PLOD2 Is Essential to Functional Activation of Integrin β1 for Invasion/Metastasis in Head and Neck Squamous Cell Carcinomas

Abstract

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 in vivo, 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.

Keywords: Cancer; Molecular Biology.

Graphical abstract

Figure 1

Expression of the Various Hydroxylases in Oral SCC Cells (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). (B) Protein expression of PLOD family in SCC lines and HaCaT by immunoblotting. (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. (D) RNA interference (siRNA)-mediated knockdown of PLOD in oral SCCs demonstrated the attenuated protein expression by immunoblotting. (E) GFP-expressing SCC cells were transfected with control siRNA (siCtrl) or with PLOD-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.

Figure 2

PLOD2 Is Essential for Stabilization of Integrin β1 (A) Immunofluorescence revealed expression, and localization of CDH1 was not affected by siPLOD2-treatment in SCC cells. (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. (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. (D) Semiquantitative expression of integrin β1 mRNA by RT-PCR with or without siPLOD2-treatement. (E) Comparative ratio of integrin β1 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). (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. (G) Effect of PLOD2 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. (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).

Figure 3

Specific Coupling of Integrin β1 with PLOD2 (A) The construction of PLOD2 expressor fused Monti-red tag (PLOD2-Red) and integrin β1 expressor fused Ash tag (Integrin β1-Ash) (left panel). Schematic representation of mechanism for intracellular fluorescent dot formation in response to specific protein-protein interaction (right panel). (B) Marked aggregation of fluorescent dots was detected only in the HeLa cells cotransfected with PLOD2-Red and Integrin β1-Ash. Scale bar = 20 μm. (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. (D) The fluorescent dots were observed at lamellipodia of the PLOD2-Red-Integrin β1-Ash cotransfected cells or the Integrin β1-Red-PLOD2-Ash cotransfected cells. Hatched box indicated hyperview field in the right. Scale bar = 20 μm. (E) Domain organization of the FLAG epitope tagged-Integrin β1 expressor, Myc epitope-tagged PLOD2 mutant expressor lacking PKHD domain (ΔPKHD) used in the immunoprecipitation assay. (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.

Figure 4

Hydroxylation of Integrin β1 in Presence of PLOD2 and Its Significance in Intracellular Localization of Integrin β1 (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. (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-PLOD2-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). (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. (D) Hydroxylation reaction of PLOD2 was carried out in vitro as described in Methods. 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). (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). (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.

Figure 5

Deficiency of PLOD2 Inhibited Metastasis of SCC Cells In Vivo (A) In vivo 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. (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.

Figure 6

Expression of PLOD2 and Integrin β1 in Head and Neck SCCs from the Patients 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.

Figure 7

Intracellular Dynamics of Integrin β1 Mediated by PLOD2 in SCC Cells 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).