Porcupine inhibitor LGK‑974 inhibits Wnt/β‑catenin signaling and modifies tumor‑associated macrophages resulting in inhibition of the malignant behaviors of non‑small cell lung cancer cells

Abstract

Tumor‑associated macrophages (TAMs) are critical components of the tumor microenvironment that are tightly associated with malignancies in human cancers, including lung cancer. LGK‑974, a small molecular inhibitor of Wnt secretion, was reported to block Wnt/β‑catenin signaling and exert anti‑inflammatory effects by suppressing pro‑inflammatory gene expression in cancer cells. Although it was reported that Wnt/β‑catenin was critical in regulating TAMs, it is still largely unknown whether LGK‑974 regulates tumor malignancies by regulating TAMs. The present study firstly verified that the polarization of TAMs was regulated by LGK‑974. LGK‑974 increased M1 macrophage functional markers and decreased M2 macrophage functional markers. The addition of Wnt3a and Wnt5a, two canonical Wnt signaling inducers, reversed the decrease in M1 macrophage functional markers, including mannose receptor, IL‑10 and Arg1, by activating Wnt/β‑catenin signaling. Conditioned medium from LGK‑974‑modified TAMs inhibited the malignant behaviors in A549 and H1299 cells, including proliferation, colony formation and invasion, by blocking Wnt/β‑catenin signaling. LGK‑974‑modified TAMs blocked the cell cycle at the G₁/G₀ phase, which was reversed by the addition of Wnt3a/5a, indicating that LGK‑974 regulates the microenvironment by blocking Wnt/β‑catenin signaling. Taken together, the results indicate that LGK‑974 indirectly inhibited the malignant behaviors of A549 and H1299 cells by regulating the inflammatory microenvironment by inhibiting Wnt/β‑catenin signaling in TAMs.

Keywords: LGK‑974; Wnt/β‑catenin signaling; lung cancer; tumor microenvironment; tumor‑associated macrophages.

Figure 1.

Addition of LGK-974 regulates TAMs and its inflammation-associated cytokines. After 24-h co-incubation with 16HBE, A549 or H1299, total RNA or protein was isolated and the expression levels of cell markers for pan-macrophage CD68 (A), for M1-macrophage Arginase 1, CD86 and HLA-DR (B), and for M2-macrophage CD163 and CD206 (C) were determined. (D-G) Expression levels of functional markers for M1-macrophage (TNF-α, IL-12 and iNOS) and for M2-macrophage (MR, IL-10 and Arg1) were measured by reverse transcription-quantitative PCR. *P<0.05 vs. mock group; ^#P<0.05 vs. mock group/16HBE. TAMS, tumor-associated macrophages; iNOS, inducible nitric oxide synthase; MR, mannose receptor; Arg1, argininase-1.

Figure 2.

LGK-974 regulates functional markers for macrophage via blocking Wnt/β-catenin signaling. 16HBE, A549 or H1299 cells were co-cultured with LGK-974 and/or Wnt3a/5aA for 24 h. Then, total RNA was isolated and the expression levels of functional markers for MR, IL-10 and Arg1 (A), and functional markers for M1-macrophage (TNF-α, IL-12 and iNOS) (B) were measured by performing reverse transcription-quantitative PCR. Arg1 protein level was detected by western blotting and IL-10 section in supernatant was measured by performing ELISA (A, right panel). TNF-α and IL-12 section in supernatant was measured by performing ELISA (B, right panel). *P<0.05 vs. mock group. MR, mannose receptor; iNOS, inducible nitric oxide synthase; Arg1, argininase-1.

Figure 3.

LGK-974 regulates Wnt/β-catenin signaling in tumor-associated macrophages. (A) In 16HBE, A549 and H1299 cells treated with LGK-974, mRNA levels of Wnt receptors, including Fzd4, Fzd7 and Fzd9, were determined. Then, the downstream genes of Wnt signaling including β-catenin, Axin2 and c-Myc (B) were determined by reverse transcription-quantitative PCR. *P<0.05 vs. alone; ^#P<0.05 vs. LGK-974 group. Fzd, Frizzled.

Figure 4.

LGK-974 modifies TAMs-inhibited malignant behaviors of A549 and H1299. (A) The cell viability of A549 or H1299 cultured in conditioned media from PMA-THP1, TAMs or TAMs + LGK-974 was measured from day 1 to 5. Then, colony formation, (B) migration (C) and invasion (D) were measured. Magnification, ×40. *P<0.05 vs. TAMs group. TAMs, tumor-associated macrophages; PMA, phorbol 12-myristate 13-acetate.

Figure 5.

LGK-974 modifies TAMs via blocking Wnt/β-catenin signaling. (A) The effect of LGK-974 on cell viability was measured by Cell Counting Kit-8 assay. Then, the addition of Wnt3a/5a on reversing this effect was measured. (B) Cell cycle phase distribution was measured by performing PI staining followed by flow cytometry. *P<0.05 vs. mock group; ^#P<0.05 vs. TAMs + LGK-974 group. TAMs, tumor-associated macrophages.