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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">PLOD2 Is Essential to Functional Activation of Integrin β1 for Invasion/Metastasis in Head and Neck Squamous Cell Carcinomas</title><author><name sortKey="Ueki, Yushi" sort="Ueki, Yushi" uniqKey="Ueki Y" first="Yushi" last="Ueki">Yushi Ueki</name><affiliation><nlm:aff id="aff1">Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Department of Otolaryngology, Head and Neck Surgery, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Saito, Ken" sort="Saito, Ken" uniqKey="Saito K" first="Ken" last="Saito">Ken Saito</name><affiliation><nlm:aff id="aff1">Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Iioka, Hidekazu" sort="Iioka, Hidekazu" uniqKey="Iioka H" first="Hidekazu" last="Iioka">Hidekazu Iioka</name><affiliation><nlm:aff id="aff1">Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Sakamoto, Izumi" sort="Sakamoto, Izumi" uniqKey="Sakamoto I" first="Izumi" last="Sakamoto">Izumi Sakamoto</name><affiliation><nlm:aff id="aff3">ACROSCALE Inc., Kokubun-cho, Aoba-ku, Sendai, Japan</nlm:aff></affiliation></author><author><name sortKey="Kanda, Yasuhiro" sort="Kanda, Yasuhiro" uniqKey="Kanda Y" first="Yasuhiro" last="Kanda">Yasuhiro Kanda</name><affiliation><nlm:aff id="aff4">Department of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Sakaguchi, Masakiyo" sort="Sakaguchi, Masakiyo" uniqKey="Sakaguchi M" first="Masakiyo" last="Sakaguchi">Masakiyo Sakaguchi</name><affiliation><nlm:aff id="aff5">Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikata-cho, Kita-ku, Okayama, Japan</nlm:aff></affiliation></author><author><name sortKey="Horii, Arata" sort="Horii, Arata" uniqKey="Horii A" first="Arata" last="Horii">Arata Horii</name><affiliation><nlm:aff id="aff2">Department of Otolaryngology, Head and Neck Surgery, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Kondo, Eisaku" sort="Kondo, Eisaku" uniqKey="Kondo E" first="Eisaku" last="Kondo">Eisaku Kondo</name><affiliation><nlm:aff id="aff1">Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">32058962</idno><idno type="pmc">6997870</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997870</idno><idno type="RBID">PMC:6997870</idno><idno type="doi">10.1016/j.isci.2020.100850</idno><date when="2020">2020</date><idno type="wicri:Area/Pmc/Corpus">000847</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000847</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">PLOD2 Is Essential to Functional Activation of Integrin β1 for Invasion/Metastasis in Head and Neck Squamous Cell Carcinomas</title><author><name sortKey="Ueki, Yushi" sort="Ueki, Yushi" uniqKey="Ueki Y" first="Yushi" last="Ueki">Yushi Ueki</name><affiliation><nlm:aff id="aff1">Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Department of Otolaryngology, Head and Neck Surgery, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Saito, Ken" sort="Saito, Ken" uniqKey="Saito K" first="Ken" last="Saito">Ken Saito</name><affiliation><nlm:aff id="aff1">Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Iioka, Hidekazu" sort="Iioka, Hidekazu" uniqKey="Iioka H" first="Hidekazu" last="Iioka">Hidekazu Iioka</name><affiliation><nlm:aff id="aff1">Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Sakamoto, Izumi" sort="Sakamoto, Izumi" uniqKey="Sakamoto I" first="Izumi" last="Sakamoto">Izumi Sakamoto</name><affiliation><nlm:aff id="aff3">ACROSCALE Inc., Kokubun-cho, Aoba-ku, Sendai, Japan</nlm:aff></affiliation></author><author><name sortKey="Kanda, Yasuhiro" sort="Kanda, Yasuhiro" uniqKey="Kanda Y" first="Yasuhiro" last="Kanda">Yasuhiro Kanda</name><affiliation><nlm:aff id="aff4">Department of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Sakaguchi, Masakiyo" sort="Sakaguchi, Masakiyo" uniqKey="Sakaguchi M" first="Masakiyo" last="Sakaguchi">Masakiyo Sakaguchi</name><affiliation><nlm:aff id="aff5">Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikata-cho, Kita-ku, Okayama, Japan</nlm:aff></affiliation></author><author><name sortKey="Horii, Arata" sort="Horii, Arata" uniqKey="Horii A" first="Arata" last="Horii">Arata Horii</name><affiliation><nlm:aff id="aff2">Department of Otolaryngology, Head and Neck Surgery, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author><author><name sortKey="Kondo, Eisaku" sort="Kondo, Eisaku" uniqKey="Kondo E" first="Eisaku" last="Kondo">Eisaku Kondo</name><affiliation><nlm:aff id="aff1">Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</nlm:aff></affiliation></author></analytic><series><title level="j">iScience</title><idno type="eISSN">2589-0042</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Summary</title><p>Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin β1, enabling integrin β1 to initiate tumor invasion/metastasis. Integrin β1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis <italic>in vivo</italic>, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin β1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin β1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Bonner, J A" uniqKey="Bonner J">J.A. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">iScience</journal-id><journal-id journal-id-type="iso-abbrev">iScience</journal-id><journal-title-group><journal-title>iScience</journal-title></journal-title-group><issn pub-type="epub">2589-0042</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">32058962</article-id><article-id pub-id-type="pmc">6997870</article-id><article-id pub-id-type="publisher-id">S2589-0042(20)30033-X</article-id><article-id pub-id-type="doi">10.1016/j.isci.2020.100850</article-id><article-id pub-id-type="publisher-id">100850</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>PLOD2 Is Essential to Functional Activation of Integrin β1 for Invasion/Metastasis in Head and Neck Squamous Cell Carcinomas</article-title></title-group><contrib-group><contrib contrib-type="author" id="au1"><name><surname>Ueki</surname><given-names>Yushi</given-names></name><xref rid="aff1" ref-type="aff">1</xref><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author" id="au2"><name><surname>Saito</surname><given-names>Ken</given-names></name><email>kens@med.niigata-u.ac.jp</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="cor1" ref-type="corresp">∗</xref></contrib><contrib contrib-type="author" id="au3"><name><surname>Iioka</surname><given-names>Hidekazu</given-names></name><xref rid="aff1" ref-type="aff">1</xref></contrib><contrib contrib-type="author" id="au4"><name><surname>Sakamoto</surname><given-names>Izumi</given-names></name><xref rid="aff3" ref-type="aff">3</xref></contrib><contrib contrib-type="author" id="au5"><name><surname>Kanda</surname><given-names>Yasuhiro</given-names></name><xref rid="aff4" ref-type="aff">4</xref></contrib><contrib contrib-type="author" id="au6"><name><surname>Sakaguchi</surname><given-names>Masakiyo</given-names></name><xref rid="aff5" ref-type="aff">5</xref></contrib><contrib contrib-type="author" id="au7"><name><surname>Horii</surname><given-names>Arata</given-names></name><xref rid="aff2" ref-type="aff">2</xref></contrib><contrib contrib-type="author" id="au8"><name><surname>Kondo</surname><given-names>Eisaku</given-names></name><email>ekondo@med.niigata-u.ac.jp</email><xref rid="aff1" ref-type="aff">1</xref><xref rid="fn1" ref-type="fn">6</xref><xref rid="cor2" ref-type="corresp">∗∗</xref></contrib></contrib-group><aff id="aff1"><label>1</label>Division of Molecular and Cellular Pathology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</aff><aff id="aff2"><label>2</label>Department of Otolaryngology, Head and Neck Surgery, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</aff><aff id="aff3"><label>3</label>ACROSCALE Inc., Kokubun-cho, Aoba-ku, Sendai, Japan</aff><aff id="aff4"><label>4</label>Department of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori, Chuo-ku, Niigata, Japan</aff><aff id="aff5"><label>5</label>Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikata-cho, Kita-ku, Okayama, Japan</aff><author-notes><corresp id="cor1"><label>∗</label>Corresponding author <email>kens@med.niigata-u.ac.jp</email></corresp><corresp id="cor2"><label>∗∗</label>Corresponding author <email>ekondo@med.niigata-u.ac.jp</email></corresp><fn id="fn1"><label>6</label><p id="ntpara0010">Lead Contact</p></fn></author-notes><pub-date pub-type="pmc-release"><day>18</day><month>1</month><year>2020</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="collection"><day>21</day><month>2</month><year>2020</year></pub-date><pub-date pub-type="epub"><day>18</day><month>1</month><year>2020</year></pub-date><volume>23</volume><issue>2</issue><elocation-id>100850</elocation-id><history><date date-type="received"><day>25</day><month>5</month><year>2019</year></date><date date-type="rev-recd"><day>20</day><month>9</month><year>2019</year></date><date date-type="accepted"><day>14</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© 2020 The Author(s)</copyright-statement><copyright-year>2020</copyright-year><license license-type="CC BY-NC-ND" xlink:href="http://creativecommons.org/licenses/by-nc-nd/4.0/"><license-p>This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).</license-p></license></permissions><abstract id="abs0010"><title>Summary</title><p>Identifying the specific functional regulator of integrin family molecules in cancer cells is critical because they are directly involved in tumor invasion and metastasis. Here we report high expression of PLOD2 in oropharyngeal squamous cell carcinomas (SCCs) and its critical role as a stabilizer of integrin β1, enabling integrin β1 to initiate tumor invasion/metastasis. Integrin β1 stabilized by PLOD2-mediated hydroxylation was recruited to the plasma membrane, its functional site, and accelerated tumor cell motility, leading to tumor metastasis <italic>in vivo</italic>, whereas loss of PLOD2 expression abrogated it. In accordance with molecular analysis, examination of oropharyngeal SCC tissues from patients corroborated PLOD2 expression associated with integrin β1 at the invasive front of tumor nests. PLOD2 is thus implicated as the key regulator of integrin β1 that prominently regulates tumor invasion and metastasis, and it provides important clues engendering novel therapeutics for these intractable cancers.</p></abstract><abstract abstract-type="graphical" id="abs0015"><title>Graphical Abstract</title><fig id="undfig1" position="anchor"><graphic xlink:href="fx1"></graphic></fig></abstract><abstract abstract-type="author-highlights" id="abs0020"><title>Highlights</title><p><list list-type="simple" id="ulist0010"><list-item id="u0010"><label>•</label><p id="p0010">PLOD2 specifically regulates intracellular localization of integrin β1</p></list-item><list-item id="u0015"><label>•</label><p id="p0015">The hydroxylation on integrin β1 by PLOD2 is critical for its stability</p></list-item><list-item id="u0020"><label>•</label><p id="p0020">PLOD2 and integrin β1 expression colocalized at the invasive front of SCC tissues</p></list-item><list-item id="u0025"><label>•</label><p id="p0025">PLOD2 is necessary for tumor invasion/metastasis through the integrin β1 maturation</p></list-item></list></p></abstract><abstract abstract-type="teaser" id="abs0025"><p>Molecular Biology; Cancer</p></abstract><kwd-group id="kwrds0010"><title>Subject Areas</title><kwd>Molecular Biology</kwd><kwd>Cancer</kwd></kwd-group></article-meta><notes><p id="misc0010">Published: February 21, 2020</p></notes></front><body><sec id="sec1"><title>Introduction</title><p id="p0030">Head and neck cancers are the sixth most common malignant tumors worldwide. They are mainly treated with extended resection or radiation/chemotherapy, but functional deteriorations in swallowing, vocalization, and respiration lead to clinical decline in quality of life (QOL). Even in cases of combined surgical treatment with radiation therapy and chemotherapy, local recurrence including primary lesion and surrounding lymph nodes, is usually observed in approximately 30% of the patients. As distant metastases, as in the lung, can be seen in 25% of cases, recurrence and therapeutic resistance are very conspicuous. The 5-year survival rate in advanced cases of stage III and above is about 40%; thus the patients still present a poor prognosis (<xref rid="bib23" ref-type="bibr">Laramore et al., 1992</xref>). Recently, cetuximab, a molecular targeting agent, has been administered in combination with cisplatin to patients with head and neck malignancies, showing some benefit to outcomes. However, it remains that subjects suffer from serious side effects such as dermatitis and mucositis, and novel avenues for cancer therapeutics are needed for improved antitumor properties and patient QOL (<xref rid="bib1" ref-type="bibr">Bonner et al., 2006</xref>).</p><p id="p0035">Previous studies revealed that 2-oxoglutarate and the iron-dependent dioxygenases superfamily function as a hydroxylase/demethylase and that they hydroxylate or demethylate molecules such as transcription factor, histones, and DNA as substrates. Indeed, it has been reported that these enzymes play various roles in cell cycle and gene expression and control of invasion/metastasis of cancer cells in multiple cell lines via modified molecules (<xref rid="bib26" ref-type="bibr">Markolovic et al., 2015</xref>).</p><p id="p0040">There are some reports that procollagen lysyl hydroxylase (PLOD), which belongs to this superfamily, is involved in the hydroxylation of collagen molecules as the only target molecule identified to date, and it is considered to be an essential enzyme for cross-linking reaction between collagen molecules outside the cell (<xref rid="bib44" ref-type="bibr">Wu et al., 2006</xref>). PLOD is composed of an N-terminal endoplasmic reticulum-body translocation signal, a hydroxyl-catalyzed domain at the C-terminus, and hydroxylates lysine residues located in the terminal region of the collagen precursor in the ER.</p><p id="p0045">Currently, three members of the PLOD family molecular group are found to share high homology: PLOD1, PLOD2, and PLOD3 (<xref rid="bib41" ref-type="bibr">Valtavaara et al., 1997</xref>, <xref rid="bib30" ref-type="bibr">Passoja et al., 1998a</xref>). Tissue expression analysis by Northern blot showed that PLOD2 and PLOD3 had tissue-specific expression unlike the broad expression of PLOD1. In particular, PLOD2 has been reported to be induced in response to differentiation of bone marrow stromal cells (<xref rid="bib42" ref-type="bibr">Valtavaara et al., 1998</xref>, <xref rid="bib39" ref-type="bibr">Uzawa et al., 1999</xref>). Gene mutations in PLOD1 and PLOD3 have been identified in the case of Ehlers-Danlos syndrome (fragile skin, hematomas, and joint hypermobility), whereas PLOD2 mutations are clinically reported as involved in the Bruck syndrome characterized by imperfect bone formation (<xref rid="bib43" ref-type="bibr">Walker et al., 2004</xref>, <xref rid="bib40" ref-type="bibr">van der Slot et al., 2003</xref>).</p><p id="p0050">Furthermore, based on the previous studies, PLOD2 has been reported to be specifically induced by activation of transcription factor HIF-1α in response to hypoxia and TGF-β1 stimulation on tumor stroma, cancer cells (breast cancer, hepatocellular carcinoma), and sarcoma (mouse sarcoma model) (<xref rid="bib3" ref-type="bibr">Chen et al., 2015</xref>, <xref rid="bib9" ref-type="bibr">Gilkes et al., 2013a</xref>, <xref rid="bib10" ref-type="bibr">Gilkes et al., 2013b</xref>, <xref rid="bib5" ref-type="bibr">Eisinger-Mathason et al., 2013</xref>).</p><p id="p0055">It is known that collagen secretion and remodeling of extracellular matrix (ECM) are accelerated especially in cancer stromal cells, leading to invasion and metastases of cancer cells (<xref rid="bib9" ref-type="bibr">Gilkes et al., 2013a</xref>). The association of HIF-1α-dependent increase in expression of PLOD2 and epithelial mesenchymal transition (EMT) has been established in human glioma cells, and glioma patients with high expression of PLOD2 showed a poor prognosis (<xref rid="bib45" ref-type="bibr">Xu et al., 2017</xref>). However, the intracellular substrate and tumorigenic activity of PLOD2 expressed in cancer cells remains unclear, especially in refractory cancers, such as oral, head, and neck cancers.</p><p id="p0060">In this study, apart from tumor microenvironmental factors, we focused on biological function of PLOD2 on cancer cell itself using oral squamous cell carcinoma (SCC) lines as a model because they are high in PLOD2 expression, especially in tumor invasion/metastasis highlighting specific interaction between PLOD2 and integrin β1. We have found that PLOD2 induces the hydroxylation of integrin β1, which plays an essential role in the stability and cellular localization of integrin β1 for its functional activation. The integrin β1 requires specific interaction with PLOD2, which will lead to greater understanding of molecular mechanisms for cancer invasion/metastasis and foster innovation in therapies against refractory malignancies.</p></sec><sec id="sec2"><title>Results</title><sec id="sec2.1"><title>Specific Upregulation of PLOD2 and Its Effect on Cellular Motility of Oral SCC Cells</title><p id="p0065">Since the 2-oxoglutarate and iron-dependent dioxygenases have been classified into several groups according to the target amino acid residue, such as proline hydroxylase, asparagine/aspartate hydroxylase, etc., we first examined their expression in each category to identify specific hydroxylases highly expressed only in SCC lines from the oral cavity, in contrast to the non-neoplastic cellular counterpart (non-tumorigenic immortalized keratinocyte), HaCaT. Three representative human oral SCC lines (HSC-2, HSC-3, Ca9-22) derived from different patients were examined in comparison with HaCaT, and gene expression of typical hydroxylases for each category was examined by a semiquantitative RT-PCR method (<xref rid="mmc1" ref-type="supplementary-material">Figure S1</xref>A). The expression of <italic>JMJD2A</italic>, <italic>JMJD6</italic>, <italic>EGLN2</italic>, <italic>MINA53</italic>, <italic>NO66</italic>, <italic>HIF1AN</italic>, <italic>FTO</italic>, and <italic>TET1</italic> did not show a significant difference between the tumor cells and HaCaT. On the other hand, the expression of <italic>PLOD2</italic>, <italic>EGLN1</italic>, <italic>EGLN3</italic>, <italic>OGFOD1</italic>, <italic>ASPH</italic>, and <italic>ALKBH3</italic> revealed certain differences in expression among these cell lines, especially in that the mRNA level of <italic>PLOD2</italic> in the SCC lines showed the most prominent and consistent increase at four to eight times compared with HaCaT (<xref rid="mmc1" ref-type="supplementary-material">Figures S1</xref>A and <xref rid="fig1" ref-type="fig">1</xref>A). This upregulation of <italic>PLOD2</italic> mRNA in tumor cells was also corroborated by protein levels in immunoblot analysis even among the lysyl hydroxylase family including PLOD1 and PLOD3 (<xref rid="fig1" ref-type="fig">Figure 1</xref>B). Immunofluorescence analysis with anti-PLOD2 antibody revealed that endogenous PLOD2 was predominantly localized to the ER which was confirmed by GFP-labeled ER marker (<xref rid="fig1" ref-type="fig">Figure 1</xref>C). Thus, tumor-specific upregulation of the expression was observed only in PLOD2 among the examined hydroxylase and demethylase family, and the mRNA and protein levels of PLOD1 and PLOD3 were not specifically elevated in the tumor cells although they are categorized to the same family.<fig id="fig1"><label>Figure 1</label><caption><p>Expression of the Various Hydroxylases in Oral SCC Cells</p><p>(A) The expression level of mRNAs in oral SCC cells was determined by quantitative PCR compared with that of HaCaT. Data are means ± s.d. from three biological replicates (*p < 0.05, Student's t-test).</p><p>(B) Protein expression of PLOD family in SCC lines and HaCaT by immunoblotting.</p><p>(C) Immunofluorescence of PLOD2 in oral SCC lines (HSC-2, HSC-3, and Ca9-22) and non-neoplastic keratinocyte (HaCaT). Colocalization of PLOD2 with ER marker (ER-GFP) was indicated by arrowhead. Nuclei were stained with Hoechst 33258. Scale bar = 20 μm.</p><p>(D) RNA interference (siRNA)-mediated knockdown of <italic>PLOD</italic> in oral SCCs demonstrated the attenuated protein expression by immunoblotting.</p><p>(E) GFP-expressing SCC cells were transfected with control siRNA (siCtrl) or with <italic>PLOD</italic>-siRNA (siPLOD1, siPLOD2, and siPLOD3). Cell migration was evaluated by wound healing assay. Images were taken at 0 and 24 h after wound formation (scale bar = 400 μm). The wound width was estimated using fluorescence microscopic images. Each symbol represents siCtrl (circle, black), siPLOD1 (square, blue), siPLOD2 (triangle, red), and siPLOD3 (cross, green). Asterisk indicates p < 0.05 as compared with siCtrl. Data are means ± s.d. from three technical replicates for one biological replicate.</p></caption><graphic xlink:href="gr1"></graphic></fig></p><p id="p0070">Based on these results, we next studied the cellular motility of the SCC cells, HSC-2, HSC-3, and Ca9-22, treated with specific siRNA against <italic>PLOD2</italic> (si<italic>PLOD2</italic>) in comparison with the cells treated with si<italic>PLOD1</italic> or si<italic>PLOD3</italic>. Specificity of the siRNA for each PLOD isoform (si<italic>PLOD1</italic>, si<italic>PLOD2</italic>, and si<italic>PLOD3</italic>) was confirmed by immunoblotting, which demonstrated that knockdown by these siRNAs did not affect other isotypes (<xref rid="mmc1" ref-type="supplementary-material">Figures S1</xref>B and <xref rid="fig1" ref-type="fig">1</xref>D). In the wound-healing assay, each stable GFP-expressing clone established from parental HSC-2, HSC-3, and Ca9-22 was employed for evaluation. Mobilization of each clone, visualized by fluorescence microscope 24 h after treatment with the siRNA, revealed that only attenuated expression of PLOD2 significantly affected cellular migration among these three SCC cells, whereas neither <italic>PLOD1</italic>-knockdown nor <italic>PLOD3</italic>-knockdown critically disturbed migration of the tumor cells (<xref rid="fig1" ref-type="fig">Figure 1</xref>E, upper images and <xref rid="mmc1" ref-type="supplementary-material">Figure S1</xref>B). Inhibition of wound closure was over 50% in si<italic>PLOD2</italic>-treated cells at 24 h following siRNA treatment (<xref rid="fig1" ref-type="fig">Figure 1</xref>E, lower graphs); however, the MTT assay indicated that tumor cell proliferation was not affected by siRNA-treatment in all <italic>PLOD</italic> isoforms (<xref rid="mmc1" ref-type="supplementary-material">Figure S1</xref>C). These data implied that PLOD2 might be deeply involved in regulating tumor cell motility.</p></sec><sec id="sec2.2"><title>Crosstalk between PLOD 2 and Integrin β1 in Cellular Motility</title><p id="p0075">On the basis of these findings, we focused on the specific role of PLOD2 in tumor cell motility. Generally, acceleration of cell mobility is closely related to invasive properties of tumor cells, and we examined whether expression of E-cadherin (CDH1) as a marker of epithelial-mesenchymal transition (EMT) was altered with or without si<italic>PLOD2</italic>-treatment of the SCC cells. We found that <italic>PLOD2</italic>-knockdown did not affect the expression and membrane localization of CDH1 for all SCC cells (<xref rid="fig2" ref-type="fig">Figures 2</xref>A and <xref rid="mmc1" ref-type="supplementary-material">S2</xref>A). Because all of these three SCC lines highly express integrin β1 as one of the critical motor molecules for invasion, we next examined the effect of PLOD2 on integrin β1 therein. In si<italic>PLOD2</italic>-treated tumor cells, lack of filopodia development was observed as a morphological change as confirmed by phalloidin staining, and a marked decrease in integrin β1 was revealed, in contrast to the cells treated with control siRNA (<xref rid="fig2" ref-type="fig">Figures 2</xref>B, <xref rid="mmc1" ref-type="supplementary-material">S2</xref>B, S2C, and <xref rid="mmc1" ref-type="supplementary-material">S3</xref>). This phenomenon was not seen in si<italic>PLOD1-</italic> or si<italic>PLOD3</italic>-treated cells (<xref rid="mmc1" ref-type="supplementary-material">Figure S1</xref>D). The si<italic>PLOD2</italic>-treated tumor cells with decreased integrin β1 showed no altered expression of CDH1 or SNAIL, suggesting the PLOD2 seemed not to be involved in EMT at least in these SCC cells (<xref rid="fig2" ref-type="fig">Figures 2</xref>C and <xref rid="mmc1" ref-type="supplementary-material">S4</xref>A). Involvement of integrin β1 in migration of these SCC cells was demonstrated by knockdown assay using si<italic>Integrin β1</italic> (<xref rid="mmc1" ref-type="supplementary-material">Figures S4</xref>B and S4C). Taken together, our data indicate that integrin β1 appears directly regulated by PLOD2 for these tumor cells in an EMT-independent manner.<fig id="fig2"><label>Figure 2</label><caption><p>PLOD2 Is Essential for Stabilization of Integrin β1</p><p>(A) Immunofluorescence revealed expression, and localization of CDH1 was not affected by siPLOD2-treatment in SCC cells.</p><p>(B) Expression and intracellular localization of integrin β1 of the SCC cells was examined at 48 h after treatment with siPLOD2. Cytoskeleton and nuclei were stained with phalloidin and Hoechst, respectively. Scale bar = 20 μm.</p><p>(C) Expression of integrin β1, CDH1, and SNAIL in the siPLOD2-transfected cells by immunoblotting using anti-PLOD2, anti-integrin β1, anti-CDH1, and anti-SNAIL Ab, respectively.</p><p>(D) Semiquantitative expression of <italic>integrin β1</italic> mRNA by RT-PCR with or without siPLOD2-treatement.</p><p>(E) Comparative ratio of <italic>integrin β1</italic> mRNA in siPLOD2-treated cells based on the quantitative PCR results. Quantitative results are mean ± s.d. from three biological replicates (n.s. = not significant, Student's t-test).</p><p>(F) Restoration of integrin β1 by treatment with MG132 and chloroquine (CHQ). HSC-2 cells pretreated with siPLOD2 were examined for integrin β1 expression 18 h after treatment with MG132 (1 nM) or CHQ (50 μM), respectively. Expression of integrin β1 protein by immunoblotting (upper panel), intracellular localization of integrin β1 by immunofluorescence using anti-integrin β1 Ab (lower panel). Integrin β1 (red) was merged with lysosome marker (Lyso-GFP). Scale bar = 20 μm.</p><p>(G) Effect of <italic>PLOD2</italic> mutant lacking the catalytic domain (ΔPKHD) to integrin β1. Integrin β1 of the HSC-2 transfected with myc-tagged PLOD2 lacking the hydroxylase domain (ΔPKHD) compared with that of the cells transfected with the WT. Reduction of integrin β1 detected by immunoblotting (upper panel) and the loss of plasma membrane localization indicated by arrowhead with immunofluorescence (lower panel). Scale bar = 20 μm.</p><p>(H) Wound healing assay revealed cell migration was affected in the ΔPKHD-transfected cells as shown in the graph (upper panel) and migratory images (lower panel). Each symbol in the graph represents empty vector (circle, black), PLOD2 WT (square, blue), and PLOD2 ΔPKHD mutant (triangle, red). Data are means ± s.d. from three technical replicates for one biological replicate (*p < 0.05, Student's t-test as compared with empty vector).</p></caption><graphic xlink:href="gr2"></graphic></fig></p><p id="p0080">Next, to clarify whether PLOD2 affects induction of <italic>integrin β1</italic> mRNA, or directly modifies the integrin β1 protein, RT-PCR was first performed to examine fluctuations in mRNA levels. Ultimately, no significant alteration in <italic>integrin β1</italic> mRNA expression with or without si<italic>PLOD2</italic> introduction was detected in SCC cells, which was further confirmed by qPCR (<xref rid="fig2" ref-type="fig">Figures 2</xref>D and 2E). Therefore, si<italic>PLOD2</italic> did not affect induction of <italic>integrin β1</italic> mRNA, i.e. the result suggested that integrin β1 protein might be persistently produced in tumor cells but may require a certain modification by PLOD2 for stabilization.</p><p id="p0085">Following the recent study reporting degradation of integrin α at lysosome post-ubiquitination (<xref rid="bib24" ref-type="bibr">Lobert et al., 2010</xref>), we examined the effects of proteasome inhibitor, MG132, and chloroquine (CHQ)—which is known to interfere with endocytosis processes and with ligand delivery to the lysosome (<xref rid="bib6" ref-type="bibr">Erbacher et al., 1996</xref>)—as to whether they might restore the integrin β1 protein in si<italic>PLOD2</italic>-treated tumor cells. Immunoblotting demonstrated that both MG132 and CHQ successfully restore integrin β1 to the level of the control siRNA-treated cell as shown in <xref rid="fig2" ref-type="fig">Figure 2</xref>F (upper panel) and <xref rid="mmc1" ref-type="supplementary-material">Figure S5</xref>A and that intracellular localization of integrin β1 was observed in the si<italic>PLOD2</italic>-introduced cells treated with MG132 and with CHQ, whereas membranous integrin β1 was only restored in MG132-treated cells (<xref rid="mmc1" ref-type="supplementary-material">Figure S5</xref>B). The incidence of lysosome localization of integrin β1 remains at the same level in all cases (<xref rid="fig2" ref-type="fig">Figure 2</xref>F lower panel and <xref rid="mmc1" ref-type="supplementary-material">Figure S5</xref>C). Based on this finding, the role of PLOD2 as a hydroxylase was examined with respect to stability of integrin β1 in the wild-type <italic>PLOD2</italic>-transfected HSC-2 vs. the ΔPKHD-<italic>PLOD2</italic>-transfected cell (ΔPKHD; ΔLysyl hydroxylase/prolyl 4-hydroxylase domain, inactive form lacking the catalytic domain of <italic>PLOD2</italic>) (<xref rid="bib32" ref-type="bibr">Pirskanen et al., 1996</xref>, <xref rid="bib31" ref-type="bibr">Passoja et al., 1998b</xref>, <xref rid="bib14" ref-type="bibr">Heikkinen et al., 2000</xref>, <xref rid="bib34" ref-type="bibr">Ruotsalainen et al., 2006</xref>, <xref rid="bib18" ref-type="bibr">Kati et al., 2007</xref>). Significant enhancement of integrin β1 was not observed in the wild-type <italic>PLOD2</italic>-transfected HSC-2 but its membrane expression (filopodial localization) was prominently enhanced (<xref rid="fig2" ref-type="fig">Figure 2</xref>G, lower panel images) due to the stabilization of integrin β1 protein by sufficient PLOD2. On the other hand, immunoblotting revealed reduction of integrin β1 in the ΔPKHD-<italic>PLOD2</italic>-transfected cells compared with empty vector or wild-type <italic>PLOD2</italic>-transfected HSC2 (WT), and loss of the membranous integrin β1 was observed in contrast to the WT and the vector cells (<xref rid="fig2" ref-type="fig">Figure 2</xref>G). In this regard, decrease of integrin β1 in ΔPKHD-PLOD2-transfected cells was supposed to be due to destabilization of integrin β1 lacking active PLOD2, which was explained by MG132 assay shown in <xref rid="mmc1" ref-type="supplementary-material">Figure S5</xref>D. Cellular motility of the ΔPKHD-<italic>PLOD2</italic>-transfected cells was significantly attenuated as shown by wound-healing assay (<xref rid="fig2" ref-type="fig">Figure 2</xref>H). These data imply that hydroxylase activity of PLOD2 was required for protein stability and functional localization of integrin β1.</p></sec><sec id="sec2.3"><title>PLOD2 Regulates Intracellular Localization of Integrin β1 Through Their Coupling</title><p id="p0090">To examine whether PLOD2 specifically regulates intracellular localization of integrin β1, "Fluorescent based technology detecting Protein-Protein Interaction in living cells" (the Fluoppi system) was employed to visualize the movement of integrin β1 in the presence of PLOD2 (<xref rid="fig3" ref-type="fig">Figure 3</xref>A). When the two proteins of interest specifically bind to each other, it yields aggregated fluorescence foci (red colored dots) inside cells under the Fluoppi system. As shown in <xref rid="fig3" ref-type="fig">Figure 3</xref>B, large fluorescence foci in HeLa cotransfected with <italic>PLOD2</italic>-Red and <italic>integrin β1</italic>-Ash demonstrated specific binding of PLOD2 to integrin β1 within cells. Further we did semiquantification of fluorescent dot intensity on Fluoppi specimens between <italic>PLOD2</italic>-alone-expressing cells and <italic>integrin β1</italic>–<italic>PLOD2</italic>-coexpressing cells (<xref rid="mmc1" ref-type="supplementary-material">Figures S6</xref>A and S6B). We also repeated Fluoppi assay to obtain clear image of fluorescent dot formation only on the <italic>integrin β1</italic>–<italic>PLOD2</italic> cotransfection into the PLOD2-knockout cells (<xref rid="mmc1" ref-type="supplementary-material">Figure S6</xref>C). Tracking the PLOD2-integrin β1 complex by Fluoppi system using integrin β1-Red expressor in combination with PLOD2-Ash, the fluorescent foci were first colocalized with ER-GFP in the early phase of transfection, suggesting they were at the ER (<xref rid="fig3" ref-type="fig">Figures 3</xref>C and 3D) and then Monti-red-labeled integrin β1 protein transited to filopodia and the plasma membrane in the cotransfected cells (<xref rid="fig3" ref-type="fig">Figure 3</xref>D). Immunoprecipitation assay using anti-Flag Ab on the cotransfected HEK293T cell lysates indicated binding of integrin β1 both to full-length PLOD2 and ΔPKHD-PLOD2, which demonstrated direct binding of these two proteins, and integrin β1 bound to the site outside of the PKHD domain of PLOD2 (<xref rid="fig3" ref-type="fig">Figures 3</xref>E and 3F). Further immunoprecipitation experiments of endogenous PLOD2 in HSC-2 cells revealed an interaction with the immature integrin β1 (<xref rid="mmc1" ref-type="supplementary-material">Figure S6</xref>D).<fig id="fig3"><label>Figure 3</label><caption><p>Specific Coupling of Integrin β1 with PLOD2</p><p>(A) The construction of PLOD2 expressor fused Monti-red tag (<italic>PLOD2</italic>-Red) and integrin β1 expressor fused Ash tag (<italic>Integrin β1</italic>-Ash) (left panel). Schematic representation of mechanism for intracellular fluorescent dot formation in response to specific protein-protein interaction (right panel).</p><p>(B) Marked aggregation of fluorescent dots was detected only in the HeLa cells cotransfected with <italic>PLOD2</italic>-Red and <italic>Integrin β1</italic>-Ash. Scale bar = 20 μm.</p><p>(C) The fluorescent dots were merged with GFP-labeled ER marker (ER-GFP). Arrowhead indicated each PLOD2, ER-GFP, and colocalized proteins in the transfected HeLa cells, respectively. Scale bar = 20 μm.</p><p>(D) The fluorescent dots were observed at lamellipodia of the <italic>PLOD2</italic>-Red-<italic>Integrin β1</italic>-Ash cotransfected cells or the <italic>Integrin β1</italic>-Red-<italic>PLOD2</italic>-Ash cotransfected cells. Hatched box indicated hyperview field in the right. Scale bar = 20 μm.</p><p>(E) Domain organization of the FLAG epitope tagged-<italic>Integrin β1</italic> expressor, Myc epitope-tagged <italic>PLOD2</italic> mutant expressor lacking PKHD domain (ΔPKHD) used in the immunoprecipitation assay.</p><p>(F) Integrin β1 was coprecipitated both with WT-PLOD2 and ΔPKHD-PLOD2 in HEK293T cells. Expression of each PLOD2 expressor and EGFP expressor as a control in whole cell lysates (left), immunoprecipitated PLOD2, and its variant (probed with anti-Myc Ab) using anti-Flag Ab for precipitation (right). IgG heavy chain (H.C.) and light chain (L.C.) are shown.</p></caption><graphic xlink:href="gr3"></graphic></fig></p></sec><sec id="sec2.4"><title>Direct Hydroxylation of Integrin β1 by PLOD2 for Its Activation</title><p id="p0095">Because the result of the specific binding between PLOD2 and integrin β1 would indicate that integrin β1 might be a substrate for PLOD2, hydroxylation of the integrin β1 protein purified from the lysate of the 293T transfected with wild-type (WT) <italic>PLOD2</italic> was analyzed by mass spectrometry (LC/MS) using the integrin β1 derived from <italic>ΔPKHD-PLOD2</italic> transfected cells. The main spike, indicated with the hatched box in the overviewed LC/MS chromatogram of integrin β1 from the cotransfected cells with <italic>integrin β1</italic> and <italic>WT-PLOD2</italic> (<xref rid="fig4" ref-type="fig">Figure 4</xref>A), contained the unique spike (indicated by arrow) representing the molecular weight of triple lysine hydroxylation (encoded amino acids between 651 a.a. and 658 a.a., “AFN<bold><underline>K</underline></bold>GE<bold><underline>KK</underline></bold>”), which was not observed in the integrin β1 protein from the <italic>integrin β1</italic>-<italic>ΔPKHD-PLOD2</italic> cotransfected sample (<xref rid="fig4" ref-type="fig">Figure 4</xref>B; hyperview of 4A). The shift of the integrin β1 peak indicated by the arrow in the <italic>WT-PLOD2</italic> cotransfected sample is two-fold higher than that of the integrin β1 in the <italic>ΔPKHD-PLOD2</italic> cotransfected sample (<xref rid="fig4" ref-type="fig">Figure 4</xref>B). Additionally, we performed an experiment monitoring the activity of PLOD2 employing substrate peptides according to the previous reports (<xref rid="bib38" ref-type="bibr">Takaluoma et al., 2007</xref>, <xref rid="bib13" ref-type="bibr">Guo et al., 2017</xref>). The result showed specific increase of succinate derived from α-ketoglutarate and substrate (integrin β1 substrate peptide.; AFNKGEKK) incubated with purified PLOD2 as well as positive control sample using collagen peptide (IKGIKGIKG) sample (<xref rid="fig4" ref-type="fig">Figures 4</xref>C and 4D). The stability of integrin β1 harboring the single Lys-replacement with Ala (K564A, K657A, and K658A) did not seem significantly impaired inside cells transfected with each integrin β1 mutant; however, triple Lys-mutated integrin β1 (K564A + K657A + K658A) seemed markedly unstable (<xref rid="fig4" ref-type="fig">Figure 4</xref>E). Moreover, the triple Lys-mutative integrin β1 was not recruited to the plasma membrane in the transfected HSC-2 cells, in contrast to the cells with the single Lys-mutative integrin β1, which still retained its membranous localization as well as in those with WT-integrin β1 (<xref rid="fig4" ref-type="fig">Figure 4</xref>F). In this regard, flow cytometry analysis revealed that approximately 20% of the WT-transfected BHK cells showed plasma membrane expression of integrin β1, whereas no significant number of cells showed membrane expression of integrin β1 in the triple K-A mutant-transfected cells, which indicated loss of membrane localization without PLOD2 activity (<xref rid="mmc1" ref-type="supplementary-material">Figures S7</xref>A and S7B). Thus, the magnitude of hydroxylation on integrin β1 by PLOD2 might be critical for its intracellular stability.<fig id="fig4"><label>Figure 4</label><caption><p>Hydroxylation of Integrin β1 in Presence of PLOD2 and Its Significance in Intracellular Localization of Integrin β1</p><p>(A) LC/MS of integrin β1 purified from lysate of the 293T cells coexpressing integrin β1-Flag and WT-PLOD2 in comparison with that from the cells expressing integrin β1-Flag and ΔPKHD-PLOD2. The main peaks of integrin β1 were highlighted with the hatched box.</p><p>(B) Hyperview of the highlighted peaks. Arrowhead represented the fragment of integrin β1 (position of #651-658 a.a. containing three lysines; AFNKGEKK) from the WT-<italic>PLOD2</italic>-transfected or form the ΔPKHD-PLOD2-transfected lysate, which showed shift of the peak between these two integrin β1 (spectra; 484.5 to 486 m/z).</p><p>(C) Recombinant PLOD2-6xHis protein from HEK293T was purified in 150 mM imidazole using cobalt resin. Purification of PLOD2 was confirmed by Coomassie staining and immunoblotting.</p><p>(D) Hydroxylation reaction of PLOD2 was carried out <italic>in vitro</italic> as described in <xref rid="sec4" ref-type="sec">Methods</xref>. Collagen peptides or no peptide substrates were used as controls for reaction. Data are means ± s.d. from three technical replicates for one biological replicate (*p < 0.05, Student's t-test).</p><p>(E) Expression of integrin β1 mutants replacing Lys#654 to Ala (K654A), Lys#657 to Ala (K657A), Lys#658 to Ala (K658A), or the triple Ala-substituted mutant replacing the lysine (3KA).</p><p>(F) Intracellular localization of the integrin β1 of these three mutants on HSC-2. Arrowhead indicated integrin β1 at the plasma membrane. Only in the transfectant with triple Ala mutant did integrin β1 lack its membranous localization. Scale bar = 20 μm.</p></caption><graphic xlink:href="gr4"></graphic></fig></p></sec><sec id="sec2.5"><title>PLOD2 Facilitates Metastasis of SCC Cells <italic>In Vivo</italic></title><p id="p0100">Based on these findings, we examined whether PLOD2-affected invasion/metastasis of SCC cells <italic>in vivo</italic> using the intrathoracic metastatic mouse model xenografted with HSC-2 cells. Stably GFP-expressing HSC-2 cells from the <italic>PLOD2</italic>-knockout clone (<italic>PLOD2</italic>-KO; KO#31, <xref rid="mmc1" ref-type="supplementary-material">Figure S8</xref>A) were xenografted into the thoracic cavity of athymic nude (nu/nu) mice and evaluated for development of metastatic foci 40 days post-implant in comparison with mice xenografted with parental HSC-2 cells (PLOD2-WT). As to cellular behaviors, <italic>PLOD2</italic>-KO (KO#31) carrying the heterozygous knockout of <italic>PLOD2</italic> did not show significant differences in cellular growth compared with parent HSC-2 (<xref rid="mmc1" ref-type="supplementary-material">Figure S8</xref>B), but endogenous expression of integrin β1 was seriously attenuated (<xref rid="mmc1" ref-type="supplementary-material">Figure S8</xref>C). In the KO#31 clone, plasma membrane localization of integrin β1 was also disturbed, which suggested impaired function of integrin β1 (<xref rid="mmc1" ref-type="supplementary-material">Figure S8</xref>D). Reflecting these results, cellular motility of KO#31 was also seriously decreased in comparison with the parental HSC-2 cells (<xref rid="mmc1" ref-type="supplementary-material">Figure S8</xref>E, image and graphs). Whereas, using <italic>PLOD2</italic>-KO clone (#31) of HSC-2, we performed rescue experiment by reintroducing wild-type PLOD2. The result showed the revertant (revPLOD2) restored cellular motility, which was similar to the original HSC-2 by regaining expression of maturated integrin β1 (<xref rid="mmc1" ref-type="supplementary-material">Figures S9</xref>A and S9B). This corroborated requirement of integrin β1 expression for invasive property of HSC-2 cells, and it was regulated by PLOD2. Further we performed the time-dependent chase experiment for integrin β1 under presence of PLOD2 on <italic>PLOD2</italic>-KO clone with or without <italic>WT-PLOD2</italic> and <italic>ΔPKHD-PLOD2</italic> transfection. Result showed significant induction of mature integrin β1 was detected in response to time-dependent increase of WT-PLOD2 (<xref rid="mmc1" ref-type="supplementary-material">Figure S10</xref>). Whereas overexpression of ΔPKHD-PLOD2, deficient form of hydroxylation activity (<xref rid="fig4" ref-type="fig">Figure 4</xref>B), induced the instability of integrin β1, which suggested the necessity of hydroxylation for integrin β1 maturation. Consequently, in the tumor metastasis mouse model, implant of the <italic>PLOD2</italic>-KO (KO#31) HSC-2 clone showed drastic elimination of metastatic lesions inside the thoracic cavity <italic>in vivo</italic> except for local growth of lung tumors, which was clearly different from the <italic>PLOD2</italic>-WT cell-implanted mice with multiple metastatic foci on pleura (<xref rid="fig5" ref-type="fig">Figures 5</xref>A and <xref rid="mmc1" ref-type="supplementary-material">S11</xref>). Immunohistochemistry of the tumors derived from the PLOD2-WT cell-xenograft and KO#31 cell-xenograft was corroborative with tumor cells with or without coexpression of PLOD2 and functional integrin β1 (<xref rid="fig5" ref-type="fig">Figure 5</xref>B).<fig id="fig5"><label>Figure 5</label><caption><p>Deficiency of PLOD2 Inhibited Metastasis of SCC Cells <italic>In Vivo</italic></p><p>(A) <italic>In vivo</italic> development of metastatic foci inside thoracic cavity in mice xenografted with GFP-expressing HSC-2 bearing PLOD2-WT (left panel) and the PLOD2-deficient HSC-2 (PLOD2-KO; right panel). Fluorescence imaging was performed 40 days post-implant.</p><p>(B) Histological examination of the metastatic tumor with wild type of PLOD2 (PLOD2-WT) and of that without PLOD2 (PLOD2-KO) in mice tumor models. Immunohistochemistry using anti-PLOD2 Ab and anti-integrin β1 Ab were performed in the same tumor. Box at the HE images (upper panel) indicated the field shown as hyperview in the middle and lower panel. Scale bar = 50 μm.</p></caption><graphic xlink:href="gr5"></graphic></fig></p></sec><sec id="sec2.6"><title>Coexpression of PLOD2 and Integrin β1 in Oral, Pharyngeal, and Laryngeal SCC Tissues from the Patients</title><p id="p0105">Finally, endogenous expression of PLOD2 and integrin β1 in SCC tissues from patients was examined. As shown in <xref rid="fig6" ref-type="fig">Figure 6</xref>, enhanced expression of PLOD2 was detected in SCC cells from different origins such as oral cavity (case 1, case 2: well-differentiated type; case 3: moderately differentiated type), pharynx (case 4: well-differentiated type; case 5: moderately differentiated type), and larynx (case 6: moderately differentiated type), which was consistent and coincident with integrin β1 expression, especially at the marginal region and invasive front of the tumor nests (<xref rid="mmc1" ref-type="supplementary-material">Figure S12</xref>A). Moreover, we demonstrated coexpression of these two proteins at invasive cancer nest using double immunofluorescence (<xref rid="mmc1" ref-type="supplementary-material">Figure S12</xref>B).<fig id="fig6"><label>Figure 6</label><caption><p>Expression of PLOD2 and Integrin β1 in Head and Neck SCCs from the Patients</p><p>Immunohistochemistry using anti-PLOD2 Ab and anti-integrin β1 Ab were performed on the SCC tissues derived from oral cavity, pharynx, and larynx, respectively. Box at the HE images (left column) indicated the field shown as hyperview in the middle and right column. Scale bar = 50 μm.</p></caption><graphic xlink:href="gr6"></graphic></fig></p></sec></sec><sec id="sec3"><title>Discussion</title><p id="p0110">Intracellular molecules undergoing hydroxylation by various hydroxylases have been reported to significantly alter cellular functions by regulation of protein degradation, resulting in translational activation or repression of particular proteins in various cancer cells. These intracellular events consequently affect cell-cycle progression, gene expression and activation, and the triggering of cancer invasion/metastases (<xref rid="bib33" ref-type="bibr">Ploumakis and Coleman, 2015</xref>, <xref rid="bib16" ref-type="bibr">Ivan et al., 2001</xref>, <xref rid="bib49" ref-type="bibr">Zhang et al., 2009</xref>, <xref rid="bib37" ref-type="bibr">Singleton et al., 2014</xref>). Previous studies have shown that PLOD2, one of the lysine hydroxylases, which is highly expressed in cancer cells, plays a major role to process collagens as substrate to form a suitable tumor ECM (<xref rid="bib3" ref-type="bibr">Chen et al., 2015</xref>, <xref rid="bib9" ref-type="bibr">Gilkes et al., 2013a</xref>, <xref rid="bib10" ref-type="bibr">Gilkes et al., 2013b</xref>, <xref rid="bib5" ref-type="bibr">Eisinger-Mathason et al., 2013</xref>). Accordingly, previous reports on the biological significance of PLOD2 in tumors focused on ECM regulation through its interaction with collagen fibers comprising the tumor matrix.</p><p id="p0115">In this study, we found an additional functional aspect of PLOD2 as a direct regulator of cancer invasion/metastasis through its specific interaction with a major integrin family molecule, integrin β1. Namely, PLOD2 is a key regulator for activation of integrin β1 expressed in head and neck SCCs, which stabilizes and activates integrin β1 by hydroxylation via specific binding. This event leads to accelerated cellular motility needed to form metastatic sites for the SCC cells <italic>in vivo</italic>. Based on results of our analysis, tumor invasion via the PLOD2-mediated activation of integrin β1 seems not to be mediated by the EMT (epithelial-mesenchymal transition) process in cancer cells but by the direct effect of interactions of these two molecules. Marked increase in cellular motility of SCC cells was observed by integrin β1-PLOD2 protein-protein interaction without altering CDH1 or SNAIL expression (<xref rid="bib7" ref-type="bibr">Friedl, 2004</xref>, <xref rid="bib8" ref-type="bibr">Friedl et al., 2004</xref>, <xref rid="bib2" ref-type="bibr">Canel et al., 2013</xref>) as shown in <xref rid="fig2" ref-type="fig">Figure 2</xref>C, and the increase appeared strongly affected by catalytic activity of PLOD2 (<xref rid="fig2" ref-type="fig">Figures 2</xref>G, 2H, and <xref rid="fig4" ref-type="fig">4</xref>D). Importantly, instability of integrin β1 protein from knockdown of PLOD2 (downregulation of PLOD2) was observed in cancer cells of diverse origins such as esophageal SCC cells (KYSE30), lung adenocarcinoma cells (A549), uterine cervical SCC cells (HeLa), hepatocellular carcinoma cells (KYN-2), and breast invasive ductal adenocarcinoma cells (MCF7) as well (<xref rid="mmc1" ref-type="supplementary-material">Figure S13</xref>A). In addition, we examined change of integrin β6 and integrin α5 expression by treating with PLOD2-siRNA in HSC-2, HSC-3, and Ca9-22 cells, because integrin β6 and α5 were reported to be expressed, in addition to β1 on head/neck SCCs (<xref rid="bib21" ref-type="bibr">Koivistro et al., 2000</xref>). The result was shown as <xref rid="mmc1" ref-type="supplementary-material">Figure S13</xref>B, which demonstrated significant attenuation of integrin β6 and α5 by PLOD2-siRNA treatment in these cells. As an exception, in HSC-3 cells, unlike other two SCC lines, decrease of α5 was not clearly observed, whereas integrin β6 was decreased as well as β1. This might be because of the HSC-3's different cellular property from HSC-2 and Ca9-22 (both were cloned from primary SCC lesion), which was cloned from the lymph nodal metastasis and with highly expressed Vimentin (<xref rid="fig2" ref-type="fig">Figure 2</xref>C). These observations imply that PLOD2 might play a pivotal role for regulation of integrin β1 involved in invasion/metastasis of tumors with diverse lineages. Additionally, integrin β1 knockdown had less of an effect on migration in <xref rid="mmc1" ref-type="supplementary-material">Figure S4</xref>B, than siPLOD2 throughout. PLOD2 may in fact provide an approach to target integrin β1 along with some other invasion-promoting targets. A large screening for PLOD2 targets may be required in the future.</p><p id="p0120">We demonstrated that PLOD2 hydroxylase modified integrin β1 protein as a direct substrate. In the intracellular molecular binding assay (Fluoppi assay), PLOD2 and integrin β1 formed complex at the ER (ER) as shown in <xref rid="fig3" ref-type="fig">Figures 3</xref>B, 3C, and <xref rid="mmc1" ref-type="supplementary-material">S6</xref>, then the integrin β1 moved to filopodia and the plasma membrane (<xref rid="fig3" ref-type="fig">Figure 3</xref>D). ER localization of PLOD2 has been reported in the previous studies (<xref rid="bib4" ref-type="bibr">Chen et al., 2017</xref>, <xref rid="bib12" ref-type="bibr">Gjaltema et al., 2016</xref>), which is consistent with our present result, indicating that PLOD2 first hydroxylizes integrin β1 at the ER, following which the integrin β1 is recruited to the cell surface functional site after the modification.</p><p id="p0125">In previous studies on modification of collagen by PLOD2, hydroxylation of lysine within the X-K-G motif was reported to be essential for polymerization of collagen fibers (<xref rid="bib20" ref-type="bibr">Kivirikko and Pihlajaniemi, 1998</xref>, <xref rid="bib29" ref-type="bibr">Myllyharju and Kivirikko, 2004</xref>, <xref rid="bib38" ref-type="bibr">Takaluoma et al., 2007</xref>, <xref rid="bib40" ref-type="bibr">van der Slot et al., 2003</xref>). Based on earlier findings, the putative lysine-rich motif for hydroxylation was found at position of #651-658 a.a., “AFNKGEKK,” in integrin β1 (<xref rid="mmc1" ref-type="supplementary-material">Figure S13</xref>C), which may point to a target motif for PLOD2. In fact, the mass spectrometry result revealed “AFNKGEKK” as the supershifted peak among integrin β1 fragments, which appeared only in the active PLOD2-treated sample (<xref rid="fig4" ref-type="fig">Figures 4</xref>A and 4B), and coincidently, the triple Lys substitution with Ala (K654A/K657A/K658A) abolished membrane localization of integrin β1 (<xref rid="fig4" ref-type="fig">Figures 4</xref>F and <xref rid="mmc1" ref-type="supplementary-material">S7</xref>). Taking these together, the hydroxylation of these lysine within AFNKGEKK sequence by PLOD2 is critically implicated in gain of integrin β1 function <italic>in vivo</italic>.</p><p id="p0130">We generated the <italic>PLOD2</italic>-knockout HSC-2 clone, PLOD2-KO (KO#31; heterozygous genomic knockout of PLOD2, <italic>PLOD2</italic><sup>−/+</sup>), and tracked its dynamics <italic>in vivo</italic> in comparison with those of the parental HSC-2 (wild type; PLOD2-WT) using the mouse xenografted model. Intrathoracic implant of PLOD2-KO cells showed loss of pleural dissemination and pulmonary metastasis; it only showed local growth of tumor, whereas PLOD2-WT cells developed multiple metastatic foci covering the entire thoracic cavity. These data suggested that cancer cell motility was critically regulated by the presence of PLOD2. Immunohistochemistry demonstrated coexpression of PLOD2 and integrin β1 at invasive cancer nests in the metastatic tumor tissue derived from the mice implanted with PLOD2-WT cells, whereas integrin β1 expression in cancer nests was markedly attenuated by defect in PLOD2. This histological finding corroborated the significant relationship between integrin β1 and PLOD2 <italic>in vivo</italic> as was proven, further, by <italic>in vitro</italic> molecular assay. Finally, we confirmed that the expression pattern of integrin β1 and PLOD2 in SCC tissues surgically obtained from patients was similar to that of tumors in the mouse model.</p><p id="p0135">Therefore, we conclude that integrin β1, a critical motor molecule for SCC invasion/metastasis, requires PLOD2 for its stabilization and functional cellular localization via their specific interaction through hydroxylation (<xref rid="fig7" ref-type="fig">Figure 7</xref>). Our results indicate that specific suppression of PLOD2 activity may provide one of the chief clues for inhibiting cancer invasion/metastasis in future cancer therapeutics.<fig id="fig7"><label>Figure 7</label><caption><p>Intracellular Dynamics of Integrin β1 Mediated by PLOD2 in SCC Cells</p><p>Head and neck squamous cell carcinomas retain high-level expression of PLOD2, which induces hydroxylation of integrin β1. Integrin β1 protein is stabilized by the hydroxylation and recruited to cell membrane as its functional site, which contributes to invasion/metastasis of SCCs. Loss of PLOD2 by contrast causes instability of integrin β1, which results in loss of tumor metastasis. PM (plasma membrane), ER (endoplasmic reticulum), integrin α (yellow), and β1 (red).</p></caption><graphic xlink:href="gr7"></graphic></fig></p><sec id="sec3.1"><title>Limitations of the Study</title><p id="p0140">This study provides a crosstalk between PLOD 2 and integrin β1 in cellular motility, and the hydroxylation on integrin β1 by PLOD2 is critical for its intracellular stability. However, we could not optimize the <italic>in vitro</italic> reaction of integrin β1 hydroxylation, because of an insoluble integrin β1 mutant substrate, and we were unable to directly detect the function of PLOD2 and integrin β1 in patient-derived primary SCC samples. 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Transparent Methods, Figures S1–S13, and Table S1</title></caption><media xlink:href="mmc1.pdf"></media></supplementary-material></p></sec><ack id="ack0010"><title>Acknowledgments</title><p>We thank Dr. K. Honma and Dr. T. Kawasaki (Niigata Cancer Center Hospital) for pathological informations. The collaboration was approved both by Niigata University ethical committee and Niigata Cancer Center ethical committee. We thank Dr. K. Myrick for critical reading of the manuscript. We are also grateful to Ms. A. Ageishi and Ms. N. Sumi for kind assistance for office procedures. This work was supported by <funding-source id="gs1">Grant-in-Aid for Challenging Exploratory Research</funding-source> (Grant Number JP 24659170; K.S.), JAPAN SOCIETY FOR THE PROMOTION OF SCIENCE (JSPS).</p><sec id="sec6"><title>Author Contributions</title><p id="p0160">Conceptualization, K.S., E.K.; Methodology, Y.U., K.S.; Investigation, Y.U., K.S., H.I., I.S., Y.K., M.S.; Writing—Original Draft, K.S., E.K.; Writing—Review & Editing, E.K.; Funding Acquisition, Y.U., K.S., E.K.; Resources, Y.U., K.S, A.H.; Supervision, E.K.</p></sec><sec sec-type="COI-statement" id="sec7"><title>Declaration of Interests</title><p id="p0165">The authors declare no competing interests.</p></sec></ack><fn-group><fn id="appsec1" fn-type="supplementary-material"><p id="p0170">Supplemental Information can be found online at <ext-link ext-link-type="uri" xlink:href="https://doi.org/10.1016/j.isci.2020.100850" id="intref0010">https://doi.org/10.1016/j.isci.2020.100850</ext-link>.</p></fn></fn-group></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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