PLOD3 promotes lung metastasis via regulation of STAT3

Abstract

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD3), a membrane-bound homodimeric enzyme, hydroxylates lysyl residues in collagen-like peptides; however, its role in lung cancer is unknown. This study aimed to investigate the role of PLOD3 as a pro-metastatic factor and to elucidate the underlying mechanism. First, we experimentally confirmed the release of PLOD3 in circulation in animal models, rendering it a potential serum biomarker for lung cancer in humans. Thereafter, we investigated the effects of PLOD3 overexpression and downregulation on cancer cell invasion and migration in vitro and in vivo, using human lung cancer cell lines and a mouse tumor xenograft model, respectively. Further, PLOD3 levels were determined in lung tissue samples from lung cancer patients. Functional analyses revealed that PLOD3 interacts with STAT3, thereby expressing matrix metalloproteinases (MMP-2 and MMP-9) and with urokinase plasminogen activator (uPA) to enhance tumor metastasis. PLOD3 and the STAT3 pathway were significantly correlated in the metastatic foci of lung cancer patients; PLOD3-STAT3 levels were highly correlated with a poor prognosis. These results indicate that PLOD3 promotes lung cancer metastasis in a RAS-MAP kinase pathway-independent manner. Therefore, secreted PLOD3 serves as a potent inducer of lung cancer metastasis and a potential therapeutic target to enhance survival in lung cancer.

Fig. 1. Endogenous PLOD3 plays an important role in lung cancer.

a Representative microscopic images of lung cancers and their normal tissue counterparts stained with an anti-PLOD3 antibody (left panel, scale bar, 100 μm). Staining intensity was scored as follows in all patient samples (n = 59). The scores are calculated by multiplying the staining intensity with the percentage of stained cells in primary lung cancer relative to the paired normal tissue. Statistical significance was determined by Student’s t-test. ***P < 0.001. b PLOD3 upregulation is positively correlated with the stage of lung cancer in all patient samples (n = 59). c Box plot analysis of the PLOD3 mRNA levels obtained from ONCOMINE from lung cancer patients. Gene: PLOD3; Analysis type: cancer vs normal analysis; Data type: mRNA; Sample type: clinical specimen; Lung. d The effects of PLOD3 on the overall survival of lung cancer patients, using Kaplan–Meier plotter analysis. e R-H460 and A549 cells were cultured up to 80% confluence, followed by changing of the medium to fresh serum-free RPMI1640. After 6 h, collected supernatants were analyzed via immunoblotting with anti-PLOD3 antibody (left panel) and enzyme-linked immunosorbent assay (ELISA) at 450 nm (right panel). Statistical significance was determined by Student’s t-test. ***P < 0.001. f R-H460 and A549 cells (1 × 10⁶ cells) were inoculated subcutaneously into the backs of nude mice. After tumorigenesis, mice were killed. Serum collected from each group was analyzed via ELISA. Statistical significance was determined by Student’s t-test. **P < 0.01

Fig. 2. PLOD3 possesses greater potential to promote lung cancer malignancy.

a PLOD3 protein expression was assessed via western blot analysis, following transfection of HA-PLOD3 in H460 cells for 48 h. b A transwell assay was performed to evaluate the motility of PLOD3-overexpressing H460 cells. PLOD3 displayed superior potential to promote cell migration. The relative numbers of migratory cells were determined and presented as the mean ± SD values from three independent experiments. Statistical significance was determined by Student’s t-test. ***P < 0.001. c The invasion assay revealed different cell motilities in PLOD3-overexpressing lung cancer cells. PLOD3 expression promoted the invasion of H460 cells. The relative numbers of invasive cells were determined and presented as the mean ± SD values from three independent experiments. Statistical significance was determined by Student’s t-test. ***P < 0.001. d PLOD3 protein and mRNA levels were determined via western blotting (lower) and quantitative reverse transcription polymerase chain reaction (qRT-PCR; upper), respectively, after the indicated concentration of siPLOD3 was transfected in R-H460 cells for 24 h. The qRT-PCR data are expressed as the mean ± SD values *P < 0.05, **P < 0.01. e The cell viability assay for siPLOD3-treated cells for 24 h; x-axis: siCON, siPLOD3 (concentration). f A transwell assay was conducted to evaluate the motility of PLOD3 knockdown R-H460 cells at 12 h. Statistical significance was determined by Student’s t-test. **P < 0.01. g Cell invasion assay revealed that siPLOD3 had a limited effect on pro-metastasis at 24 h in R-H460 cells. Statistical significance was determined by Student’s t-test. **P < 0.01. h R-H460 cells were treated with siPLOD3 and then incubated for 24 and 48 h. The cells were then scraped with a sterile 200-μl pipette tip for the scratch assay

Fig. 3. Loss of PLOD3 suppressed R-H460 tumor metastasis in vivo.

a Representative micrographs with metastatic nodules are shown via Bouin’s staining. b Hematoxylin and eosin staining were performed using xenografts. c, d The number and volume of metastatic nodules were determined microscopically in PLOD3 xenograft tumors. Statistical significance was determined by Student’s t-test. *P < 0.05, **P < 0.01. e, f Estimation of hPLOD3 mRNA and PLOD3 protein levels in the lung nodules. Statistical significance was determined by ANOVA analysis. ***P < 0.001

Fig. 4. MMP-2/9 and uPA expression in siPLOD3- or HA-PLOD3-treated lung cancer cells.

a hMMP-2/9 mRNA levels were determined after siPLOD3 treatment in vivo. Statistical significance was determined by Student’s t-test. *P < 0.05; **P < 0.01. b In vivo immunofluorescence of siCON- and siPLOD3-treated groups. c hMMP-2/9 and hu-PA mRNA levels after the indicated concentration of HA-PLOD3 was transfected were determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). Statistical significance was determined by Student’s t-test. *P < 0.05. d H460 cells were transfected with siCON or the indicated concentration of siPLOD3 for 24 h. hMMP-2/9 and hu-PA mRNA levels were determined via RT-qPCR. Statistical significance was determined by ANOVA analysis. **P < 0.01, ***P < 0.001. e–g Correlation of hPLOD3 with hMMP-2 or hMMP-9 or hu-PA transcripts in the cbioportal datasets of lung cancer patients

Fig. 5. PLOD3 regulated the malignant phenotype via STAT3 signaling.

a Immunostaining of PLOD3, pS-STAT3, and STAT3 in PLOD3 knockdown xenograft tumors. b Protein levels of PLOD3, pS-STAT3, pY-STAT3, and STAT3 were determined via western blotting in PLOD3 xenograft tumors. Statistical significance was determined by Student’s t-test. *P < 0.05, **P < 0.01. c Levels of the indicated proteins were determined via western blotting after siPLOD3 treatment in R-H460 cells. d Levels of the indicated proteins were determined via western blotting in HA-PLOD3-overexpressing H460 cells. e, f The HA-PLOD3 and STAT3 inhibitor S3I-201-treated H460 cells were analyzed via a transwell assay to evaluate cell motility and invasiveness. Statistical significance was determined by Student’s t-test. *P < 0.05; **P < 0.01

Fig. 6. Correlation between the PLOD3-STAT3 signaling pathway and the survival and prognosis of lung cancer patients.

a The in situ proximity ligation assay was performed for R-H460 cells stained with STAT3, pS-STAT3, and pY-STAT3. Statistical significance was determined by Student’s t-test. **P < 0.01; ***P < 0.001. b Immunoblotting analysis of lysates after immunoprecipitation from R-H460 cells with expressing PLOD3 and STAT3. c Representative microscopic images of specimens of lung cancer patients stained with an anti-PLOD3, anti-STAT3, anti-pS-STAT3, and anti-pY-STAT3 antibodies. d Correlation between PLOD3 and STAT3 transcripts in the cbioportal datasets of lung cancer patients

Fig. 7. PLOD3-induced metastasis is independent of the RAS-MAPK pathway.

a Ras expression assessed via western blot analysis following transfection with siPLOD3 in R-H460 cells (upper). Ras activity was determined via an enzyme-linked immunosorbent assay-based activity assay after siPLOD3 treatment in R-H460 cells (lower, x-axis: siCON, siPLOD3). b Protein levels of MAP kinases were determined via western blotting after siPLOD3 treatment in R-H460 cells. c A cell invasion assay showing that SB and SP have a limited effect on pro-metastatic PLOD3 in R-H460 cells. (upper). Protein levels of indicated proteins determined via western blotting after treatment with siPLOD3 and MAP kinase inhibitor (p38 inhibitor, SB203580 (SB); JNK inhibitor, SP600125 (SP)) in R-H460 cells (lower). Statistical significance was determined by Student’s t-test. ***P < 0.001