FDPS promotes glioma growth and macrophage recruitment by regulating CCL20 via Wnt/β-catenin signalling pathway

Abstract

Glioma is one of the most lethal tumours and common malignant in the central nervous system (CNS), which exhibits diffuse invasion and aggressive growth. Several studies have reported the association of FDPS to tumour development and progression. However, the role of FDPS in progression of glioma and macrophage recruitment is not well-elucidated. In the current study, a remarkable enhancement in FDPS level was observed in glioma tissues and associated with poor prognosis, contributed to tumour growth. FDPS was correlated with macrophage infiltration in glioma and pharmacological deletion of macrophages largely abrogated the oncogenic functions of FDPS in glioma. Mechanistically, FDPS activated Wnt/β-catenin signalling pathway and ultimately facilitates macrophage infiltration by inducing CCL20 expression. In conclusion, overexpressed FDPS exhibits an immunomodulatory role in glioma. Therefore, targeting FDPS may be an effective therapeutic strategy for glioma.

Keywords: CCL20; FDPS; TAMs; Wnt/β-catenin; glioma.

FIGURE 1

FDPS is expressed at high levels in glioma tissues. The expression of FDPS in glioma tissue and adjacent non‐tumour tissue from same patient was assessed by immunohistochemistry (A), real‐time PCR (B) and Western blotting (C). (D) Kaplan‐Meier curves showing the overall survival of glioma patients with low versus high FDPS expression (P = 0.025). Error bars represent the SD, **P < 0.01 (Scale bars, 50 μm)

FIGURE 2

FDPS regulates glioma cell growth. A, The protein expression levels of FDPS in human NHAs and glioma cell lines determined by real‐time PCR. B, The protein expression levels of FDPS in human NHAs and glioma cell lines determined by Western blotting. C, A172 and H4 cells with FDPS overexpression were established. The level of FDPS in these established cell lines was verified by Western blotting. D and E, Cell proliferation was examined by MTS in A172 and H4 cells with FDPS overexpression. F, EdU assay of indicated cells with FDPS overexpression. G, Colony formation assays of indicated cells with FDPS overexpression. H, U87 cells with stable expression of shCon/shFDPS were subcutaneous injected into NSG mice. Tumour size was measured every 2 days. I, The weight of tumours formed at day 17th. J, The FDPS expression in tumours was analysed by Western blotting. K, The expression of Ki67 was evaluated by IHC staining. Error bars represent the SD, *P < 0.05; **P < 0.01; ***P < 0.001 (Scale bars, 50 μm)

FIGURE 3

FDPS promotes glioma invasion and migration in vitro. A, The invasion capacity of FDPS in A172 and H4 cells with FDPS overexpression was examined by Matrigel invasion assay. B, The invasion capacity of FDPS in U87 and U251 cells with FDPS silencing was examined by Matrigel invasion assay. C, The migration capacity of FDPS in A172 and H4 cells with FDPS overexpression was examined by wound healing assay. D, The migration capacity of FDPS in U87 and U251 cells with FDPS silencing was examined by wound healing assay. E, The indicated protein level was analysed by Western blotting in A172 and H4 cells with FDPS overexpression. F, The indicated protein level was analysed by Western blotting in U87 and U251 cells with FDPS silencing. Error bars represent the SD, **P < 0.01

FIGURE 4

FDPS promotes tumour growth in a macrophage‐dependent manner. A, The level of FDPS in GL261 cells was analysed by Western blotting. B and C, Representative immunohistochemistry of F4/80+ cells in sections from glioma tumours obtained from C57BL/6J mice treated with clodronate liposomes or PBS liposomes. D, Volume of GL261 tumours treated as indicated. Error bars represent the SD, *P < 0.05; **P < 0.01 (Scale bars, 50 μm)

FIGURE 5

CCL20 mediates the promoting role of macrophage infiltration induced by FDPS. A, The mRNA level of indicated chemokines in the conditional medium from U87 cells with or without FDPS silencing was analysed by real‐time PCR. B, The mRNA level of CCL20 in the conditional medium from U251 cells with or without FDPS silencing was analysed by real‐time PCR. C, The level of CCL20 in the conditional medium from U87 and U251 cells with or without FDPS silencing was analysed by ELISA. D, The level of CCL20 in the conditional medium from A172 and H4 cells with or without FDPS overexpression was analysed by ELISA. E, Volume of parental and FDPS‐overexpressing GL261 tumours treated as indicated. F, F4/80+ cells in sections from indicated glioma tumours. Error bars represent the SD, *P < 0.05; **P < 0.01; ***P < 0.001

FIGURE 6

FDPS regulates Wnt/β‐catenin signalling pathway activation. (A) mRNA level of indicated genes in U87 and U251 cells with or without FDPS silencing was analysed by real‐time PCR. (B) Protein level of indicated genes in U87 and U251 cells with or without FDPS silencing was analysed by Western blotting. (C) mRNA level of indicated genes in A174 and H4 cells with or without FDPS overexpression was analysed by real‐time PCR. (D) Protein level of indicated genes in A172 and H4 cells with or without FDPS overexpression was analysed by Western blotting. (E) Protein level of p‐β‐catenin in U87 and U251 cells with or without FDPS silencing was analysed by Western blotting. (F) Protein level of p‐β‐catenin in A172 and H4 cells with or without FDPS overexpression was analysed by Western blotting. The activity of TCF/β‐catenin reporter (TOP/FOP Flash) in FDPS knockdown (G) and FDPS‐overexpressing cells (H). Error bars represent the SD, *P < 0.05; **P < 0.01

FIGURE 7

Wnt/β‐catenin signalling pathway is required for FDPS‐mediated CCL20 induction. A, mRNA level of CCL20 in the conditional medium from A172 FDPS‐overexpressing cells with or without XAV‐939 pre‐treatment was analysed by real‐time PCR. B, mRNA level of CCL20 in the conditional medium from H4 FDPS‐overexpressing cells with or without XAV‐939 pre‐treatment was analysed by real‐time PCR. C, The level of CCL20 in the conditional medium from A172 FDPS‐overexpressing cells with or without XAV‐939 pre‐treatment was analysed by ELISA. D, The level of CCL20 in the conditional medium from H4 FDPS‐overexpressing cells with or without XAV‐939 pre‐treatment was analysed by ELISA. E, The level of CCL20 in the conditional medium from U87 FDPS knockdown cells with or without β‐catenin overexpression was analysed by ELISA. F, The level of CCL20 in the conditional medium from U251 FDPS knockdown cells with or without β‐catenin overexpression was analysed by ELISA. G, CCL20 was analysed by IHC in the shcon and shFDPS U87 tumours. H, CCL20 was analysed by IHC in the vector and FDPS GL261 tumours. I, p‐β‐catenin was analysed by IHC in the shcon and shFDPS U87 tumours. J, p‐β‐catenin was analysed by IHC in the vector and FDPS GL261 tumours. K, Volume of parental and FDPS‐overexpressing GL261 tumours treated as indicated. L, F4/80+ cells in sections from indicated glioma tumours. Error bars represent the SD, *P < 0.05; **P < 0.01