Magnesium cantharidate suppresses hepatocellular carcinoma progression and modulates tumor-associated macrophage polarization: potential involvement of ERK1/2 signaling and de novo serine synthesis

Background Hepatocellular carcinoma (HCC) is characterized by an immunosuppressive tumor microenvironment in which M2-like tumor-associated macrophages (TAMs) contribute to tumor progression. De novo serine synthesis is increasingly recognized as an important regulator of macrophage polarization. Magnesium cantharidate (MC) exhibits anti-HCC activity; however, whether MC regulates TAM polarization through the de novo serine synthesis pathway remains unclear. Methods HCC-derived TAMs were generated using conditioned media from HCC cells and treated with MC. Macrophage polarization, metabolomic and transcriptomic alterations, de novo serine synthesis and activation of the ERK signaling pathway were evaluated. Pharmacological inhibition and siRNA-mediated knockdown of PHGDH were used for functional validation. TAM-conditioned media were used to assess their effects on HCC cells, and key findings were further validated in a Hep3B/TAMs xenograft model. Results MC significantly inhibited the growth of Hep3B/TAM xenografts. Additionally, MC significantly reduced CD206 + TAMs and decreased the expression of the M2-associated markers Arg-1, IL-10 and TGFB1 in vivo and in vitro . Integrated metabolomic and transcriptomic analyses showed that MC altered serine-associated metabolic programs in TAMs. Moreover, MC reduced the expression of key enzymes involved in de novo serine synthesis (PHGDH, PSAT1 and PSPH) in vivo and in vitro . Pharmacological inhibition or knockdown of PHGDH further enhanced the suppressive effect of MC on M2-like TAM polarization. Moreover, conditioned media from MC-treated TAMs inhibited HCC cell proliferation, migration and invasion, and these effects were further strengthened by PHGDH inhibition or knockdown. Molecular docking suggested favorable binding potential between MC and ERK1/2. MC reduced ERK1/2 phosphorylation in xenograft tumors and in TAMs. Additionally, pharmacological ERK1/2 inhibition further decreased PHGDH, PSAT1 and PSPH expression and potentiated the inhibitory effect of MC on the expression of key enzymes involved in de novo serine synthesis. Conclusion MC could suppress HCC xenograft growth and modulate M2-like TAM-associated phenotypes, accompanied by alterations in serine metabolism and ERK1/2 signaling. These findings suggested the potential involvement of ERK1/2 signaling and de novo serine synthesis-related pathways in the antitumor effects of MC.