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Over‐expression of USP15/MMP3 predict poor prognosis and promote growth, migration in non-small cell lung cancer cells

  • Author Footnotes
    ⁎ These authors contributed equally to this work.
    Weiwei Chen
    Footnotes
    ⁎ These authors contributed equally to this work.
    Affiliations
    Medical School of Nantong University, Nantong, 226007, China

    Department of Oncology, Affiliated Hospital of Nantong University, Nantong, 226001, China

    Department of Radiotherapy, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Author Footnotes
    ⁎ These authors contributed equally to this work.
    Daguang Ni
    Footnotes
    ⁎ These authors contributed equally to this work.
    Affiliations
    Department of Radiotherapy, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Author Footnotes
    ⁎ These authors contributed equally to this work.
    Hailin Zhang
    Footnotes
    ⁎ These authors contributed equally to this work.
    Affiliations
    Department of Pneumology, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Xia Li
    Affiliations
    Department of General Medicine, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Youqin Jiang
    Affiliations
    Department of Radiotherapy, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Jixiang Wu
    Affiliations
    Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Yan Gu
    Affiliations
    Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Mingcheng Gao
    Affiliations
    Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Woda Shi
    Correspondence
    Corresponding authors.
    Affiliations
    Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Jianxiang Song
    Correspondence
    Corresponding authors.
    Affiliations
    Department of Cardiothoracic Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng Third People's Hospital, The Yancheng School of Clinical Medicine of Nanjing Medical University, Yancheng 224002, China
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  • Wenyu Shi
    Correspondence
    Corresponding authors.
    Affiliations
    Medical School of Nantong University, Nantong, 226007, China

    Department of Oncology, Affiliated Hospital of Nantong University, Nantong, 226001, China
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  • Author Footnotes
    ⁎ These authors contributed equally to this work.
Open AccessPublished:January 06, 2023DOI:https://doi.org/10.1016/j.cancergen.2023.01.001

      Highlights

      • USP15 was highly expressed in NSCLC cancerous tissues and cells.
      • USP15 knockdown reduced NSCLC progression in vitro and in vivo.
      • USP15 knockdown inhibited the expression of MMP3 and MMP9 in NSCLC cells.
      • High USP15 and MMP3 expression associated with poor prognosis.

      Abstract

      Aberrant ubiquitin modifications caused by an imbalance in the activities of ubiquitinases and de-ubiquitinases are emerging as important mechanisms underlying non-small cell lung cancer (NSCLC) progression. The deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) has been identified as an important factor in oncogenesis and a potential therapeutic target. However, the expression profile and function of USP15 in NSCLC remain elusive. In the present study, we investigated the expression pattern and the potential biological functions of USP15 in NSCLC both in cells and animal models. Our data revealed that USP15 was highly expressed in NSCLC tissues and cells compared with normal counterpart. We subsequently knocked down USP15 expression in two NSCLC cell lines, which significantly suppressed cell proliferation. In addition, knocking down USP15 expression reduced NSCLC cell migration and invasion according to the results from Matrigel-Transwell analysis. NSCLC animal model results showed that USP15 knockdown also reduced NSCLC size. Biochemical analysis revealed that USP15 knockdown inhibited matrix metalloproteinase (MMP)3 and MMP9 expression. Furthermore, high levels of USP15 and MMP3 expression were associated with poor prognosis in NSCLC. In conclusion, the results from the present study suggest that the high expression of USP15 promotes NSCLC tumorigenesis. Therefore, it is proposed that USP15 and MMPs may represent novel biomarkers for NSCLC progression and prognosis.

      Keywords

      Introduction

      Lung cancer has been one of the main causes of cancer-related mortality worldwide over the past few decades [

      IARC Globocan 2012. Cancer Fact Sheet: Lung Cancer Mortality Worldwide in 2012..

      ]. In total, ∼80% of the patients with lung cancer are histopathologically diagnosed with the non-small cell lung cancer (NSCLC) subtype [
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      ]. Therefore, it remains an urgent priority to understand the molecular physiology of NSCLC to develop novel therapeutic targets for NSCLC treatment.
      Ubiquitin modification of proteins is a multifaceted and dynamic process that is involved in nearly all aspects of eukaryotic biology [
      • Kulathu Y
      • Komander D.
      Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages.
      ]. Aberrant ubiquitin modifications in tumors have been previously found and are currently a subject of extensive research [
      • Kulathu Y
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      Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages.
      ,
      • Chen ZJ
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      ]. Ubiquitin modification is a reversible process that is mediated by de-ubiquitinases (DUBs), of which ∼95 members exist in the human genome [
      • D'Andrea A.
      • Pellman D.
      Deubiquitinating enzymes: a new class of biological regulators.
      ,
      • Nijman S.M.
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      • Velds A.
      • Brummelkamp T.R.
      • Dirac A.M.
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      Bernards, R. A genomic and functional inventory of deubiquitinating enzymes.
      ,
      • Fraile JM
      • Quesada V
      • Rodriguez D
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      Deubiquitinases in cancer: new functions and therapeutic options.
      ,
      • Weathington N.M.
      • Mallampalli R.K.
      Emerging therapies targeting the ubiquitin proteasome system in cancer.
      ]. However, the downstream function mediated by the majority of DUBs remains poorly understood. DUBs belong to a family of proteases that are ubiquitin-specific [
      • Baek K.H.
      Cytokine-regulated protein degradation by the ubiquitination system.
      ]. First discovered in 1999, ubiquitin-specific peptidase 15 (USP15) is a ubiquitously-expressed DUB that contains a zinc finger domain critical for its de-ubiquitinase activity [
      • Hetfeld BK
      • Helfrich A
      • Kapelari B
      • Scheel H
      • Hofmann K
      • Guterman A
      • et al.
      The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1.
      ]. USP15 has been found to deubiquitinate inhibitor of nuclear factor κB (NF-κB)-α, which resulted in the reduction of NF-κB activity [
      • Schweitzer K
      • Bozko PM
      • Dubiel W
      • Naumann M.
      CSN controls NF-kappaB by deubiquitinylation of IkappaBalpha.
      ]. In addition, USP15 can recognize receptor-activated SMADs to enhance SMAD complex function [
      • Inui M
      • Manfrin A
      • Mamidi A
      • Martello G
      • Morsut L
      • Soligo S
      • et al.
      USP15 is a deubiquitylating enzyme for receptor-activated SMADs.
      ]. In another study, USP15 was shown to specifically deubiquitinate kelch like ECH associated protein 1 and increase nuclear factor-erythroid factor 2-related factor 2 expression, which enhanced chemoresistance in breast cancer [
      • Villeneuve NF
      • Tian W
      • Wu T
      • Sun Z
      • Lau A
      • Chapman E
      • et al.
      USP15 negatively regulates Nrf2 through deubiquitination of Keap1.
      ]. Recent studies have also revealed a de novo stabilizing function of USP15 on newly synthesized proteins, which sheds new light on the potentially multifaceted roles of DUBs [
      • Villeneuve NF
      • Tian W
      • Wu T
      • Sun Z
      • Lau A
      • Chapman E
      • et al.
      USP15 negatively regulates Nrf2 through deubiquitination of Keap1.
      ,
      • Faronato M
      • Patel V
      • Darling S
      • Dearden L
      • Clague MJ
      • Urbe S
      • et al.
      The deubiquitylase USP15 stabilizes newly synthesized REST and rescues its expression at mitotic exit.
      ].
      Matrix metalloproteinases (MMPs) have the ability to degrade extracellular matrix (ECM) proteins and expose binding sites within the matrix molecules to facilitate tumor progression and metastasis [
      • Foda HD
      • Zucker S.
      Matrix metalloproteinases in cancer invasion, metastasis and angiogenesis.
      ,
      • Egeblad M
      • Werb Z.
      New functions for the matrix metalloproteinases in cancer progression.
      ]. MMP2, MMP3 and MMP9 expression have all been found to be upregulated in lung cancer compared to normal counterparts [
      • Han L
      • Sheng B
      • Zeng Q
      • Yao W
      • Jiang Q.
      Correlation between MMP2 expression in lung cancer tissues and clinical parameters: a retrospective clinical analysis.
      ,
      • Mehner C
      • Miller E
      • Nassar A
      • Bamlet WR
      • Radisky ES
      • Radisky DC.
      Tumor cell expression of MMP3 as a prognostic factor for poor survival in pancreatic, pulmonary, and mammary carcinoma.
      ,
      • Zhang H
      • Zhao B
      • Zhai ZG
      • Zheng JD
      • Wang YK
      • Zhao YY.
      Expression and clinical significance of MMP-9 and P53 in lung cancer.
      ]. In particular, MMP3 was revealed to be highly associated with poor survival in pancreatic, pulmonary and breast cancer [
      • Zhang H
      • Zhao B
      • Zhai ZG
      • Zheng JD
      • Wang YK
      • Zhao YY.
      Expression and clinical significance of MMP-9 and P53 in lung cancer.
      ].
      In the present study, we found that compared with that in the normal control tissues and cell lines, USP15 was highly expressed in NSCLC tumor tissues and cells. USP15 knockdown significantly reduced the proliferation and invasion of NSCLC cells by downregulating the expression of MMP3 and MMP9. In addition, results from the NSCLC animal models showed that USP15 knockdown inhibited NSCLC growth. High levels of USP15 and MMP3 expression were also associated with poor prognosis. Therefore, this study suggests that upregulation of USP15 is important for NSCLC pathogenesis by regulating the expression of matrix metalloproteinases, rendering USP15 to be a novel target for NSCLC treatment.

      Materials and methods

      Tissues

      NSCLC and healthy control tissues (30 cases, 14 male and 16 female) were collected from patients at The First Affiliated Hospital of Soochow University (Soochow, China) between Jan 2017 to Mar 2018. This project obtained the subjects’ written informed consent and institutional review board approval of The First Affiliated Hospital of Soochow University.

      Cells and plasmids

      The lung epithelial cell line BEAS-2B and lung cancer cell A549, were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and maintained in DMEM (Hyclone, USA) supplemented with 10 % heat-inactivated fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc., USA) and penicillin and streptomycin (100 μg/ml). Lung cancer cell H1299 were also purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and maintained in RPMI-1640 (Hyclone, USA) medium supplemented with 10 % FBS and penicillin and streptomycin (100 μg/ml). All cells were cultured at 37˚C in a humidified atmosphere of 5% CO2 and were confirmed to be free of mycoplasma contamination.
      The USP15 knockdown lentivirus was purchased from Hanyin Biotech, Co. Ltd. (Shanghai, china). To obtain stable cell lines, lentivirus supernatant was added to the NSCLC cells (MOI:10), followed by puromycin (1 μg/ml) selection after 2 weeks at 37˚C in a humidified atmosphere of 5% CO2.

      Immunofluorescence

      Immunofluorescence experiments were performed. The slides were fixed with 4 % paraformaldehyde and blocked with PBS containing 0.3 % TritonX-100 and 5 % bovine serum albumin (BSA) for 30 min. Antibodies were incubated at 4°C overnight. The slides were washed 6 times with PBST (PBS containing 0.3 % TritonX-100) for a total of an hour and incubated with secondary antibodies for 1 hour at room temperature. The samples were observed with a microscope (Nikon).

      Cell Counting Kit-8 (CCK-8) assay

      A CCK-8 assay was conducted according to the manufacturer's protocols (Dojindo, Kumamoto, Japan). Briefly, NSCLC cells with or without USP15 knockdown were seeded into 96-well plates (2,000 cells/ well) and detected for 5 days at 37˚C in a humidified atmosphere of 5% CO2. Following the incubation, 10 µl CCK-8 reagent (5 mg/ml) diluted in the culture medium was added to the cells and cultured for 2 h at 37˚C. The absorbance in each well was detected at 490 nm using a microplate reader (Biotek ELx800; BioTek Instruments, Inc.).

      Matrigel-Transwell assay

      Cell invasion after USP15 knockdown was examined. Briefly, NSCLC cells were suspended in serum-free DMEM or 1640 medium and plated into the upper chamber of Matrigel (BD)-coated Transwell inserts (8-μm). At the same time, medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 24 h at 37˚C, cells on the upper surface of the insert were gently removed with a cotton swab. Cells that had invaded onto the lower surface of the insert were fixed with 4% paraformaldehyde for 20min and stained with 1% crystal violet for 30 mins at room temperature. Stained cells were visualized in five randomly selected microscopic fields of view.

      Western blot analysis

      The cells were lysed with radio immunoprecipitation assay buffer (RIPA, Beyotime, China) containing protease inhibitors (Roche, Complete Mini). The total protein was quantitated by bicinchoninic acid assay kit (Beyotime, Haimen, China). Proteins (30-50 μg/lane) were isolated and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% fat-free milk at room temperature for 60 min and incubated with primary antibodies (1:1,000 dilution) overnight at 4˚C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1h at room temperature (Jackson lab, 1:10,000 dilution) and detected by chemiluminescence (ECL, millipore). The following antibodies were used: Rabbit anti-human USP15 (cat. no. GTX104644; GeneTex, Inc.), mouse anti-β-actin (cat. no. 3700P; Cell Signaling Technology, Inc.), rabbit anti-human MMP2 (cat. no. ab192082; Abcam), rabbit anti-human MMP3 (cat. no. ab53015; Abcam) and rabbit anti-human MMP9 (cat. no. 3852; Cell Signaling Technology, Inc.).

      Animal models

      16 male-nude mice (6 to 8 weeks-old) purchased from Lingchang Biotech (Shanghai, China) were maintained and treated in accordance to the Institutional Animal Care and Use Committee of the Soochow University. H1299 cells (1 × 106 suspended in 1640) with or without USP15 knockdown were injected into the tail veins of 6–8-week-old nude mice (6-8 mice/group). After 6 weeks, mice were euthanized using CO2 before the lungs were removed and fixed in Bouin's solution overnight at room temperature. The number of nodes that were >0.5 mm in diameter in every lobe of lung were counted.

      Hematoxylin-Eosin (H&E) Staining

      For H&E staining, paraffin embedded sections are dyed with hematoxylin for 5 minutes after they are deparaffinized and hydrated. Then washed with soft running water, treated with 1 % hydrochloric acid alcohol for seconds and quickly turn blue with 0.6 % ammonia. After that the slices are dyed in the eosin dye solution for 3min. After dehydration, use neutral resin to seal, and the images are captured by a microscope (Nikon).

      Statistical analysis

      Data were presented as mean±SD. One-way ANOVA followed by the Student-Newman-Keuls tests was performed to determine any differences among the treatment groups using SPSS 13.0 software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference, Repeat each experiment three times.

      Results

      USP15 expression is upregulated in NSCLC tissues and cell lines

      The expression of the USP15 gene has been previously found to be increased in glioblastoma, breast and ovarian cancer compared with normal counterpart [
      • Han L
      • Sheng B
      • Zeng Q
      • Yao W
      • Jiang Q.
      Correlation between MMP2 expression in lung cancer tissues and clinical parameters: a retrospective clinical analysis.
      ,
      • Mehner C
      • Miller E
      • Nassar A
      • Bamlet WR
      • Radisky ES
      • Radisky DC.
      Tumor cell expression of MMP3 as a prognostic factor for poor survival in pancreatic, pulmonary, and mammary carcinoma.
      ,
      • Zhang H
      • Zhao B
      • Zhai ZG
      • Zheng JD
      • Wang YK
      • Zhao YY.
      Expression and clinical significance of MMP-9 and P53 in lung cancer.
      ]. We therefore measured USP15 expression in NSCLC and adjacent normal tissues. We found that the USP15 expression level was significantly higher in NSCLC tissues compared with that in adjacent normal tissues (Fig. 1A and B). We next measured USP15 expression in two different NSCLC cell lines, A549 and H1299, and in the lung normal epithelial cell line, BEAS-2B. Western blot analysis revealed that USP15 expression was higher in the NSCLC cells compared with that in BEAS-2B cells (Fig. 1C).
      Fig. 1
      Fig. 1Expression of USP15 in NSCLC tissues and cells. A, Immunofluorescence analysis of USP15 expression in NSCLC and adjacent control normal tissues. B, Western blot analysis of USP15 expression in NSCLC and adjacent normal tissues (n=5). C, Western blot analysis of USP15 expression in the normal lung epithelial cell line BEAS-2B and in NSCLC cell lines. USP15, ubiquitin-specific peptidase 15; NSCLC, non-small cell lung cancer. Repeat each experiment three times.
      To explore the possible function of USP15 during NSCLC progression, we constructed lentivirus particles encoding short hairpin RNA (shRNA) targeting USP15 to knock down USP15 expression in A549 and H1299 cells. USP15 protein expression was significantly decreased in cells transfected with the lentivirus expressing USP15-shRNA compared to negative control (Fig. 1D and 1E).

      USP15 inhibition reduces the proliferation and invasion of NSCLC cell lines

      To explore the possible physiological function of USP15 in NSCLC, we performed CCK-8 assays to assess the viability of the NSCLC cell lines, A549 (Fig. 2A) and H1299 (Fig. 2B). The CCK-8 assay results showed that the viability of the NSCLC cells was reduced following USP15 knockdown (Fig. 2C).
      Fig. 2
      Fig. 2Knockdown of USP15 reduces NSCLC cell proliferation and invasion. A, Western blotting data following USP15 knockdown in A549 cells. B, Western blotting data following USP15 knockdown in H1299 cells. C, CCK-8 analysis of cell proliferation in negative control and USP15 knockdown A549 cells. D, CCK-8 analysis of cell proliferation in negative control and USP15 knockdown H1299 cells. E and F, Matrigel-Transwell assay in A549 and H1299 cells after USP15 knockdown. G and H, Semi-quantification of the Transwell assay results in E and F. CCK-8, Cell Counting Kit-8; USP15, ubiquitin-specific peptidase 15; NSCLC, non-small cell lung cancer. Repeat each experiment three times.
      The effect of USP15 knockdown on cell migration and invasion was subsequently evaluated using Matrigel-Transwell assays. Knocking down USP15 expression significantly decreased cell migration and invasion (Fig. 2C-2F). These results suggest that USP15 serves an active role in NSCLC cell proliferation and invasion.

      Inhibition of USP15 reduces the progression of NSCLC in vivo

      To study the functional role of USP15 in NSCLC progression further, we injected H1299 cells into the tail vein of nude mice to induce pulmonary tumor nodules (Fig. 3). Compared with that in mice injected with control cells, knockdown of USP15 resulted in the significantly reduced formation of tumor nodules (Fig. 3C). Consistently, the tumor weight was also found to be significantly lower in the USP15-knockdown group compared with that in the control group (Fig. 3D).
      Fig. 3
      Fig. 3Knockdown of USP15 reduces the progression of NSCLC in vivo.
      A, Tumor formation ability of H1299 cells (1 × 106) after injection into each mouse was examined after USP15 knockdown.
      B, Hematoxylin and eosin staining analysis of tissues from each group are shown.
      C, The numbers of tumor nodules was measured.
      D, Tumor weight was measured.
      USP15, ubiquitin-specific peptidase 15; NSCLC, non-small cell lung cancer.Repeat each experiment three times.

      USP15 knockdown suppresses the expression of MMPs in NSCLC cells

      Since MMP-mediated degradation of the basement membrane is a pivotal process for tumor metastasis [
      • Kessenbrock K.
      • Plaks V.
      • Werb Z.
      Matrix metalloproteinases: regulators of the tumor microenvironment.
      ,
      • Zhang M.
      • Dai C.
      • Zhu H.
      • Chen S.
      • Wu Y.
      • Li Q.
      • Zeng X.
      • Wang W.
      • Zuo J.
      • Zhou M.
      • Xia Z.
      • Ji G.
      • Saiyin H.
      • Qin L.
      • Yu L.
      Cyclophilin A promotes human hepatocellular carcinoma cell metastasis via regulation of MMP3 and MMP9.
      ], we determined whether MMPs can regulate USP15-mediated NSCLC cell proliferation and invasion. Western blotting results showed that the knockdown of USP15 significantly decreased the expression of MMP3 and MMP9 in NSCLC cells compared to negative control (Fig. 4A and 4B).
      Fig. 4
      Fig. 4Knockdown of USP15 inhibits the expression of MMPs in NSCLC.
      A, Western blotting and subsequent quantification analysis of MMP2, MMP3 and MMP9 expression in A549 cells after USP15 knockdown.
      B, Western blotting and subsequent quantification analysis of MMP2, MMP3 and MMP9 expression in H1299 after USP15 knockdown.
      USP15, ubiquitin-specific peptidase 15; NSCLC, non-small cell lung cancer; MMP, matrix metalloproteinase.Repeat each experiment three times.

      Higher levels USP15 and MMP3 expression are associated with poor prognosis

      We then investigated the effects of USP15 and the associated MMPs on the prognosis of patients with NSCLC using the public Gene Expression Profiling Interactive Analysis (GEPIA) database (https://gepia.cancer-pku.cn/detail.php?gene). The results showed that high USP15 and MMP3 expression levels were associated with a poor disease-free survival (P=0.041; n=481; Fig. 5B). However, USP15 and MMP2 or MMP9 were not significantly associated with disease-free survival (Fig. 5A and 5C).
      Fig. 5
      Fig. 5High USP15 and MMP3 expression is associated with poor prognosis in terms of NSCLC.
      A, Kaplan-Meier analysis of the effects of USP15 and MMP2 expression on the prognosis of patients with NSCLC using the public GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene).
      B, Kaplan-Meier analysis of the effects of USP15 and MMP3 expression on the prognosis of patients with NSCLC using the public GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene).
      C, Kaplan-Meier analysis of the effects of USP15 and MMP9 expression on the prognosis of patients with NSCLC using the public GEPIA database (http://gepia.cancer-pku.cn/detail.php?gene).
      USP15, ubiquitin-specific peptidase 15; NSCLC, non-small cell lung cancer; MMP, matrix metalloproteinase.
      D, The amount of USP15 protein in shUSP15 after overexpression of MMP9 in rescue experiment*** P < 0.001, * * P < 0.01, Repeat each experiment three times.
      Altogether, the results from our study suggest that USP15 promotes the growth and invasion of NSCLC by regulating MMP expression, which may provide a novel target for NSCLC treatment.

      Discussion

      USP15 is a deubiquitinating enzyme that was first discovered in 1999 and has been reported to serve active roles in various types of malignancies, including brain tumors, colorectal cancer and several other types of cancer [
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      ,
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      ]. In particular, USP15 has been reported to serve a direct role in the pathogenesis of glioblastoma [
      • Srihari S
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      Systematic tracking of dysregulated modules identifies novel genes in cancer.
      ]. Although USP15 is predominantly localized to the cytoplasm [
      • Urbé S
      • Liu H
      • Hayes SD
      • Heride C
      • Rigden DJ
      • Clague MJ.
      Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions.
      ], it has also been observed to perform specific functions in the nucleus [
      • Long L
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      • Furgason M
      • Haj-Yahya M
      • Brik A
      • Cheng D
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      The U4/U6 recycling factor SART3 has histone chaperone activity and associates with USP15 to regulate H2B deubiquitination.
      ], mitochondria [
      • Cornelissen T
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      ] or in polysomes [
      • Faronato M
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      • et al.
      The deubiquitylase USP15 stabilizes newly synthesized REST and rescues its expression at mitotic exit.
      ]. In the present study, USP15 was revealed to be highly expressed in NSCLC, suggesting that USP15 may be used as a beneficial biomarker for NSCLC.
      In a previous study, USP15 gene expression was found to be significantly reduced in docetaxel-resistant gastric cancer cells compared with that in their corresponding parental cells using microarray analysis [
      • Xie L
      • Wei J
      • Qian X
      • Chen G
      • Yu L
      • Ding Y
      • et al.
      CXCR4, a potential predictive marker for docetaxel sensitivity in gastric cancer.
      ]. These observations were subsequently validated using reverse transcription-quantitative PCR[36]. Further analysis was conducted in 11 cancer cell lines of the digestive system, which demonstrated that USP15 expression levels were significantly correlated with docetaxel sensitivity [
      • Xie L
      • Wei J
      • Qian X
      • Chen G
      • Yu L
      • Ding Y
      • et al.
      CXCR4, a potential predictive marker for docetaxel sensitivity in gastric cancer.
      ]. However, the function and underlying mechanism of USP15 in NSCLC remain to be elucidated. The results of the present study showed that USP15 knockdown significantly reduced the tumorigenic ability of NSCLC cells. In addition, USP15 regulated the migration and invasion of NSCLC cells, which were mediated by modulating MMP3 and MMP9 expression. MMPs are proteolytic enzymes that have been documented to contribute to most stages of tumor progression, including the later stages of invasion and metastasis [
      • Kessenbrock K.
      • Plaks V.
      • Werb Z.
      Matrix metalloproteinases: regulators of the tumor microenvironment.
      ]. MMP2, MMP3 and MMP9 have been found to promote NSCLC and hepatocellular carcinoma cell invasion [
      • Dong QZ
      • Wang Y
      • Tang ZP
      • Fu L
      • Li QC
      • Wang ED
      • Wang EH.
      Derlin-1 is overexpressed in non-small cell lung cancer and promotes cancer cell invasion via EGFR-ERK-mediated up-regulation of MMP-2 and MMP-9.
      ,
      • Zhang M.
      • Dai C.
      • Zhu H.
      • Chen S.
      • Wu Y.
      • Li Q.
      • Zeng X.
      • Wang W.
      • Zuo J.
      • Zhou M.
      • Xia Z.
      • Ji G.
      • Saiyin H.
      • Qin L.
      • Yu L.
      Cyclophilin A promotes human hepatocellular carcinoma cell metastasis via regulation of MMP3 and MMP9.
      ]. The expression of MMP3 in tumor cells has been previously reported to be a prognostic predictor of poor survival in pancreatic, pulmonary and mammary carcinoma [
      • Mehner C
      • Miller E
      • Nassar A
      • Bamlet WR
      • Radisky ES
      • Radisky DC.
      Tumor cell expression of MMP3 as a prognostic factor for poor survival in pancreatic, pulmonary, and mammary carcinoma.
      ]. Our in vivo results found that USP15 regulates NSCLC tumor growth and invasion. However, although USP6 expression was previously found to induce MMPs by activating NF-κB [
      • Ye Y
      • Pringle LM
      • Lau AW
      • Riquelme DN
      • Wang H
      • Jiang T
      • Lev D
      • Welman A
      • Blobel GA
      • Oliveira AM
      • Chou MM.
      TRE17/USP6 oncogene translocated in aneurysmal bone cyst induces matrix metalloproteinase production via activation of NF-kappaB.
      ], the precise mechanism underlying the USP15-mediated regulation of MMPs in NSCLC requires further study.
      In recent years, efforts have been made to target DUBs in cancer [
      • Vanneman M
      • Dranoff G.
      Combining immunotherapy and targeted therapies in cancer treatment.
      ]. USP15 may prove to be an efficient target for preventing NSCLC progression.

      Data availability

      • Data will be made available on request.

      Declaration of Competing Interest

      None.

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