CTHRC1 induces non-small cell lung cancer (NSCLC) invasion through upregulating MMP-7/MMP-9

Abstract

Background: The strong invasive and metastatic nature of non-small cell lung cancer (NSCLC) leads to poor prognosis. Collagen triple helix repeat containing 1 (CTHRC1) is involved in cell migration, motility and invasion. The object of this study is to investigate the involvement of CTHRC1 in NSCLC invasion and metastasis.

Methods: A proteomic analysis was performed to identify the different expression proteins between NSCLC and normal tissues. Cell lines stably express CTHRC1, MMP7, MMP9 were established. Invasion and migration were determined by scratch and transwell assays respectively. Clinical correlations of CTHRC1 in a cohort of 230 NSCLC patients were analysed.

Results: CTHRC1 is overexpressed in NSCLC as measured by proteomic analysis. Additionally, CTHRC1 increases tumour cell migration and invasion in vitro. Furthermore, CTHRC1 expression is significantly correlated with matrix metalloproteinase (MMP)7 and MMP9 expression in sera and tumour tissues from NSCLC. The invasion ability mediated by CTHRC1 were mainly MMP7- and MMP9-dependent. MMP7 or MMP9 depletion significantly eradicated the pro-invasive effects mediated by CTHRC1 on NSCLC cells. Clinically, patients with high CTHRC1 expression had poor survival.

Conclusions: CTHRC1 serves as a pro-metastatic gene that contributes to NSCLC invasion and metastasis, which are mediated by upregulated MMP7 and MMP9 expression. Targeting CTHRC1 may be beneficial for inhibiting NSCLC metastasis.

Keywords: CTHRC1; Invasion/metastasis; Lung cancer; MMP7; MMP9.

Fig. 1

Differential protein expression in NSCLC and corresponding adjacent nontumor tissues (ANTs) samples and the correlation with tumor metastasis. Thirty-four differential protein spots were identified from NSCLC (a, right) and ANT (a, left) samples in the representative silver-stained 2D gel image, and the outlined areas show CTHRC1 upregulation in NSCLC tissues (n = 20). b MS identification analysis of CTHRC1. The red arrow marks the specific peak corresponding to the CTHRC1 protein. c, d Comparative CTHRC1 protein quantification in paired primary NSCLC tissues (T) and their corresponding ANTs in the left panel (n = 20) as measured by western blotting and IHC (**p < 0.01). e CTHRC1 levels increase as tumour grade (I–IV) progresses, as determined by IHC staining (n = 230). Representative western blot bands and IHC images are presented. Three independent experiments were performed

Fig. 2

CTHRC1 inhibits adhesion and promotes NSCLC cell migration and invasion of in vitro and metastasis in vivo. a, d CTHRC1 overexpression inhibited cell adhesion; CTHRC1 knockdown increased cell adhesion; (b-f) Transwell assay results indicated that ectopic overexpression of CTHRC1 promoted cell invasion; knockdown of endogenous CTHRC1 inhibited cell invasion. Migratory cells were stained with HE and qualitatively assessed, as summarized in the bar graphs. g-j A wound assay indicated CTHRC1 overexpression increased tumour cell migration; CTHRC1 knockdown inhibited tumour cell migration. Migration distances were measured and are summarized in the bar graphs; *p < 0.05 and **p < 0.01

Fig. 3

CTHRC1 regulates MMP7 and MMP9 expression in vitro and in vivo. a The heat map shows the concentration distributions of CTHRC1 and diverse MMPs in the sera of NSCLC patients as measured by ELISA (n = 92). b mRNA levels of CTHRC1 and diverse MMPs in NSCLC tissues were further analysed by reverse dot blot hybridization (n = 20). c, d CTHRC1 expression was positively correlated with MMP7 and MMP9 in both sera and primary tumour tissues. e, f Western blot analysis demonstrated increased MMP7 and MMP9 production accompanying the ectopic overexpression of CTHRC1; CTHRC1 knockdown downregulated MMP7 and MMP9 expression. g, h The western blot results were semi-quantified and shown in bar graph form. Representative images were shown and all the experiments had been repeated 3 times. *p < 0.05 and **p < 0.01

Fig. 4

The regulation of CTHRC1 on adhesion, migration, invasion and metastasis of tumor cells was mediated by MMP7 and MMP9. a Tumour cell adhesion ability decreased in CTHRC1-overexpressing cells. This decreased adhesion ability was elevated when either MMP7 or MMP9 was knocked down. b, c HE staining of cells invading through the Transwell gel demonstrated increased tumour cell invasion accompanying the ectopic overexpression of CTHRC1, which was inhibited by knocking down either MMP7 or MMP9. d-f A wound assay showing increased migration distance accompanying CTHRC1 overexpression. Knocking down either MMP7 or MMP9 inhibited the increased migration distance mediated by CTHRC1. *p < 0.05 and **p < 0.01

Fig. 5

CTHRC1 transcriptionally modulates MMP7 expression through c-JUN. a CTHRC1 overexpression increased MMP7 promoter activity. b MMP7 promoter activity was inhibited when CTHRC1 was downregulated. c The MMP7 promoter region was cloned as three fragments (P1 to P3). d Transactivating activity of CTHRC1 on serial MMP7 promoter fragments, as indicated in NCI-H1975 cells. CTHRC1 overexpression enhanced promoter activity in P1 and P2. CTHRC1 knockdown weakened promoter activity in P1 and P2. e Schematic illustration showing the PCR-amplified fragments of the MMP7 promoter. f Regions of the MMP7 promoter that were physically associated with CTHRC1 were analysed in a ChIP assay. IgG was used as a negative control. PCR amplification indicated the binding efficiency to region 2 was significantly decreased in NCI-H1975-AP-1/c-Jun siRNA cells. g CTHRC1 overexpression upregulated the expression of MMP7, and the upregulation of MMP7 was abolished when c-JUN was knocked down as measured by western blot. Representative bands are shown. h The western blot results were semi-quantified and are shown in bar graph form. Experiments had been repeated three times. *p < 0.05, **p < 0.01

Fig. 6

CTHRC1 transcriptionally modulates MMP9 expression through c-JUN and NF-κB signals. a CTHRC1 overexpression increased MMP9 promoter activity. b MMP9 promoter activity was inhibited when CTHRC1 was downregulated. c The promoter region was cloned as seven fragments (P1 to P7). d Transactivating activity of CTHRC1 on serial MMP9 promoter fragments as indicated in NCI-H1975 cells. CTHRC1 overexpression enhanced the promoter activity in P1–5, while CTHRC1 knockdown weakened promoter activity in P1–5. e Schematic illustration showing the PCR-amplified fragments of the MMP9 promoter. f Regions of the MMP9 promoter that were physically associated with CTHRC1 were analysed in a ChIP assay. IgG was used as a negative control. PCR amplification indicated the binding efficiencies to region 1 and region 4 were decreased in NCI-H1975-AP-1/c-Jun siRNA cells, and binding efficiency to region 1 was significantly decreased in NCI-H1975-NF-κB siRNA cells. g Western blotting revealed upregulated MMP9 expression accompanying the ectopic overexpression of CTHRC1; this MMP9 upregulation was abolished when either NF-κB or c-JUN was knocked down. Knockdown of NF-κB and c-JUN together further decreased MMP9 expression. Representative bands are shown. h The western blot results were semi-quantified and are shown in bar graph form. Experiments had been repeated 3 times. *p < 0.05, **p < 0.01

Fig. 7

Clinical relevance of CTHRC1 expression in NSCLC patients. a, b Circulating tumour cells (CTC) were detected using the NanoVelcro system. The CTHRC1 concentration positively correlated with the number of CTCs. c, d The cut-off value for CTHRC1 had optimal sensitivity and specificity for metastasis, with an area under the curve of 0.97 (95% CI: 0.941–1.000; p < 0.001), and optimal sensitivity and specificity for recurrence, with an area under the curve of 0.691 (95% CI: 0.604–0.788; p < 0.001). e IHC data indicating the post-surgery survival rate was significantly lower in the group of CTHRC1-high patients. f, g CTHRC1-low patients had much higher post-surgery survival rates, at both the early and late stages. h Summary of overall survival with high or low CTHRC1 expression. i Compared to low CTHRC1, MMP7 and MMP9 expression, the combined high expression of CTHRC1, MMP7 and MMP9 was associated with a significantly lower post-surgery survival rate. j Summary of overall survival with high or low combinatorial CTHRC1, MMP7 and MMP9 expression