Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis
Identifieur interne : 000835 ( Pmc/Corpus ); précédent : 000834; suivant : 000836Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis
Auteurs : Wenshu Chen ; Qiong Wang ; Xiuling Xu ; Bryanna Saxton ; Mathewos Tessema ; Shuguang Leng ; Swati Choksi ; Steven A. Belinsky ; Zheng-Gang Liu ; Yong LinSource :
- Translational Oncology [ 1936-5233 ] ; 2019.
Abstract
Url:
DOI: 10.1016/j.tranon.2019.09.001
PubMed: 31760267
PubMed Central: 6883318
Links to Exploration step
PMC:6883318Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis</title><author><name sortKey="Chen, Wenshu" sort="Chen, Wenshu" uniqKey="Chen W" first="Wenshu" last="Chen">Wenshu Chen</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Wang, Qiong" sort="Wang, Qiong" uniqKey="Wang Q" first="Qiong" last="Wang">Qiong Wang</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Xu, Xiuling" sort="Xu, Xiuling" uniqKey="Xu X" first="Xiuling" last="Xu">Xiuling Xu</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Saxton, Bryanna" sort="Saxton, Bryanna" uniqKey="Saxton B" first="Bryanna" last="Saxton">Bryanna Saxton</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Tessema, Mathewos" sort="Tessema, Mathewos" uniqKey="Tessema M" first="Mathewos" last="Tessema">Mathewos Tessema</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Leng, Shuguang" sort="Leng, Shuguang" uniqKey="Leng S" first="Shuguang" last="Leng">Shuguang Leng</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Choksi, Swati" sort="Choksi, Swati" uniqKey="Choksi S" first="Swati" last="Choksi">Swati Choksi</name><affiliation><nlm:aff id="aff2">Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr., Bethesda, MD, 20892, USA</nlm:aff></affiliation></author><author><name sortKey="Belinsky, Steven A" sort="Belinsky, Steven A" uniqKey="Belinsky S" first="Steven A." last="Belinsky">Steven A. Belinsky</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Liu, Zheng Gang" sort="Liu, Zheng Gang" uniqKey="Liu Z" first="Zheng-Gang" last="Liu">Zheng-Gang Liu</name><affiliation><nlm:aff id="aff2">Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr., Bethesda, MD, 20892, USA</nlm:aff></affiliation></author><author><name sortKey="Lin, Yong" sort="Lin, Yong" uniqKey="Lin Y" first="Yong" last="Lin">Yong Lin</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31760267</idno><idno type="pmc">6883318</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883318</idno><idno type="RBID">PMC:6883318</idno><idno type="doi">10.1016/j.tranon.2019.09.001</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000835</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000835</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis</title><author><name sortKey="Chen, Wenshu" sort="Chen, Wenshu" uniqKey="Chen W" first="Wenshu" last="Chen">Wenshu Chen</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Wang, Qiong" sort="Wang, Qiong" uniqKey="Wang Q" first="Qiong" last="Wang">Qiong Wang</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Xu, Xiuling" sort="Xu, Xiuling" uniqKey="Xu X" first="Xiuling" last="Xu">Xiuling Xu</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Saxton, Bryanna" sort="Saxton, Bryanna" uniqKey="Saxton B" first="Bryanna" last="Saxton">Bryanna Saxton</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Tessema, Mathewos" sort="Tessema, Mathewos" uniqKey="Tessema M" first="Mathewos" last="Tessema">Mathewos Tessema</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Leng, Shuguang" sort="Leng, Shuguang" uniqKey="Leng S" first="Shuguang" last="Leng">Shuguang Leng</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Choksi, Swati" sort="Choksi, Swati" uniqKey="Choksi S" first="Swati" last="Choksi">Swati Choksi</name><affiliation><nlm:aff id="aff2">Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr., Bethesda, MD, 20892, USA</nlm:aff></affiliation></author><author><name sortKey="Belinsky, Steven A" sort="Belinsky, Steven A" uniqKey="Belinsky S" first="Steven A." last="Belinsky">Steven A. Belinsky</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author><author><name sortKey="Liu, Zheng Gang" sort="Liu, Zheng Gang" uniqKey="Liu Z" first="Zheng-Gang" last="Liu">Zheng-Gang Liu</name><affiliation><nlm:aff id="aff2">Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr., Bethesda, MD, 20892, USA</nlm:aff></affiliation></author><author><name sortKey="Lin, Yong" sort="Lin, Yong" uniqKey="Lin Y" first="Yong" last="Lin">Yong Lin</name><affiliation><nlm:aff id="aff1">Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</nlm:aff></affiliation></author></analytic><series><title level="j">Translational Oncology</title><idno type="eISSN">1936-5233</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><italic>BACKGROUND:</italic> Escaping cell death pathways is an important event during carcinogenesis. We previously identified anti-TNFα-induced apoptosis (ATIA, also known as vasorin) as an antiapoptotic factor that suppresses reactive oxygen species (ROS) production. However, the role of vasorin in lung carcinogenesis has not been investigated. <italic>METHODS:</italic> Vasorin expression was examined in human lung cancer tissues with immunohistochemistry and database analysis. Genetic and pharmacological approaches were used to manipulate protein expression and autophagy activity in human bronchial epithelial cells (HBECs). ROS generation was measured with fluorescent indicator, apoptosis with release of lactate dehydrogenase, and cell transformation was assessed with colony formation in soft agar. <italic>RESULTS:</italic> Vasorin expression was increased in human lung cancer tissues and cell lines, which was inversely associated with lung cancer patient survival. Cigarette smoke extract (CSE) and benzo[a]pyrene diol epoxide (BPDE)–induced vasorin expression in HBECs. Vasorin knockdown in HBECs significantly suppressed CSE-induced transformation in association with enhanced ROS accumulation and autophagy. Scavenging ROS attenuated autophagy and cytotoxicity in vasorin knockdown cells, suggesting that vasorin potentiates transformation by impeding ROS-mediated CSE cytotoxicity and improving survival of the premalignant cells. Suppression of autophagy effectively inhibited CSE-induced apoptosis, suggesting that autophagy was pro-apoptotic in CSE-treated cells. Importantly, blocking autophagy strongly potentiated CSE-induced transformation. <italic>CONCLUSION:</italic> These results suggest that vasorin is a potential lung cancer–promoting factor that facilitates cigarette smoke–induced bronchial epithelial cell transformation by suppressing autophagy-mediated apoptosis, which could be exploited for lung cancer prevention.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Siegel, R L" uniqKey="Siegel R">R.L. Siegel</name></author><author><name sortKey="Miller, K D" uniqKey="Miller K">K.D. Miller</name></author><author><name sortKey="Jemal, A" uniqKey="Jemal A">A. Jemal</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wingo, P A" uniqKey="Wingo P">P.A. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Transl Oncol</journal-id><journal-id journal-id-type="iso-abbrev">Transl Oncol</journal-id><journal-title-group><journal-title>Translational Oncology</journal-title></journal-title-group><issn pub-type="epub">1936-5233</issn><publisher><publisher-name>Neoplasia Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31760267</article-id><article-id pub-id-type="pmc">6883318</article-id><article-id pub-id-type="publisher-id">S1936-5233(19)30353-5</article-id><article-id pub-id-type="doi">10.1016/j.tranon.2019.09.001</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original article</subject></subj-group></article-categories><title-group><article-title>Vasorin/ATIA Promotes Cigarette Smoke–Induced Transformation of Human Bronchial Epithelial Cells by Suppressing Autophagy-Mediated Apoptosis</article-title></title-group><contrib-group><contrib contrib-type="author" id="au1"><name><surname>Chen</surname><given-names>Wenshu</given-names></name><xref rid="aff1" ref-type="aff">∗</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au2"><name><surname>Wang</surname><given-names>Qiong</given-names></name><xref rid="aff1" ref-type="aff">∗</xref><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au3"><name><surname>Xu</surname><given-names>Xiuling</given-names></name><xref rid="aff1" ref-type="aff">∗</xref></contrib><contrib contrib-type="author" id="au4"><name><surname>Saxton</surname><given-names>Bryanna</given-names></name><xref rid="aff1" ref-type="aff">∗</xref></contrib><contrib contrib-type="author" id="au5"><name><surname>Tessema</surname><given-names>Mathewos</given-names></name><xref rid="aff1" ref-type="aff">∗</xref></contrib><contrib contrib-type="author" id="au6"><name><surname>Leng</surname><given-names>Shuguang</given-names></name><xref rid="aff1" ref-type="aff">∗</xref></contrib><contrib contrib-type="author" id="au7"><name><surname>Choksi</surname><given-names>Swati</given-names></name><xref rid="aff2" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="au8"><name><surname>Belinsky</surname><given-names>Steven A.</given-names></name><xref rid="aff1" ref-type="aff">∗</xref></contrib><contrib contrib-type="author" id="au9"><name><surname>Liu</surname><given-names>Zheng-Gang</given-names></name><xref rid="aff2" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="au10"><name><surname>Lin</surname><given-names>Yong</given-names></name><email>ylin@lrri.org</email><xref rid="aff1" ref-type="aff">∗</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib></contrib-group><aff id="aff1"><label>∗</label>Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA</aff><aff id="aff2"><label>†</label>Laboratory of Immune Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Dr., Bethesda, MD, 20892, USA</aff><author-notes><corresp id="cor1"><label>∗</label>Address all correspondence to: Yong Lin, MD, PhD, Molecular Biology and Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest DR. SE, Albuquerque, NM, 87108, USA. <email>ylin@lrri.org</email></corresp><fn id="fn1"><label>1</label><p id="ntpara0010">Equal contribution.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>21</day><month>11</month><year>2019</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="collection"><month>1</month><year>2020</year></pub-date><pub-date pub-type="epub"><day>21</day><month>11</month><year>2019</year></pub-date><volume>13</volume><issue>1</issue><fpage>32</fpage><lpage>41</lpage><history><date date-type="received"><day>28</day><month>6</month><year>2019</year></date><date date-type="rev-recd"><day>29</day><month>8</month><year>2019</year></date><date date-type="accepted"><day>3</day><month>9</month><year>2019</year></date></history><permissions><copyright-statement>© 2019 The Authors</copyright-statement><copyright-year>2019</copyright-year><license license-type="CC BY-NC-ND" xlink:href="http://creativecommons.org/licenses/by-nc-nd/4.0/"><license-p>This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).</license-p></license></permissions><abstract id="abs0010"><p><italic>BACKGROUND:</italic> Escaping cell death pathways is an important event during carcinogenesis. We previously identified anti-TNFα-induced apoptosis (ATIA, also known as vasorin) as an antiapoptotic factor that suppresses reactive oxygen species (ROS) production. However, the role of vasorin in lung carcinogenesis has not been investigated. <italic>METHODS:</italic> Vasorin expression was examined in human lung cancer tissues with immunohistochemistry and database analysis. Genetic and pharmacological approaches were used to manipulate protein expression and autophagy activity in human bronchial epithelial cells (HBECs). ROS generation was measured with fluorescent indicator, apoptosis with release of lactate dehydrogenase, and cell transformation was assessed with colony formation in soft agar. <italic>RESULTS:</italic> Vasorin expression was increased in human lung cancer tissues and cell lines, which was inversely associated with lung cancer patient survival. Cigarette smoke extract (CSE) and benzo[a]pyrene diol epoxide (BPDE)–induced vasorin expression in HBECs. Vasorin knockdown in HBECs significantly suppressed CSE-induced transformation in association with enhanced ROS accumulation and autophagy. Scavenging ROS attenuated autophagy and cytotoxicity in vasorin knockdown cells, suggesting that vasorin potentiates transformation by impeding ROS-mediated CSE cytotoxicity and improving survival of the premalignant cells. Suppression of autophagy effectively inhibited CSE-induced apoptosis, suggesting that autophagy was pro-apoptotic in CSE-treated cells. Importantly, blocking autophagy strongly potentiated CSE-induced transformation. <italic>CONCLUSION:</italic> These results suggest that vasorin is a potential lung cancer–promoting factor that facilitates cigarette smoke–induced bronchial epithelial cell transformation by suppressing autophagy-mediated apoptosis, which could be exploited for lung cancer prevention.</p></abstract></article-meta></front><body><sec id="sec1"><title>Introduction</title><p id="p0010">Lung cancer is a major health concern that is closely associated with cigarette smoke exposure [<xref rid="bib1" ref-type="bibr">1</xref>,<xref rid="bib2" ref-type="bibr">2</xref>]. While cigarette smoke carcinogens induce lung cancer via damaging DNA [<xref rid="bib3" ref-type="bibr">3</xref>], only a small fraction of DNA-damaged cells become malignant, partly because apoptosis eliminates precancerous cells to prevent tumor formation and growth. Meanwhile, carcinogens and proliferation cues activate cell survival mechanisms to counteract cell death. As the success of carcinogenesis depends on the balance of cell death and survival pathways within the premalignant and cancerous cells, evading apoptosis significantly contributes to carcinogenesis [<xref rid="bib2" ref-type="bibr">2</xref>,<xref rid="bib4" ref-type="bibr">4</xref>]. However, tremendous efforts in tackling currently known apoptosis pathways have had limited improvement for cancer prevention [<xref rid="bib5" ref-type="bibr">5</xref>]. Thus, elucidating novel apoptosis evasion mechanisms in cancer is highly significant for reducing cancer incidence and mortality.</p><p id="p0015">Carcinogens induce production of reactive oxygen species (ROS). Mitochondria are the main site of ROS production during the process of electron leakage along the mitochondrial respiratory chain for energy production. While ROS serve as second messengers for cellular signaling [<xref rid="bib6" ref-type="bibr">6</xref>], they also damage DNA, lipids, and proteins, contributing to the pathogenesis of cancer. Particularly, DNA damage may generate somatic gene mutations that lead to cancer development. However, excessive ROS are highly toxic, resulting in extensive damage of cellular components and eventually cell death through apoptosis or necrosis [<xref rid="bib7" ref-type="bibr">7</xref>]. This type of ROS-mediated cell death is assumed to be a protective mechanism against cancer [<xref rid="bib8" ref-type="bibr">8</xref>,<xref rid="bib9" ref-type="bibr">9</xref>]. Therefore, restraining ROS in a nontoxic range in premalignant and cancerous cells is crucial for carcinogenesis [<xref rid="bib7" ref-type="bibr">7</xref>]. Although ROS scavenging by reductases such as superoxide dismutase, catalase, and the cellular redox buffer system GSH/GSSH has been extensively studied [<xref rid="bib6" ref-type="bibr">6</xref>,<xref rid="bib10" ref-type="bibr">10</xref>], how ROS is regulated during cigarette smoke–induced lung carcinogenesis is not yet completely elucidated.</p><p id="p0020">We recently identified anti-TNFα-induced apoptosis (ATIA), also known as vasorin, as an antiapoptotic factor that protects cells against TNFα- and hypoxia-induced apoptosis [<xref rid="bib11" ref-type="bibr">11</xref>]. While it is expressed on the cell membrane and can be secreted [<xref rid="bib12" ref-type="bibr">12</xref>,<xref rid="bib13" ref-type="bibr">13</xref>], vasorin also translocates to the mitochondria where it binds to thioredoxin-2 and suppresses ROS production [<xref rid="bib11" ref-type="bibr">11</xref>]. We and others have previously reported that vasorin is overexpressed and promotes growth in glioblastoma [<xref rid="bib11" ref-type="bibr">11</xref>,<xref rid="bib14" ref-type="bibr">14</xref>], while an oncogenic role in hepatoma was also recently proposed [<xref rid="bib15" ref-type="bibr">15</xref>,<xref rid="bib16" ref-type="bibr">16</xref>]. However, the role of vasorin in lung carcinogenesis has never been examined. Thus, we hypothesized that vasorin may play an oncogenic role in cells with cigarette smoke–induced genomic damage through suppression of excessive ROS production. This hypothesis was tested by examining vasorin expression in human lung cancer tissues and cell lines and investigating the role of vasorin in cigarette smoke extract (CSE)-induced transformation of human bronchial epithelial cells. The results suggest that vasorin is a potential lung cancer–promoting factor that facilitates cigarette smoke–induced bronchial epithelial cell transformation by suppressing ROS-mediated autophagy and apoptosis.</p></sec><sec id="sec2"><title>Materials and Methods</title><sec id="sec2.1"><title>Reagents and Antibodies</title><p id="p0025">Synthesized benzo[a]pyrene diol epoxide (BPDE) was kindly provided by Dr. Shantu Amin (Department of Pharmacology, Penn State College of Medicine, Hershey, PA) [<xref rid="bib17" ref-type="bibr">17</xref>] and dissolved in anhydrous dimethyl sulfoxide. CSE was prepared as described previously [<xref rid="bib18" ref-type="bibr">18</xref>] and expressed as total particulate material (μg/mL) for treating cells. Chloroquine diphosphate salt (Cat. No. C6628), wortmannin (W1628), and 3-methyladenine (M9281) were obtained from Sigma (St. Louis, MO). Recombinant human transform growth factor-β (TGF-β) was purchased from eBioscience (San Diego, CA). Primary Antibodies used were anti-vasorin/vasorin (MAB2140; R&D Systems, Minneapolis, MN), ATG-7 (PA5-17216; Thermo Fisher Scientific, Grand Island, NY), β-actin (A2103; Sigma), β-tubulin (T8328; Sigma), LC3B (L7543; Sigma), p62 (610833; BD Biosciences, San Jose, CA), PARP1 (BML-SA248; Enzo Life Sciences, Farmingdale, NY), phospho-Smad2 (3101; Cell Signaling, Danvers, MA), Smad2 (3103; Cell Signaling), and GAPDH (sc-32233; Santa Cruz Technologies, Santa Cruz, CA).</p></sec><sec id="sec2.2"><title>Cell Culture</title><p id="p0030">Immortalized human bronchial epithelial cell (HBEC) lines HBEC-1, HBEC-2, HBEC-13, and small airway epithelial cell (SAEC) line SAEC-30 were kindly provided by Drs. Jerry W. Shay and John D. Minna (University of Texas Southwestern Medical Center, Dallas, TX) [<xref rid="bib19" ref-type="bibr">19</xref>] and authenticated by short tandem repeat DNA profiling (Genetica DNA Laboratories, Burlington, NC). HBECs were maintained in keratinocyte serum-free medium (K-SFM) (Cat. No. 17005042; Thermo Fisher Scientific) supplemented with 5 μg/L of human recombinant epidermal growth factor and 50 mg/L of bovine pituitary extract and antibiotics (100 U/ml of penicillin and 100 μg/ml of streptomycin) in plates coated with FNC Coating Mix (0407; AthenaES, Baltimore, MD). The immortalized human bronchial cell line BEAS-2B was purchased from American Type Culture Collection (ATCC, Manassas, VA) and were cultured in K-SFM with supplements and antibiotics. All lung cancer cell lines were obtained from ATCC and authenticated by Genetica DNA Laboratories.</p></sec><sec id="sec2.3"><title>Establishment of Vasorin Knockdown Stable BEAS-2B Cell Lines</title><p id="p0035">The vasorin shRNA-expressing plasmid was constructed by inserting a synthetic oligonucleotide encoding a hairpin sequence with a 19-nucleotide stem that is homologous to the target sequence of human vasorin, GGCCGGCAACACCCGCATT, and a 9-base loop sequence into pSilencer 4.1-CMV hygro at the BamHI site (OligoEngine). BEAS-2B cells were transfected with pSilencer 4.1-CMV-hygro-vasorinshRNA using FuGENE HD transfection reagent (Promega, Madison, WI) following the manufacturer's instruction. Cell clones with stable vasorin knockdown were selected with hygromycin (25 μg/mL) and confirmed by western blot. Cells stably transfected with pSilencer 4.1-CMV-hygro were pooled after hygromycin selection and used as negative control.</p></sec><sec id="sec2.4"><title>Small Interference RNA (siRNA) Transfection</title><p id="p0040">Small interfering RNAs (siRNA; SiGenome SMARTpool) for human vasorin, Atg-7, and control were purchased from Dharmacon (Lafayette, CO). Cells were seeded in 12-well plates at about 60–70% confluence overnight and then were transfected with siRNAs using INTERFERin siRNA transfection reagent (Polyplus-transfection, New York, NY) according to the manufacturer's instructions.</p></sec><sec id="sec2.5"><title>Western Blot</title><p id="p0045">Cells were harvested in M2 buffer (20 mM Tris–HCl, pH 7.6, 0.5% Nonidet P-40, 250 mM NaCl, 3 mM ethylenediaminetetraacetic acid, 3 mM ethylene glycol-<italic>bis</italic>(2-aminoethylether)-<italic>N</italic>,<italic>N</italic>,<italic>N</italic>′,<italic>N</italic>′-tetraacetic acid A, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20 mM β-glycerophosphate, 1 mM sodium vanadate, and 1 μg/ml leupeptin) to obtain total cell lysate. The protein concentration was determined with Bio-Rad protein quantification kit (Bia-Rad, Hercules, CA). Western blot was carried out according to standard protocol with 10%–12% sodium dodecyl sulfate–polyacrylamide gels and polyvinylidene difluoride membranes. The protein signal was detected by enhanced chemiluminescence reagent (Millipore). The band intensity was quantified using NIH ImageJ software with normalization to respective loading controls and expressed as fold changes with the control sample as 1. For LC3B expression, only LC3B-II was normalized to loading controls and quantified.</p></sec><sec id="sec2.6"><title>Detection of LC3B Puncta</title><p id="p0050">pEGFP-LC3B–expressing plasmid was a gift from Dr. Han-Ming Shen (National University of Singapore). BEAS-2B cells were seeded on tissue slide cover glass and transfected with the plasmid using FuGENE HD. The next day, cells were further transfected with control or vasorin siRNA. Twenty hours after siRNA transfection, cells were treated with CSE for 2 hours, washed with cold phosphate saline buffer and fixed with cold methanol for 5 min in room temperature. Cells were then observed and photographed using a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany). The GFP puncta were counted and the results expressed as puncta/cells.</p></sec><sec id="sec2.7"><title>Detection of ROS</title><p id="p0055">ROS detection using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA 5 μM; Invitrogen) as an indicator was done as described previously [<xref rid="bib10" ref-type="bibr">10</xref>]. The indicator was added to cell culture medium for 30 min before the cells were lysed with M2 buffer. ROS levels were measured in cell lysates with a fluorescence plate reader using a fluorescein filter. The readings were normalized to respective protein concentrations.</p></sec><sec id="sec2.8"><title>Cell Death Assay</title><p id="p0060">Cell death was detected by the release of lactate dehydrogenase (LDH) in culture medium using CytoTox 96 Non-Radioactive Cytotoxicity Assay kit from Promega [<xref rid="bib20" ref-type="bibr">20</xref>]. After removing a portion of the media, the lysis buffer from the kit was added to the wells to release total LDH. The average basal readings (OD490) from control cells were subtracted from all the readings and then the results expressed as percentage against individual total LDH reading set as 100. The experiments were carried out in triplicate.</p></sec><sec id="sec2.9"><title>Colony Formation Assay</title><p id="p0065">BEAS-2B cells (10<sup>4</sup> cells/well in a 6-well plate) were treated with CSE (20 μg/ml) or BPDE (0.2 μM) for 1 hour and then incubated in fresh medium. The treatment was repeated every two days for a total of three treatments. Cells were trypsinized and seeded in soft agar (12-well plate) in duplicate. After two weeks of incubation, colonies in the agar were photographed under microscope in six random and nonoverlaid fields and counted. Colony formation was expressed as colonies/field [<xref rid="bib10" ref-type="bibr">10</xref>].</p></sec><sec id="sec2.10"><title>Tissue Array and Immunohistochemistry</title><p id="p0070">Immunohistochemistry staining using VECTASTAIN ABC Kit and DAB (3,3′-diaminobenzidine) Peroxidase Substrate Kit (Vector Laboratories, Burlingame, CA) and result assessment of human lung cancer tissue array (Imgenex; Novus Biologicals, Centennial, CO) has been described previously [<xref rid="bib10" ref-type="bibr">10</xref>]. Briefly, the paraffin-embedded tissue slide was deparaffinized and rehydrated, and then antigens were heat-retrieved followed by 3% H<sub>2</sub>O<sub>2</sub> treatment and blocking. After overnight incubation of primary antibody (goat anti-vasorin, R&D systems, 1:100) at 4 °C, further incubation was performed with ABC Kit and color was developed with DAB according to manufacturer's instructions. The staining of vasorin was compared between tumor and corresponding normal bronchial epithelial cells, and a tumor tissue was considered to have “normal expression” when its staining was comparable with that of bronchial epithelial cells, or as “increased expression” when its staining was higher.</p></sec><sec id="sec2.11"><title>Patient Survival Analysis with TCGA and Online Database</title><p id="p0075">RNA-seq data for human NSCLC (<italic>n</italic> = 957) were retrieved from The Cancer Genome Atlas (TCGA) database. The corresponding patients were followed up to 150 months. Data were analyzed for vasorin expression and patient survival by Cox regression with adjustment for covariates that included age, sex, smoking status, and tumor stage. Estimation of the survival function between high versus low gene expression was done with Kaplan–Meier estimator. For the online data (Kaplan–Meier Plotter, <ext-link ext-link-type="uri" xlink:href="http://kmplot.com" id="intref0010">kmplot.com</ext-link>) that combine 7 cohorts (<italic>n</italic> = 673, GSE19188, GSE29013, GSE30219, GSE31210, GSE3141, GSE37745, and GSE50081), the association of vasorin expression and lung cancer patient survival was analyzed by the Kaplan–Meier plotter online analysis tool [<xref rid="bib21" ref-type="bibr">21</xref>,<xref rid="bib22" ref-type="bibr">22</xref>].</p></sec><sec id="sec2.12"><title>Statistics</title><p id="p0080">All quantitative data were expressed as mean ± standard deviation. Comparison of two means was performed with two-tailed Student's <italic>t</italic> test. For analyzing tissue array results, Fisher's exact test was used. <italic>P</italic> < 0.05 was considered statistically significant.</p></sec></sec><sec id="sec3"><title>Results</title><sec id="sec3.1"><title>Vasorin is Overexpressed in Lung Cancer and Transformed HBECs</title><p id="p0085">We first examined vasorin expression in human NSCLC by using immunohistochemistry in tissue arrays containing lung cancer and normal lung tissues. A total of 192 NSCLC cases, including 100 squamous cell carcinomas (SqCC), 74 adenocarcinomas (AC), and 18 other types of NSCLC were evaluated. The results revealed that vasorin was overexpressed in cancers compared with normal lung tissue (<xref rid="fig1" ref-type="fig">Figure 1</xref><italic>A</italic>). Overall, 34.4% of all evaluated NSCLC cases regardless of tumor histology showed increased expression (<xref rid="fig1" ref-type="fig">Figure 1</xref><italic>B</italic>). Notably, vasorin overexpression was significantly more prevalent in lung AC (52.7%) compared with lung SqCC (21.0%; <italic>P</italic> < 0.0001, Fisher's exact test, <xref rid="fig1" ref-type="fig">Figure 1</xref><italic>B</italic>). This may suggest that vasorin abnormality is involved in the pathogenesis of both lung cancer types, although it is more commonly affected in lung AC. Consistently, vasorin expression was increased (two- to five-fold) in 7 of 9 tested NSCLC cell lines as compared with two immortalized HBEC lines. The increased expression of vasorin was detected for both the 110 kD and 72 kD isoforms (both antiapoptotic) we have previously reported [<xref rid="bib11" ref-type="bibr">11</xref>] (<xref rid="fig1" ref-type="fig">Figure 1</xref><italic>C</italic>).<fig id="fig1"><label>Figure 1</label><caption><p><bold>Increased vasorin expression in human lung cancer and HBECs exposed to tobacco carcinogens</bold>. (A) Tissue arrays were stained for vasorin with immunohistochemistry. The normal airway and alveolar epithelial cells are weakly positive while the tumor cells are strongly positive. (B) Results of tissue arrays with comparison of the staining between vasorin and normal lung epithelial cells. (C) Vasorin was examined in nontransformed (HBEC-1 and -2) and lung cancer cell lines. Relative vasorin fold increase (HBEC-2 was set to 1) was shown. (D) BEAS-2B and HBEC-2 cells were treated with CSE for 24 hours. (E) HBEC-2B (BPDE-transformed) cells were compared with HBEC-2 cells for vasorin expression. Protein was detected by western blot in C to E with β-actin as loading control.</p></caption><alt-text id="alttext0030">Figure 1</alt-text><graphic xlink:href="gr1"></graphic></fig></p></sec><sec id="sec3.2"><title>Increased Vasorin Expression is Associated With Poor Survival of Patients With Lung Adenocarcinoma</title><p id="p0090">The clinical significance of increased vasorin expression in lung cancer was investigated using data from TCGA. Cox regression analysis of lung cancer cases followed up for up to 150 months revealed a significant inverse association between vasorin expression and survival of these patients after adjustment for age, sex, smoking status, and tumor stage [hazard ratio (HR) = 1.34, 95% confidence interval (CI) 1.02–1.76, <italic>P</italic> = 0.0356, <xref rid="appsec1" ref-type="sec">Figure S1A</xref>]. A similar trend was also observed in adenocarcinomas and squamous cell carcinomas from TCGA when analyzed separately using an online analytical tool [<ext-link ext-link-type="uri" xlink:href="https://www.proteinatlas.org/" id="intref0015">https://www.proteinatlas.org/</ext-link> [<xref rid="bib25" ref-type="bibr">25</xref>,<xref rid="bib26" ref-type="bibr">26</xref>]]. With an independent online resource validation [Kaplan–Meier Plotter [<xref rid="bib21" ref-type="bibr">21</xref>,<xref rid="bib22" ref-type="bibr">22</xref>]], the inverse association between vasorin expression and survival in patients with lung adenocarcinoma was detected (HR = 1.64, 95% CI = 1.28–2.03, <italic>P</italic> = 6.9e-5, <xref rid="appsec1" ref-type="sec">Figure S1B</xref>), although the association in squamous cell carcinomas was unclear. In addition, vasorin overexpression indicates a worse prognosis in both smokers (HR = 1.58, 95% CI = 1.05–2.39, <italic>P</italic> = 0.028) and never smokers (HR = 2.22, 95% CI = 0.95–5.19, <italic>P</italic> = 0.05, <xref rid="appsec1" ref-type="sec">Figure S1B</xref>) with lung adenocarcinomas [<xref rid="bib22" ref-type="bibr">22</xref>]. These results suggest that vasorin is a potential tumor-promoting oncoprotein in human lung adenocarcinoma.</p></sec><sec id="sec3.3"><title>Vasorin Expression in HBECs was Induced by Cigarette Smoke Extract and BPDE</title><p id="p0095">To explore the role and mechanism of vasorin in lung carcinogenesis, we first examined if cigarette smoke affected its expression in HBECs. CSE strongly induced vasorin expression in a dose-dependent manner in BEAS-2B and HBEC-2 cells (<xref rid="fig1" ref-type="fig">Figure 1</xref><italic>D</italic>). Similarly, BPDE, the active metabolite of cigarette smoke carcinogen benzo[a]pyrene that potently transforms HBECs [<xref rid="bib10" ref-type="bibr">10</xref>,<xref rid="bib18" ref-type="bibr">18</xref>], strongly induced vasorin expression (<xref rid="appsec1" ref-type="sec">Figure S2</xref>). In addition, HBEC-2B cells (HBEC-2 cells transformed by BPDE [<xref rid="bib23" ref-type="bibr">23</xref>,<xref rid="bib24" ref-type="bibr">24</xref>]), showed a marked constitutively increased vasorin expression (<xref rid="fig1" ref-type="fig">Figure 1</xref><italic>E</italic>), indicating that the transient increase in vasorin expression by CSE and BPDE was stabilized during transformation and in lung cancer cell lines. These results suggest that cigarette smoke carcinogens induce vasorin expression, which may be involved in the early steps of lung cancer development.</p></sec><sec id="sec3.4"><title>Knockdown of Vasorin Expression Enhances CSE-Induced Cytotoxicity</title><p id="p0100">Because vasorin was identified as an anti-apoptosis factor [<xref rid="bib11" ref-type="bibr">11</xref>], we examined if it is involved in suppressing CSE-induced cytotoxicity. Stable vasorin knockdown was established in BEAS-2B cells (<xref rid="fig2" ref-type="fig">Figure 2</xref><italic>A</italic>). While the cytotoxicity of CSE to BEAS-2B cells in general was increased in a dose-dependent manner, the cells with vasorin knockdown showed significantly increased cell death in each of the various CSE doses evaluated (<xref rid="fig2" ref-type="fig">Figure 2</xref><italic>B</italic> and <italic>C</italic>). The CSE-induced cytotoxicity was suppressed by the pan-caspase inhibitor z-VAD (<xref rid="fig2" ref-type="fig">Figure 2</xref><italic>D</italic>), indicating that vasorin inhibits CSE-induced apoptosis in human lung epithelial cells.<fig id="fig2"><label>Figure 2</label><caption><p><bold>Vasorin knockdown potentiates CSE-induced cytotoxicity and cigarette smoke carcinogen-induced transformation</bold>. (A) Stable knockdowns of vasorin in BEAS-2B cells were generated with shRNA and expression was measured by western blot using GAPDH as loading control. (B, C, D) Cells were treated with 20 μg/ml of CSE (B), or various concentrations of CSE (C) for 48 hours, or cells were left untreated or pretreated with z-VAD (20 μM) followed by CSE (20 μg/ml) treatment for 48 hours (D). Cell death was measured by LDH release assay in triplicate samples. (E, F) Control and two clones of BEAS-2B vasorin knockdown cells (10<sup>4</sup> cells each) were untreated or exposed to CSE (20 μg/ml; E) or BPDE (0.2 μM; F) every two days for a total of three times and then seeded in soft agar in duplicate. Two weeks later, the colonies were photographed at 6 random fields per well and counted. *<italic>P</italic> < 0.05.</p></caption><alt-text id="alttext0035">Figure 2</alt-text><graphic xlink:href="gr2"></graphic></fig></p></sec><sec id="sec3.5"><title>Knockdown of Vasorin Expression Suppresses CSE-Induced HBEC Transformation</title><p id="p0105">Transformation of lung epithelial cells represents an early step of lung carcinogenesis, and the capacity to grow in an anchorage-independent manner is an indicator of transformation. Thus, we used the soft-agar colony formation assay and stable vasorin knockdown cells to investigate whether vasorin plays a role in CSE-induced transformation [<xref rid="bib18" ref-type="bibr">18</xref>,<xref rid="bib23" ref-type="bibr">23</xref>]. While CSE and BPDE effectively induced control cells to form colonies in soft agar, vasorin knockdown significantly suppressed this effect (<xref rid="fig2" ref-type="fig">Figure 2</xref><italic>E</italic> and <italic>F</italic> and <xref rid="appsec1" ref-type="sec">Figure S3</xref>). Together with the observation that vasorin inhibits CSE-induced apoptosis, these results suggest that vasorin potentiates CSE-induced HBEC transformation by suppressing apoptotic cell death.</p></sec><sec id="sec3.6"><title>Vasorin Knockdown Potentiates CSE-Induced Autophagy Through Cellular ROS Accumulation</title><p id="p0110">Cigarette smoke exposure of lung epithelial cells activates autophagy and mediates apoptosis or survival under certain circumstances [<xref rid="bib24" ref-type="bibr">[24]</xref>, <xref rid="bib25" ref-type="bibr">[25]</xref>, <xref rid="bib26" ref-type="bibr">[26]</xref>, <xref rid="bib27" ref-type="bibr">[27]</xref>, <xref rid="bib28" ref-type="bibr">[28]</xref>, <xref rid="bib29" ref-type="bibr">[29]</xref>]. Thus, we investigated if vasorin knockdown impacts CSE-induced autophagy. Indeed, exposure of BEAS-2B cells to CSE strongly induced autophagy activation, as demonstrated by the increased LC3B-II and decreased p62 expression (<xref rid="fig3" ref-type="fig">Figure 3</xref><italic>A</italic>) and GFP-LC3B puncta (<xref rid="appsec1" ref-type="sec">Figures 3F and S4</xref>). Suppressing lysosomal protein degradation with chloroquine (CQ) confirmed the increased activity of autophagy by CSE treatment (<xref rid="fig3" ref-type="fig">Figure 3</xref><italic>B</italic> and <italic>F</italic>). A similar effect of BPDE on autophagy was also detected (<xref rid="fig3" ref-type="fig">Figure 3</xref><italic>C</italic>). Of note, while the detection of LC3-I is not very reproducible, which may partly be due to the fact that the anti-LC3 antibody preferably detects LC3-II and that LC3-I is relatively unstable [<xref rid="bib30" ref-type="bibr">30</xref>], the induction of LC3-II is reproducibly detected in our experiments. These findings, consistent with previous reports [<xref rid="bib26" ref-type="bibr">[26]</xref>, <xref rid="bib27" ref-type="bibr">[27]</xref>, <xref rid="bib28" ref-type="bibr">[28]</xref>, <xref rid="bib29" ref-type="bibr">[29]</xref>,<xref rid="bib31" ref-type="bibr">[31]</xref>, <xref rid="bib32" ref-type="bibr">[32]</xref>, <xref rid="bib33" ref-type="bibr">[33]</xref>], indicate that CSE and BPDE activate autophagy in HBECs.<fig id="fig3"><label>Figure 3</label><caption><p><bold>Vasorin inhibits autophagy</bold>. (A) BEAS-2B cells were exposed to increasing concentration of CSE for 4 hours. (B) BEAS-2B cells were treated with CSE (20 μg/ml) for 4 hours with or without pretreatment of chloroquine (CQ, 20 μM for 30 min). (C) BEAS-2B cells were treated with increasing concentrations of BPDE for 4 hours. (D, E) Control or stable vasorin knockdown cells were treated with various concentrations of CSE for 4 hours (D), or with 20 μg/ml of CSE for 4 hours without or with CQ (20 μM) pretreatment for 30 min (E). (A, B, C, D, E) Proteins were detected with western blot with β-actin or β-tubulin as loading control. (F) BEAS-2B cells were transfected with pEGFP-LC3B plasmid for 24 hours, and then were further transfected with control or vasorin siRNA (10 nM). The next day, cells were left untreated or treated with 20 μM of CQ for 30 min before CSE (20 μg/ml) exposure for 2 hours. Cells were observed and images recorded under a fluorescence microscope and LC3 puncta counted. *<italic>P</italic> < 0.05.</p></caption><alt-text id="alttext0040">Figure 3</alt-text><graphic xlink:href="gr3"></graphic></fig></p><p id="p0115">Interestingly, vasorin knockdown strongly increased CSE-induced autophagy, which was detected biochemically and morphologically (<xref rid="fig3" ref-type="fig">Figure 3</xref><italic>D</italic>–<italic>F</italic>). Vasorin suppresses ROS accumulation in other cell types such as fibroblasts, and excessive ROS suppresses CSE carcinogen-induced HBEC transformation [<xref rid="bib10" ref-type="bibr">10</xref>,<xref rid="bib11" ref-type="bibr">11</xref>]. Thus, we further examined if ROS is involved in vasorin-mediated autophagy regulation in HBEC cells. While the basal ROS level was significantly increased in vasorin knockdown cells, the CSE-induced ROS accumulation was much higher when vasorin was suppressed (<xref rid="fig4" ref-type="fig">Figure 4</xref><italic>A</italic>), suggesting that vasorin plays a role in retaining ROS at low levels in cells. The ROS scavenger N-acetylcysteine effectively reduced CSE-induced LC-3B-II expression to basal levels in control and vasorin knockdown cells (<xref rid="fig4" ref-type="fig">Figure 4</xref><italic>B</italic>), suggesting that CSE induces autophagy depending on ROS and that vasorin deficiency promotes CSE-induced autophagy through enhanced ROS accumulation.<fig id="fig4"><label>Figure 4</label><caption><p><bold>Involvement of ROS in vasorin-mediated autophagy</bold>. (A) BEAS-2B cells transfected with control or vasorin siRNA (10 nM) for 24 hours were left untreated or treated with CSE for 4 hours. At the last 30 min of treatment, CM-H2DCFDA (5 μM) was added to the cells. The cells were then harvested for ROS detection with a fluorescent plate reader. The readings of fluorescence were normalized to corresponding protein concentrations. Experiments were carried out in triplicate, *<italic>P</italic> < 0.05. (B) BEAS-2B cells without (control) or with stable vasorin knockdown were treated with NAC (3 mM) 30 min before CSE (20 μg/ml) incubation for 4 hours. Western blot was carried out to detect LC3B with β-actin as loading control. NAC, N-acetylcysteine.</p></caption><alt-text id="alttext0045">Figure 4</alt-text><graphic xlink:href="gr4"></graphic></fig></p></sec><sec id="sec3.7"><title>Suppressing Autophagy Inhibits CSE-Induced Cytotoxicity and Potentiates Transformation</title><p id="p0120">We then examined the role of autophagy in CSE-induced cytotoxicity and transformation. Pharmacological (CQ, wortmannin [WTM] and 3-methyladinine [3-MA]) or genetic (siRNA against Atg-7) autophagy suppression effectively reduced CSE-induced cytotoxicity (<xref rid="fig5" ref-type="fig">Figure 5</xref><italic>A</italic>)<bold>,</bold> and this was associated with decreased apoptosis as shown by decreased PARP cleavage (<xref rid="fig6" ref-type="fig">Figure 6</xref><italic>B</italic>). Similarly, BPDE-induced apoptosis was inhibited by autophagy suppression (<xref rid="fig5" ref-type="fig">Figure 5</xref><italic>C</italic>). These results suggest that autophagy plays a proapoptosis role in the context of CSE-induced cytotoxicity in HBECs. In addition, autophagy inhibition with either CQ and 3-MA or Atg7 siRNA effectively increased CSE-induced transformation (<xref rid="fig6" ref-type="fig">Figure 6</xref><italic>A</italic>–<italic>D</italic>). Together with data that show vasorin suppresses CSE-induced autophagy and apoptosis, these results suggest that vasorin promotes transformation, at least in part, by inhibiting autophagy-mediated apoptosis.<fig id="fig5"><label>Figure 5</label><caption><p><bold>Autophagy contributes to CSE-induced cell death</bold>. (A) BEAS-2B cells treated with autophagy inhibitors CQ (20 μM), WTM (1 μM) or 3-MA (10 mM) for 30 min followed by incubation of CSE (20 μg/ml) for 48 hours. Cell death was determined in triplicate samples by LDH release assay. *<italic>P</italic> < 0.05. (B, C) BEAS-2B cells were transfected with control or Atg-7 siRNA (10 nM) for 24 hours, then treated with CSE (20 μg/ml, B) or BPDE (0.4 μM) for 4 hours (C). PARP cleavage and Atg-7 levels were examined by western blot with β-tubulin as a loading control.</p></caption><alt-text id="alttext0050">Figure 5</alt-text><graphic xlink:href="gr5"></graphic></fig><fig id="fig6"><label>Figure 6</label><caption><p><bold>Inhibition of autophagy increases CSE-induced transformation</bold>. (A, B) Representative images (A) of colonies in soft agar of BEAS-2B cells untreated, treated with CQ (20 μM), CSE (20 μg/ml), or their combination. Colonies were counted after two weeks of seeding (B). (C and D) HBEC-13 cells were untreated or treated with 3-MA (10 mM, C), or transfected with control or Atg-7 siRNA before the treatment of CSE (20 μg/ml, D). Cells were then seeded in soft agar and colonies counted as in A and B. *<italic>P</italic> < 0.05.</p></caption><alt-text id="alttext0055">Figure 6</alt-text><graphic xlink:href="gr6"></graphic></fig></p></sec></sec><sec id="sec4"><title>Discussion</title><p id="p0125">This study provides evidence that vasorin is a potential lung cancer–promoting factor that facilitates cigarette smoke–induced bronchial epithelial cell transformation. Vasorin expression was increased in a number of human lung cancer tissues, which was inversely associated with lung cancer patient survival. Vasorin expression was also increased in lung cancer cell lines and transformed HBECs. Vasorin knockdown in HBECs significantly suppressed CSE-induced transformation, which was associated with enhanced ROS accumulation and cytotoxicity. Mechanistically, vasorin knockdown potentiated CSE-induced autophagy and apoptosis in an ROS-dependent manner. Suppression of autophagy effectively suppressed CSE-induced apoptotic cell death and potentiated CSE-induced transformation. These results suggest that vasorin is a potential lung cancer–promoting factor, which suppresses autophagy-mediated apoptosis to facilitate CS-induced bronchial epithelial cell transformation. Taken together, our findings establish a novel lung carcinogenesis mechanism involving vasorin, the vasorin/ROS/autophagy/apoptosis pathway, which could be exploited for preventing lung carcinogenesis.</p><p id="p0130">Although an increasing number of oncogenic gene mutations are being identified in lung cancer, about one-third of patients with lung cancer have no known mutations or aberrant activation of cancer driver genes. Cellular signaling pathways regulating DNA repair, cell proliferation, survival, and death are involved in cancer development. In addition, there are potential cofactors or promoting factors that facilitate driver-mediated carcinogenesis [<xref rid="bib3" ref-type="bibr">3</xref>]. Our findings suggest vasorin as an endogenous tumor-promoting factor. Vasorin is induced by cigarette smoke to counteract apoptosis, which maintains survival of premalignant cells during cigarette smoke-induced lung epithelial cell transformation. Notably, the inverse association between vasorin expression and patient survival seen in both smokers and nonsmokers suggests that the role of vasorin in human lung cancer development may also apply to lung cancer caused by secondhand cigarette smoke or other environmental carcinogen exposures.</p><p id="p0135">Vasorin suppresses ROS and apoptosis found in mouse embryonic fibroblasts exposed to TNFα or hypoxia [<xref rid="bib11" ref-type="bibr">11</xref>]. Consistent with this role, vasorin suppressed CSE-induced and ROS-mediated apoptosis in HBECs. It should be noted that vasorin was shown to function as a decoy ligand to suppress TGF-β signaling in vascular smooth muscle and some tumor cells [<xref rid="bib12" ref-type="bibr">12</xref>,<xref rid="bib34" ref-type="bibr">34</xref>,<xref rid="bib35" ref-type="bibr">35</xref>]. TGF-β functions as a tumor suppressor in the early stages of carcinogenesis [<xref rid="bib36" ref-type="bibr">36</xref>,<xref rid="bib37" ref-type="bibr">37</xref>]. However, TGF-β-induced signaling was not affected by vasorin knockdown in lung cancer cells (data not shown). Thus, inhibition of TGF-β signaling is unlikely to be involved in vasorin's function in cigarette smoke–induced cell transformation. While our current results suggest an ROS-related tumor-promoting mechanism, it does not exclude the possibility that the ROS and TGF-β pathways cooperatively contribute to vasorin's cancer-promoting function. Future <italic>in vivo</italic> studies are needed to clarify this possibility.</p><p id="p0140">Autophagy is a cellular process for degradation of exhausted, redundant, and unwanted cell components, including proteins and organelles [<xref rid="bib24" ref-type="bibr">24</xref>,<xref rid="bib38" ref-type="bibr">38</xref>], which can lead to either cell survival or death depending on context. In general, moderate autophagy maintains cell homeostasis and survival, whereas excessive autophagy exhausts cell components to kill cells [<xref rid="bib24" ref-type="bibr">24</xref>,<xref rid="bib38" ref-type="bibr">38</xref>]. In parallel with its contradictory functions in regulating cell survival or death, the role of autophagy in carcinogenesis is believed to be complex: to promote or suppress cancer development [<xref rid="bib39" ref-type="bibr">39</xref>]. Although cell type- and insult-specific functions of autophagy are observed, it is highly likely that autophagy plays different roles in carcinogenesis in different organs. Although autophagy has been widely studied in lung cancer cell lines, there is no report to date on the role of autophagy in lung epithelial cell transformation and lung cancer initiation. Prior studies suggest the premise that cigarette smoke induces autophagy in lung epithelial cells [<xref rid="bib25" ref-type="bibr">[25]</xref>, <xref rid="bib26" ref-type="bibr">[26]</xref>, <xref rid="bib27" ref-type="bibr">[27]</xref>, <xref rid="bib28" ref-type="bibr">[28]</xref>, <xref rid="bib29" ref-type="bibr">[29]</xref>,<xref rid="bib31" ref-type="bibr">[31]</xref>, <xref rid="bib32" ref-type="bibr">[32]</xref>, <xref rid="bib33" ref-type="bibr">[33]</xref>], and our results further show that autophagy suppresses cell transformation, an early step in lung carcinogenesis. This observation is consistent with the general assumption that autophagy suppresses carcinogenesis at early stages [<xref rid="bib40" ref-type="bibr">[40]</xref>, <xref rid="bib41" ref-type="bibr">[41]</xref>, <xref rid="bib42" ref-type="bibr">[42]</xref>, <xref rid="bib43" ref-type="bibr">[43]</xref>, <xref rid="bib44" ref-type="bibr">[44]</xref>]. Similarly, a most recent report strongly suggests that autophagy restricts chromosomal instability during replicative crisis, and that loss of autophagy function is required for the initiation of cancer [<xref rid="bib45" ref-type="bibr">45</xref>]. However, it is also believed that autophagy promotes cancer progression at later stages [<xref rid="bib40" ref-type="bibr">[40]</xref>, <xref rid="bib41" ref-type="bibr">[41]</xref>, <xref rid="bib42" ref-type="bibr">[42]</xref>, <xref rid="bib43" ref-type="bibr">[43]</xref>, <xref rid="bib44" ref-type="bibr">[44]</xref>]. How vasorin in concert with autophagy activation contributes to lung carcinogenesis needs further careful delineation with temporal autophagy suppression in vasorin knockout animals.</p><p id="p0145">As the so-called autophagic death is uncommon, autophagy can mediate either apoptosis or necrosis in different contexts and circumstances [<xref rid="bib46" ref-type="bibr">46</xref>,<xref rid="bib47" ref-type="bibr">47</xref>]. Our observations that vasorin knockdown strongly potentiated autophagy and suppressing autophagy effectively attenuated CSE-induced apoptosis suggest that autophagy mediates cytotoxicity induced by CSE and cigarette smoke carcinogens such as BPDE through apoptosis to inhibit transformation.</p><p id="p0150">Our results establish a potentially oncogenic role of vasorin in lung carcinogenesis, and shed lights on the mechanism by which vasorin facilitates carcinogen-induced transformation. Further studies are warranted to map the vasorin-mediated pathway in lung cancer development, which may identify targets that could be exploited for lung cancer prevention and/or therapy.</p></sec><sec id="sec5"><title>Conflict of interest</title><p id="p0155">The authors declare no conflict of interest.</p></sec><sec sec-type="data-availability" id="sec6"><title>Data availability statement</title><p id="p0160">The data that support the findings of this study are available from the corresponding author on reasonable request.</p></sec></body><back><ref-list id="cebib0010"><title>References</title><ref id="bib1"><label>1</label><element-citation publication-type="journal" id="sref1"><person-group person-group-type="author"><name><surname>Siegel</surname><given-names>R.L.</given-names></name><name><surname>Miller</surname><given-names>K.D.</given-names></name><name><surname>Jemal</surname><given-names>A.</given-names></name></person-group><article-title>Cancer 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S1</label><caption><p>Relationship of vasorin expression in lung cancer cells and patient survival. (A) RNA-seq data were downloaded from TCGA and were analyzed for vasorin expression and patient survival by Cox regression with adjustment for covariates that included age, sex, smoking status, and tumor stage. Estimation of the survival function between high versus low gene expression was done with Kaplan–Meier estimator. (B) Survival curves and statistics were generated using online tools in Kaplan–Meier plotter database that combine 7 cohorts (<italic>n</italic> = 673, GSE19188, GSE29013, GSE30219, GSE31210, GSE3141, GSE37745, and GSE50081).1</p></caption><alt-text id="alttext0010">Figure S1</alt-text><graphic xlink:href="figs1"></graphic></fig><fig id="dfig2" position="anchor"><label>Figure S2</label><caption><p>BPDE-induced vasorin expression in a time-dependent manner in BEAS-2B cells. The cells were treated with 0.3 μM of BPDE for various time points. Vasorin protein expression and β-tubulin as a loading control were determined with western blot.</p></caption><alt-text id="alttext0015">Figure S2</alt-text><graphic xlink:href="figs2"></graphic></fig><fig id="dfig3" position="anchor"><label>Figure S3</label><caption><p>Vasorin knockdown in HBECs impairs CSE-induced transformation. Representative images of cell colonies in soft agar.3</p></caption><alt-text id="alttext0020">Figure S3</alt-text><graphic xlink:href="figs3"></graphic></fig><fig id="dfig4" position="anchor"><label>Figure S4</label><caption><p>Vasorin knockdown increased CSE-induced autophagy. BEAS-2B cells were transfected with pEGFP-LC3B for 24 hours, followed with control or vasorin siRNA (10 nM) transfection for another 24 hours. Cells were then treated with CSE (20 μM) for 2 hours and were observed and photographed using a fluorescence microscope.</p></caption><alt-text id="alttext0025">Figure S4</alt-text><graphic xlink:href="figs4"></graphic></fig></p></sec><ack id="ack0010"><title>Acknowledgments</title><p>The authors thank Dr. Shantu Amin (Hershey College of Medicine, Penn State University) for providing BPDE, and Dr. Han-Ming Shen (National University of Singapore) for providing pEGFP-LC3B-expressing plasmid. The vasorin expression and lung cancer patient survival association results are based on data generated by the TCGA Research Network (<ext-link ext-link-type="uri" xlink:href="https://cancergenome.nih.gov/" id="intref0020">http://cancergenome.nih.gov/</ext-link>) and Kaplan Meier Plotter (<ext-link ext-link-type="uri" xlink:href="http://kmplot.com/analysis/index.php?p=service&cancer=lung" id="intref0025">http://kmplot.com/analysis/index.php?p=service&cancer=lung</ext-link>). This study was supported in part by grants from <funding-source id="gs1">National Cancer Institute, National Institutes of Health, USA</funding-source> (1R21CA193633 and P30CA11800).</p></ack><fn-group><fn id="appsec2" fn-type="supplementary-material"><label>Appendix A</label><p id="p0175">Supplementary data to this article can be found online at <ext-link ext-link-type="uri" xlink:href="https://doi.org/10.1016/j.tranon.2019.09.001" id="intref0030">https://doi.org/10.1016/j.tranon.2019.09.001</ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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