A novel STAT3/ NFκB p50 axis regulates stromal-KDM2A to promote M2 macrophage-mediated chemoresistance in breast cancer

Abstract

Background: Lysine Demethylase 2A (KDM2A) plays a crucial role in cancer cell growth, differentiation, metastasis, and the maintenance of cancer stemness. Our previous study found that cancer-secreted IL-6 can upregulate the expression of KDM2A to promote further the transition of cells into cancer-associated fibroblasts (CAFs). However, the molecular mechanism by which breast cancer-secreted IL-6 regulates the expression of KDM2A remains unclear. Therefore, this study aimed to elucidate the underlying molecular mechanism of IL-6 in regulating KDM2A expression in CAFs and KDM2A-mediated paclitaxel resistance in breast cancer.

Methods: The ectopic vector expression and biochemical inhibitor were used to analyze the KDM2A expression regulated by HS-578 T conditioned medium or IL-6 in mammary fibroblasts. Immunoprecipitation and chromatin immunoprecipitation assays were conducted to examine the interaction between STAT3 and NFκB p50. M2 macrophage polarization was assessed by analyzing M2 macrophage-specific markers using flow cytometry and RT-PCR. ESTIMATE algorithm was used to analyze the tumor microenvironment-dominant breast cancer samples from the TCGA database. The correlation between stromal KDM2A and CD163 + M2 macrophages was analyzed using the Pearson correlation coefficient. Cell viability was determined using trypan blue exclusion assay.

Results: IL-6 regulates gene expression via activation and dimerization of STAT3 or collaboration of STAT3 and NFκB. However, STAT3, a downstream transcription factor of the IL-6 signaling pathway, was directly complexed with NFκB p50, not NFκB p65, to upregulate the expression of KDM2A in CAFs. Enrichment analysis of immune cells/stromal cells using TCGA-breast cancer RNA-seq data unveiled a positive correlation between stromal KDM2A and the abundance of M2 macrophages. CXCR2-associated chemokines secreted by KDM2A-expressing CAFs stimulated M2 macrophage polarization, which in turn secreted CCL2 to increase paclitaxel resistance in breast cancer cells by activating CCR2 signaling.

Conclusion: This study revealed the non-canonical molecular mechanism of IL-6 secreted by breast cancer upregulated KDM2A expression in CAFs via a novel STAT3/NFκB p50 axis, which STAT3 complexed with NFκB p50 in NFκB p50 binding motif of KDM2A promoter. KDM2A-expressing CAFs dominantly secreted the CXCR2-associated chemokines to promote M2 macrophage polarization and enhance paclitaxel resistance in breast cancer. These findings underscore the therapeutic potential of targeting the CXCR2 or CCR2 pathway as a novel strategy for paclitaxel-resistant breast cancer.

Keywords: Cancer-associated fibroblasts; Lysine demethylase 2A; Paclitaxel resistance; Tumor-associated macrophage.

Fig. 1

IL-6 upregulated the expression of KDM2A in mammary fibroblasts by activating STAT3. A Overexpression of constitutively active STAT3 by pSTAT3 plasmid increased the expression of KDM2A in RMF-EG cells. The protein and mRNA levels of KDM2A were analyzed by Western blotting analysis and real-time PCR, respectively. B RMF-EG cells were transfected with dominant-negative STAT3 plasmids and then treated with or without 10 ng/ml IL-6 for 48 h. The protein and mRNA levels of KDM2A were analyzed by Western blotting analysis and real-time PCR, respectively. The pcDNA3.1 vector was used as the control vector. C RMF-EG cells were incubated with HS-578 T CM for 48 h in the presence or absence of 5 μM stattic. The protein and mRNA levels of KDM2A were analyzed by Western blotting analysis and real-time PCR, respectively. IL-6 expression in serum-free medium (SF) or HS-578 T CM was analyzed by Western blotting analysis. D RMF-EG cell was treated with 20 ng/ml IL-6 for 48 h in the presence or absence of 5 μM stattic. The protein and mRNA levels of KDM2A expression were analyzed by Western blotting analysis and real-time PCR, respectively. Differences were found to be statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001

Fig. 2

IL-6 upregulated the expression of KDM2A in mammary fibroblasts through the STAT3/NFκB p50 axis. A ChIP assay was performed to pull down the STAT3 proteins-chromatin complexes in IL-6-treated mammary fibroblasts using anti-STAT3 antibody. STAT4 and NFκB p50 binding motifs on the KDM2A promoter region were amplified by PCR. Each experiment was performed in triplicate and repeated three times independently. Data are expressed as the fold change relative to non-treated control cells. The upper region of the histogram shows the putative STAT4 and NFκB p50 binding motif on the KDM2A promoter region. B RMF-EG cells were incubated with HS-578 T CM for 48 h in the presence and absence of JSH-23. The protein and mRNA levels of KDM2A were analyzed by Western blotting analysis and real-time PCR, respectively. C RMF-EG cells were treated IL-6 for 48 h in the presence and absence of 30 μM JSH-23. The protein and mRNA levels of KDM2A were analyzed by Western blotting analysis and real-time PCR, respectively. D Ectopic pSTAT3-expressing RMF-EG cells were treated with or without JSH-23 for 48 h. KDM2A protein level was analyzed by Western blotting analysis. E Immunoprecipitation assay was performed in IL-6-treated RMF-EG cells. STAT3 bound protein complexes were pulled down using anti-STAT3 antibody and analyzed by immunoblotting with anti-NFκB p65 and anti-NFκB p50 antibodies. Differences were found to be statistically significant at *p < 0.05, ** p < 0.01, and *** p < 0.001

Fig. 3

Stromal KDM2A was associated with infiltrated CD163 + M2 macrophages in the breast tumor microenvironment. A Flowchart of analyzing gene expression profiles of breast tumor samples obtained from the TCGA database. The abundance of stromal and infiltrated immune cells in each breast tumor sample was assessed using ESTIMATEScore, StromalScore, and ImmuneScore. B According to ESTIMATEScore, the samples were divided into tumor-dominant group and TME-dominant group, and the relative expression levels of CD163 in the two groups were analyzed. C The abundance of CD163 + cells was analyzed between the two groups and further stratified by ImmuneScore. D The correlation between the stromal KDM2A and CD163⁺ M2 macrophage was evaluated by the Person’s correlation coefficient in the stromal and immune cell-high group. The I:S ratio was calculated as the ratio of ImmuneScore to StromalScore and used to represent immune and stromal content in the TME-dominant group. Differences were found to be statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001

Fig. 4

KDM2A-expressing mammary fibroblasts promoted M2 macrophage polarization. A PMA-pretreated THP-1 cells were co-cultured with RMF-EG cells or KDM2A-expressing RMF-EG cells (K2A-1), and the relative expression of M2-macrophage markers CCL2, IL-10, and VEGF-A were determined by real-time PCR. B Relative mRNA levels of tumor-associated fibroblast surface marker CD206 and CD163 in THP-1 cells co-cultured with RMF-EG or KDM2A-expressing RMF-EG (K2A-1) cells were determined by real-time PCR. C The relative expression levels of CD206 and CD163 on the cell surface of THP-1 cells co-cultured with RMF-EG or KDM2A-expressing RMF-EG (K2A-1) cells were determined and quantified by flow cytometry. Each experiment was performed in triplicate and repeated three times independently. Differences were found to be statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001

Fig. 5

KDM2A-expressed fibroblasts stimulated CCL2 released from macrophage through CXCR2 to increase the paclitaxel resistance in breast cancer. A PMA-pretreated THP-1 was treated with 100 ng/ml CXCL2, CXCL5, or IL-8, and relative mRNA and protein levels of CCL2 were determined by Real-time PCR and western blot analysis. B PMA-pretreated THP-1 cells were incubated with RMF-CM or K2A-CM in the presence and absence of CXCR2 inhibitor SB225002 (25 nM). Relative mRNA and protein levels of CCL2 were analyzed by Real-time PCR and western blot analysis. C Cell viability analysis of paclitaxel-treated HS-578 T cells. HS-578 T cells were treated with conditioned medium TRMF-CM or TRK2A-CM supplemented with additional 25 nM paclitaxel for 48 h. D Cell viability of paclitaxel-treated HS-578 T cells co-treated with TRK2A-CM in the presence and absence of CCR2 inhibitor INCB3344 (5 nM) for 48 h. Cell viability was determined and counted using the trypan blue exclusion assay. Each experiment was performed in triplicate and was repeated three times independently. Differences were found to be statistically significant at * p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 6

A schematic diagram of the molecular mechanism by which IL-6 secreted by breast cancer upregulates KDM2A in mammary fibroblast that can promote M2 macrophage polarization and increase paclitaxel resistance. Breast cancer cells secret IL-6, which triggers the activation of STAT3 in mammary fibroblasts. Active STAT3 then forms a complex with NFκB p50 and binds to the -154 bp to -144 bp region of the KDM2A promoter, leading to increased KDM2A expression in these fibroblasts. Elevated KDM2A levels in fibroblasts result in the release of CXCR2-assocciated chemokines, which stimulate the polarization of M2 macrophages via the CXCR2 signaling pathway. Finally, M2 macrophage release CCL2, contributing to increase resistance of breast cancer to paclitaxel. The mechanism highlights potential targets for therapeutic intervention in paclitaxel-resistant breast cancer