Promotion of epithelial-mesenchymal transformation by hepatocellular carcinoma-educated macrophages through Wnt2b/β-catenin/c-Myc signaling and reprogramming glycolysis

Background: Tumour-associated macrophages (TAMs) in the tumour microenvironment (TME) can promote the progression of hepatocellular carcinoma (HCC). Some tumours can be suppressed by targeting Wnt2b in tumour cells. However, the role of Wnt2b in HCC is still unknown. In particular, the role of Wnt2b-mediated signal activation in macrophage polarization in the HCC microenvironment, and the regulatory effect between Wnt and glycolysis in TAMs has not been described.

Methods: The expression of Wnt2b in TAMs was detected by qPCR and immunofluorescence. Wnt2b/β-catenin interference in HCC-TAMs was performed by lentivirus carrying targeted shRNA or TLR9 agonist. Markers related to macrophage polarization and the changes of key glycolytic enzymes expression were detected by flow cytometry and qPCR. ECAR was analysed by Seahorse analyser. MTT assay, wound healing assay, western blotting were used to evaluate the promoting effect of different HCC-TAMs on the proliferation, migration and EMT of HCC in vitro. Tumour cells and different HCC-TAMs were injected via subcutaneously into immunodeficient mice to assess the effects of CpG ODN, Wnt2b, or β-catenin on HCC-TAMs in tumour growth in vivo.

Results: Polarization-promoting factors derived from HCC cells upregulated the expression of Wnt2b in macrophages, which promoted the polarization of TAMs to M2-like macrophages by activating Wnt2b/β-catenin/c-Myc signalling. Furthermore, this process was associated with the activation of glycolysis in HCC-TAMs. These HCC-TAMs could promote the development of EMT, proliferation, and migration of HCC. In addition to silencing Wnt2b or β-catenin expression, TLR9 agonist CpG ODN downregulated the level of glycolysis and inhibited the M2 polarization of HCC-TAMs, reversing the tumour-promoting effects of TAMs in vitro and vivo.

Conclusions: As a potential target for HCC therapy, Wnt2b may play an important regulatory role for the functions of TAMs in the TME. Moreover, the TLR9 agonist CpG ODN might act as a Wnt2b signal inhibitor and can potentially be employed for HCC therapy by disturbing Wnt2b/β-catenin/c-Myc and inhibiting glycolysis in HCC-TAMs.