Matrix metalloproteinase-21 promotes metastasis via increasing the recruitment and M2 polarization of macrophages in HCC

Abstract

MMP-21 is a newly identified member of the matrix metalloproteinase family and has been reported to regulate both embryonic development and tumor progression. However, the roles of MMP-21 in hemofiltrate C-C chemokine (HCC) remain largely unclear. In this study, we used western blot, qPCR and immunohistochemistry (IHC) to determine the upregulation of MMP-21 in HCC tissues, and showed that the increase in MMP-21 was associated with vascular invasion and poor prognosis. Although changing levels of MMP-21 in HCC cell lines had no significant effect on cell migration or invasion abilities in in vitro transwell tests, both IHC analysis and in vivo mouse models proved that upregulated MMP-21 promoted metastasis. Functional enrichments of MMP-21 using The Cancer Genome Atlas (TCGA) data suggested that MMP-21 might regulate metastasis via macrophages. Further experiments proved that MMP-21 enhanced macrophage recruitment by increasing CCL-14 levels and promoted M2-type polarization of macrophage by elevating the expression of CSF-1 and FGF-1. Taken together, this study revealed that MMP-21 controlled the tumor microenvironment remodeling and functional regulation of macrophages to regulate HCC metastasis.

Keywords: CCL-14; CSF-1; FGF-1; HCC; MMP-21; macrophage; metastasis.

FIGURE 1

MMP‐21 is upregulated in HCC and related to poor prognosis. (A) Relative mRNA expression of MMP‐21 in HCC tissues and adjacent non‐tumor tissues in public data sets. (B, C) MMP‐21 expression in 30 paired HCC tissues and adjacent non‐tumor tissues. T, HCC tissue, N, adjacent non‐tumor tissue. (D) Statistical data of western blot. (E) Representative IHC staining images in non‐tumor tissue and HCC tissues. (F) Statistical data of the IHC staining score. (G, H) Correlation between MMP‐21 and vascular invasion, TNM stage in IHC cohort. (I) Overall survival of HCC patients with different MMP‐21 expression levels

FIGURE 2

MMP‐21 promotes EMT in vivo, whereas it confers no significant function in vitro. (A, B) Representative IHC staining images of MMP‐21 and E‐cadherin, N‐cadherin in human HCC. (C, D) Representative IHC staining images of MMP‐21 and E‐cadherin, N‐cadherin in liver orthotopic xenografts from mouse. (E) In vivo effect of MMP‐21 in orthotopic liver transplanted mice. (F) In vivo effect of MMP‐21 in mouse lung metastasis models. (G) Representative images of hematoxylin‐eosin staining. The white arrows point to the tumor region. (H) Kaplan–Meier analysis for overall survival of mouse lung metastasis models. (I) Kaplan–Meier analysis for overall survival of orthotopic liver transplanted mice. *p < 0.05, **p < 0.01

FIGURE 3

MMP‐21 promotes the recruitment of macrophages. (A) Representative IHC staining images of MMP‐21 and CD68 in human HCC tissues. (B) The correlation between MMP‐21 and CD68‐positive macrophages. (C) Representative IHC staining images of MMP‐21 and F4/80 in liver orthotopic xenografts from tumor‐bearing mouse. (D) Cell count of macrophages. (E, F) Transwell assays to determine macrophage recruitment. (G, H) Transwell assays to determine macrophage recruitment using culture medium from HCC cells. *p < 0.05, **p < 0.01. [Correction added on 21 September 2022, after first online publication: in Figure 3, parts E, F, G and H have been replaced with the correct images].

FIGURE 4

CCL‐14 is the key secreted molecule from HCC cells in MMP21‐mediated macrophage recruitment. (A) Functional KEGG pathway enrichment from a TCGA‐LIHC data set. (B) The correlation between MMP‐21 and CCL‐14 from a TCGA‐LIHC data set. (C) Protein levels of MMP‐21 and CCL‐14 in HCC tissues. (D) Correlation between MMP‐21 and CCL‐14 for mRNA levels in HCC tissues. (E) IHC analysis of MMP‐21, CCL‐14, and CD68 in HCC tissues. (F) CCL‐14 expression in 7402 cells with increasing MMP‐21. (G) CCL‐14 expression in 7721 cells with decreasing MMP‐21. (H) Knockdown efficiency of CCL‐14 in 7402 cells. (I) Transwell test to determine the macrophage recruitment using 7402 cells with CCL‐14 knockdown. (J) Overexpression efficiency of MMP21 in a CCL14‐knockdown 7402 stable cell line. (K) Macrophage recruitment assessment using the CCL14‐knockdown 7402 stable cell line with MMP‐21 overexpression. *p < 0.05, **p < 0.01; ns, no significance. [Correction added on 21 September 2022, after first online publication: in Figure 4, part G has been replaced with the correct image].

FIGURE 5

MMP‐21 regulates the expression of FGF‐1 and CSF‐1 to promote macrophage polarization toward M2‐type. (A) Genes strongly correlated with MMP‐21 in the three pathways (R ≥ 0.3). (B) Correlations of CSF‐1, FGF‐1, and MMP‐21 from the TCGA‐LIHC data set. (C) Relative mRNA expression of CSF‐1 and FGF‐1 in HCC cell lines with overexpression or knockdown of MMP‐21. (D) Relative mRNA expression of M1 and M2 markers in THP1 cells incubated with culture medium from HCC cell lines. (E) IHC analysis of CSF‐1, FGF‐1 and CD206 in human HCC tissues with different MMP‐21 levels. (F) Statistical data for IHC staining score and the cell count of CD206‐positive macrophages in human HCC tissues. *p < 0.05; ns, no significance

FIGURE 6

Schematic describing how MMP‐21 functions in HCC