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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Cell Dev. Biol.</journal-id>
<journal-title>Frontiers in Cell and Developmental Biology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Cell Dev. Biol.</abbrev-journal-title>
<issn pub-type="epub">2296-634X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="publisher-id">745554</article-id>
<article-id pub-id-type="doi">10.3389/fcell.2021.745554</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Cell and Developmental Biology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>PPAR&#x3b3;/SOD2 Protects Against Mitochondrial ROS-Dependent Apoptosis via Inhibiting ATG4D-Mediated Mitophagy to Promote Pancreatic Cancer Proliferation</article-title>
<alt-title alt-title-type="left-running-head">Nie et&#x20;al.</alt-title>
<alt-title alt-title-type="right-running-head">PPAR&#x3b3; Inhibits ATG4D-Mediated Mitophagy</alt-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Nie</surname>
<given-names>Shuang</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="fn" rid="fn1">
<sup>&#x2020;</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/837320/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Shi</surname>
<given-names>Zhao</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="fn" rid="fn1">
<sup>&#x2020;</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1574581/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Shi</surname>
<given-names>Mengyue</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="fn" rid="fn1">
<sup>&#x2020;</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1635216/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Li</surname>
<given-names>Hongzhen</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1062569/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Qian</surname>
<given-names>Xuetian</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/963311/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Peng</surname>
<given-names>Chunyan</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Ding</surname>
<given-names>Xiwei</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhang</surname>
<given-names>Shu</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1636161/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Lv</surname>
<given-names>Ying</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1139163/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Wang</surname>
<given-names>Lei</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1636647/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Kong</surname>
<given-names>Bo</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff4">
<sup>4</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/91503/overview"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Zou</surname>
<given-names>Xiaoping</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1299403/overview"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Shen</surname>
<given-names>Shanshan</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<uri xlink:href="https://loop.frontiersin.org/people/985233/overview"/>
</contrib>
</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>Department of Gastroenterology</institution>, <institution>the Affiliated Drum Tower Hospital of Nanjing University Medical School</institution>, <addr-line>Nanjing</addr-line>, <country>China</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Nanjing University Institute of Pancreatology</institution>, <addr-line>Nanjing</addr-line>, <country>China</country>
</aff>
<aff id="aff3">
<sup>3</sup>
<institution>Department of Gastroenterology</institution>, <institution>Nanjing Drum Tower Hospital</institution>, <institution>Clinical College of Nanjing Medical University</institution>, <addr-line>Nanjing</addr-line>, <country>China</country>
</aff>
<aff id="aff4">
<sup>4</sup>
<institution>Department of Surgery</institution>, <institution>Ulm University Hospital</institution>, <institution>Ulm University</institution>, <addr-line>Ulm</addr-line>, <country>Germany</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>
<bold>Edited by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/959608/overview">Yongchao Zhao</ext-link>, Institute of Translational Medicine, Zhejiang University, China</p>
</fn>
<fn fn-type="edited-by">
<p>
<bold>Reviewed by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/1416994/overview">Danrui Cui</ext-link>, First Affiliated Hospital, Zhejiang University, China</p>
<p>
<ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/1418534/overview">Ranjit Mehta</ext-link>, University of Michigan, United&#x20;States</p>
</fn>
<corresp id="c001">&#x2a;Correspondence: Xiaoping Zou, <email>zouxp@nju.edu.cn</email>; Shanshan Shen, <email>shenshanshan@njglyy.com</email>
</corresp>
<fn fn-type="equal" id="fn1">
<label>
<sup>&#x2020;</sup>
</label>
<p>These authors have contributed equally to this&#x20;work</p>
</fn>
<fn fn-type="other">
<p>This article was submitted to Cell Death and Survival, a section of the journal Frontiers in Cell and Developmental Biology</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>02</day>
<month>02</month>
<year>2022</year>
</pub-date>
<pub-date pub-type="collection">
<year>2021</year>
</pub-date>
<volume>9</volume>
<elocation-id>745554</elocation-id>
<history>
<date date-type="received">
<day>22</day>
<month>07</month>
<year>2021</year>
</date>
<date date-type="accepted">
<day>17</day>
<month>11</month>
<year>2021</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2022 Nie, Shi, Shi, Li, Qian, Peng, Ding, Zhang, Lv, Wang, Kong, Zou and Shen.</copyright-statement>
<copyright-year>2022</copyright-year>
<copyright-holder>Nie, Shi, Shi, Li, Qian, Peng, Ding, Zhang, Lv, Wang, Kong, Zou and Shen</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these&#x20;terms.</p>
</license>
</permissions>
<abstract>
<p>Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease with poor prognosis. Our previous study found that peroxisome proliferator activated receptor gamma (PPAR&#x3b3;) was capable of enhancing glycolysis in PDAC cells. However, whether PPAR&#x3b3; could promote PDAC progression remains unclear. In our present study, PPAR&#x3b3; was positively associated with tumor size and poor prognosis in PDAC patients. Functional assays demonstrated that PPAR&#x3b3; could promote the proliferation of pancreatic cancer cells <italic>in&#x20;vitro</italic> and <italic>in vivo</italic>. Additionally, flow cytometry results showed that PPAR&#x3b3; decreased mitochondrial reactive oxygen species (mitochondrial ROS) production, stabilized mitochondrial membrane potential (MMP) and inhibited cell apoptosis <italic>via</italic> up-regulating superoxide dismutase 2 (SOD2), followed by the inhibition of ATG4D-mediated mitophagy. Meanwhile, the activation of PPAR&#x3b3; might reduce pancreatic cancer cell stemness to improve PDAC chemosensitivity <italic>via</italic> down-regulating ATG4D. Thus, these results revealed that PPAR&#x3b3;/SOD2 might protect against mitochondrial ROS-dependent apoptosis <italic>via</italic> inhibiting ATG4D-mediated mitophagy to promote pancreatic cancer proliferation, further improving PDAC chemosensitivity.</p>
</abstract>
<kwd-group>
<kwd>PPAR&#x3b3;</kwd>
<kwd>SOD2</kwd>
<kwd>ATG4D</kwd>
<kwd>mitophagy</kwd>
<kwd>pancreatic ductal adenocarcinoma</kwd>
</kwd-group>
</article-meta>
</front>
<body>
<sec id="s1">
<title>Introduction</title>
<p>Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a high mortality rate, the 5-year survival rate of which is only 10% (<xref ref-type="bibr" rid="B35">Siegel et&#x20;al., 2021</xref>). It is probably due to the fact that PDAC is a complex and heterogenic disease with extensive variations in genetic, clinical and histological profiles. It is of great importance to elucidate the molecular mechanisms and to identify new therapeutic strategies for PDAC. We previously generated several novel genetically engineered mouse models (GEMMs) of PDAC entities (<xref ref-type="bibr" rid="B20">Kong et&#x20;al., 2015</xref>; <xref ref-type="bibr" rid="B11">Cheng et&#x20;al., 2016</xref>; <xref ref-type="bibr" rid="B21">Kong et&#x20;al., 2016</xref>; <xref ref-type="bibr" rid="B19">Kong et&#x20;al., 2018</xref>). In a PDAC subtype with poor prognosis characterized by elevated level of ALDH1A3 (aldehyde dehydrogenase family 1, subfamily A3) (<xref ref-type="bibr" rid="B21">Kong et&#x20;al., 2016</xref>; <xref ref-type="bibr" rid="B30">Nie et&#x20;al., 2020</xref>), PPAR&#x3b3; (peroxisome proliferator activated receptor &#x3b3;) was significantly upregulated, leading to activation of the PI3K/AKT/mTOR signaling pathway and accelerated glycolysis (<xref ref-type="bibr" rid="B30">Nie et&#x20;al., 2020</xref>). However, the specific effects of PPAR&#x3b3; on PDAC malignant behaviors remain unclarified in our previous studies (<xref ref-type="bibr" rid="B30">Nie et&#x20;al., 2020</xref>). PPAR&#x3b3;, as a nuclear receptor transcription factor, regulates mitochondrial function and participates in cancer cell metabolism, oxidative redox and biosynthesis (<xref ref-type="bibr" rid="B8">Calvier et&#x20;al., 2017</xref>; <xref ref-type="bibr" rid="B38">Tseng et&#x20;al., 2019</xref>; <xref ref-type="bibr" rid="B43">Zou et&#x20;al., 2019</xref>). The functions of PPAR&#x3b3; in cancer development is ambiguous, and its roles in PDAC carcinogenesis and progression remain unclear (<xref ref-type="bibr" rid="B28">Nakajima et&#x20;al., 2008</xref>; <xref ref-type="bibr" rid="B33">Reka et&#x20;al., 2010</xref>; <xref ref-type="bibr" rid="B25">Lv et&#x20;al., 2019</xref>; <xref ref-type="bibr" rid="B43">Zou et&#x20;al., 2019</xref>). Thus, it is necessary to further explore the role of PPAR&#x3b3; in PDAC, so as to provide a theoretical basis for therapeutic strategies.</p>
<p>As a lysosomal-dependent degradation pathway, mitophagy selectively targets mitochondria for elimination and renewal (<xref ref-type="bibr" rid="B22">Kubli and Gustafsson, 2012</xref>). The regulation of mitochondrial function largely depends on mitophagy (<xref ref-type="bibr" rid="B23">Lesmana et&#x20;al., 2016</xref>; <xref ref-type="bibr" rid="B16">Humpton et&#x20;al., 2019</xref>). On one hand, mitophagy abrogates cancer cell proliferation <italic>via</italic> inducing oxidative stress (<xref ref-type="bibr" rid="B6">Boyle et&#x20;al., 2018</xref>; <xref ref-type="bibr" rid="B34">Shen et&#x20;al., 2018</xref>). On the other hand, cancer cells can utilize mitophagy to adapt particular metabolic stress, leading to therapy resistances (<xref ref-type="bibr" rid="B12">Ferro et&#x20;al., 2020</xref>). A previous study showed that the loss of PPAR&#x3b3; in platelet is closely associated with mitophagy activation, resulting in increased mitochondrial electron transport chain complex activity and enhanced mitochondrial ROS production (<xref ref-type="bibr" rid="B42">Zhou et&#x20;al., 2017</xref>). However, the function and underlying mechanism of PPAR&#x3b3;-associated mitophagy during PDAC development remains largely unknown. Here, we aim to investigate the role of PPAR&#x3b3; on mitophagy and its involvement in the progression of&#x20;PDAC.</p>
</sec>
<sec sec-type="methods" id="s2">
<title>Methods</title>
<sec id="s2-1">
<title>Oncomine and TCGA Database Using</title>
<p>Expressions of PPAR&#x3b3; in normal pancreas and PDAC tissues were collected and analyzed using datasets deposited in Oncomine database (<ext-link ext-link-type="uri" xlink:href="https://www.oncomine.org">https://www.oncomine.org</ext-link>). The Cancer Type was defined as Pancreatic cancer and Data Type was mRNA, and Analysis Type was Cancer vs. Normal Analysis.</p>
<p>The expression levels of PPAR&#x3b3;, SOD2, ATG4D and survival data for TCGA pancreatic adenocarcinoma (provisional) patients (<italic>n</italic>&#x20;&#x3d; 176) were downloaded from The Human Protein Atlas (<ext-link ext-link-type="uri" xlink:href="https://www.proteinatlas.org/">https://www.proteinatlas.org/</ext-link>). The best cutoff value for PPAR&#x3b3;, SOD2 and ATG4D mRNA expression level (FPKM) was 3.64, 11.98 and 6.65 respectively.</p>
</sec>
<sec id="s2-2">
<title>Human PDAC Tissue Array Analysis</title>
<p>The study was conducted in accordance with International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS). The clinical part of study was approved by the Research Ethics Committee of Drum Tower Hospital, School of Medicine, Nanjing University. The patient cohort of human PDAC tissue array contained 59 PDAC specimens from May 2004 to November 2016 obtained from Drum Tower Hospital (School of medicine, Nanjing University). Patients had not received radiotherapy, chemotherapy or other related anti-tumor therapies before surgery.</p>
</sec>
<sec id="s2-3">
<title>Immunohistochemistry</title>
<p>IHC of PDAC tissues was performed as described previously (<xref ref-type="bibr" rid="B30">Nie et&#x20;al., 2020</xref>). Specific antibodies used for immunohistochemistry were: PPAR&#x3b3; (1:200, Proteintech, &#x23;16643-1-AP). PPAR&#x3b3; was localized mainly (but not always) in the nucleus. Only the staining of nucleus was counted and analyzed in this study, which revealed the prognostic effect of PPAR&#x3b3; on PDAC patients. The proportion of nucleus stained was evaluated as follows: 0 for &#x3c;5%, 1 for 5&#x2013;25%, 2 for 25&#x2013;50%, 3 for 50&#x2013;75%, and 4 for &#x2265; 75%. The intensity of staining was scored as 0, 1, 2 and 3 for the representation of no color, yellow, brown and dark brown. The final scores were obtained by multiplying the extent of positivity and intensity scores. Final score &#x2265; 3 was defined as positive. The stained slides were evaluated by two experienced pathologists independently.</p>
</sec>
<sec id="s2-4">
<title>Cell Culture and Reagents</title>
<p>Human pancreatic cancer cell lines AsPC-1, BxPC3, Capan2, CFPAC-1, HPAC, MIAPaCa-2, PANC-1, SW1990 were gifts from Klinikum rechts der Isar, Technical University of Munich. All cell lines were cultured in suggested medium according to ATCC protocols. HPAC, SW1990 and PANC-1 cell lines were cultured with DMEM (Bio-Channel).</p>
<p>Rosiglitazone (APExBIO, &#x23;A4303), a therapeutic drug for diabetes, was used as an agonist of PPAR&#x3b3;. T0070907 (Selleck Chemicals, &#x23;S2871) was used as an antagonist of PPAR&#x3b3;. Cells were treated with Rosiglitazone (0, 10 or 20&#xa0;&#x3bc;M) or T0070907 (0, 5 or 10&#xa0;&#x3bc;M) for 72&#xa0;h 10&#x3bc;M Chloroquine (CQ) (MedChemExpress, &#x23;HY-17589), gemcitabine (MedChemExpress, &#x23;HY-17026) was used for research.</p>
</sec>
<sec id="s2-5">
<title>Luciferase Reporter Assay</title>
<p>The PPAR response element (PPRE) X3-TK-Luc plasmid is a reporter construct containing three copies of PPRE (PPRE X3) upstream of a thymidine kinase (TK) promoter fused to a luciferase gene (<xref ref-type="bibr" rid="B37">Tsai et&#x20;al., 2014</xref>). The DNA sequence of this commercialized plasmid was downloaded from <ext-link ext-link-type="uri" xlink:href="https://www.addgene.org">https://www.addgene.org</ext-link>. (Addgene, &#x23;1015) and constructed by Genechem (Shanghai, China). The plasmid was transfected into HPAC and SW1990 cells after treated with Rosiglitazone (0, 10 or 20&#xa0;&#x3bc;M) or T0070907 (0, 5 or 10&#xa0;&#x3bc;M) for 48&#xa0;h. After another 24&#xa0;h, the luciferase Assay System Kit (E1910, Promega, United&#x20;States) was used to detect PPRE-driven luciferase activity.</p>
</sec>
<sec id="s2-6">
<title>RNA Extraction and Quantitative Real-Time PCR</title>
<p>Total RNA was isolated using Trizol reagent (Takara) according to the manufacturer&#x2019;s instructions. Reverse transcription reactions and Quantitative PCR were carried out as described previously. Reactions were run in triplicate in three independent experiments. The 2<sup>&#x2212;&#x394;&#x394;CT</sup>method was used to determine the relative levels of mRNA expression between experimental samples and controls. Primers were listed as following: ATG4A forward: TTC&#x200b;CCT&#x200b;TGA&#x200b;GTG&#x200b;CTG&#x200b;ACA&#x200b;CA; ATG4A reverse: ATT&#x200b;TGG&#x200b;TTT&#x200b;ATG&#x200b;CCC&#x200b;AGG&#x200b;CG. ATG4B forward: CAC&#x200b;CAG&#x200b;ATA&#x200b;GCG&#x200b;CAA&#x200b;ATG&#x200b;GG; ATG4B reverse: CTC&#x200b;CAC&#x200b;GTA&#x200b;TCG&#x200b;AAG&#x200b;ACA&#x200b;GCA. ATG4C forward: TGG&#x200b;ACT&#x200b;TCC&#x200b;CAC&#x200b;ACT&#x200b;GTC&#x200b;AAA; ATG4C reverse: AGG&#x200b;GGG&#x200b;AAT&#x200b;CAC&#x200b;CAA&#x200b;ACC&#x200b;AA; ATG4D forward: 5&#x2032;-TGG&#x200b;TGT&#x200b;ACG&#x200b;TTT&#x200b;CTC&#x200b;AGG&#x200b;ACT-3&#x2032;; ATG4D reverse: 5&#x2032;-CAC&#x200b;ATA&#x200b;CAC&#x200b;GGG&#x200b;GTT&#x200b;GAG&#x200b;AGT-3&#x2032;. SOD2 forward: GTC&#x200b;AAC&#x200b;CAT&#x200b;CAA&#x200b;AGA&#x200b;GGT&#x200b;CTG&#x200b;C; SOD2 reverse: GAC&#x200b;TGG&#x200b;AGA&#x200b;TAC&#x200b;AGG&#x200b;TCT&#x200b;TGG&#x200b;T. &#x3b2;-actin forward: 5&#x2032;-CTA&#x200b;CGT&#x200b;CGC&#x200b;CCT&#x200b;GGA&#x200b;CTT&#x200b;CGA&#x200b;GC-3&#x2032;; &#x3b2;-actin reverse: 5&#x2032;-GAT&#x200b;GGA&#x200b;GCC&#x200b;GCC&#x200b;GAT&#x200b;CCA&#x200b;CAC&#x200b;GG-3&#x2032;. CD24 forward: CAT&#x200b;GGG&#x200b;CAG&#x200b;AGC&#x200b;AAT&#x200b;GGT&#x200b;G; CD24 reverse: TAG&#x200b;TTG&#x200b;GAT&#x200b;TTG&#x200b;GGG&#x200b;CCA&#x200b;ACC. CD44 forward: TAC&#x200b;AGC&#x200b;ATC&#x200b;TCT&#x200b;CGG&#x200b;ACG&#x200b;GA; CD44 reverse: GCA&#x200b;GGT&#x200b;CTC&#x200b;AAA&#x200b;TCC&#x200b;GAT&#x200b;GC. CD 90 forward: GCA&#x200b;GAA&#x200b;GGT&#x200b;GAC&#x200b;CAG&#x200b;CCT&#x200b;AA; CD90 reverse: TGG&#x200b;TGA&#x200b;AGT&#x200b;TGG&#x200b;TTC&#x200b;GGG&#x200b;AG. CD133 forward: CAC&#x200b;TAC&#x200b;CAA&#x200b;GGA&#x200b;CAA&#x200b;GGC&#x200b;GT; CD133 reverse: TCC&#x200b;AAC&#x200b;GCC&#x200b;TCT&#x200b;TTG&#x200b;GTC&#x200b;TC. ESA forward: CTG&#x200b;GCC&#x200b;GTA&#x200b;AAC&#x200b;TGC&#x200b;TTT&#x200b;GT; ESA reverse: AGC&#x200b;CCA&#x200b;TCA&#x200b;TTG&#x200b;TTC&#x200b;TGG&#x200b;AGG.</p>
</sec>
<sec id="s2-7">
<title>Western Blot</title>
<p>Cell and tissues lysates were collected as previously described (<xref ref-type="bibr" rid="B30">Nie et&#x20;al., 2020</xref>). Protein concentrations were determined using BCA Assay (Beyotime Biotechnology). Equal amounts of protein were separated with 8&#x2013;12% SDS-PAGE and then electrophoretically transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, United&#x20;States). TBST containing with 5% nonfat milk or bovine serum albumin was used to block nonspecific binding for 2&#xa0;h at room temperature. Then, membranes were incubated with primary antibodies according to the instructions overnight at 4&#xb0;C followed by appropriate secondary antibodies. Signals generated by enhanced chemiluminescence (Millipore) were recorded with a CCD camera (Tanon, Shanghai). Primary and secondary antibodies included: PPAR&#x3b3; (1:1,000, Santa cruze, &#x23;sc-7273), BCL-XL (1:1,000, Cell Signaling Technology, &#x23;2764), BAX (1:1,000, Cell Signaling Technology, &#x23;5023), BNIP3 (1:500, Cell Signaling Technology, &#x23;44060), LC3B (1:1,000, Abcam, &#x23;ab51520), P62 (1:1,000, Abcam, &#x23;ab109012), mTOR (1:1,000, Cell Signaling Technology, &#x23;2983), p-mTOR<sup>Ser2448</sup> (1:1,000, Cell Signaling Technology, &#x23;5536), ULK1 (1:1,000, Cell Signaling Technology, &#x23;6439), p-ULK1<sup>Ser317</sup> (1:1,000, Cell Signaling Technology, &#x23;12753), S6 (1:1,000, Cell Signaling Technology, &#x23;2217), P-S6<sup>Ser235/236</sup> (1:1,000, Cell Signaling Technology, &#x23;2211), ATG4D (1:400, Zen-bioscience, &#x23;507842), SOD2 (1:1,000, proteintech, &#x23;66474-1-Ig), GAPDH (1:5,000, Proteintech, &#x23;60004-1-Ig), CD44 (1:1,000, Cell Signaling Technology, &#x23;5640), CD133 (1:1,000, Sigma-Aldrich, &#x23;4300882).</p>
</sec>
<sec id="s2-8">
<title>Cell Proliferation Analysis</title>
<p>HPAC, SW1990 and PANC-1 cells were plated into 96-well plates at a concentration of 10&#x5e;3 cells per well in 100&#xa0;&#x3bc;L complete growth medium. Rosiglitazone (0, 10 or 20&#xa0;&#x3bc;M) or T0070907 (0, five or 10&#xa0;&#x3bc;M) was added to the cells 24&#xa0;h after seeding. Cell viability was analyzed 1, 2, 3 and 4&#x20;days after cell seeding with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer&#x2019;s instructions. Gemcitabine (0, 1, 2, 5, 10, 20, 50, 100, 200&#xa0;&#x3bc;M) combined with 10&#xa0;&#x3bc;M Rosiglitazone or not was added to the cells 24&#xa0;h after seeding. Cell viability was analyzed 3&#x20;days after cell seeding with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer&#x2019;s instructions.</p>
</sec>
<sec id="s2-9">
<title>Colony-Formation Assay</title>
<p>Cells were plated in six-well plates in 2&#xa0;ml complete medium with Rosiglitazone (0, 10 or 20&#xa0;&#x3bc;M) or T0070907 (0, 5 or 10&#xa0;&#x3bc;M). Numbers of cells per well were 1,000 for HPAC and SW1990 cells. The culture media with reagent was replaced by complete medium (2&#xa0;ml) 4&#xa0;days after cell seeding. After 14&#xa0;days, colonies were fixed in methanol and stained with 0.5% crystal violet. The number of colonies was calculated by ImageJ.</p>
</sec>
<sec id="s2-10">
<title>
<italic>In Vivo</italic> Tumor Xenograft Study</title>
<p>Five-week-old male BALB/c nu/nu mice were purchased from CAVENS lab animal corporation. HPAC and PANC-1 cells were inoculated subcutaneously (1 &#xd7; 10<sup>6</sup> cells) into the left flank of each mouse. Six days after inoculation of HPAC, 14&#xa0;days after inoculation of PANC-1, the mice were randomly divided into three groups and treated with Rosiglitazone (100&#xa0;mg/kg) or T0070907 (5&#xa0;mg/kg) <italic>via</italic> gavage three times a week. The tumor volumes were measured and calculated by the following formula (A&#x2a;B<sup>2</sup>)/2, where A is the length and B is the width of the two dimensions of tumor. After animals were sacrificed, the weights of the tumor mass were measured.</p>
</sec>
<sec id="s2-11">
<title>Mitochondria ROS Detection by Flow Cytometry</title>
<p>Mitochondria ROS (mito-ROS) was measured by using MitoSOX red (Yeasen, 40778ES50) according to the manufacturer&#x2019;s instructions, at a concentration of 2&#xa0;&#x3bc;M and incubated at 37&#xb0;C with 5% CO<sub>2</sub> for 20&#xa0;min. Quantification of mito-ROS was carried out by flow cytometry.</p>
</sec>
<sec id="s2-12">
<title>JC-1 Analysis for Mitochondrial Membrane Potential</title>
<p>Mitochondrial membrane depolarization was monitored by changes in the tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) (Beyotime, C2006) green: red fluorescence ratio, where an increased ratio is indicative of elevated mitochondrial membrane potential (MMP). And the increased ratio could be a landmark of the early stage of apoptosis. Cells were incubated with JC-1 (1:1,000 dilution) for 20&#xa0;min at 37&#xb0;C. Then cells were harvested, washed twice with 1&#x20;&#xd7; washing buffer and mixed in 100&#xa0;&#x3bc;L of 1&#x20;&#xd7; washing buffer. The fluorescence intensity was measured by flow cytometry.</p>
</sec>
<sec id="s2-13">
<title>Flow Cytometric Analysis for Apoptosis</title>
<p>Cells with various culture reagent were seeded in 6-well plates. Cells were harvested, washed twice with PBS, and mixed in 100&#xa0;&#x3bc;L of 1&#x2a;binding buffer. After culturing in Annexin-V/PI (BD Biosciences) double staining liquid for 15&#xa0;min at room temperature in the dark, the cells were examined by flow cytometry.</p>
</sec>
<sec id="s2-14">
<title>Mitophagy Assay</title>
<p>For quantification of mitophagic flux, cells were treated with Mtphagy dye (Mitophagy Detection Kit, Dojindo Molecular Technologies, &#x23;MD01) according to the manufacturer&#x2019;s instruction. The fluorescence intensity of Mtphagy dye (excitation 530&#xa0;nm; emission 700&#xa0;nm) was measured by flow cytometry. Increased Mtphagy dye fluorescence intensity indicated the progression of mitophagy.</p>
</sec>
<sec id="s2-15">
<title>Immunofluorescence</title>
<p>Cells treated with different reagents were seeded in 24-well plates at a density of 1&#x20;&#xd7; 10<sup>3</sup> cells per well. At 72&#xa0;h after seeding, cells were fixed with 4% paraformaldehyde for 15&#xa0;min at room temperature and permeabilized with 0.2% Triton X-100 for 15&#xa0;min. After blocking with 5% BSA for 1&#xa0;h at room temperature, cells were incubated with LC3B (1:100, Cell Signaling Technology, &#x23;3868), TOM20 (1:200, Santa cruze, &#x23;17764) or ATG4D (1:100, Zen-bioscience, &#x23;507842) antibodies overnight at 4&#xb0;C. Cells were washed 3&#x20;times and then incubated with Alexa Fluor<sup>&#xae;</sup> 488 (Abcam, ab150077) or Alexa Fluor<sup>&#xae;</sup> 647 (Abcam, 150115) for 1&#xa0;hour at room temperature in the dark. Then, cells were incubated with DAPI (Beyotime, C1005) for 20&#xa0;min. Cells were visualized by a fluorescence microscope (Olympus, Tokyo, Japan).</p>
</sec>
<sec id="s2-16">
<title>Electron Microscopy</title>
<p>After collecting cells for different treatment, cells were centrifuged and pellets were fixed with 0.1&#xa0;M cacodylate buffer with a pH of 7.4 at RT and sections were processed by the Electron Microscopy unit per standard protocols. Pictures were taken with a Hitachi transmission electron microscope (TEM) system.</p>
</sec>
<sec id="s2-17">
<title>Chromatin Immunoprecipitation Assay</title>
<p>The chromatin immunoprecipitation (ChIP) assay was performed as instructions from Magna ChIP Kit (Merck, &#x23;17-10085). Precleared cell lysates were incubated with the antibodies at 4&#xb0;C overnight. Immune complexes were recovered with salmon sperm DNA/protein A/G agarose slurry. After washing and elution, genomic DNA was extracted with phenol/chloroform for PCR analysis. The PCR primer of SOD2 was following. Forward: 5&#x2032;- AGT&#x200b;ACC&#x200b;TCC&#x200b;TGC&#x200b;TGA&#x200b;GAC&#x200b;GA- 3&#x2032;. Reverse: 5&#x2032;- TGG&#x200b;GAA&#x200b;AAC&#x200b;AGT&#x200b;CAG&#x200b;GCG&#x200b;AA- 3&#x2032;.</p>
</sec>
<sec id="s2-18">
<title>siRNA Transfection</title>
<p>Cells were transfected with either two siRNAs against SOD2, ATG4D or one non-targeting siRNA and cultured in 6-well plates according to the manufacturer&#x2019;s instructions. The target sequences of oligo siRNAs were as follows: siPPAR&#x3b3;&#x23;1: forward: ACU&#x200b;CCA&#x200b;CAU&#x200b;UAC&#x200b;GAA&#x200b;GAC&#x200b;ATT, reverse: UGU&#x200b;CUU&#x200b;CGU&#x200b;AAU&#x200b;GUG&#x200b;GAG&#x200b;UTT. siPPAR&#x3b3;&#x23;2: forward: CUG&#x200b;GCC&#x200b;UCC&#x200b;UUG&#x200b;AUG&#x200b;AAU&#x200b;ATT, reverse: UAU&#x200b;UCA&#x200b;UCA&#x200b;AGG&#x200b;AGG&#x200b;CCA&#x200b;GTT. siSOD2&#x23;1: forward: CUG&#x200b;GGA&#x200b;GAA&#x200b;UGU&#x200b;AAC&#x200b;UGA&#x200b;A, reverse: UUC&#x200b;AGU&#x200b;UAC&#x200b;AUU&#x200b;CUC&#x200b;CCA&#x200b;G. siSOD2&#x23;2: forward: CAC&#x200b;GCU&#x200b;UAC&#x200b;UAC&#x200b;CUU&#x200b;CAG&#x200b;U, reverse: ACU&#x200b;GAA&#x200b;GGU&#x200b;AGU&#x200b;AAG&#x200b;CGU&#x200b;G. siATG4D&#x23;1: forward: GGC&#x200b;AGA&#x200b;UUG&#x200b;UGU&#x200b;CCU&#x200b;GGU&#x200b;UTT, reverse: AAC&#x200b;CAG&#x200b;GAC&#x200b;ACA&#x200b;AUC&#x200b;UGC&#x200b;CTT. siATG4D&#x23;2: forward: GGA&#x200b;AGG&#x200b;AGU&#x200b;UUG&#x200b;AGA&#x200b;CAC&#x200b;UTT, reverse: GGA&#x200b;AGG&#x200b;AGU&#x200b;UUG&#x200b;AGA&#x200b;CAC&#x200b;UTT. Negative control: forward: UUC&#x200b;UCC&#x200b;GAA&#x200b;CGU&#x200b;GUC&#x200b;ACG&#x200b;UTT, reverse: UUC&#x200b;UCC&#x200b;GAA&#x200b;CGU&#x200b;GUC&#x200b;ACG&#x200b;UTT.</p>
</sec>
<sec id="s2-19">
<title>Sphere-Formation Assay</title>
<p>HPAC cell spheres were generated and expanded in DMEM-F12 (Invitrogen) supplemented with 20&#xa0;nM epidermal growth factor (EGF) (Sigma-Aldrich, &#x23;E4127), 20&#xa0;nM basic fibroblast growth factor (bFGF) (Gibco, &#x23;aa1-155) and 3% FBS. One thousand cells/ml/well were seeded in ultra-low attachment 24-well plates as described previously (<xref ref-type="bibr" rid="B13">Gallmeier et&#x20;al., 2011</xref>). For serial passaging, spheres were harvested at day 7 using a 40-&#xb5;m cell strainer, dissociated to single cells with trypsin, and then re-grown in the same conditions for 7&#xa0;days (14&#xa0;days total). Spheres were defined as morphologically characteristic three-dimensional structures of approximately &#x2265; 75&#xa0;&#x3bc;m (<xref ref-type="bibr" rid="B26">Mani et&#x20;al., 2008</xref>). Diameters and numbers of spheres were determined by an inverted microscope (Thermo Fisher Scientific) using a &#xd7;10, &#xd7;20 objective with phase contrast.</p>
</sec>
<sec id="s2-20">
<title>Statistical Analyses</title>
<p>Data were analyzed using GraphPad Prism v7.0. All experiments were repeated at least three times, with the mean and standard deviation (S.D.) being reported where appropriate. The repeated results were used as data points for statistical tests. Differences between treatments were evaluated using ANOVA or Student&#x2019;s <italic>t</italic>&#x20;test. Correlations were analyzed by the Pearson method. Log-rank tests were performed on Kaplan-Meier survival curves to determine any significant relationships between gene expression and patient outcomes. Differences were considered significant at <italic>p</italic>&#x20;&#x3c;&#x20;0.05.</p>
</sec>
</sec>
<sec sec-type="results" id="s3">
<title>Results</title>
<sec id="s3-1">
<title>High Expression of PPAR&#x3b3; in Nucleus is Correlated With Poor Prognosis in PDAC</title>
<p>Two previously published datasets from Oncomine were initially analyzed to determine the expression pattern of PPAR&#x3b3; in pancreatic cancer and normal tissues. The results revealed that PPAR&#x3b3; expression level was upregulated in cancer tissues compared to that in the corresponding adjacent non-tumor tissues (<xref ref-type="fig" rid="F1">Figure&#x20;1A</xref>). We then analyzed TCGA dataset and found that the mRNA expression of PPAR&#x3b3; was correlated with overall survival and tumor stage. PDAC patients with high PPAR&#x3b3; expression exhibited advanced tumor stages and poor prognosis compared to those with low PPAR&#x3b3; expression (<xref ref-type="fig" rid="F1">Figures&#x20;1B,C</xref>).</p>
<fig id="F1" position="float">
<label>FIGURE 1</label>
<caption>
<p>High expression of PPAR&#x3b3; in nuleus is correlated with poor prognosis in PDAC. <bold>(A)</bold> The expression of PPAR&#x3b3; increased in pancreatic cancer tissue compared to adjacent normal pancreatic tissue by Oncomine datasets analysis. <bold>(B)</bold> In TCGA dataset, high expression of PPAR&#x3b3; in cancer tissues was associated with shorter overall survival time in the PDAC patients (<italic>p &#x3d;</italic> 0.0165). <bold>(C)</bold> In TCGA dataset, PPAR&#x3b3; expression level was positively correlated with tumor stages (<italic>p &#x3d;</italic> 0.0107). <bold>(D)</bold> PPAR&#x3b3; immunostaining signal in pancreatic cancer tissues was primarily detected in nucleus. The scale bar was 20&#xa0;&#x3bc;m. <bold>(E)</bold> High expression level of PPAR&#x3b3; in pancreatic cancer tissues was associated with shorter overall survival time in the PDAC patients (<italic>p &#x3d;</italic> 0.0290). <bold>(F)</bold> PPAR&#x3b3; expression level was positively correlated with tumor size (<italic>p &#x3d;</italic> 0.0105).</p>
</caption>
<graphic xlink:href="fcell-09-745554-g001.tif"/>
</fig>
<p>To further examine the expression and clinical relevance of PPAR&#x3b3; in PDAC, we detected the expression of PPAR&#x3b3; in 59 human PDAC tissues from Nanjing Drum Tower Hospital. Immunohistochemical staining on tissues was performed, and the results showed that the immunostaining signal of PPAR&#x3b3; was mainly located in nucleus (<xref ref-type="fig" rid="F1">Figure&#x20;1D</xref>) and only the expression level of nuclear PPAR&#x3b3; was correlated with overall survival. Kaplan-Meier analysis revealed that the positive expression of nuclear PPAR&#x3b3; in cancer tissues was associated with short overall survival time in PDAC patients (<italic>p</italic>&#x20;&#x3d; 0.0290) (<xref ref-type="fig" rid="F1">Figure&#x20;1E</xref>). In addition, the nuclear PPAR&#x3b3; expression level was significantly related to tumor size (<italic>p</italic>&#x20;&#x3d; 0.0105) (<xref ref-type="fig" rid="F1">Figure&#x20;1F</xref>).</p>
</sec>
<sec id="s3-2">
<title>PPAR&#x3b3; Promotes Pancreatic Cancer Cells Proliferation <italic>in Vitro</italic> and <italic>in Vivo</italic>
</title>
<p>To investigate the biological functions of PPAR&#x3b3; in PDACs, human PDAC cell lines were used for further studies. Firstly, the mRNA and protein expression levels of PPAR&#x3b3; were detected in different human pancreatic cancer cell lines. HPAC and SW1990 cells with positive expression of PPAR&#x3b3; were selected for subsequent study (<xref ref-type="fig" rid="F2">Figure&#x20;2A</xref>).</p>
<fig id="F2" position="float">
<label>FIGURE 2</label>
<caption>
<p>PPAR&#x3b3; promotes pancreatic cancer cell proliferation <italic>in&#x20;vitro</italic> and <italic>in vivo.</italic> <bold>(A)</bold> The expression status of PPAR&#x3b3; was detected in eight human pancreatic cancer cell lines at mRNA and protein levels. <bold>(B)</bold> The PPRE-driven luciferase reporter gene assay showed Rosiglitazone could activate, while T0070907 could inhibit PPAR&#x3b3; transcriptional activity. <bold>(C)</bold> Relative cell viability of HPAC and SW1990 cells treated with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) for 0, 24, 48 and 72&#xa0;h. <bold>(D)</bold> Formation of colonies of HPAC and SW1990 cells treated with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) for 72&#xa0;h <bold>(E&#x2013;G)</bold> Compared with the control group, administrating nude mice with Rosiglitazone (100&#xa0;mg/kg) or T0070907 (5&#xa0;mg/kg) reduced or increased the tumor weight <bold>(E)</bold> and the tumor volume <bold>(F)</bold> 4&#xa0;weeks after HPAC cells injection, without the influence on body weight of mice <bold>(G)</bold>. Experiments were repeated at least three times, with statistical analyses being reported appropriate.</p>
</caption>
<graphic xlink:href="fcell-09-745554-g002.tif"/>
</fig>
<p>PPAR&#x3b3; is a member of the nuclear receptor superfamily that functions as a ligand-activated transcription factor (<xref ref-type="bibr" rid="B38">Tseng et&#x20;al., 2019</xref>). Here, we used PPAR&#x3b3; agonist Rosiglitazone or PPAR&#x3b3; antagonist T0070907 to intervene the PPAR&#x3b3; pathway and to investigate whether pharmacological activation or inhibition of PPAR&#x3b3; would affect the cancer cell survival. To confirm the effects of agonist or antagonist on the transcriptional activity of PPAR&#x3b3;, PPRE-driven luciferase activity was detected in HPAC and SW1990 treated with Rosiglitazone, T0070907 or not. The results showed that Rosiglitazone could enhance, while T0070907 could weaken the transcriptional activity of PPAR&#x3b3; significantly (<xref ref-type="fig" rid="F2">Figure&#x20;2B</xref>). <italic>In vitro</italic> CCK8 assay and colony-formation assay results showed that Rosiglitazone could promote, while T0070907 could inhibit the proliferation and colony-formation capacity of HPAC and SW1990 cells (<xref ref-type="fig" rid="F2">Figures 2C,D</xref>), which was further confirmed in nude mice models. After injecting 1&#x20;&#xd7; 10<sup>6</sup> HPAC cells into nude mice to construct tumors <italic>in vivo,</italic> solvent or Rosiglitazone (100&#xa0;mg/kg) or T0070907 (5&#xa0;mg/kg) was given to treat mice three times a week, respectively. Results revealed that Rosiglitazone promoted, while T0070907 inhibited tumor growth without influencing the weight of nude mice (<xref ref-type="fig" rid="F2">Figures 2E&#x2013;G</xref>).</p>
</sec>
<sec id="s3-3">
<title>PPAR&#x3b3; Inhibits Mitochondrial ROS-Dependent Apoptosis in Pancreatic Cancer Cells</title>
<p>To detect the mechanisms underlying the role of PPAR&#x3b3; on pancreatic cancer cell survival, we determined the early stage of apoptosis by JC-1 assay in the presence or absence of Rosiglitazone or T0070907. An increase in green fluorescence and the concomitant damage of red fluorescence (increased green: red ratio) were observed in cells treated with T0070907, and an inversed ratio was observed in cells treated with Rosiglitazone, indicating the effect of PPAR&#x3b3; on maintaining the mitochondrial membrane potential (MMP) (<xref ref-type="fig" rid="F3">Figure&#x20;3A</xref>). To further assess the apoptotic rate upon drug treatment, flow cytometry analysis was performed after FITC Annexin V/PI staining, which showed that PPAR&#x3b3; significantly protected against pancreatic cancer cell apoptosis (<xref ref-type="fig" rid="F3">Figure&#x20;3B</xref>). In addition, there were increased levels of pro-apoptotic factors&#x2014;BAX and BNIP3 in HPAC and SW1990 cells treated with T0070907, coupled with decreased level of anti-apoptotic factor BCL-XL, and it showed the opposite results in cells treated with Rosiglitazone (<xref ref-type="fig" rid="F3">Figure&#x20;3C</xref>). These data suggested that the activation of PPAR&#x3b3; could effectively promote PDAC proliferation <italic>via</italic> inhibiting pancreatic cancer cell apoptosis.</p>
<fig id="F3" position="float">
<label>FIGURE 3</label>
<caption>
<p>PPAR&#x3b3; inhibits mitochondrial ROS-dependent apoptosis in pancreatic cancer cells. <bold>(A)</bold> Treating HPAC or SW1990 cells with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) for 72&#xa0;h affected mitochondrial membrane potential (MMP) determined by JC-1 fluorescent intensity. <bold>(B)</bold> Treating HPAC or SW1990 cells with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) for 72&#xa0;h affected cell apoptosis determined by flow cytometry with Annexin V-FITC/PI staining. <bold>(C)</bold> The molecular changes in apoptosis determined by Western blot with antibodies against BCL-XL, BAX and BNIP3. <bold>(D)</bold> Treating HPAC or SW1990 cells with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) for 72&#xa0;h affected mitochondrial ROS levels determined and quantitated by MitoSOX staining. Experiments were repeated at least three times, with statistical analyses being reported appropriate.</p>
</caption>
<graphic xlink:href="fcell-09-745554-g003.tif"/>
</fig>
<p>Elevated intracellular levels of ROS induces oxidative stress, leading to cell death. In view of the potential function of PPAR&#x3b3; on mitochondrial metabolism, the MitoSOX red fluorescent dye was used to detect the effect of PPAR&#x3b3; on mitochondria-originating ROS alterations. This assay showed a significant decrease of fluorescence in cells with Rosiglitazone treatment, and an increase in cells with T0070907 treatment (<xref ref-type="fig" rid="F3">Figure&#x20;3D</xref>).</p>
<p>Besides, to confirm the influence of PPAR&#x3b3; agonist-Rosiglitazone or antagonist-T0070907 was in a PPAR&#x3b3;-dependent way, PANC-1 cell line with PPAR&#x3b3;-negative expression was chosen for <italic>in&#x20;vitro</italic> functional assays as well. Though the statistical analysis results showed that T0070907 could inhibit cell proliferation after 96&#xa0;h treatment <italic>in&#x20;vitro</italic>, it had no effect on cell growth and the body weight of mice <italic>in vivo</italic> (<xref ref-type="sec" rid="s11">Supplementary Figures S1A&#x2013;D</xref>). Besides the results showed that only 10&#xa0;&#x3bc;M T0070907 could induce increased mitochondria membrane potential and apoptosis, while Rosiglitazone and low-dose of T0070907 had no effect on mitochondrial ROS production, mitochondria membrane potential and apoptosis in PANC-1 cells (<xref ref-type="sec" rid="s11">Supplementary Figures S1E&#x2013;G</xref>). We supposed that high-dose of T0070907 might induce apoptosis <italic>via</italic> oxidative stress in a PPAR&#x3b3;-independent manner (<xref ref-type="bibr" rid="B17">Kawahara et&#x20;al., 2013</xref>). Thus, in HPAC and SW1990 cells (with PPAR&#x3b3;-positive expression), the growth-promoting role of Rosiglitazone or -inhibiting role of T0070907 were primarily and specifically dependent on PPAR&#x3b3;.</p>
<p>Additionally, the gene-knockdown assay in HPAC and SW1990 cell lines was performed to further verify the function of PPAR&#x3b3; in regulating tumor cell survival. The knockdown efficiency of PPAR&#x3b3; in HPAC and SW1990 cells was detected both on mRNA and protein levels (<xref ref-type="sec" rid="s11">Supplementary Figure S2A</xref>). The transcriptional activity of PPAR&#x3b3; was impaired after interfering PPAR&#x3b3; expression (<xref ref-type="sec" rid="s11">Supplementary Figure S2B</xref>). Moreover, there were elevated levels of pro-apoptotic factors&#x2014;BAX and BNIP3, depressed level of anti-apoptotic factor BCL-XL in PPAR&#x3b3;-knockdown cells compared to negative control cells (<xref ref-type="sec" rid="s11">Supplementary Figure S2C</xref>). And the results of flow-cytometry also revealed that PPAR&#x3b3;-knockdown could contribute to increased mitochondrial membrane potential (<xref ref-type="sec" rid="s11">Supplementary Figure S2D</xref>) and apoptosis (<xref ref-type="sec" rid="s11">Supplementary Figure S2E</xref>) in HPAC and SW1990&#x20;cells.</p>
</sec>
<sec id="s3-4">
<title>PPAR&#x3b3; Regulates the mTOR-ULK1 Signaling Pathway to Inhibit Mitophagy in Pancreatic Cancer Cells</title>
<p>Mitophagy has crucial effects on controlling mitochondrial quality and function (<xref ref-type="bibr" rid="B32">Pickles et&#x20;al., 2018</xref>). To precisely assess the effect of PPAR&#x3b3; on mitophagy, we quantified mitophagic flux using Mtphagy dye after treating cells with T0070907. Mtphagy dye stains mitochondria, and its fluorescence intensity depends on the pH. When mitochondria are transported to lysosomes by mitophagy, Mtphagy dye exhibits higher fluorescence intensity. We found that the activation of PPAR&#x3b3; by Rosiglitazone, or the inhibition of PPAR&#x3b3; by T0070907 in HPAC and SW1990 cells could inhibit, or activate the mitophagic flux, respectively (<xref ref-type="fig" rid="F4">Figure&#x20;4A</xref>). The activation of different steps of autophagy (the autophagosome formation or the lysosome-autophagosome fusion) would lead to the dynamic change on LC3B-II expression level. Since p62 accumulates when autophagy is inhibited, and decreased levels can be observed when autophagy is induced, p62 is used as a marker to study autophagic flux (<xref ref-type="bibr" rid="B5">Bj&#xf8;rk&#xf8;y et&#x20;al., 2009</xref>). Thus, the expression level of autophagy marker was further confirmed by treating cells with chloquine, an inhibitor of lysosome function. We found that there was no difference in trends of expression level of P62 in Rosiglitazone- and T0070907-treated groups with or without CQ, that Rosiglitazone increasing and T0070907 decreasing the expression level of P62 (<xref ref-type="fig" rid="F4">Figure&#x20;4B</xref>). Furthermore, under the condition of lysosomal degradation-blocking, LC3B-II, another hallmark of autophagy activation, accumulating significantly in T0070907-treated cells compared to negative control group cells. The above results revealed that PPAR&#x3b3; had a strong effect on autophagic flux (<xref ref-type="fig" rid="F4">Figure&#x20;4B</xref>). Results from cell immunofluorescence also showed the green fluorescent signal of LC3B increased in T0070907-treated cells and it co-localized with the red fluorescent signal of TOM20, a mitochondrial outer membrane protein (<xref ref-type="fig" rid="F4">Figure&#x20;4C</xref>), indicating the occurrence of mitophagy. Evidence of T0070907-induced mitophagy in HPAC cells was determined by direct observation of the formation of mitophagosomes using electron microscopy (<xref ref-type="fig" rid="F4">Figure&#x20;4D</xref>). Additionally, the protein expression level of LC3B-II in nude mice transplanting tumors treated with T0070907 was elevated significantly as well (<xref ref-type="fig" rid="F4">Figure&#x20;4E</xref>).</p>
<fig id="F4" position="float">
<label>FIGURE 4</label>
<caption>
<p>PPAR&#x3b3; regulates the mTOR-ULK1 signaling pathway to inhibit mitophagy in pancreatic cancer cells. <bold>(A)</bold> The inhibition of PPAR&#x3b3; activated mitophagic flux detected by flow cytometry in HPAC and SW1990 cells. <bold>(B)</bold> Treating HPAC or SW1990 cells with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) in the absence or presence autophagy inhibitor CQ (10&#xa0;&#x3bc;M), LC3B-II and P62 was detected by Western blot to indicate the autophagic flux. <bold>(C)</bold> The effects of T0070907 on mitophagy indicated by LC3B-II expression was further confirmed by cell Immunofluorescence. The scale bar was 20&#xa0;&#x3bc;m. <bold>(D)</bold> Representative TEM graphs showed the existence of mitophagosome after T0070907 treatment in HPAC cells. Black triangle: mitochondria. Black arrow: autophagosome. The scale bar was 10&#xa0;&#x3bc;m. <bold>(E)</bold> The protein expression level of LC3B-II was elevated in the mice tumor tissues treated with T0070907 compared to negative control group. <bold>(F)</bold> Treating HPAC or SW1990 cells with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) for 72&#xa0;h changed the mTOR/ULK1 signaling pathway. Experiments were repeated at least three times, with statistical analyses being reported appropriate.</p>
</caption>
<graphic xlink:href="fcell-09-745554-g004.tif"/>
</fig>
<p>The mTOR pathway commonly participates in autophagic process. On one hand, mTOR could contribute to regulating termination of autophagy and reformation of lysosomes (<xref ref-type="bibr" rid="B40">Yu et&#x20;al., 2010</xref>). On the other hand, mTOR could inhibit the autophagosome formation <italic>via</italic> ULK1 ubiquitylation (<xref ref-type="bibr" rid="B29">Nazio et&#x20;al., 2013</xref>). The mammalian orthologue of yeast Atg1, the serine/threonine kinase ULK1, plays a key role in autophagy induction (<xref ref-type="bibr" rid="B15">Hosokawa et&#x20;al., 2009</xref>). Thus, it prompts us to examine the alteration of mTOR pathway in cells treated with Rosiglitazone or T0070907. As shown, the phosphorylation levels of mTOR and S6 (the classical downstream target of mTOR) were increased in cells with Rosiglitazone treatment, while decreased in cells with T0070907 treatment (<xref ref-type="fig" rid="F4">Figure&#x20;4F</xref>), indicating the activated process of regulating termination of autophagy and reformation of lysosomes. Unc-51 Like Autophagy Activating Kinase 1 (ULK1) regulates the initiation of autophagy by recruiting downstream autophagy-related proteins (ATGs) to autophagy formation site. And the phosphorylation of ULK1 would be inhibited by mTOR complex 1 (mTORC1) activation, thereby inhibiting the autophagy occurrence. Thus, we further determined the status of ULK1 in cells treated with Rosiglitazone or T0070907. The phosphorylation level of ULK1 markedly increased along with the decrease of phospho-mTOR in HPAC and SW1990 cells treated with T0070907 compared to negative control group cells, the situation of which was opposite in cells treated with Rosiglitazone (<xref ref-type="fig" rid="F4">Figure&#x20;4F</xref>). In this part, we figured out that PPAR&#x3b3; might regulate mTOR-mediated degradation of ULK1, linked to impaired mitophagy in pancreatic cancer&#x20;cells.</p>
</sec>
<sec id="s3-5">
<title>PPAR&#x3b3; Downregulates ATG4D-Mediated Mitophagy to Inhibit Pancreatic Cancer Cell Apoptosis</title>
<p>ATG4D, one member of autophagy-related gene 4 (ATG4) family, is able to re-localize to damaged mitochondria, contributing to targeted mitophagy. Additionally, ATG4D could expose the BH3 domain allowing for interaction with BCL-2 family members and induction of cell apoptosis. Notably, mRNA expression level of ATG4D in TCGA dataset was positively correlated with the overall survival of PDAC patients (<xref ref-type="fig" rid="F5">Figure&#x20;5A</xref>). The expression level of ATG4 isoforms (ATG4A, ATG4B, ATG4C and ATG4D) were all detected in HPAC and SW1990 cells treated with Rosiglitazone or T0070907, while only ATG4D was dose-dependent on PPAR&#x3b3; in HPAC and SW1990 cells (<xref ref-type="sec" rid="s11">Supplementary Figure S3A</xref>, <xref ref-type="fig" rid="F5">Figures 5B,C</xref>). Thus, we focused on detecting the role of ATG4D in cells with different PPAR&#x3b3; status. Results from cell immunofluorescence further confirmed that ATG4D mainly expressed on mitochondria when it was activated by T0070907 (<xref ref-type="fig" rid="F5">Figure&#x20;5D</xref>). It suggested that ATG4D could be down-regulated by PPAR&#x3b3; to inhibit autophagy especially located on mitochondria.</p>
<fig id="F5" position="float">
<label>FIGURE 5</label>
<caption>
<p>PPAR&#x3b3; downregulates ATG4D-mediated mitophagy to decrease mitochondrial ROS-dependent apoptosis. <bold>(A)</bold> In TCGA dataset, the mRNA expression level of ATG4D was negatively correlated with PDAC patients&#x2019; overall survival time (<italic>p</italic>&#x20;&#x3d; 0.0010). <bold>(B)</bold> Treating HPAC or SW1990 cells with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) for 72&#xa0;h affected the mRNA expression level of ATG4D. <bold>(C)</bold> Treating HPAC or SW1990 cells with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) for 72&#xa0;h affected the protein expression level of ATG4D. <bold>(D)</bold> The expression and location of ATG4D was mainly changed on mitochondria after treating HPAC or SW1990 cells with negative control, Rosiglitazone (10&#xa0;&#x3bc;M) or T0070907 (5&#xa0;&#x3bc;M) for 72&#xa0;h. <bold>(E)</bold> Knockdown of ATG4D by siRNA in HPAC and SW1990 cells decreased mitophagic flux with or without Rosiglitazone (10 &#x03BC;M) or T0070907 (5 &#x03BC;M). <bold>(F)</bold> Knockdown of ATG4D by siRNA in HPAC and SW1990 cells stabilized mitochondrial membrane potential with or without Rosiglitazone (10&#xa0;&#x3bc;M) or T0070907 (5&#xa0;&#x3bc;M). <bold>(G)</bold> Knockdown of ATG4D by siRNA in HPAC and SW1990 cells decreased cell apoptosis with or without Rosiglitazone (10&#xa0;&#x3bc;M) or T0070907 (5&#xa0;&#x3bc;M). Experiments were repeated at least three times, with statistical analyses being reported appropriate.</p>
</caption>
<graphic xlink:href="fcell-09-745554-g005.tif"/>
</fig>
<p>Studies have found that mitochondrial ATG4D sensitizes cells to death in the presence of the mitochondrial uncoupler, CCCP. And during the mitochondrial clearance phase in differentiating primary human erythroblasts stably expressing ATG4D, these cells have elevated levels of mitochondrial ROS (<xref ref-type="bibr" rid="B4">Betin et&#x20;al., 2012</xref>). To further figure out the role of ATG4D-mediated mitophagy on mitochondrial ROS production, mitochondrial membrane potential and cell apoptosis, the downregulation of ATG4D <italic>via</italic> siRNA was performed in HPAC and SW1990 cells treated with Rosiglitazone, T0070907 or not. Firstly, knockdown efficiency of ATG4D was confirmed on mRNA and protein levels (<xref ref-type="sec" rid="s11">Supplementary Figures S3B,C</xref>), and ATG4D-knockdown did inhibit the expression of LC3B-II (<xref ref-type="sec" rid="s11">Supplementary Figure S3C</xref>). Additionally, we quantified mitophagic flux using Mtphagy dye after treating cells with ATG4D knockdown, and the results showed that downregulating ATG4D could block the role of T0070907 on mitophagy activation to inhibit mitophagic flux (<xref ref-type="fig" rid="F5">Figure&#x20;5E</xref>). Furthermore, after inhibiting the expression of ATG4D by siRNA in cells, mitochondrial membrane potential (<xref ref-type="fig" rid="F5">Figure&#x20;5F</xref>) and cell apoptosis (<xref ref-type="fig" rid="F5">Figure&#x20;5G</xref>) were all decreased, no matter whether Rosiglitazone or T0070907 was used to treat cancer cells or not. These results revealed that PPAR&#x3b3; inhibited mitophagy <italic>via</italic> regulating ATG4D, decreasing the mitochondrial ROS-dependent cell apoptosis.</p>
</sec>
<sec id="s3-6">
<title>PPAR&#x3b3; Inhibits Mitochondrial ROS-ATG4D-Mediated Mitophagy <italic>via</italic> Upregulating SOD2</title>
<p>SOD2, the primary mitochondrial oxidative scavenger, plays a crucial role during the regulation of mitochondrial ROS by catalyzing O2<sup>&#x2212;</sup> conversation to H<sub>2</sub>O<sub>2</sub>. The expression of SOD2 in TCGA dataset was negatively correlated to PDAC patients&#x2019; overall survival time (<xref ref-type="fig" rid="F6">Figure&#x20;6A</xref>). Notably, the TCGA dataset revealed that there was a negative correlation between SOD2 and ATG4D mRNA expression level (<xref ref-type="fig" rid="F6">Figure&#x20;6B</xref>). The effect of SOD2 expression on PPAR&#x3b3;-inhibiting mitochondrial ROS production was further investigated. <italic>In silico</italic> analysis predicted that the promoter region of SOD2 gene contained PPAR&#x3b3; binding sites, moreover one of which contained the PPRE binding site (<xref ref-type="fig" rid="F6">Figure&#x20;6C</xref>). Consistently, ChIP assay results demonstrated that PPAR&#x3b3; could directly bind to the PPRE in the promoter region of <italic>SOD2</italic> in HPAC and SW1990 cells treated with Rosiglitazone (<xref ref-type="fig" rid="F6">Figure&#x20;6D</xref>). The role of PPAR&#x3b3; on modulating SOD2 expression was also confirmed on the protein level (<xref ref-type="fig" rid="F6">Figure&#x20;6E</xref>). To access if SOD2 could influence the ATG4D-mediated mitophagy, mitophagic markers, MitoSOX, JC-1 and cell apoptosis assays were detected after the inhibition of SOD2 by siRNA transfection. The blockade of SOD2 increased the protein expression level of ATG4D, as well as the accumulation of LC3B-II on mitochondria, the increased level of BNIP3 (<xref ref-type="fig" rid="F6">Figures&#x20;6F,G</xref>).</p>
<fig id="F6" position="float">
<label>FIGURE 6</label>
<caption>
<p>PPAR&#x3b3; inhibits ATG4D-mediated mitophagy <italic>via</italic> upregulating SOD2. <bold>(A)</bold> In TCGA dataset, The PDAC patients with SOD2 high expression suffered from shorter overall survival time than those with SOD2 low expression (<italic>p</italic>&#x20;&#x3d; 0.0368). <bold>(B)</bold> Analysis of TCGA data showed the expression of SOD2 and ATG4D had significant negative correlation (<italic>r</italic>&#x20;&#x3d;&#x2014;0.3268, <italic>p</italic>&#x20;&#x3c; 0.0001). <bold>(C)</bold> The promoter regions of SOD2 contained PPAR response element (PPRE) (red letter). <bold>(D)</bold> ChIP assay confirmed that PPAR&#x3b3; regulated directly the transcription of SOD2 in HPAC and SW1990 cells. <bold>(E)</bold> Treating HPAC or SW1990 cells with negative control, Rosiglitazone (10, 20&#xa0;&#x3bc;M) or T0070907 (5, 10&#xa0;&#x3bc;M) for 72&#xa0;h affected the expression level of SOD2. <bold>(F)</bold> Knockdown of SOD2 by siRNA in HPAC and SW1990 cells increased the expression of ATG4D, LC3B-II and the apoptosis-related protein expression-BNIP3. <bold>(G)</bold> Knockdown of SOD2 by siRNA in HPAC and SW1990 cells increased the expression of LC3B-II on mitochondria by cell immunofluorescence. The scale bar was 20&#xa0;&#x3bc;m. <bold>(H)</bold> Knockdown of SOD2 by siRNA in HPAC and SW1990 cells increased mitochondrial ROS production. <bold>(I)</bold> Knockdown of SOD2 by siRNA in HPAC and SW1990 cells stabilized mitochondrial membrane potential. <bold>(J)</bold> Knockdown of SOD2 by siRNA in HPAC and SW1990 cells increased cell apoptosis. Experiments were repeated at least three times, with statistical analyses being reported appropriate.</p>
</caption>
<graphic xlink:href="fcell-09-745554-g006.tif"/>
</fig>
<p>Moreover, MitoSOX-based measurement revealed that, the inhibition of SOD2 by siRNA in HPAC and SW1990 cells could increase the mitochondrial ROS level (<xref ref-type="fig" rid="F6">Figure&#x20;6H</xref>). Furthermore, SOD2 siRNA treatment also increase MMP as shown in JC-1 assay (<xref ref-type="fig" rid="F6">Figure&#x20;6I</xref>) and cell apoptosis assay (<xref ref-type="fig" rid="F6">Figure&#x20;6J</xref>) in HPAC and SW1990 cells. Taken together, these results suggested that PPAR&#x3b3; might inhibit ATG4D-mediated mitophagy <italic>via</italic> upregulating SOD2, to reduce mitochondrial ROS-dependent cell apoptosis.</p>
<p>The above results were also verified <italic>via</italic> PPAR&#x3b3;-knockdown assay in HPAC and SW1990 cells. The flow-cytometry result showed that PPAR&#x3b3;-knockdown in HPAC and SW1990 cells could activate mitophagic flux (<xref ref-type="sec" rid="s11">Supplementary Figure S4A</xref>). And the expression of ATG4D and SOD2 was confirmed to be regulated by PPAR&#x3b3; in HPAC and SW1990 cells (<xref ref-type="sec" rid="s11">Supplementary Figures S4B&#x2013;D</xref>). Additionally, PPAR&#x3b3; knockdown in cells did influence mTOR-ULK1 pathway (<xref ref-type="sec" rid="s11">Supplementary Figure S4D</xref>), to activate the expression of LC3B-II and induce mitophagy, which was also verified by cell immunofluorescence (<xref ref-type="sec" rid="s11">Supplementary Figures S4D,E</xref>).</p>
</sec>
<sec id="s3-7">
<title>PPAR&#x3b3;/SOD2 Decreases the Potential Stemness of PDAC <italic>via</italic> Inhibiting ATG4D-Mediated Mitophagy</title>
<p>Notably, pancreatic cancer stem cells (PaCSCs) could use mitophagy for particular adaptation of metabolic stress, contributing to better survive in tumor micro-environment (<xref ref-type="bibr" rid="B12">Ferro et&#x20;al., 2020</xref>). Emerging evidence suggests that PaCSCs, marked by CD44, CD24, ESA, CD133, or c-Met proteins, characterize a subset of PDAC with distinct stemness features that permit them to drive tumor heterogeneity, metastasis and resistance to the current chemotherapy and radiation (<xref ref-type="bibr" rid="B36">Subramaniam et&#x20;al., 2018</xref>). A previous study found that Rosiglitazone and Gemcitabine in combination reduces immune suppression in pancreatic cancer, participating in chemotherapy resistance (<xref ref-type="bibr" rid="B7">Bunt et&#x20;al., 2013</xref>). To investigate whether the enhanced PDAC proliferation <italic>via</italic> PPAR&#x3b3;-inhibited mitophagic pathway has effects on stemness of pancreatic cancer cells and chemotherapy sensitivity, we detected the expression of PaCSCs&#x2019; markers. The results showed that Rosiglitazone could decrease, while T0070907 increase both the mRNA and protein expression levels of CD44 and CD133 in HPAC and SW1990 cells (<xref ref-type="fig" rid="F7">Figure&#x20;7A</xref>). To confirm whether the effect of PPAR&#x3b3; on the expression of PaCSCs markers was dependent on ATG4D-mediated mitophagy, we detected the markers of PaCSCs expression after inhibiting the expression of ATG4D in HPAC and SW1990 cells. We found that the mRNA and protein expression levels of CD44 and CD133 decreased after downregulating ATG4D by siRNA in HPAC and SW1990 cells (<xref ref-type="fig" rid="F7">Figures 7B,C</xref>). Sphere formation assay is a key method to reveal the self-renew and differentiation ability of PaCSCs. Thus, we measured self-renewal capacity and observed significantly increased sphere-forming capacity for HPAC cells treated with T0070907 compared to Rosiglitazone and negative control cells (<xref ref-type="fig" rid="F7">Figure&#x20;7D</xref>).</p>
<fig id="F7" position="float">
<label>FIGURE 7</label>
<caption>
<p>PPAR&#x3b3; inhibits the capacity of pancreatic cancer cell stemness and induces the sensitivity of Gemcitabine treatment. <bold>(A)</bold> Treating HPAC or SW1990 cells with negative control, Rosiglitazone (10&#xa0;&#x3bc;M) or T0070907 (5&#xa0;&#x3bc;M) for 72&#xa0;h affected the CD44 and CD133 mRNA expression levels. <bold>(B)</bold> Knockdown of ATG4D by siRNA in HPAC and SW1990 cells decreased the CD44 and CD133 mRNA expression levels. <bold>(C)</bold> PPAR&#x3b3; activation or inhibition, or ATG4D downregulation affected CD44 and CD133 protein expression levels. <bold>(D)</bold> Rosiglitazone could promote, while T0070907 could inhibit the sphere-formation capacity of HPAC cells compared to negative control groups. <bold>(E)</bold> Combination of Rosiglitazone and Gemcitabine in HPAC and SW1990 cells significantly inhibited tumor cell viability compared with Gemcitabine alone. Experiments were repeated at least three times, with statistical analyses being reported appropriate.</p>
</caption>
<graphic xlink:href="fcell-09-745554-g007.tif"/>
</fig>
<p>Additionally, we detected the cell viability after treating HPAC or SW1990 cells with Gemcitabine combined with Rosiglitazone or not. The results indicated that combination of Rosiglitazone and Gemcitabine in HPAC and SW1990 cells significantly inhibited tumor cell viability compared to Gemcitabine alone (<xref ref-type="fig" rid="F7">Figure&#x20;7E</xref>). These results gave us some hints that the pro-proliferation role of PPAR&#x3b3; in PDAC might improve the chemotherapy sensitivity of PDAC <italic>via</italic> inhibiting mitophagy-regulated cancer stemness.</p>
</sec>
</sec>
<sec sec-type="discussion" id="s4">
<title>Discussion</title>
<p>PPAR&#x3b3; has been implicated in the carcinogenesis and progression of various solid tumors. Emerging evidence has shown that PPAR&#x3b3; plays an oncogenic role <italic>via</italic> mitochondrial anti-oxidative function (<xref ref-type="bibr" rid="B9">Cao et&#x20;al., 2018</xref>; <xref ref-type="bibr" rid="B43">Zou et&#x20;al., 2019</xref>). The inhibition of mitochondrial anti-oxidative function produces ROS, damaging the electron transport chain. Then, decreased MMP leads to the collapse of mitochondrial structure and function to induce cell apoptosis (<xref ref-type="bibr" rid="B1">Abdel Hadi et&#x20;al., 2021</xref>). The mitochondrial anti-oxidative function of PPAR&#x3b3; on PDAC progression remains unclear. Our study found that high expression of nuclear PPAR&#x3b3; in pancreatic cancer tissues was correlated positively with tumor size and predicted poor prognosis in patients. <italic>In vitro</italic> and <italic>in vivo</italic> studies further confirmed that PPAR&#x3b3; activation could promote cell proliferation <italic>via</italic> stabilizing the MMP and inducing the mitochondrial redox capability to inhibit mitochondrial ROS-dependent cell apoptosis. SOD2, the primary mitochondrial oxidative scavenger, over-expresses in a variety of tumors including PDAC. It could influence the malignant behaviors of tumors <italic>via</italic> exerting mitochondrial anti-oxidative function (<xref ref-type="bibr" rid="B14">Hart et&#x20;al., 2015</xref>; <xref ref-type="bibr" rid="B24">Li et&#x20;al., 2015</xref>). In our study, SOD2 expression could be directly regulated by the transcription factor PPAR&#x3b3;, inhibiting mitochondrial ROS-dependent cell apoptosis. These results indicated that PPAR&#x3b3;/SOD2 pathway could protect against mitochondrial ROS-dependent apoptosis to promote the proliferation of pancreatic cancer&#x20;cells.</p>
<p>Mitophagy plays an important role in the quality and function of mitochondria, influencing tumor cell metabolism, oxidative stress and biosynthesis process (<xref ref-type="bibr" rid="B22">Kubli and Gustafsson, 2012</xref>). mTOR pathway, the metabolism-related classical pathway, participating in autophagic process, regulates termination of autophagy and reformation of lysosomes (<xref ref-type="bibr" rid="B40">Yu et&#x20;al., 2010</xref>). ULK1 modulates the initiation of autophagy by recruiting autophagy-related genes (ATGs), and the phosphorylation of ULK1 would be activated by mTOR blockade, thereby inducing the autophagy occurrence (<xref ref-type="bibr" rid="B18">Kim et&#x20;al., 2011</xref>). Consistently, our study suggested that the inhibition of PPAR&#x3b3;, followed by SOD2 blockade, could activate mitophagy process <italic>via</italic> the mTOR/ULK1 signaling pathway. Notably, autophagic degradation and the removal of damaged mitochondria might aid cellular response to mitochondrial oxidative stress (<xref ref-type="bibr" rid="B2">Ashrafi and Schwarz, 2013</xref>). Mitochondrial ROS under oxidative stress could be regulated by mitophagy contributing to cell apoptosis (<xref ref-type="bibr" rid="B10">Chen et&#x20;al., 2012</xref>; <xref ref-type="bibr" rid="B41">Zhou et&#x20;al., 2019</xref>). The molecular mechanism between mitophagy regulated by PPAR&#x3b3;/SOD2 and apoptosis remains unknown. ATG4D, one of the ATG4 family members, plays an important regulatory role during the formation of autophagosome. The sequence of ATG4D contains mitochondrial targeting sequences (MTS) and is located on the downstream of the caspase cleavage site. The ATG4D fragment (&#x394;N63 Atg4D) could be cleaved by the caspase, then translocated to the damaged mitochondrial (<xref ref-type="bibr" rid="B4">Betin et&#x20;al., 2012</xref>). The fragment expresses BH3 domain, inducing interaction with BCL-2 family members to induce apoptosis (<xref ref-type="bibr" rid="B3">Betin and Lane, 2009</xref>). In our study, we found that PPAR&#x3b3;/SOD2 pathway activation could downregulate the expression of ATG4D. Furthermore, after the&#x20;blockade of PPAR&#x3b3;/SOD2 pathway, ATG4D was located in mitochondria to activate the process of mitophagy, contributing to the increase of mitochondrial ROS production, damage of MMP stabilization and promotion of cancer cell apoptosis. Therefore, we conclude that PPAR&#x3b3;/SOD2 could protect against mitochondrial ROS-dependent apoptosis <italic>via</italic> inhibiting ATG4D-mediated mitophagy to promote PDAC proliferation.</p>
<p>Notably, mitophagy could be used by PaCSCs for particular adaptation of metabolic stress, as a major limitation of anti-cancer treatments, contributing to chemotherapy resistance (<xref ref-type="bibr" rid="B12">Ferro et&#x20;al., 2020</xref>). PaCSCs present more intracellular active mitophagic flux than non-stem cancer cells (<xref ref-type="bibr" rid="B39">Valle et&#x20;al., 2020</xref>). Previous studies found that PPAR&#x3b3; activation could induce the differentiation of cancer stem cells to mature cancer cells, promoting the proliferation capability of breast cancer (<xref ref-type="bibr" rid="B27">Moon et&#x20;al., 2014</xref>; <xref ref-type="bibr" rid="B31">Papi et&#x20;al., 2014</xref>). Rosiglitazone combined with gemcitabine reduces immune suppression and modulates T&#x20;cell function in pancreatic cancer (<xref ref-type="bibr" rid="B7">Bunt et&#x20;al., 2013</xref>). In our study, we found that PPAR&#x3b3; might diminish the PDAC stemness <italic>via</italic> decreasing the expression level of CD44 and CD133 in pancreatic cancer cells, enhancing the killing effect of Gemcitabine on pancreatic cancer cells. The weakened ATG4D-mediated mitophagy dominated by PPAR&#x3b3; might play a role in the&#x20;process of inhibiting pancreatic cancer cell stemness. Thus, PPAR&#x3b3; agonists combined with mitophagy inhibitors probably promote PDAC proliferation to improve chemosensitivity, defining a novel therapeutic target of PDAC. In our future study, we will explore the mechanisms of specific interaction between PPAR&#x3b3;, mitophagy and pancreatic cancer stemness.</p>
</sec>
</body>
<back>
<sec id="s5">
<title>Data Availability Statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="sec" rid="s11">Supplementary Material</xref>, further inquiries can be directed to the corresponding authors.</p>
</sec>
<sec id="s6">
<title>Ethics Statement</title>
<p>The studies involving human participants were reviewed and approved by the Ethical Committee of Medical Research, the Affiliated Drum Tower Hospital of Nanjing University Medical School. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.</p>
</sec>
<sec id="s7">
<title>Author Contributions</title>
<p>Study concept and design: SS, XZ, BK, and SN. Acquisition of data: SN, MS, CP, XD, and SZ. Analysis and interpretation of data: SN, MS, HL, XQ, and ZS. Drafting and editing of the manuscript: SN. Critical revision of the manuscript: YL, LW, and BK. Administrative and material support, critical revision of the manuscript: SS and XZ. All authors gave final approval of the version to be published, and agree to be accountable for all aspects of the&#x20;work.</p>
</sec>
<sec id="s8">
<title>Funding</title>
<p>This work was supported by the National Natural Science Foundation of China (81871947, 82072651, 82072652), the Sino-German mobility Programme (M-0251), China Postdoctoral Science Foundation (2018M642224), Jiangsu Planned Projects for Postdoctoral Research Funds (2018K076B), Nanjing Medical Science and technique Development Foundation (QRX17117).</p>
</sec>
<sec sec-type="COI-statement" id="s9">
<title>Conflict of Interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec sec-type="disclaimer" id="s10">
<title>Publisher&#x2019;s Note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s11">
<title>Supplementary Material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fcell.2021.745554/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fcell.2021.745554/full&#x23;supplementary-material</ext-link>
</p>
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