PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2
Identifieur interne : 000745 ( Pmc/Corpus ); précédent : 000744; suivant : 000746PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2
Auteurs : Xiao Liu ; Chengyuan Li ; Yunfeng Fu ; Jing LiuSource :
- OncoTargets and therapy [ 1178-6930 ] ; 2020.
Abstract
Treatment of bortezomib (BTZ) improves the clinical outcomes of patients with multiple myeloma (MM). However, primary resistance and acquired resistance to BTZ frequently develop in patients with MM. PH domain leucine-rich repeat protein phosphatase (PHLPP) plays an important role in chemoresistance in a number of cancers. However, the role of PHLPP on MM remains unclear. In this study, we investigated the role of PHLPP in BTZ-resistant MM cells.
BrdU assays, immunoprecipitation, flow cytometry analyses, and immunofluorescence assays were performed.
PHLPP and lysosome-associated membrane protein 2 (LAMP2) levels were downregulated in BTZ-resistant MM cells compared with BTZ-sensitive MM cells, accompanied by inactivation of autophagy pathway evaluated by a reduction in Beclin1, Atg5 and LC3B and increase in p62. Gain- and loss-of-function experiments revealed that PHLPP partially re-sensitized MM cells to BTZ. In addition, PHLPP overexpression increased whereas PHLPP knockdown reduced LAMP2 expression, subsequently regulating the autophagy pathway in MM cells. Further findings demonstrated that LAMP2 knockdown reversed PHLPP-mediated cell apoptosis and autophagy activation in MM cells.
This study demonstrated that PHLPP is a potential strategy for overcoming BTZ resistance in patients with MM.
Url:
DOI: 10.2147/OTT.S237343
PubMed: 32021285
PubMed Central: 6969690
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2</title><author><name sortKey="Liu, Xiao" sort="Liu, Xiao" uniqKey="Liu X" first="Xiao" last="Liu">Xiao Liu</name><affiliation><nlm:aff id="AFF0001"><institution>Department of Hematology, The Third Xiangya Hospital of Central South University</institution>,<addr-line>Changsha</addr-line><addr-line>410013</addr-line>,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Li, Chengyuan" sort="Li, Chengyuan" uniqKey="Li C" first="Chengyuan" last="Li">Chengyuan Li</name><affiliation><nlm:aff id="AFF0001"><institution>Department of Hematology, The Third Xiangya Hospital of Central South University</institution>,<addr-line>Changsha</addr-line><addr-line>410013</addr-line>,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Fu, Yunfeng" sort="Fu, Yunfeng" uniqKey="Fu Y" first="Yunfeng" last="Fu">Yunfeng Fu</name><affiliation><nlm:aff id="AFF0001"><institution>Department of Hematology, The Third Xiangya Hospital of Central South University</institution>,<addr-line>Changsha</addr-line><addr-line>410013</addr-line>,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Jing" sort="Liu, Jing" uniqKey="Liu J" first="Jing" last="Liu">Jing Liu</name><affiliation><nlm:aff id="AFF0001"><institution>Department of Hematology, The Third Xiangya Hospital of Central South University</institution>,<addr-line>Changsha</addr-line><addr-line>410013</addr-line>,<country>People’s Republic of China</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">32021285</idno><idno type="pmc">6969690</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969690</idno><idno type="RBID">PMC:6969690</idno><idno type="doi">10.2147/OTT.S237343</idno><date when="2020">2020</date><idno type="wicri:Area/Pmc/Corpus">000745</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000745</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2</title><author><name sortKey="Liu, Xiao" sort="Liu, Xiao" uniqKey="Liu X" first="Xiao" last="Liu">Xiao Liu</name><affiliation><nlm:aff id="AFF0001"><institution>Department of Hematology, The Third Xiangya Hospital of Central South University</institution>,<addr-line>Changsha</addr-line><addr-line>410013</addr-line>,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Li, Chengyuan" sort="Li, Chengyuan" uniqKey="Li C" first="Chengyuan" last="Li">Chengyuan Li</name><affiliation><nlm:aff id="AFF0001"><institution>Department of Hematology, The Third Xiangya Hospital of Central South University</institution>,<addr-line>Changsha</addr-line><addr-line>410013</addr-line>,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Fu, Yunfeng" sort="Fu, Yunfeng" uniqKey="Fu Y" first="Yunfeng" last="Fu">Yunfeng Fu</name><affiliation><nlm:aff id="AFF0001"><institution>Department of Hematology, The Third Xiangya Hospital of Central South University</institution>,<addr-line>Changsha</addr-line><addr-line>410013</addr-line>,<country>People’s Republic of China</country></nlm:aff></affiliation></author><author><name sortKey="Liu, Jing" sort="Liu, Jing" uniqKey="Liu J" first="Jing" last="Liu">Jing Liu</name><affiliation><nlm:aff id="AFF0001"><institution>Department of Hematology, The Third Xiangya Hospital of Central South University</institution>,<addr-line>Changsha</addr-line><addr-line>410013</addr-line>,<country>People’s Republic of China</country></nlm:aff></affiliation></author></analytic><series><title level="j">OncoTargets and therapy</title><idno type="eISSN">1178-6930</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec id="S2001" sec-type="intro"><title>Introduction</title><p>Treatment of bortezomib (BTZ) improves the clinical outcomes of patients with multiple myeloma (MM). However, primary resistance and acquired resistance to BTZ frequently develop in patients with MM. PH domain leucine-rich repeat protein phosphatase (PHLPP) plays an important role in chemoresistance in a number of cancers. However, the role of PHLPP on MM remains unclear. In this study, we investigated the role of PHLPP in BTZ-resistant MM cells.</p></sec><sec id="S2002"><title>Methods</title><p>BrdU assays, immunoprecipitation, flow cytometry analyses, and immunofluorescence assays were performed.</p></sec><sec id="S2003"><title>Results</title><p>PHLPP and lysosome-associated membrane protein 2 (LAMP2) levels were downregulated in BTZ-resistant MM cells compared with BTZ-sensitive MM cells, accompanied by inactivation of autophagy pathway evaluated by a reduction in Beclin1, Atg5 and LC3B and increase in p62. Gain- and loss-of-function experiments revealed that PHLPP partially re-sensitized MM cells to BTZ. In addition, PHLPP overexpression increased whereas PHLPP knockdown reduced LAMP2 expression, subsequently regulating the autophagy pathway in MM cells. Further findings demonstrated that LAMP2 knockdown reversed PHLPP-mediated cell apoptosis and autophagy activation in MM cells.</p></sec><sec id="S2004"><title>Conclusion</title><p>This study demonstrated that PHLPP is a potential strategy for overcoming BTZ resistance in patients with MM.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Onco Targets Ther</journal-id><journal-id journal-id-type="iso-abbrev">Onco Targets Ther</journal-id><journal-id journal-id-type="publisher-id">OTT</journal-id><journal-id journal-id-type="pmc">ott</journal-id><journal-title-group><journal-title>OncoTargets and therapy</journal-title></journal-title-group><issn pub-type="epub">1178-6930</issn><publisher><publisher-name>Dove</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">32021285</article-id><article-id pub-id-type="pmc">6969690</article-id><article-id pub-id-type="publisher-id">237343</article-id><article-id pub-id-type="doi">10.2147/OTT.S237343</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Research</subject></subj-group></article-categories><title-group><article-title>PHLPP Sensitizes Multiple Myeloma Cells to Bortezomib Through Regulating LAMP2</article-title><alt-title alt-title-type="running-authors">Liu et al</alt-title><alt-title alt-title-type="running-title">Liu et al</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Xiao</given-names></name><xref ref-type="aff" rid="AFF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Chengyuan</given-names></name><xref ref-type="aff" rid="AFF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Fu</surname><given-names>Yunfeng</given-names></name><xref ref-type="aff" rid="AFF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Jing</given-names></name><xref ref-type="corresp" rid="AN0001"></xref><xref ref-type="aff" rid="AFF0001">1</xref></contrib><aff id="AFF0001"><label>1</label><institution>Department of Hematology, The Third Xiangya Hospital of Central South University</institution>,<addr-line>Changsha</addr-line><addr-line>410013</addr-line>,<country>People’s Republic of China</country></aff></contrib-group><author-notes><corresp id="AN0001">Correspondence: Jing Liu; Yunfeng Fu <institution>The Third Xiangya Hospital of Central South University</institution>, <addr-line>Tongzipo Road 138</addr-line>, <addr-line>Changsha</addr-line><addr-line>410013</addr-line>, <country>People’s Republic of China</country><phone>Tel/Fax + 86-073188618511</phone> Email liujing3571@163.com; fuyunfeng@csu.edu.cn</corresp></author-notes><pub-date pub-type="epub"><day>14</day><month>1</month><year>2020</year></pub-date><pub-date pub-type="collection"><year>2020</year></pub-date><volume>13</volume><fpage>401</fpage><lpage>411</lpage><history><date date-type="received"><day>05</day><month>11</month><year>2019</year></date><date date-type="accepted"><day>24</day><month>12</month><year>2019</year></date></history><permissions><copyright-statement>© 2020 Liu et al.</copyright-statement><copyright-year>2020</copyright-year><copyright-holder>Liu et al.</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/"><license-p>This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at <ext-link ext-link-type="uri" xlink:href="https://www.dovepress.com/terms.php">https://www.dovepress.com/terms.php</ext-link> and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc/3.0/">http://creativecommons.org/licenses/by-nc/3.0/</ext-link>). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (<ext-link ext-link-type="uri" xlink:href="https://www.dovepress.com/terms.php">https://www.dovepress.com/terms.php</ext-link>).</license-p></license></permissions><abstract><sec id="S2001" sec-type="intro"><title>Introduction</title><p>Treatment of bortezomib (BTZ) improves the clinical outcomes of patients with multiple myeloma (MM). However, primary resistance and acquired resistance to BTZ frequently develop in patients with MM. PH domain leucine-rich repeat protein phosphatase (PHLPP) plays an important role in chemoresistance in a number of cancers. However, the role of PHLPP on MM remains unclear. In this study, we investigated the role of PHLPP in BTZ-resistant MM cells.</p></sec><sec id="S2002"><title>Methods</title><p>BrdU assays, immunoprecipitation, flow cytometry analyses, and immunofluorescence assays were performed.</p></sec><sec id="S2003"><title>Results</title><p>PHLPP and lysosome-associated membrane protein 2 (LAMP2) levels were downregulated in BTZ-resistant MM cells compared with BTZ-sensitive MM cells, accompanied by inactivation of autophagy pathway evaluated by a reduction in Beclin1, Atg5 and LC3B and increase in p62. Gain- and loss-of-function experiments revealed that PHLPP partially re-sensitized MM cells to BTZ. In addition, PHLPP overexpression increased whereas PHLPP knockdown reduced LAMP2 expression, subsequently regulating the autophagy pathway in MM cells. Further findings demonstrated that LAMP2 knockdown reversed PHLPP-mediated cell apoptosis and autophagy activation in MM cells.</p></sec><sec id="S2004"><title>Conclusion</title><p>This study demonstrated that PHLPP is a potential strategy for overcoming BTZ resistance in patients with MM.</p></sec></abstract><kwd-group kwd-group-type="author"><title>Keywords</title><kwd>multiple myeloma</kwd><kwd>PHLPP</kwd><kwd>bortezomib</kwd><kwd>resistance</kwd><kwd>chaperone-mediated autophagy</kwd><kwd>LAMP2</kwd></kwd-group><funding-group><funding-statement>This work was supported by the National Natural Science Foundation of China (No. 81670203) and Natural Science Foundation of Hunan Provincial China (No. 2018JJ3776 and No. 2017JJ3463).</funding-statement></funding-group><counts><fig-count count="5"></fig-count><ref-count count="29"></ref-count><page-count count="11"></page-count></counts></article-meta></front><body><sec sec-type="intro" id="S0001"><title>Introduction</title><p>Multiple myeloma (MM) is a common hematologic malignancy of plasma cells (accounting for ~1% of hematologic malignancy).<xref rid="CIT0001" ref-type="bibr">1</xref> Recently, treatments with immunomodulatory drugs, monoclonal antibodies, and second-generation proteasome inhibitors, such as bortezomib (BTZ), have increased recurrence-free survival and overall survival rates for patients with MM.<xref rid="CIT0002" ref-type="bibr">2</xref>,<xref rid="CIT0003" ref-type="bibr">3</xref> However, most patients who have primary or acquired resistance to BTZ will suffer a relapse.<xref rid="CIT0004" ref-type="bibr">4</xref></p><p>PH domain leucine-rich repeat protein phosphatase (PHLPP) is a Ser/Thr protein phosphatase that plays a critical role in maintaining cellular homeostasis by directly regulating Akt, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK).<xref rid="CIT0005" ref-type="bibr">5</xref> Loss of PHLPP expression is associated with tumor progression, and increasing evidence indicates that PHLPP is a tumor suppressor and a potential therapeutic target.<xref rid="CIT0006" ref-type="bibr">6</xref> Patients with lung adenocarcinoma that have low PHLPP levels have poorer survival rates than those with high PHLPP.<xref rid="CIT0007" ref-type="bibr">7</xref> In addition, hypoxia-induced chemoresistance is associated with the downregulation of PHLPP in colon cancer cells,<xref rid="CIT0008" ref-type="bibr">8</xref> suggesting that PHLPP plays an important role in chemoresistance. However, the role of PHLPP on MM remains unclear.</p><p>Lysosomal associated membrane proteins (LAMPs) family is characterized by an evolutionary-conserved membrane-proximal LAMP domain and composed of five known members: LAMP1, LAMP2, LAMP3, LAMP4 and LAMP5.<xref rid="CIT0009" ref-type="bibr">9</xref> LAMP2 has been well documented in a variety of cellular processes including autophagy, specifically chaperone-mediated autophagy (CMA), a process that targets specific proteins to degradation by lysosomes.<xref rid="CIT0010" ref-type="bibr">10</xref> LAMP2 can affect the lysosomes localization and the autophagic flux, and LAMP2 deficiency was responsible for Azacytidine resistance in MDS/AML cells.<xref rid="CIT0011" ref-type="bibr">11</xref> However, the expression and roles of LAMP2 in BTZ-resistant MM cells are unknown.</p><p>In this study, we found that PHLPP levels were decreased in bortezomib-resistant cells and bone marrow samples of patients with MM, and overexpression of PHLPP partially sensitized MM cells to BTZ by increasing LAMP2. Our study indicates that the rescue of PHLPP is a potential strategy for the management of acquired BTZ resistance in multiple myeloma.</p></sec><sec id="S0002"><title>Materials and Methods</title><sec id="S0002-S2001"><title>Clinical Samples Collection</title><p>Three newly diagnosed with MM and three BTZ-resistant MM patients were enrolled in this study. This study was approved by the Ethics Committee of the Third Xiangya Hospital of Central South University. Written informed consent was obtained from each subject in accordance with the Declaration of Helsinki. Bone marrow tissues were obtained from these patients. Cell flow cytometry was used to sort CD138<sup>+</sup> cells.</p></sec><sec id="S0002-S2002"><title>Cell Culture</title><p>The MM cell line U266 was purchased from Cell Bank (Chinese Academy of Sciences, Beijing, China). Cells were grown in Roswell Park Memorial Institute (RPMI)-1640 medium (GlutaMAX™-I, cat no. 72400120, Gibco). 10% fetal bovine serum (Gibco, CA, USA) was added in the medium. Cells were cultured under the condition in a 37°C humidified atmosphere of 5% CO<sub>2</sub>.</p></sec><sec id="S0002-S2003"><title>Generation of BTZ-Resistant U266 Cells</title><p>The bortezomib-resistant U266 cells (U266-R) were established as follows: U266 cells were treated with the initial concentration of bortezomib (2 nM). The medium was refreshed once every 2 days. After cultured for 2 weeks under the same concentration, the dose was increased for two folds, and continuously maintained for 2 weeks. The cycles were repeated 10 times, so that the U266-R cells can be resistant to bortezomib up to 20 nM. The cells were kept under 10 nM bortezomib pressure before experimental usage.</p></sec><sec id="S0002-S2004"><title>Cell Transfection and Treatment</title><p>Lentivirus mediated PHLPP (shRNA) and LAMP2 (shRNA) downregulation, and PHLPP and LAMP2 overexpression were obtained from Genefullen (Guangzhou, China). The lentivirus-containing scramble sequences were used as control. Lentivirus infection was performed according to the manufacture’s protocol. The cells were then co-treated with rapamycin (RAP) or hydroxychloroquine (HCQ), or BTZ (5 nM or 40 nM) for 48 hrs and then used for further analysis.</p></sec><sec id="S0002-S2005"><title>BrdU Assay</title><p>BrdU Cell Proliferation ELISA kit (Abcam, Shanghai, China) was used in this experiment. After indicated treatment, the cells were added BrdU and incubate for 12 hrs to incorporate BrdU into their DNA. The cells then were fixed with Fixing Solution and incubate at room temperature. Add the TMB Solution to each well for 5 mins and then add the Stop Solution to stop the reaction. The color development was recorded immediately.</p></sec><sec id="S0002-S2006"><title>Apoptosis Analysis</title><p>MEBCYTO Apoptosis Kit (MBL Beijing Biotech Co., Ltd, Beijing, China) was used in this experiment. After treatment, cells were collected and then resuspended in binding buffer. Annexin V-FITC (5 μL) and Propidium Iodide (5 μL) were added and mixed well. The cells were incubated with the mixed solution for 15 mins. Finally, flow cytometric analysis was performed in Attune NxT (Thermofisher, Shanghai, China).</p></sec><sec id="S0002-S2007"><title>Western Blot</title><p>Protein was extracted using RIPA (Beyotime, Hangzhou, China) and their concentrations were determined by Enhanced BCA Protein Assay Kit (Beyotime, Hangzhou, China). Sixty microgram proteins were separated in 10% SDS/PAG and transferred on PVDF membrane. The membranes were incubated with primary antibody: anti-LAMP2 antibody (1:1000, cat no. 49067, Cell Signaling Technology), anti-LAMP1 antibody (1:1000, cat no. ab13523, Abcam), anti-LAMP2A antibody (1:1000, cat no. ab125068, Abcam), anti-LAMP2B antibody (1:1000, cat no. ab18529, Abcam), anti-AKT antibody (1:1000, cat no. ab18785, Abcam), anti-phospho-Akt (Ser473) antibody (1:1000, cat no. 4058, Cell Signaling Technology), anti-PHLPP antibody (1:1000, cat no. ab62830, Abcam); anti-Beclin1 antibody (1:1000, cat no. ab210498, Abcam); anti-Atg5 antibody (1:1000, cat no. ab228668, Abcam); anti-p62 antibody (1:1000, cat no. ab56416, Abcam); anti-LC3B antibody (1:1000, cat no. ab51520, Abcam). GAPDH (1:1000, cat no. ab181602, Abcam) was used as control. After being washed, the membranes were reacted with peroxidase-conjugated secondary antibody. The signals were visualized by enhanced chemiluminescence reagents (GE, Fairfield, CT, USA).</p><p>Immunoprecipitation was performed to determine the interaction between PHLPP and LAMP2. Ice-cold cell lysis buffer (500 μL) was added to each plate and incubate for 5 mins. The cells were collected to microcentrifuge tubes. The samples were sonicated on ice three times for 5 s each and micro-centrifuged for 10 mins at 14000 g, 4°C. 200 μL cell lysate was incubated with PHLPP or LAMP2 primary antibodies with rocking overnight at 4°C. Protein A agarose beads (Beyotime, Hangzhou, China) were added and incubated with for 3 hrs at 4°C. After microcentrifuge, the pellets were washed with cell lysis buffer and were resuspended with SDS sample buffer and heated to 98°C for 5 mins. 30 μL of the sample was loaded on SDS-PAGE gel (12%) for Western blotting analysis.</p></sec><sec id="S0002-S2008"><title>Immunofluorescence Assay</title><p>After blocked with 5% BSA, the cells were incubated with primary antibodies (anti-LAMP2 antibody, 1:200 and anti-PHLPP, 1:500) at 4°C overnight. The cells were incubated with appropriate secondary antibodies at 37°C for 1 hr. The cells were stained with 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (1:1000, Beyotime, Hangzhou, China) for 5 mins and mounted on slides. Immunofluorescence images were captured by a fluorescence microscope (BX51, Olympus, Japan).</p></sec><sec id="S0002-S2009"><title>Lysotracker-Red Staining</title><p>Lysosome was stained with Lysotracker-Red DND-99 (Molecular Probes, Eugene, USA). After infected with PHLPP lentivirus (labelled with EGFP), U266 cells were incubated in the presence of 50 nM Lysotracker-Red for 30 mins at 37°C. Immunofluorescence images were captured by a fluorescence microscope (BX51, Olympus, Japan).</p></sec><sec id="S0002-S2010"><title>Animal Experiment</title><p>Animal experiments were approved by the Ethics Committee for Animal Research of the Third Xiangya Hospital of Central South University following the National Research Council’s Guide for the Care and Use of Laboratory Animals (Eight Edition). The male, 2-month-old nude mice were purchased from Animal Center of Central South University. U266 were transfected with PHLPP or negative control for 48 hrs. Transfected U266 cells (1 × 10<sup>6</sup>) were then subcutaneously injected into nude mice (N = 5/each group). Tumor volume was calculated as 0.5 × L × W,<sup>2</sup> where L and W are long and short diameters of the tumor, respectively.</p></sec><sec id="S0002-S2011"><title>Statistical Analysis</title><p>GraphPad Prism software (GraphPad Software Inc., La Jolla, CA) was used for statistical analyses. Student’s <italic>t</italic>-test for two-group and one-way ANOVA with post hoc Bonferroni test for three or more groups were used to assess the statistical significance. P < 0.05 was considered statistically significant.</p></sec></sec><sec id="S0003"><title>Results</title><sec id="S0003-S2001"><title>PHLPP and LAMP2 Levels are Increased in BTZ-Resistant MM Cells</title><p>To investigate the role of PHLPP and LAMP2 in BTZ-resistant MM, we measured their expression levels in plasma cell from patients with MM and BTZ-resistant MM cell lines. We found that LAMP2 and PHLPP expression levels in BTZ-resistant plasma cells of human samples were downregulated compared with BTZ-sensitive samples (<xref rid="F0001" ref-type="fig">Figure 1A</xref>). Their expressions were also lower in BTZ-resistant U266 cells compared with parental U266 cells (<xref rid="F0001" ref-type="fig">Figure 1B</xref>). However, the expression of another member of LAMP family, LAMP1 was comparable between BTZ-sensitive and BTZ-resistant patients with MM. The expression of LAMP2 isoform LAMP2A, not LAMP2B, was significantly downregulated in BTZ-resistant patients with MM compared the BTZ-sensitive patients with MM (<underline><ext-link ext-link-type="uri" xlink:href="https://www.dovepress.com/get_supplementary_file.php?f=237343.pdf">supplementary Figure 1A</ext-link></underline>). In addition, downregulation of PHLPP and LAMP2 was accompanied by inactivation of autophagy, as determined by decreased Beclin1 and Atg5 levels, and ratio of LC3B-II/LC3B-I, and increased p62 expression in plasma cells from BTZ-resistant MM and U266-R cells (<xref rid="F0001" ref-type="fig">Figure 1</xref>). These results suggest that PHLPP downregulation is associated with LAMP2 and chaperone-mediated autophagy and involved in BTZ resistance.<fig id="F0001" fig-type="figure" orientation="portrait" position="float"><label>Figure 1</label><caption><p>The expression of PHLPP in bortezomib-resistant MM cells. (<bold>A</bold>) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in bone marrow samples from patients with MM (upper), and quantification of the bands (lower). (<bold>B</bold>) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in U266 cells and bortezomib-resistant U266 cells (upper), and quantification of the bands (lower). MM-S, BTZ-sensitive MM; MM-R, BTZ-resistant MM; U266-R, BTZ-resistant U266 cells. *<italic>P</italic> < 0.05.</p></caption><graphic content-type="print-only" xlink:href="OTT-13-401-g0001"></graphic></fig></p></sec><sec id="S0003-S2002"><title>PHLPP Sensitizes MM Cells to BTZ</title><p>PHLPP was knocked-down in U266 cells and was overexpressed in U266-R cells (<xref rid="F0002" ref-type="fig">Figure 2A</xref>). PHLPP knockdown significantly promoted U266 cell proliferation, and inhibited cell apoptosis following BTZ treatment (<xref rid="F0002" ref-type="fig">Figure 2B</xref> and C). However, PHLPP overexpression significantly inhibited U266-R cell proliferation, and induced cell apoptosis following BTZ treatment (<xref rid="F0002" ref-type="fig">Figure 2B</xref> and C). These results suggest that PHLPP sensitizes MM cells to BTZ treatment.<fig id="F0002" fig-type="figure" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Overexpression of PHLPP sensitizes MM cells to BTZ. (<bold>A</bold>) Western blot analyses of PHLPP expression in U266 cells and BTZ-resistant U266 cells after lentivirus infection. (<bold>B</bold>) BrdU assays were used to determine cell viability after sh-PHLPP or PHLPP lentivirus infection in U266 and U266-R cells, respectively. (<bold>C</bold>) Flow cytometry was used to determine apoptosis after knockdown or overexpression of PHLPP under BTZ treatment. (<bold>D</bold>) U266 cells were infected with PHLPP lentivirus and were then injected into nude mice. Tumor volumes were measured weekly. (<bold>E</bold>) PHLPP and LAMP2 expression in tumor sections were evaluated using immunohistochemistry (IHC); Magnification, 100X; *<italic>P</italic> < 0.05.</p></caption><graphic content-type="print-only" xlink:href="OTT-13-401-g0002"></graphic></fig></p></sec><sec id="S0003-S2003"><title>PHLPP Suppresses MM Cells Growth in vivo</title><p>Furthermore, we performed xenografted tumor experiments in nude mice using PHLPP-expressing U266 cells to examine the effects of PHLPP on tumor growth in vivo. PHLPP overexpression slowed down tumor growth in vivo (<xref rid="F0002" ref-type="fig">Figure 2D</xref>). Immunohistochemical staining showed that PHLPP and LAMP2 expression were upregulated in tumor tissues (<xref rid="F0002" ref-type="fig">Figure 2E</xref>).</p></sec><sec id="S0003-S2004"><title>PHLPP Interacts with LAMP2</title><p>Given that PHLPP expression was associated with LAMP2 expression, we investigated whether PHLPP interacts physically with LAMP2. Immunofluorescence assays showed that PHLPP and LAMP2 were co-localized in U266 cells (<xref rid="F0003" ref-type="fig">Figure 3A</xref>). Co-immunoprecipitation (co-IP) experiments further confirmed that PHLPP interacts with LAMP2 (<xref rid="F0003" ref-type="fig">Figure 3B</xref>), and they were co-expressed in the lysosome (<xref rid="F0003" ref-type="fig">Figure 3C</xref>). In addition, we found that knockdown of PHLPP decreased LAMP2 expression (<xref rid="F0003" ref-type="fig">Figure 3D</xref>). Knockdown of PHLPP also reduced Beclin1 and Atg5 levels and ratio of LC3B-II/LC3B-I, and increased p-AKT(ser473) and p62 expression, suggesting autophagy signaling inactivation in U266 cells, whereas overexpression of PHLPP increased the expression of LAMP2 and LAMP2A, but did not alter the expression of LAMP1 and LAMP2B (<underline><ext-link ext-link-type="uri" xlink:href="https://www.dovepress.com/get_supplementary_file.php?f=237343.pdf">supplementary Figure 1B</ext-link></underline>) and inhibited phosphorylation of AKT, activating autophagy signaling in U266-R cells (<xref rid="F0003" ref-type="fig">Figure 3D</xref>).<fig id="F0003" fig-type="figure" orientation="portrait" position="float"><label>Figure 3</label><caption><p>PHLPP positively regulates LAMP2 expression. (<bold>A</bold>) Immunofluorescence assays were performed to investigate the interactions between PHLPP and LAMP2 in U266 cells. (<bold>B</bold>) Immunoprecipitation confirmed the interactions between PHLPP and LAMP2 in U266 cells; (<bold>C</bold>) EGFP-PHLPP was expressed in U266 cells for 48 hrs and loaded with lysotracker-Red DND-99 for 30 mins at 37°C. Cells were fixed and analyzed by confocal microscopy. (<bold>D</bold>) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in U266 and U266-R cells after infection with sh-PHLPP or PHLPP lentivirus. (<bold>E</bold>) Quantification of the bands in (<bold>D</bold>). *<italic>P</italic> < 0.05.</p></caption><graphic content-type="print-only" xlink:href="OTT-13-401-g0003"></graphic></fig></p></sec><sec id="S0003-S2005"><title>PHLPP Partially Sensitizes MM Cells to BTZ Through LAMP2 and Autophagy</title><p>We next tested the role of LAMP2 in BTZ-induced cell apoptosis. We found that LAMP2 overexpression enhanced while LAMP2 knockdown attenuated BTZ-induced growth inhibition and cell apoptosis (<underline><ext-link ext-link-type="uri" xlink:href="https://www.dovepress.com/get_supplementary_file.php?f=237343.pdf">supplementary Figure 2</ext-link></underline>). To investigate the role of LAMP2 in PHLPP-mediated BTZ sensitization, LAMP2 was knocked down by shRNA in U266-R cells (<xref rid="F0004" ref-type="fig">Figure 4A</xref>) and overexpressed in U266 cells (<xref rid="F0004" ref-type="fig">Figure 4B</xref>). Under BTZ treatment, LAMP2 knockdown reversed PHLPP-mediated autophagy activation as determined by downregulation of Beclin1 and Atg5 levels and the ratio of LC3B-II/LC3B-I and upregulation of p-AKT(ser473) and p62 (<xref rid="F0004" ref-type="fig">Figure 4A</xref>), proliferation inhibition (<xref rid="F0004" ref-type="fig">Figure 4C</xref>), and apoptosis (<xref rid="F0004" ref-type="fig">Figure 4D</xref>) in U266-R cells. LAMP2 overexpression rescued the effects of shPHLPP treatment on autophagy (<xref rid="F0004" ref-type="fig">Figure 4B</xref>), cell proliferation (<xref rid="F0004" ref-type="fig">Figure 4E</xref>), and cell apoptosis (<xref rid="F0004" ref-type="fig">Figure 4F</xref>) in U266 cells, suggesting that LAMP2 is required for PHLPP in re-sensitizing MM cells to BTZ. Moreover, pharmacological activator of autophagy RAP treatment significantly decreased cell proliferation and enhanced cell apoptosis compared with shPHLPP transfection alone in U266 cells (<xref rid="F0005" ref-type="fig">Figure 5A</xref> and <xref rid="F0005" ref-type="fig">B</xref>); the autophagy inhibitor hydroxychloroquine (HCQ) treatment dramatically promoted cell proliferation and reduced cell apoptosis compared with PHLPP transfection alone in U266-R cells (<xref rid="F0005" ref-type="fig">Figure 5C</xref> and <xref rid="F0005" ref-type="fig">D</xref>). These results suggest that PHLPP sensitizes MM cells to BTZ through LAMP2 and the autophagy pathway.<fig id="F0004" fig-type="figure" orientation="portrait" position="float"><label>Figure 4</label><caption><p>LAMP2 knockdown reverses the inhibitory effects of PHLPP in U266-R cells. (<bold>A</bold>) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in U266-R cells infected with sh-LAMP2 or PHLPP lentivirus under 40 nM BTZ treatment (left), and quantification of the bands (right). (<bold>B</bold>) Western blot analyses of the expression of PHLPP, LAMP2, and key autophagy signaling molecules in U266 cells that were infected with LAMP2 or shPHLPP lentivirus under 5 nM BTZ treatment (left), and quantification of the bands (right). (<bold>C</bold>) Cell proliferation was evaluated using BrdU assay in U266-R cells that were infected with sh-LAMP2 or PHLPP lentivirus under 40 nM BTZ treatment. (<bold>D</bold>) Apoptosis was evaluated using flow cytometry in U266-R cells that were infected with sh-LAMP2 or PHLPP lentivirus under 40 nM BTZ treatment. (<bold>E</bold>) Cell proliferation was evaluated using BrdU assay in U266 cells that were infected with LAMP2 or shPHLPP lentivirus under 5 nM BTZ treatment. (<bold>F</bold>) Apoptosis was evaluated using flow cytometry in U266 cells that were infected with LAMP2 or shPHLPP lentivirus under 5 nM BTZ treatment. *<italic>P</italic> < 0.05.</p></caption><graphic content-type="print-only" xlink:href="OTT-13-401-g0004"></graphic></fig><fig id="F0005" fig-type="figure" orientation="portrait" position="float"><label>Figure 5</label><caption><p>The effects of pharmacological inhibitor of autophagy on PHLPP-mediated cell apoptosis. (<bold>A</bold>) Cell proliferation was evaluated using BrdU assay in U266 cells that were infected with shPHLPP lentivirus and treated with rapamycin (RAP) under 5 nM BTZ treatment. (<bold>B</bold>) Apoptosis was evaluated using flow cytometry in U266 cells that were infected with shPHLPP lentivirus and treated with rapamycin (RAP) under 5 nM BTZ treatment. (<bold>C</bold>) Cell proliferation was evaluated using BrdU assay in U266-R cells that were infected with PHLPP lentivirus and treated with hydroxychloroquine (HCQ, 25 μM) under 40 nM BTZ treatment. (<bold>D</bold>) Apoptosis was evaluated using flow cytometry in U266-R cells that were infected with PHLPP lentivirus and treated with hydroxychloroquine (HCQ, 25 μM) under 40 nM BTZ treatment. *<italic>P</italic> < 0.05.</p></caption><graphic content-type="print-only" xlink:href="OTT-13-401-g0005"></graphic></fig></p></sec></sec><sec id="S0004"><title>Discussion</title><p>In this study, we found that PHLPP and LAPM2 were downregulated in BTZ-resistant MM cells. Induced PHLPP expression partially re-sensitized MM cells to BTZ through activating the autophagy pathway.</p><p>MM is a common hematologic malignancy, constituting 1% of all malignancies. Although treatment regimens have greatly improved, MM remains incurable because of resistance to drugs such as dexamethasone and bortezomib.<xref rid="CIT0012" ref-type="bibr">12</xref> Drug resistance involves the dysregulation of various signaling pathways, including the JAK/STAT, MEK/MAPK, and PI3K/AKT.<xref rid="CIT0013" ref-type="bibr">13</xref> AKT phosphorylation activates AKT signaling, which is associated with anti-apoptotic effects of plasma cell-derived growth factor against the effects of dexamethasone.<xref rid="CIT0014" ref-type="bibr">14</xref> MM cells produce the growth factor IGF-1, and high levels of IGF1 and its receptor (IGF-1R) are associated with poor survival and BTZ resistance in patients with MM.<xref rid="CIT0015" ref-type="bibr">15</xref> The IGF-1R inhibitor OSI-906 can increase the cytotoxicity of BTZ in MM cells, and PI3K and AKT are the downstream targets of IGF1.<xref rid="CIT0016" ref-type="bibr">16</xref> AKT inhibition also causes BTZ-resistant cell death.<xref rid="CIT0017" ref-type="bibr">17</xref> Therefore, combination bortezomib and OSI-906 therapy could be a useful treatment for patients with BTZ resistance.</p><p>PHLPP negatively regulates AKT activity, and FK506-binding protein 51 functions as a scaffolding protein for Akt and PHLPP. PHLPP can promote dephosphorylation of AKT and enhances the efficacy of chemotherapy.<xref rid="CIT0018" ref-type="bibr">18</xref> PHLPP overexpression inactivates Akt, whereas PHLPP knockdown increases phosphorylation and activation of Akt.<xref rid="CIT0019" ref-type="bibr">19</xref> In addition, hypoxia-induced chemoresistance is associated with the downregulation of PHLPP in colon cancer cells.<xref rid="CIT0008" ref-type="bibr">8</xref> In this study, we found that PHLPP levels were reduced in BTZ-resistant MM cells, and induced PHLPP expression re-sensitized MM cells to BTZ. Interestingly, we found that PHLPP inhibited proliferation and promoted apoptosis in MM cells by enhancing CMA. CMA contributes to the maintenance of proteostasis through the degradation of cytosolic proteins in lysosomes.<xref rid="CIT0020" ref-type="bibr">20</xref> Cytosolic chaperones delivered CMA substrates to the lysosomal surface, and are internalized through a membrane translocation complex.<xref rid="CIT0021" ref-type="bibr">21</xref> It was previously found that PHLPP positively regulated CMA through inhibition of lysosomal Akt.<xref rid="CIT0022" ref-type="bibr">22</xref> However, in addition to AKT and autophagy pathways, PHLPP is also associated with activation of NF-κB signaling.<xref rid="CIT0023" ref-type="bibr">23</xref> Tang et al reported that PHLPP upregulation was mediated by NF-κB transcriptional activity.<xref rid="CIT0024" ref-type="bibr">24</xref> The NF-κB pathway regulates numerous genes, which influence the development and pathogenesis of multiple myeloma. IGF-1 was reported to be important for cell survival by AKT signaling pathways, which further mediate NF-κB activation in MM cells.<xref rid="CIT0025" ref-type="bibr">25</xref> Thus, there may be a negative feedback loop among NF-κB, PHLPP and Akt, which may lead to a mild effect on cell apoptosis. However, the precise mechanism needs more evidences to illustrate.</p><p>Low expression of LAMP2 was associated with poor overall survival in a cohort of 150 AML patients. Moreover, in Azacytidine (Aza)-treated MDS cell lines and cells from MDS/AML patients, a progressive reduction in LAMP2 expression was correlated with a loss of sensitivity to Aza.<xref rid="CIT0011" ref-type="bibr">11</xref> Bao et al found that the downregulation of LAMP2 in the lung cancer cells increased their resistance to Temozolomide.<xref rid="CIT0026" ref-type="bibr">26</xref> In LAMP1/LAMP2 double-knockout mice, autophagic vacuoles accumulated in many tissues suggesting impaired autophagosome maturation.<xref rid="CIT0027" ref-type="bibr">27</xref> In this study, we found that LAMP2 but not LAMP1 was downregulated in BTZ-resistant patients with MM and the expression of LAMP2A, not LAMP2B, was significantly downregulated in BTZ-resistant patients with MM compared the BTZ-sensitive patients with MM. LAMP2A was encoded by transcript variant A of Lamp2 gene, which is the longest and predominant form of this gene. These results indicated that LAMP2A is the executor in the regulation of CMA in MM cells. Re-expression of LAMP2 in LAMP2-deficient BTZ-resistant MM cells restored chaperone-mediated autophagy (CMA), highlighting the key role of LAMP2 in this process. CMA is a highly selective form of autophagy that allows the elimination of substrates endowed with a KFERQ motif through direct transport to the lysosome. Lysosomes can sensitize cancer cells to lysosomal membrane permeabilization and cell death.<xref rid="CIT0028" ref-type="bibr">28</xref>,<xref rid="CIT0029" ref-type="bibr">29</xref> In this study, we found that LAMP2 levels were reduced in BTZ-resistant MM cells, and knockdown of LAMP2 reversed PHLPP-mediated cell apoptosis in BTZ-resistant U266 cells. Therefore, LAMP2 contributes to BTZ resistance and cell apoptosis may be via CMA activation. Our results showed that PHLPP interacted with LAMP2, and LAMP2 is required for PHLPP-mediated autophagy. It would be reasonable that PHLPP acts primarily to interact with and stabilize LAMP2 on the lysosomes to promote the fusion between autophagosome and lysosome (ie, formation of autolysosome) and thereby removal of autophagosome.</p><p>In summary, our findings reveal for the first time that interaction of PHLPP and LAMP2 partially sensitizes MM cells to BTZ by enhancing autophagy. Rescue of PHLPP is a potential strategy for the management of BTZ resistance in patients with MM.</p></sec></body><back><ack><title>Acknowledgment</title><p>Authors would like to thank Dr Yanwei Luo for his help in collecting some of the data for this study.</p></ack><sec id="S0005"><title>Ethics Approval and Informed Consent</title><p>This study was approved by the Ethics Committee of The Third Xiangya Hospital, Central South University. Informed consent was obtained from each subject in accordance with the Declaration of Helsinki. Animal experiments were approved by the Ethics Committee for Animal Research of the Third Xiangya Hospital of Central South University following the National Research Council’s Guide for the Care and Use of Laboratory Animals (Eight Edition).</p></sec><sec id="S0006"><title>Author Contributions</title><p>All authors contributed to data analysis, drafting and revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.</p></sec><sec id="S0007" sec-type="COI-statement"><title>Disclosure</title><p>The authors declare that they have no conflicts of interest in this work.</p></sec><ref-list><title>References</title><ref id="CIT0001"><label>1.</label><mixed-citation publication-type="journal"><string-name><surname>Hu</surname><given-names>J</given-names></string-name>, <string-name><surname>Hu</surname><given-names>WX</given-names></string-name>. <article-title>Targeting signaling pathways in multiple myeloma: pathogenesis 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