VE-822 upregulates the deubiquitinase OTUD1 to stabilize FHL1 to inhibit the progression of lung adenocarcinoma

The deubiquitinase ovarian tumor domain-containing 1 (OTUD1) has been considered as a tumor suppressor in many tumors, but there is minimal research on the role of OTUD1 in lung adenocarcinoma (LUAD) pathogenesis. Bioinformatics analyses and western blot were applied for investigating OTUD1 expression in lung cancer and the drug that upregulated OTUD1. Kaplan–Meier analysis with log-rank test was used for survival analyses. IP-MS and co-IP were performed for identifying potential protein interactions with OTUD1. In vitro and in vivo assays were used for exploring the function of OTUD1 during the progression of LUAD. OTUD1 was dramatically downregulated in tumors and cell lines of human lung cancer. OTUD1 inhibited proliferation and migration of lung cancer cells in vitro. Moreover, OTUD1 inhibited growth of xenografts in nude mice and formation of primary lung tumors in urethane-induced lung cancer model. Mechanistically, we showed that OTUD1 deubiquitinated and stabilized FHL1. Furthermore, we listed and identified VE-822 as a candidate agonist for OTUD1. VE-822 inhibited proliferation of lung adenocarcinoma both in vitro and in vivo. These results indicated that the deubiquitinase OTUD1, which was upregulated by VE-822, inhibited the progression of LUAD in vitro and in vivo by deubiquitinating and stabilizing FHL1.

ligase (E3), 26 S proteasome and deubiquitinase (DUB) [6,7]. The enzymatic cascades lead to degradation of proteins by proteasome pathway or non-proteolytic outcomes such as altered protein activity or subcellular localization [8]. The ubiquitination process is a reversible post-translational modification which can be reversed by DUBs [9]. In human proteome, more than 100 DUBs have been identified, which can be divided into six subfamilies, including the ubiquitin-specific proteases (USPs), the ovarian tumor (OTU) proteases, the Machado-Joseph Disease (MJD) DUBs, the ubiquitin C-terminal hydrolases (UCHs), the motif interacting with ubiquitin (MIU)-containing novel DUB family (MINDY), the JAB1/MPN/MOV34 metalloprotease DUBs (JAMMs) [10]. Accumulative evidences demonstrated that dysregulation of the deubiquitination process frequently occurred in tumorigenesis, and consequently, many proteins were affected [11,12]. The disorder of UPS is related to various human diseases, especially cancers. Researches on the mechanism and function of UPS may provide effective clinical therapeutic targets for cancer [13,14].
The deubiquitinase ovarian tumor domain-containing 1 (OTUD1) is a member of OTU domain family [15]. Previous studies showed that OTUD1 acted as a tumor suppressor in many cancers [16,17]. It was reported that OTUD1 repressed breast cancer metastasis [18], and OTUD1 could overcome chemoresistance in esophageal squamous cell carcinoma, pancreatic ductal adenocarcinoma and NSCLC [19][20][21]. Deng et al. reported that high mRNA level of OTUD1 was associated with improved prognosis in LUAD [22]. The mechanism by which OTUD1 suppresses LUAD has not been elucidated.
Four and a half LIM domain protein 1 (FHL1), the founding member of the FHL protein family, is composed of four and a half highly conserved LIM domains with two zinc fingers arranged in tandem [23]. FHL1 positively regulates growth and migration of normal muscle cells [24]. However, FHL1 appeared to execute opposite functions in tumor cells. FHL1 suppressed growth of colorectal cancer cells and the metastasis of breast cancer cells [25,26]. In lung cancer, FHL1 expression was correlated with tumor-infiltrating immune cells, immune checkpoints and chemokine levels [27]. It is known that the function of FHL1 is regulated by post-translational modification. Phosphorylation of residues Y149 and Y272 switched FHL1 from a tumor suppressor to a cell growth promoter in LUAD [28]. Ubiquitination of FHL1 is rarely reported. Exploring the ubiquitination modification of FHL1 will help us to understand the role of FHL1 in cancers more comprehensively.
VE-822 is an ATM and Rad3-related (ATR) inhibitor which is currently in clinical trials in different cancers [29,30]. VE-822 was reported to inhibit the DNA damage repair via ATR or other mechanisms [31,32]. Recently, more targets of VE-822 have been identified [33,34]. In this study, screening of CellMiner drug database which include approved drugs in clinical trials revealed that OTUD1 expression was correlated with drug sensitivity of VE-822.
Here, we demonstrated that OTUD1 deubiquitinated and stabilized FHL1 to inhibit LUAD progression in vitro and in vivo. VE-822 synergizes with OTUD1 to inhibit proliferation and migration of lung cancer cells.

Animals
All procedures and experiments involving animals were approved by the Institution Animal Use and Care Committee of Huazhong University of Science and Technology (IACUC number: 2980). C57BL/6J WT (RRID: MGI: 5,657,312) and BALB/c nude mice (RRID: IMSR_JCL: JCL: mID-0001) were purchased from Vital River (Beijing, China) and Otud1 knockout (KO) mice (on a C57BL/6J background) were purchased from Gem Pharmatech Company (Nanjing, China). Mice were raised in specialpathogen-free (SPF) conditions in Experimental Animal Center of Huazhong University of Science and Technology. All animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology. For tumor xenograft experiments, seven BALB/c nude mice were used to explore the effect of OTUD1 on tumor cell growth, and fifteen BALB/c nude mice were used to explore the effect of OTUD1 on tumor cell growth under the VE-822 treatment. For primary lung cancer model, eighteen C57BL/6J WT were used as control and eighteen Otud1 knockout mice were used as experimental mice.

Drugs and plasmids
VE-822 was purchased from Topscience (T2669, Shanghai, China), and dissolved in DMSO. Plasmids expressing OTUD1-Flag and FHL1-Myc were generated using PCR and cloned into pHAGE and pcDNA5-Myc respectively. All primers were obtained from Tsingke (Beijing, China), and the plasmids were constructed using the forward and reverse primer sets listed in Supplementary Table 1.

Lentiviral transduction
The OTUD1-overexpression H827 and H2030 cell lines were constructed as previously described [35]. HEK293T cells cultured in a 10 cm dish were co-transfected with the 6 µg psPAX2 (RRID: Addgene, #12,260), 4 µg pMD2.G (RRID: Addgene, #12,259), and 8 µg pHAGE-OTUD1 to produce lentiviral particles. Sixty hours after transfection, the medium was collected and filtered with a 0.45 μm filter (Biosharp, BS-PES-45, Hefei, China). Viral supernatant mixed with 8 µg/mL polybrene was used to infect the target cells for 6 h and was then replaced with fresh medium. The stable cell lines were selected with treatment of 1 mg/mL G418 48 h after transfection. We produced lentiviruses containing pHAGE-control and pHAGE-OTUD1 to establish stable cell lines. The OTUD1 protein levels in different cells were determined by western blot.

Cell viability assay
H827 or H2030 cells were planted at 1 × 10 3 /well into 96-well plates. Cell Counting Kit-8 solution (Abclonal, RM02823, Wuhan, China) was added to each well at the same time for 6 consecutive days, and the OD value was determined by measuring the absorbance at 450 nm.

Wound-healing assay
H827 and H2030 cells were inoculated into 12-well plates with 2 × 10 5 and 1.5 × 10 5 cells per well respectively. The confluent cell monolayer was damaged by scraping the cells with a 1 mL pipette tip with the same strength and angle when the cell density reached 80%. Cell migration was evaluated by measuring the differences in the areas of the wounds.

Transwell assay
Cells were collected and resuspended in serum-free RPMI-1640 medium, and then 5 × 10 4 H827 or 2 × 10 4 H2030 cells were seeded in the upper chamber (Corning, 3398, New York, USA) with (invasion) or without (migration) Matrigel (Sigma-Aldrich, E1270, Darmstadt, Germany). The chamber was inserted into a 24-well plate. The lower chamber was filled with RPMI-1640 complete medium. After 24 h incubation, cells on the upper chambers were removed using a cotton swab, and cells adhered to the lower membrane surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells were photographed under an optical microscope and counted by Adobe Photoshop CS6 software (RRID: SCR_014199).

Western blot
1 × 10 6 cells were seeded into a well of 6-well plate. After 12 h or when the cell density reached 60-80%, plasmids were transfected as indicated. 48 h after transfection, proteins were taken from whole-cell extracts in RIPA buffer (Beyotime, P0013B, Shanghai, China) with 1% PMSF (Beyotime, ST506, Shanghai, China) and the protein con

Immunoprecipitation (IP) assay
3 × 10 6 HEK293T cells were seeded into a 6-cm dish. After 12 h or when the cell density reached 60-80%, plasmids were transfected as indicated. 48 h after transfection, cells were lysed by IP buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1%Triton X-100) containing protease inhibitors, mixed with Flag or Myc antibody and agarose beads (Santa PEG300 + 50% sterile PBS once per day for 4 consecutive days every week by oral gavage and lasted 3 weeks [37]. Subcutaneous tumor volumes were calculated according to the following formula: volume (mm 3 ) = 0.5 × length × width 2 , where length is the largest dimension, and the width is the point where the length is uniform in the vertical direction.

Primary lung cancer model
C57BL/6 WT and Otud1 KO male mice at 6-8 weeks of age were given intraperitoneal injections of urethane (Aladdin, U108629, Shanghai, China, 800 mg/kg) twice a week for 5 weeks [38]. Urethane was dissolved in 0.9% NaCl. Mice were sacrificed 10 weeks after the last injection. Tumors were counted manually after dissection.

Hematoxylin and eosin (H&E) staining
For histopathology analysis of lung tumors, lungs were fixed in 4% paraformaldehyde overnight at 4℃ and embedded in paraffin after dehydration in ascending concentrations of ethanol. For each sample, sections were prepared at a thickness of 2 mm at three different levels and stained using hematoxylin and eosin (H&E). Sections were observed and imaged by the Mshot microscope.

Clinical subjects and specimens
Total of 12 pairs of lung cancer tissues and corresponding adjacent tissues were collected from the Affiliated Union Hospital of Huazhong University of Science and Technology (Wuhan, China). All researches about human samples were approved by Ethics Committee of Wuhan Union Hospital and followed the principles of the Declaration of Helsinki.

Statistical analysis
Data were expressed as the mean ± SD. For tumor xenografts and primary tumor growth experiments, the number of mice in each group was indicated in each specific experiment. All statistical analyses were performed using Graph-Pad Prism 7 (RRID: SCR_002798). Comparisons between two groups were performed using a two-side Student's t test, and multiple comparisons were analysed by one-way analysis of variance (ANOVA). Specific tests were described in the respective figure legends. P values < 0.05 were considered statistically significant, with *P < 0.05, **P < 0.01, ***P < 0.001, N.S. no significant.

Denatured IP assay
The Denatured IP assay was performed as previously describe [36]. 3 × 10 6 cells were seeded into a 6-cm dish. After 12 h or when the cell density reached 60-80%, HEK293T cells were transfected with the Myc-FHL1, Flag-OTUD1 and HA-ubiquitin vectors and treated with 5 µM MG132 (Sigma-Aldrich, M7449, Darmstadt, Germany) for 12 h before collected. Forty-eight hours post-transfection, the cells were washed with phosphate-buffered saline (PBS) and lysed with 1 volume of SDS lysis buffer (10% SDS in PBS). The lysates were heated at 95 °C for 15 min, and then, 2 volumes of modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA and protease inhibitor) was added to the lysates. Then, the lysates were cooled on ice for 1 h and centrifuged at 12,000 rpm for 30 min at 4 °C. Finally, the supernatant was subjected to anti-Flag immunoprecipitation and western blot analysis.

The public databases analysis
The UALCAN database (http://ualcan.path.uab.edu) (RRID: SCR_015827) collected RNA-seq and clinical data of 31 cancer types from TCGA, and it offered a useful platform to analyse OTUD1 expression in tumor and normal tissues. Data were collected from Oncomine database (http:// www.Oncomine.org/) (RRID: SCR_007834) to analyse the mRNA levels of OTUD1 in different lung cancer databases. The prognostic values of OTUD1 specifically expressed in lung cancer samples were evaluated by overall survival (OS) using the Kaplan-Meier plotter resource (http://www. kmplot.com) (RRID: SCR_018753). Moreover, CellMiner database was used to screen clinical and preclinical drug susceptibility for association with OTUD1 (http://discover. nci.nih.gov).

Tumor xenografts
BALB/c nude mice (male and 4-6 weeks old) were purchased from Vital River (Beijing, China). H827-OTUD1 or H827-vector cells (5 × 10 6 ) were suspended in 200 µL PBS and then injected subcutaneously into the left and right flanks of BALB/c nude mice respectively. Two weeks after tumors became measurable, mice were randomly divided into PBS-treatment and VE-822-treatment groups. VE-822 was administered at a dose of 60 mg/kg in 5% DMSO + 45% Consistently, OTUD1 was downregulated in various lung cancer cell lines compared with non-tumor cells (BEAS-2B and HEK293T) (Fig. 1g). These results indicated that OTUD1 was downregulated in lung cancer and associated with patient survival.

OTUD1 inhibits the proliferation and migration of lung cancer cells
To understand the biological function of OTUD1 in lung cancer cells, we used lung adenocarcinoma cell lines HCC827 and NCI-H2030 to construct stable OTUD1 overexpression cells (H827-OTUD1 and H2030-OTUD1) and the corresponding control cells (H827-vector and H2030-vector) by lentivirus infection. The protein levels of OTUD1 in these cells were detected by western blot. (Fig. S1a).
The hallmarks of cancer include the acquired capabilities for sustaining proliferation and activating invasion and metastasis [39]. First, the Cell Counting Kit-8 (CCK-8) assay was performed to assess the effect of OTUD1 overexpression on proliferation of LUAD cells. We showed that OTUD1 overexpression significantly inhibited proliferative ability of LUAD cells (Fig. 2a-b). To further investigate the effect of OTUD1 on migration and invasion of LUAD cells,

OTUD1 is downregulated in lung cancer
To explore the expression of OTUD1 in patients with different cancers, we compared the expression of OTUD1 in tumors with corresponding normal tissues in the TCGA database. OTUD1 was significantly downregulated in many cancers, especially in lung cancer including LUAD and LUSC (Fig. 1a). To verify this result, we analysed the expression of OTUD1 in lung cancer in the Oncomine database. OTUD1 expression was also downregulated in different pathological types of lung cancer in other databases (Fig. 1b-d). Moreover, patients with relatively low levels of OTUD1 in lung tumors showed shorter overall survival than those with high levels of OTUD1 in kmplot database (Fig. 1e). These results indicated that OTUD1 was expressed at low levels in lung cancer and closely associated with poor prognosis.
To validate the downregulation of OTUD1 in public databases, western blot was performed to assess OTUD1 protein levels in 12 pairs of lung tumors and adjacent normal tissues in lung cancer patients. We found OTUD1 protein expression level was significantly lower in most lung tumors than corresponding normal tissues (Fig. 1f). subcutaneously into both flanks of BALB/c nude mice respectively, and tumors were monitored continuously after injection. Five weeks later, we anesthetized the mice and isolated tumors and found that the volume of tumors was shrunk in the H827-OTUD1 group compared with the H827-vector group (Fig. 3a-c). Consistently, the tumor growth rate decreased after overexpression of OTUD1 (Fig. 3d). In conclusion, the xenograft model showed that OTUD1 inhibited lung adenocarcinoma growth.
To understand the function of OTUD1 in LUAD tumorigenesis, the primary lung cancer model was established in C57BL/6 wildtype (WT) and Otud1 knockout (KO) mice by injecting intraperitoneally with urethane twice a week for 5 weeks. Urethane is classified as a chemical carcinogen and is widely used to induce lung tumor formation in mice [40,41]. Ten weeks after the 5-week urethane treatment, we conducted wound healing and transwell assays. The wound healing assay showed that upregulation of OTUD1 significantly reduced the migration distance in H827 and H2030 cells (Fig. 2c-d). Similarly, the results of migration and invasion assays showed that overexpression of OTUD1 significantly reduced the number of migrated H827 and H2030 cells (Fig. 2e-h and S2a-d). These results suggested that OTUD1 inhibited the proliferation, migration and invasion of LUAD cells in vitro.

OTUD1 inhibits lung adenocarcinoma growth in mice
We next assessed the effects of OTUD1 on progression of LUAD in vivo using a cell-derived xenograft model. H827-vector and H827-OTUD1 cells were injected All data values were expressed as the mean ± SD. A two-side Student's t test was used for the statistical analysis with relatively low levels of FHL1 in lung tumors showed shorter overall survival than those with high levels of FHL1 in kmplot database (Fig. 4e).
To understand the regulatory effect of OTUD1 on FHL1, the protein levels of FHL1 after ectopic expression of OTUD1 or OTUD1-CS (enzyme active site mutation, C320S) were detected by western blot. OTUD1 overexpression significantly increased FHL1 protein levels (Fig. 4f). Whereas OTUD1-CS failed to upregulate FHL1 protein levels (Fig. 4f), indicating that the ability of OTUD1 to stabilize FHL1 was dependent on its deubiquitinase activity. We further tested whether OTUD1 could act as a deubiquitinase to regulate FHL1 degradation. We treated 293T cells with protein synthesis inhibitor cycloheximide (CHX) and monitored FHL1 expression by western blot at different time points. Notably, overexpression of OTUD1 extended the half-life of FHL1 protein (Fig. 4g-h).
To validate the direct relationship between OTUD1 and FHL1, co-immunoprecipitation (co-IP) were conducted to check that FHL1-Myc interacted with OTUD1-Flag (Fig. 4i). To map the FHL1-binding region in OTUD1, we constructed truncations with every OTUD1 domain deletion (Fig. 4j). The results of co-IP assays showed that the interaction between OTUD1 and FHL1 was most significantly weakened when the OTU domain was deleted as the mice were euthanized and lung were dissected to count the number of lung tumor nodules. We found more lung tumor nodules in Otud1 KO mice than those in WT mice ( Fig. 3e-f). Hematoxylin and eosin (H&E) staining lung tissues revealed increased tumor areas in Otud1 KO mice compared with WT mice (Fig. 3g). Altogether, these results demonstrated that OTUD1 inhibited primary lung adenocarcinoma formation and lung tumor growth in vivo.

OTUD1 stabilizes FHL1 protein expression
OTUD1 inhibited the progression of lung adenocarcinoma in vitro and in vivo, but the mechanism of OTUD1 was waiting to be explored. The deubiquitinase OTUD1 was reported to remove ubiquitination from its substrates and regulate the stability and functions of its substrates. To screen the potential substrate proteins of OTUD1, we identified 122 proteins interacting with OTUD1 by immunoprecipitation and mass spectrometry (IP-MS). Four and a half LIM domain protein 1 (FHL1) was the most significantly downregulated protein in TCGA lung cancer database among all the OTUD1-interacting proteins (Fig. 4a). We also analysed the expression of FHL1 in other lung cancer databases. FHL1 expression was downregulated in different pathological types of lung cancer in other databases (Fig. 4b-d). Moreover, patients were injected into the right flank of nude mice, and H827-OTUD1 cells (5 × 10 6 ) were injected into the left flank of nude mice. After 31 days, representative images showed the two flanks' tumors in mice after anesthesia. Meantime, tumor sizes were measured at the indicated time points (n = 5-7). **P < 0.01. (e) WT and Otud1 KO mice were intraperitoneally injected with urethane twice a week for 5 weeks. Ten weeks later the urethane treatment, mice were euthanized and dissected to count the number of lung tumor nodules. Representative images of lungs with visible nodules marked with arrows (n = 9). (f) Data shown in the graph represent the tumor number ± SD. ***P < 0.001. (g) Representative H&E staining images for the lung of WT and Otud1 KO mice. Scale bar, 200 μm. All data values were expressed as the mean ± SD. A two-side Student's t test was used for the statistical analysis

OTUD1 loses the tumor suppression ability in lung cancer when FHL1 disappeared
We constructed FHL1 knockout H827 cells using the CRISP/Cas9 genome editing system and assessed the function of OTUD1 in FHL1-deficient lung cancer cells. The efficiency of knockout was confirmed by western blot (Fig. 5a). CCK-8 assay was conducted to test the effect of OTUD1 on the proliferation of H827 cells when FHL1 disappeared. truncation C (Fig. 4k). It suggested that the OTU domain was essential for the interaction between OTUD1 and FHL1. Considering OTUD1 as a deubiquitinase to remove ubiquitination from its substrates, denatured-IP assays were conducted and showed that wild-type OTUD1, but not the CS mutant, reduced the polyubiquitination of FHL1 (Fig. 4l). Taken together, OTUD1 deubiquitinated and stabilized FHL1 and is dependent on its deubiquitinase activity and the OTU domain. 293T cells were transfected with the indicated plasmids, followed by treatment with MG132 (5 µM) for 12 h prior to collection. The lysates were incubated with Myc antibody and then subjected to immunoblotting. All data values were expressed as the mean ± SD. A two-side Student's t test was used for the statistical analysis 6-mercaptopurine and Geldanamycin) whose IC50s were correlated with OTUD1 mRNA levels ( Fig. S4a-d). To verify effects of these four drugs on OTUD1, we assessed OTUD1 protein levels in H827 cells after treatment with these drugs. The results showed that VE-822 significantly upregulated OTUD1 expression, while the other drugs downregulated or did not change the expression of OTUD1 (Fig. S4e). We further assessed protein levels of OTUD1 in other lung cancer cell lines (H460, H1975, H827 and H2030) with VE-822 treatments and found the elevation of OTUD1 and FHL1 protein levels in a dose-dependent manner (Fig. S4f, 6a and S5a). Therefore, we considered VE-822 as the agonist of OTUD1 in lung cancer cells. Considering VE-822 is an anti-tumor drug tested in undergoing clinical trials, we conducted the CCK-8 assay and found that VE-822 inhibited The results showed that FHL1 deficiency promoted H827 cell proliferation and OTUD1 failed to suppress H827 cell proliferation in the absence of FHL1 (Fig. 5b). Moreover, wound healing and transwell assays showed that FHL1 deficiency promoted H827 cells migration and invasion, and OTUD1 failed to suppress H827 cells migration and invasion in the absence of FHL (Fig. 5c-e and S3a-b).

VE-822 inhibits the proliferation and migration of lung cancer cells by upregulating OTUD1
To screen drug candidate targeting OTUD1, we analysed correlation of OTUD1 mRNA levels with half maximal inhibitor concentration (IC50) of drugs in CellMiner database. We found four drugs (VE-822, Dexamethasone,

Discussion
Lung cancer was the second most diagnosed cancer and the leading cause of cancer death in 2020, but there is a lack of research on the tumor suppressor OTUD1 in lung cancer. In this study, we found the expression of deubiquitinase OTUD1 was downregulated in lung cancer clinical samples and cell lines. OTUD1 inhibited the proliferation and migration of LUAD cells and inhibited LUAD formation and progression in mice. Furthermore, we identified OTUD1 as a novel deubiquitinase stabilizing FHL1. Moreover, VE-822 inhibited LUAD progression by upregulating OTUD1 in vitro and in vivo.
OTUD1 is a tumor suppressor in many cancers, including breast cancer, esophageal squamous cell carcinoma and pancreatic ductal adenocarcinoma [18][19][20]. In lung cancer, it was reported that OTUD1 inhibited proliferation and migration of lung cancer cells by stabilizing KLF4 [42]. However, the relationship and roles of OTUD1 and FLK4 had not been fully verified in lung cancer animal models. As an improvement to this research, our primary lung cancer model in Otud1 knockout mice and xenograft model in nude mice to validate the critical role of OTUD1 in LUAD formation and progression in vivo. Moreover, we found a novel substrate protein FHL1, besides KLF4, could be deubiquitinated and stabilized by OTUD1 in lung cancer, which indicated that more substrates and functions of OTUD1 were waiting to be explored.
FHL1 was considered as a tumor suppressor in many cancers FHL1 inhibited the proliferation of colorectal cancer cells by negatively regulating the Wnt/β-catenin signaling pathway [26]. Besides, FHL1 induced apoptosis of gastric cancer cells [43]. It was reported that FHL1 mediated modulation of the G2/M checkpoint and radioresistance in breast cancer [44]. And the inhibitory effects of FHL1 on lung cancer cell growth were associated with both the G1 and the G2/M cell cycle arrest concomitant [45]. Our study found that OTUD1 depended on FHL1 to play a role in inhibiting lung adenocarcinoma tumorigenesis and growth. We identified OTUD1 as a novel deubiquitinase of FHL1 in lung cancer. Previous study reported that USP15 deubiquitinated and stabilized FHL1 and induced cardiac hypertrophy [46]. Our study about OTUD1/FHL1 in lung cancer and previous study about USP15/FHL1 in cardiomyocyte indicated that the deubiquitination of FHL1 in tumor or non-tumor tissues played distinct roles. The functional switch of FHL1 in different disease context is interesting and worth further exploration.
The phase I clinical trial of VE-822 in advanced solid tumors showed that VE-822 is safe and tolerable, and preliminary antitumor response have been observed [47]. In previous studies, VE-822 has been mostly used as an inhibitor proliferation of lung cancer cells, but not the bronchial epithelial cell (BEAS-2B) (Fig. S4g-k). Considering that VE-822 is an ATR inhibitor, we tested the effect of VE-822 on ATM/ATR signal pathway in H827 and H2030 lung cancer cell lines. The results showed that VE-822 reduced the phosphorylated protein levels of ATM and ATR (Fig. S6a). But in the H827-OTUD1 or H2030-OTUD1 cell lines, phosphorylated protein levels of ATM and ATR were not affected by the over-expression of OTUD1 (Fig. S6b). These results suggested that OTUD1 suppressing the progression of lung cancer was in an ATR-independent manner, while ATM/ ATR pathway inhibition may play a role in the treatment of lung cancer by VE-822.
To understand the relationship between VE-822 and OTUD1 in lung cancer, the CCK-8 assay was conducted to evaluate proliferation of OTUD1-overexpression and control cells after VE-822 treatment. Proliferation of H827 and H2030 was inhibited by OTUD1 overexpression and VE-822 respectively, and cell proliferation was almost completely inhibited when they were combined together ( Fig. 6b and S5b). Furthermore, wound-healing assays were conducted and showed that VE-822 reduced migration distances of H827 and H2030 cells (Fig. 6c and S5c), and migration distances of OTUD1-overexpressing cells under VE-822 treatment were even shorter ( Fig. 6d and S5d). Consistently, VE-822 suppressed the migration and invasion of H827 and H2030 cells (Fig. 6e g, S5e and S5g), and the numbers of OTUD1-overexpressing cells under VE-822 treatment were less (Fig. 6f h, S5f and S5h). The numbers of migrative and invasive cells were calculated ( Fig. S7 and S8). As a result, VE-822 inhibited the proliferation, migration and invasion of lung cancer cells by upregulating OTUD1 in vitro.

VE-822 inhibits lung adenocarcinoma growth in mice
To assess the effects of VE-822 and OTUD1 on progression of LUAD in vivo, the cell-derived xenograft model was used. H827-vector and H827-OTUD1 cell lines were injected subcutaneously into both flanks of BALB/c nude mice. Fourteen days later, mice were treated with VE-822 for 21 days by intragastric administration. Consistent with the results in vitro, VE-822 treatment significantly inhibited tumor growth in H827-vector group, and tumor growth in H827-OTUD1 group was almost completely inhibited by VE-822 (Fig. 7a-d). These findings suggested that VE-822 inhibited lung tumor growth upregulating OTUD1. Effects of VE-822 (0.1 µM) and OTUD1 overexpression on cell invasion. H827-vector and H827-OTUD1 cells were planted in Transwell chambers with matrigel. Six hours later, the medium in upper chamber was replaced with fresh medium containing 0.1 µM VE-822. Invasive potential was assessed after a 24-h incubation. Scale bar, 200 μm (left), 100 μm (right). All data values were expressed as the mean ± SD. A one-way ANOVA was used for the statistical analysis Taken together, our results demonstrated that the tumor suppressor gene OTUD1 deubiquitinated and stabilized FHL1 to inhibit the proliferation and migration of lung cancer in vitro and in vivo. Besides, we considered VE-822 as an agonist of OTUD1 to suppress lung cancer. Our research about VE-822 and OTUD1 will provide a theoretical basis for the future application of VE-822 in clinical antitumor treatment of lung cancer.

Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1007/s13402-023-00793-x. of ATR in the chemosensitization and radiosensitization of cancers [48,49]. In addition, VE-822 blocked autophagy by accumulating p62 and LC3-II in an ATR-independent manner in esophageal cancer cells [50]. In our study, we found VE-822 as an up-regulator of OTUD1 by bioinformatic analysis in CellMiner database and a series of validation experiments. The synergistic effect of VE-822 and OTUD1 significantly inhibited the proliferation and migration of lung cancer cells and LUAD tumor growth in vivo, indicating the potential application and effect of VE-822 in the treatment of lung cancer. In order to fully explore the mechanism of VE-822, the transcriptional or translational regulation of OTUD1 by VE-822 is worth studying in the further research. If VE-822 is combined with OTUD1, the binding site and mode of them are interesting research points. Besides, ubiquitination modification is believed to be closely related to autophagy pathway [51]. We are going to explore whether autophagy plays a role in the synergistic effect of VE-822 and OTUD1 in the next research. (a-b) H827-vector and H827-OTUD1 cells (5 × 10 6 ) were injected into flanks of nude mice. Two weeks later, H827-vector and H827-OTUD1 groups were respectively and randomly divided into two groups for PBS or VE-822 (60 mg/kg) treatment (n = 5-10). Thirty-five days after injection, mice were euthanized and dissected. Representative images showed the mice and tumors. (c-d) Tumor sizes were measured at the indicated time points (n = 5-10). **P < 0.01, ***P < 0.001. All data values were expressed as the mean ± SD. A one-way ANOVA was used for the statistical analysis. (e) Model showing the role of OTUD1 and VE-822 in lung cancer. OTUD1 is downregulated in lung cancer, and FHL1 is ubiquitinated and degraded, thus promoting the progression of lung cancer. VE-822 upregulates OTUD1 and results in deubiquitinating FHL1, ultimately leading to inhibit lung cancer