Counting Kit-8 was used to detect cell proliferation. A549 and H1299 were plated into each well of 96-well plates. After incubating 24 hours at 37°C in 5% CO₂, isoimperatorin(5, 10 and 20 μg/mL) and 0.05% DMSO (control) were added to each well, incubation was continued for 24, 48, 72 and 96 hours. Then, 10 μL of CCK-8 was added to each well for 2hours. The optical density(OD) was measured by a microplate reader at wavelengths of 450 nm. The percentage of growth was calculated as cell viability (%) = [OD_(treatment)_—OD_(blank)]/[OD_(control)_—OD_(blank)] × 100%.

Colony formation assay

A549 and H1299 cells were seeded into 6‐well plates at a density of 1 × 10³ cells/well and incubated for 12 hours. The cells were treated with ISO (20 and 30 μg/mL) and cultured in a 5% CO₂ atmosphere at 37°C for 15 days to allow colony formation. Cold PBS was used to wash the plate. The colonies fixed with 4% polyformaldehyde were stained with 1% crystal violet for 30 mins at room temperature. Colonies with more than 100 cells were counted under the microscope.

Wound healing assay

The wound healing assay was conducted to measure cell motility. The cells were seeded into 6-well plates and cultured to 90% confluence. Wounds were made in each well of the 6-well plates by using the tips of a 1-mL pipette. Then, the cells were treated with ISO (20 and 30 μg/mL) and allowed to migrate into the area with no cells over 48 hours. Cell migration was visualized under a microscope and photographed at 0 and 48 hours.

Transwell cell invasion assay

Cells (1×10⁴/cells) were seeded into the top transwell chamber with RPMI-1640 culture medium containing 10% FBS when 300 µg/mL Matrigel had solidified, and the bottom chamber was filled with 500 μL RPMI-1640 culture medium containing 20% FBS. After 12 hours, ISO was added to the top chamber, with the RPMI-1640 culture medium without FBS. The invading cells were fixed with 0.1% paraformaldehyde and stained with 0.1% crystal violet solution in another 12 hours. The invading cells were imaged at 200× magnification in five random fields and measured using ImageJ software.

Tunel assay

Tunel assay was carried out to detect apoptotic cell population using the Tunel Assay Kit-FITC according to the manufacture’s guidelines. Briefly, 2×10⁵ A549 cells in a 6-well plate were added in ISO at 0, 20 and 30µg/mL concentration.The cells were fixed with 4% paraformaldehyde for 20 mins. A permeabilization solution (0.2% Triton X-100) was added to both groups for 10 mins. Then, 10 μL of TdT Equilibration Buffer was added for 30 mins. 50μL labeling working solution were added to for 60 mins. Each group was treated with DAPI working solution and incubated for 5 mins. Apoptotic cells were observed and recorded under an inverted fluorescence microscope.

Flow cytometry assay

H1299 cells were collected after experimental treatment and washed with PBS. 100ul of 1×Annexin V binding buffer per 1×10⁵ cells were used to resuspend cell pellets followed by FITC Annexin V and 7-AAD staining in the dark for 15 min at room temperature and analyzed on the BD FACSCanto Flow Cytometry Machine (BD Biosciences). Flow cytometry data were analyzed using FlowJo to calculate Annexin V and 7-AAD distribution.

Tumor xenografts

The animal procedures were approved by the Research Ethics Committee of Zunyi Medical University and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. Four-week-old server combined immunodeficiency (SCID) mice (female, weight: 14-16g, SPF grade) were purchased from the Experimental Animal Company of Tengxing (Chongqing, China). To establish a cell xenograft model, SCID mice were subcutaneously injected with A549 cells (5 × 10⁶). When tumor volumes reached approximately 100 mm³, mice were randomly distributed into groups of 6 mice. The mice received treatment with vehicle control, low-dose ISO (10 mg/kg), or high-dose ISO (20 mg/kg). ISO was suspended in PBS containing dimethyl sulfoxide (DMSO), polyethylene glycol, and tween 80 and administered intraperitoneal injection once a day. Tumor size was measured every 3 days by using a vernier caliper. The average tumor volume in each group was calculated based on the equation for a prolate spheroid (tumor volume = (short diameter)2 × large diameter/2) and expressed in mm³.

Statistical analysis

Data were presented as the mean ± standard error of the mean (SEM). Data presented were representative of three independent experiments, including histological imaging and blotting. All statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software) and SPSS version 23.0 software. Statistical significance was indicated by p values of <0.05. *p < 0.05, **p < 0.01, ***p < 0.001.

YWHAZ expresses in NSCLC and enhances cell proliferation, migration, and invasion in vitro

YWHAZ expression in NSCLC and normal lung tissues were assessed using The Cancer Genome Atlas (TCGA) database. The data revealed that the YWHAZ expression was higher in NSCLC tissues than in normal tissues (Figure 1A, B). We further evaluated the prognostic significance of YWHAZ in NSCLC cases, the database indicated that high YWHAZ expression was markedly associated with a short overall survival time in LUAD patients (Figure 1C).

In our previous studies, our data showed that in the 137 clinical specimens we collected, we identified nine types of tumors, and YWHAZ was expressed in each type. The overall positive expression rate of YWHAZ was 94.2%. Specifically, the positive expression rate in lung cancer reached 96.4%. In this study, we focused on detecting YWHAZ expression in different types of lung cancer tissue, it is obvious that YWHAZ expression was higher than that of normal tissues (Figure 1D). Moreover, YWHAZ expression was demonstrated in normal tissue, paracancerous tissue and LUAD patients tissue. Our data suggested that YWHAZ expression was upregulated in LUAD tissue compared to that in paracancerous tissue and normal tissue (Figure 1E-G). Collectively, these results suggested that YWHAZ might be a key oncogene in the occurrence and development of lung cancer.

YWHAZ enhances LUAD cell proliferation, migration, and invasion in vitro

Results of the clinical tissue specimens and TCGA database analysis indicated that YWHAZ serves as an oncogene in NSCLC. To validate biological functions of YWHAZ in vitro, we selected cells with high expression of phosphorylated YWHAZ(p-YWHAZ) in cell lines Beas-2b and 16HBE from normal bronchial epithelial cells and in cell lines H358, H460, H1975, A549, H1299, and H1588 from NSCLC. The results showed that p-YWHAZ expression were lower in the two normal bronchial epithelial cell lines, whereas they were relatively higher in the LUAD cells A549 and H1299 (Figure 2A). Therefore, our study focused on LUAD. Next, we knocked down YWHAZ in the A549 and H1299 through short hairpin RNA (shRNA) interference (Figure 2B). We also investigated the effects of YWHAZ on the biological activity of LUAD cells. CCK-8 assays revealed that YWHAZ knockdown significantly suppressed the proliferation of A549 and H1299 (Figure 2C). The results of the wound healing and transwell invasion assays suggested that YWHAZ knockdown markedly impaired the migration and invasion of A549 and H1299 (Figure 2D, E). YWHAZ knockdown significantly suppressed the colony formation of A549 and H1299 (Figure 2F). Furthermore, Our data indicated that knockdown of YWHAZ in H1299 induced cell apoptosis (Figure 2G, H). Therefore, YWHAZ promoted the proliferation, migration, invasion and inhibit apoptosis of LUAD cells.

YWHAZ promotes LUAD cell growth in vivo

We further investigated the tumorigenic function of YWHAZ in vivo. A xenograft tumor model was used to evaluate the effect of YWHAZ knockdown on LUAD cell growth in vivo. We generated YWHAZ knockdown A549 cells using lentiviral vector and subcutaneously injected into the SCID mice. The xenograft tumors with YWHAZ knockdown grew more slowly and weighed less compared with the control tumors (Figure 3A, B). HE staining of the xenograft tumor revealed the nuclear division of tumor cells (Figure 3C), which indicated that the tumor tissues originated from LUAD cells. Furthermore, Ki67 staining was performed to examine tumor cell proliferation in vivo. IHC staining of tumor tissues indicated that fewer Ki67-positive cells were detected in the tumors with YWHAZ knockdown. YWHAZ knockdown increased the levels of autophagy-related proteins Beclin1 and LC3B in the xenograft tumors (Figure 3D). We then validated whether YWHAZ was associated with cellular apoptosis and autophagy in tumors. The expression of YWHAZ, Beclin1, LC3B, Bax, and Cleaved-caspase3 proteins in xenograft tumors were detected through western blotting. Compared with the control tumors, YWHAZ expression was reduced in the tumors with YWHAZ knockdown. On the contrary, the expression of autophagy-related proteins Beclin1 and LC3B and apoptosis-related proteins Bax and Cleaved-caspase3 increased in the tumors with YWHAZ knockdown (Figure 3E). Collectively, these data suggest that YWHAZ promotes LUAD cell growth in vivo, possibly through downregulating cell autophagy and apoptosis.

Screening small-molecule drugs targeting YWHAZ inhibition

Our data indicated that YWHAZ promotes LUAD cell growth both in vivo and in vitro. Therefore, YWHAZ holds significant potential as a therapeutic target for LUAD. Currently, few reported small-molecule inhibitors targeting YWHAZ are available. We here focused on identifying effective and safe YWHAZ-targeting inhibitors as candidate drugs for LUAD treatment. A dual luciferase reporter system targeting the YWHAZ promoter was established in A549 cells. Subsequently, a drug library containing 1,000 TCM small molecules was screened to identify the small molecule ISO that effectively reduces the fluorescence signal of the YWHAZ promoter (Figure 4A, B).

To confirm the targeted inhibition of YWHAZ by ISO, CCK-8 assay was conducted to evaluate the viability of A549-shNC and A549-shYWHAZ cells after ISO treatment. Our data shown that cell viability of A549-shYWHAZ was significantly higher than that of A549-shNC (Figure 4C). Furthermore, the A549 and H1299 cells were treated with ISO for 48 hours to examine the p-YWHAZ expression level. ISO dose-dependently inhibited p-YWHAZ (Figure 4D).This indicated that the anti-tumor activities of ISO were significantly attenuated in the A549 cells in which YWHAZ was knocked down through shRNA interference. ISO is a small molecule drug that can targeting inhibit YWHAZ.

ISO significantly inhibited malignant function of LUAD cells with aberrantly-active YWHAZ

YWHAZ is an oncogenic gene in lung cancer. To evaluate the antitumor effect of ISO targeting inhibit YWHAZ in cell model. CCK8 experiment was firstly used to determine the optimal time and concentration at which ISO exerted its effects. Our data indicated A549 cell proliferation was significantly inhibited by ISO in a dose-dependent and time-dependent manner. IC₅₀ values at different time points were calculated (Figure 5A). However, based on the predicted IC₅₀ value after treatment of A549 cells with ISO, we increased concentration of ISO in H1299 cells to select better concentration. ISO also inhibited H1299 cell proliferation in a new dose-dependent and time-dependent manner. IC₅₀ values at different time points were calculated (Figure 5B). In colony survival assay, ISO strongly inhibited the colony formation of A549 and H1299 cell lines at 20 and 30 μg/mL concentration (Figure 5C). Migration and invasion of tumor cell are closely related to tumor metastasis and patient mortality, ISO was further evaluated for its effect on the migration and invasion. Wound healing assay revealed that ISO significantly inhibited A549 and H1299 cell migration at 30μg/ml concentration (Figure 5D). According to the results of transwell cell invasion assay, the invasion of A549 and H1299 cells were dose-dependently inhibited by ISO (Figure 5E). To determine whether the proliferative inhibition of ISO for tumor cell was attributed to cell apoptosis, A549 cells were treated with ISO for 48 hours to examine apoptotic cells through Tunnel assay. ISO dose-dependently induced A549 cell apoptosis (Figure 5F). Taken together, ISO strongly suppressed the malignant functions of LUAD cells and promoted tumor cell apoptosis.

ISO inhibited the growth of LUAD cell xenografts

To further examine the effect of ISO for therapy of LUAD, tumor xenografts of the LUAD cell line (A549) in SCID mice subcutaneously were performed to evaluate the anti-tumor effect. ISO strongly inhibited the growth of A549 xenografts following 21-day therapy with a daily dose of 10 or 20 mg/kg administered intraperitoneally. The volumes and weights dose-dependently were decreased by ISO for A549 xenograft tumors (Figure 6A, B). Meanwhile, body weights of mice were no significant change, and signs of toxicity were not obviously noted during the therapy (Figure 6C, D).

Ki-67, YWHAZ, and autophagy-related protein LC3B in xenograft tissues were detected through IHC. Compared with the vehicle group, expression of YWHAZ and Ki-67 were significantly decreased in the ISO-treated group (Figure 6E). This indicated that ISO treatment can suppress the tumor proliferation and progression. However, the expression of the autophagy-related protein LC3B increased after ISO administration. Taken together, our data suggested that ISO prevents tumor growth by inhibiting YWHAZ expression in mouse models.

ISO targeting inhibits YWHAZ to promote Beclin1, LC3II, Bax, and Cleaved-caspase3 expression by suppressing PI3K/AKT signaling

Our studies showed that YWHAZ knockdown in LUAD cell lines or ISO treatment in A549 xenografts contributes to the expression of autophagy-related proteins and apoptosis-related proteins. YWHAZ, Beclin1, LC3II, Bax, and Cleaved-caspase3 expression in xenograft tissues were evaluated by western blotting. Compared with the vehicle group, YWHAZ expression was significantly suppressed in ISO-treated group. However, expression of the autophagy-related proteins Beclin1 and LC3II and that of the apoptosis-related proteins Bax and Cleaved-caspase3 were increased in ISO-treated group (Figure 7A). Furthermore, to determine whether the antitumor activity of ISO targeting inhibit YWHAZ is associated with autophagy and apoptosis signaling, we conducted the following studies. A549 cells were treated with ISO for 48 hour to examine its effect on YWHAZ signaling. Protein expression were evaluated by western blotting. Our data indicated that expression of Beclin1, LC3II and Bax, Cleaved-caspase3 were dose-dependently increased after ISO administration. Moreover, as an upstream signaling pathway of autophagy, expression of p-PI3K and p-AKT were dose-dependently decreased after ISO administration (Figure 7B). This suggests that the anti-tumor activity of ISO may be mediated by participating in the PI3K/AKT signaling pathway, activating autophagy in LUAD cells, and subsequently promoting apoptosis.

The identification and targeted intervention of core target molecules crucial for lung cancer pathogenesis have recently become a research hotspot^[17-20]. YWHAZ acts as an oncogene in various tumors. Although studies have reported that YWHAZ promotes occurrence and development of lung cancer, the exact pathogenic mechanism remains somewhat unknown. Moreover, research on targeting YWHAZ inhibitors for lung cancer treatment represents a new area. Here we clarified that YWHAZ is high expression in the clinical tissues of NSCLC. Subsequently, we confirmed that YWHAZ promotes the malignant biological functions of LUAD cells in vitro and enhances xenograft tumor growth in vivo. ISO was identified to selectively inhibit YWHAZ by high-throughput dual-luciferase reporter system. We confirmed the anti-tumor activity of ISO in LUAD, demonstrating that ISO inhibits malignant biological functions in vitro and xenograft tumor growth in vivo. Furthermore, We also conducted a preliminary investigation into the potential mechanisms of ISO targeting YWHAZ to inhibit LUAD development.

YWHAZ, as an oncogene, participates in various cellular activities of tumor cells. Our previous datas showed that YWHAZ was positively expressed in various tumors, with an average positive expression rate of 94.2%. The positive staining rate of YWHAZ in lung cancer cases was 96.4%, higher than the average level, which indicated that YWHAZ may be more specific and sensitive in lung cancer pathogenesis. Expression level of YWHAZ was positively correlated with tumor size, clinical stage, and metastasis in clinical patients.

Based on previous datas, we believed that it is a significant research importance for study on targeting YWHAZ in lung cancer pathogenesis. We further examined that knockdown of YWHAZ inhibit the proliferation, survival, migration, and invasion of LUAD cells while promoting cell apoptosis and the growth of LUAD xenograft tumors. We confirmed that YWHAZ is a key oncogenic factor in vitro, in vivo and in clinical tissue. Therefore, research and development of YWHAZ-targeted inhibitors have substantial clinical application value. There have been few reported YWHAZ-targeted inhibitors. Difopein is an inhibitor of YWHAZ which promotes tumor cell apoptosis and increases the sensitivity to antitumor drugs^[21]. Guggulsterone, a natural product, can also induce apoptosis of head and neck tumor cells and inhibit tumor development by regulating YWHAZ^[22]. At present, research on YWHAZ-targeted inhibitors for lung cancer has not been reported. In this study, five small-molecule drugs that can effectively reduce the fluorescence signal of the YWHAZ promoter were screened from the drug library. On consulting literatures, we noted that ISO has not been reported in LUAD research. We first verified whether ISO could selectively inhibit YWHAZ. Viability of A549 cell could be significantly inhibited by ISO. However, when YWHAZ was knocked down in A549 cell through shRNA interference, ISO may have lost its target, which resulted in less obvious inhibition of the cell viability. This suggests that ISO has a targeted inhibitory effect on YWHAZ in LUAD.

ISO is a coumarin compound with various pharmacological properties, including analgesic, antiviral, anticancer, anti-inflammatory, antibacterial, and antihypertensive characteristics. In this study, ISO inhibited the proliferation, migration, invasion and survival of LUAD cells, while inducing cell autophagy and apoptosis. The antitumor effect of ISO in vivo revealed that ISO inhibited the growth of tumor xenografts. YWHAZ protein expression decreased in the xenografts. HE staining indicated that ISO exerted no obvious toxicity to liver and kidney. Understanding the mechanism of a drug used to treat a disease will benefit patients. Some studies on the mechanisms of YWHAZ indicated that YWHAZ regulates tumor occurrence and development by participating in multiple signaling pathways^[8]. YWHAZ promotes the growth of lung cancer cells and enhances migratory and invasive capabilities by regulating downstream signaling factors such as MUC1^[23]. As an upstream regulator of autophagy, PI3K/AKT signaling pathway negatively regulates autophagy occurrence^[24]. In gastric cancer, YWHAZ knockdown promotes cell autophagy through the PI3K/AKT/mTOR signaling pathway by inhibiting cell proliferation, migration, invasion, and EMT^{[25, 26]}. Therefore, does the mechanism of ISO on lung adenocarcinoma coincide with some mechanisms related to YWHAZ? In this study, Targeting inhibited YWHAZ by ISO, resulting in increased expression of autophagy-related proteins Beclin1 and LC3II, as well as apoptosis-related proteins Bax and Cleaved-caspase3, while decreasing expression of p-PI3K and p-AKT. PI3K/AKT signaling pathway is a classical upstream pathway of autophagy. We hypothesize that the PI3K/AKT signaling pathway is one of the mechanisms through which ISO acts on lung adenocarcinoma.

Altogether, YWHAZ plays an oncogenic role as a core target molecule in LUAD. We here demonstrated that ISO, as a novel YWHAZ inhibitor, effectively targeting inhibited YWHAZ with potent anti-tumor activities in vitro and in vivo. The underlying mechanism may be mediated by participating in PI3K/AKT signaling pathway, and activating cell autophagy, and subsequently promoting cell apoptosis. This may offered a potential therapeutic strategy for LUAD patients with high YWHAZ gene expression.

Disclosure:

Ethics Statement

Approval of the research protocol by an institutional review board: This study has been approved by the Medical Ethics Committee of the First People's Hospital of Zunyi (No. lunshen2020-70).

Animal studies: The animal experiments were approved by the Laboratory Animal Welfare & Ethics Committee of Zunyi Medical University (Appl. No. ZMU21-2105-002). This study was performed in accordance with the Declaration of Helsinki.

Informed consent statement

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Competing of Interests

The authors declare that there is no competing of interest regarding the publication of this article.

Funding

This work was funded by the following grants: Guizhou Provincial Respiratory Critical Disease Clinical Research and Prevention and Treatment Talent Base Project ([2020]8), Guizhou Provincial Natural Science Foundation Project ([2018]5623), Science and Technology Bureau Project of Zunyi City (Zunshi Kehe HZ[2022]53), Science and Technology Fund of Guizhou Provincial Health Commission (gzwkj2023-294).

Author Contributions

^#These authors contributed equally to this work.

Acknowledgments

We would like to thank the School of Medicine at Nankai University for their support and assistance in the selection of ISO used in this study.

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No competing interests reported.

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A novel inhibitor isoimperatorin targeting YWHAZ suppresses human lung adenocarcinoma activity by activating autophagy

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