Reprogramming of tumor-associated macrophages by targeting β-catenin/FOSL2/ARID5A signaling: A potential treatment of lung cancer

Abstract

Tumor-associated macrophages (TAMs) influence lung tumor development by inducing immunosuppression. Transcriptome analysis of TAMs isolated from human lung tumor tissues revealed an up-regulation of the Wnt/β-catenin pathway. These findings were reproduced in a newly developed in vitro "trained" TAM model. Pharmacological and macrophage-specific genetic ablation of β-catenin reprogrammed M2-like TAMs to M1-like TAMs both in vitro and in various in vivo models, which was linked with the suppression of primary and metastatic lung tumor growth. An in-depth analysis of the underlying signaling events revealed that β-catenin-mediated transcriptional activation of FOS-like antigen 2 (FOSL2) and repression of the AT-rich interaction domain 5A (ARID5A) drive gene regulatory switch from M1-like TAMs to M2-like TAMs. Moreover, we found that high expressions of β-catenin and FOSL2 correlated with poor prognosis in patients with lung cancer. In conclusion, β-catenin drives a transcriptional switch in the lung tumor microenvironment, thereby promoting tumor progression and metastasis.

Fig. 1. Activation of Wnt/β-catenin signaling in primary and in vitro trained M2-like TAMs.

(A) Volcano plot showing differentially expressed genes (DEGs) in TAMs versus NMs, n = 5. FC, fold change. (B) Top 10 panther pathways in TAMs–up-regulated DEGs. VEGF, vascular endothelial growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor. (C) Western blot of Wnt/β-catenin signaling genes in primary TAMs and NMs. (D) Representative immunofluorescence images of donors (n = 2) and lung cancer tissues (n = 70) in lung tissue microarray. Scale bars, 50 μM. (E) Scheme depicting generation of in vitro trained TAMs. (F) Heatmaps display M1 and M2 macrophage markers’ expression in M1-like and M2-like TAMs, n = 3. (G) Enzyme-linked immunosorbent assay (ELISA)–based quantification of TNF and IL10 in M0, M1, M2, and A549-trained M1-like and M2-like TAMs, n = 4, *P < 0.05, ***P < 0.001, ****P < 0.0001 versus M0. (H) Apoptosis, (I) proliferation, and (J) migration of A549 in the presence of CM from M0, M1, M2, and A549-trained M1-like and M2-like TAMs, n = 9, ***P < 0.001, ****P < 0.0001 versus M0-CM. (K) Venn diagram showing overlap of up-regulated DEGs by combined RNA-seq analysis of primary TAMs, classical macrophages, and in vitro TAMs. (L) Western blot of Wnt/β-catenin signaling genes in M0 and A549-trained M1-like and M2-like TAMs. (M) Western blot of nuclear, cytoplasmic β-catenin, Lamin B1, and β-tubulin. (N) Relative TCF/LEF luciferase activity in M0 and A549-trained M1-like and M2-like TAMs, n = 9, ***P < 0.001 versus M0.

Fig. 2. Genetic and pharmacological ablation of β-catenin in vitro trained and primary TAMs switches M2-like TAMs to M1-like TAMs.

(A) Western blot of Wnt/β-catenin signaling genes. (B) mRNA expression of TNF and IL10 in M0, M1-like, and M2-like TAMs transfected with sh_NS, sh_EG5, and sh_β-catenin for 24 hours, n = 6. (C) Apoptosis, (D) proliferation, and (E) migration of A549 in the presence of CM from M0, M1-like, and M2-like TAMs transfected with sh_NS, sh_EG5, and sh_β-catenin for 24 hours; n = 9, ***P < 0.001, ****P < 0.0001 versus sh_NS or sh_NS-CM. mRNA expression of (F) CCND1, (G) TNF, and IL10 in ex vivo TAMs transfected with si_NS (nonsilencing control siRNA) and si_β-catenin for 24 hours, n = 6. (H) Apoptosis and proliferation of primary tumor cells in the presence of CM from ex vivo TAMs transfected with si_NS and si_β-catenin for 24 hours, n = 6, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus si_NS or si_NS-CM. mRNA expression of (I) CCND1, (J) TNF, and IL10 in ex vivo TAMs treated with XAV939 for 24 hours, n = 6. (K) Apoptosis and proliferation of primary tumor cells in the presence of CM from ex vivo TAMs treated with XAV939 for 24 hours, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001 versus ex vivo TAM or ex vivo TAM-CM.

Fig. 3. Pharmacological and macrophage-specific genetic ablation of β-catenin reduces development of lung tumors by reprogramming M2-like to M1-like TAMs in TME.

Representative pictures and images of hematoxylin-eosin-stained sections of tumor and quantification of (A) subcutaneous tumor and microscopic lung tumor nodules in (B) metastasis (C) carcinogen-induced lung tumor models. Scale bars, 20 μM, n = 5, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control. (D to F) mRNA expression of Tnf and Il10 in TAMs from mice tumor tissue treated with control (DMSO; TAM_Ctrl) and XAV939 (TAM_XAV) in (D) subcutaneous tumor, (E) metastatic, and (F) carcinogen-induced lung tumor models, n = 5, *P < 0.05, **P < 0.01, ***P < 0.001 versus TAM_Ctrl. (G to I) FACS histograms indicate mean fluorescence intensity of CD206⁺ macrophages in control and XAV939-treated tumor tissue from (G) subcutaneous tumor, (H) metastatic, and (I) carcinogen-induced lung tumor models. Representative pictures and images of hematoxylin and eosin–stained sections of lungs and quantification of microscopic lung tumor nodules in (J) carcinogen-induced and (K) bone marrow transplantation models in Lysm^Cre, Catnb^f/f, and Catnb^f/fLysm^Cre mice. Scale bars, 20 μm, n = 5, **P < 0.01, ***P < 0.001 versus Catnb^f/f. (L and M) mRNA expression of Tnf and Il10 in TAMs sorted from macrophage-specific β-catenin–deficient tumors (TAM_Catnb^f/fLysm^Cre) and WT tumors (TAM_Lysm^Cre and TAM_Catnb^f/f) in (L) carcinogen-induced and (M) bone marrow transplant (BMT) lung tumor models, n = 5, ***P < 0.001, ****P < 0.0001 versus Catnb^f/f.

Fig. 4. Reprogramming of M2-like to M1-like TAMs upon β-catenin inhibition is incompletely dependent on CCR2/β-catenin axis and TNFα.

(A) Western blot of β-catenin and CCR2 in BMDMs from WT, Lysm^Cre, Catnb^f/fLysm^Cre, and CCR2^−/− mice. mRNA expressions of Ccnd1 and Ccr2 in BMDM from (B) Lysm^Cre, Catnb^f/f Lysm^Cre, (C) WT, and CCR2^−/− mice, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 versus M0_Lysm^Cre or M0_WT. mRNA expressions of (D) Tnf, Nos2, Il1b, (E) Il10, Arg1, and Chit1 in BMDMs from WT and CCR2^−/−, n = 3, *P < 0.05, **P < 0.01 versus M0_WT. mRNA expression of (F) Tnf in M0_Lysm^Cre with si_NS and M0_ Catnb^f/fLysm^Cre with si_NS or si_TNFα for 24 hours, n = 3, *P < 0.05, **P < 0.01 versus M0_Lysm^Cre_si_NS, &&&P < 0.001 versus M0_Catnb^f/fLysm^Cre_si_NS. (G) TNF in M2-like TAMs with si_NS, si_β-catenin and si_β-catenin followed by si_TNFα, n = 6, ****P < 0.0001 versus si_NS, &&&&P < 0.0001 versus si_β-catenin. mRNA expression of (H) Il10, Mrc1, (I) Arg1, and Chit1 in M0_Lysm^Cre with si_NS, M0_ Catnb^f/fLysm^Cre with si_NS or si_TNFα for 24 hours, n = 3, **P < 0.001, ****P < 0.0001 versus M0_Lysm^Cre_si_NS, &&P < 0.01 versus M0_Catnb^f/fLysm^Cre_si_NS. (J) IL10, MRC1, (K) CD163, and ALOX15 in M2-like TAMs with si_NS, si_β-catenin and si_β-catenin followed by si_TNFα, n = 6, ****P < 0.0001 versus si_NS, &P < 0.05, &&&P < 0.001, &&&&P < 0.0001 versus si_β-catenin.

Fig. 5. Dual transcriptional role of β-catenin in the phenotypic transition to M2-like TAMs.

(A) Heatmaps display M1 and M2 macrophage markers expression in M2-like TAMs transfected with sh_Control and sh_β-catenin, n = 3. (B) DESeq normalized read count averages of genes were log₁₀ transformed and compared between sh_β-catenin and sh_Control. DEGs (light gray), TFs (annotated by JASPAR; blue and red), and nondifferential genes (dark gray) are depicted as points. (C) Left heatmap displays a row-wise Z score of RNA-seq. Right heatmap shows Pscan of TF-binding site enrichment P value. (D) mRNA expression and (E) Western blot of FOSL2 and ARID5A in undifferentiated BMDM from Lysm^Cre, Catnb^f/f, and Catnb^f/fLysm^Cre, n = 6, ****P < 0.0001 versus Catnb^Cre. (F and G) mRNA expression of Fosl2 and Arid5a in TAMs from Catnb^f/fLysm^Cre, Lysm^Cre, and Catnb^f/f in (F) carcinogen-induced and (G) BMT lung tumor models, n = 5, **P < 0.01, ****P < 0.0001 versus Catnb^f/f. Western blot of β-catenin, FOSL2, and ARID5A in (H) M0, M1, M2, (I) M0, and M1-like and M2-like TAMs. (J) Scheme showing ChIP using a β-catenin antibody. (K) Real-time PCR of FOSL2, ARID5A, IL10, and CCND1 in β-catenin ChIP assays performed in THP1-derived M2 macrophages treated with control (DMSO) and XAV939 (5 μM) for 24 hours, n = 6, ****P < 0.0001 versus Ab_β-catenin.

Fig. 6. Inhibition of Wnt/β-catenin signaling and FOSL2 and activation of ARID5A switch phenotype to M1-like TAMs; correlation of β-catenin/FOSL2/ARID5A with the survival of lung cancer patients.

Western blot of β-catenin, FOSL2, and ARID5A in M2-like TAMs transfected with (A) OE_NS, OE_β-catenin, (B) si_NS, and si_β-catenin. (C) Western blot of FOSL2. (D) mRNA expression of CD163, MRC1, IL1R1, and TGFB1 in M2-like TAMs with si_NS and si_FOSL2. (E) Western blot of ARID5A. (F) mRNA expression of TNF, IL8, CCR7, and IL6 in M2-like TAMs with OE_NS and OE_ARID5A, n = 10, **P < 0.01, ****P < 0.0001 versus si_NS or OE_NS. (G) Kaplan-Meier survival analysis of lung adenocarcinoma patients stratified by β-catenin, FOSL2, and ARID5A expression. HR, hazard ratio. (H) M2-like TAMs show up-regulation of WNT ligands (5A-7B-11), frizzled receptors (4-5-6-8-9), disheveled (2-3), and TNKS (1-2), leading to transcriptional activation of β-catenin. β-Catenin activates M2-macrophage program by binding to promoter region of M2 macrophage genes (IL10) and to TF-activating M2 macrophage genes, FOSL2 (CD163, MRC1, IL1R1, and TGFB1). In addition, β-catenin represses M1 macrophage program by binding to TF-activating M1 macrophage genes, ARID5A (TNFα, IL8, CCR7, and IL6). Therefore, in vitro trained, ex vivo cultured, and in vivo β-catenin-KO M2-like TAMs are reprogramed into M1-like TAMs by genetic and pharmacological inhibition of β-catenin, knockdown of FOSL2, and overexpression of ARID5A. These results indicate the reactivation of antitumor immunity in TME to restrict primary and metastatic lung tumor growth.