CHD4 mutation occurs frequently in endometrial cancerThe frequency of CHD4 mutations in endometrial cancer was systematically investigated by analysing data from various databases associated with endometrial carcinoma in cBioPortal (https://www.cbioportal.org/). Across a comprehensive cohort of 2692 patients, 4.4% exhibited somatic mutations in CHD4 (119 out of 2692 patients) (Fig. 1A). Notably, within specific endometrial cancer datasets—the Uterine Corpus Endometrial Carcinoma (TCGA, PanCancer Atlas), Endometrial Carcinoma (CPTAC, Cell 2020), and Uterine Corpus Endometrial Carcinoma (TCGA, Nature 2013) datasets—the CHD4 mutation frequencies were 20.98% (111 out of 529 patients), 13.58% (11 out of 81 patients), and 9.43% (35 out of 371 patients), respectively (Fig. 1B). These collective findings underscore the high prevalence of CHD4 mutations among individuals with endometrial cancer.Figure 1The CHD4 R975H mutation occurs frequently in endometrial cancer. (A) Somatic mutation frequency of CHD4 in endometrial cancer patients in cBioPortal database. (B) Alteration frequency of CHD4 in various endometrial cancer datasets. (C) The corresponding number of samples with various CHD4 mutations. (D) Location of CHD4 R975H mutation on CHD4 schematic figure. (E,F) Prediction of the impact of the CHD4 R975H mutation on protein structure and function via two bioinformatics tools. (E) Polymorphism phenotyping v2. (F) Sorting intolerant from tolerant. (G) Prediction of CHD4 structural alteration induced by R975H mutation. Upper: Overall structure of CHD4 ATPase domain lobe 2. Middle: Detailed structure and interaction between 975R and 1182E in CHD4 WT. Lower: Detailed structure of 975H and 1182E in CHD4 R975H.Upon closer examination of the mutation landscape, the missense mutation R975H emerged as the most common mutation, occurring 15 times within a pool of 271 total mutations (Fig. 1C). It locates in the lobe 2 of CHD4 ATPase domain, which is critical for DNA twisting and chromatin remodelling function of CHD4 (Fig. 1D)18. Consequently, our focus was on elucidating the impact of the CHD4 R975H mutation. Leveraging two bioinformatics analysis software—Polymorphism Phenotyping v2 (PolyPhen-2) and Sorting Intolerant from Tolerant (SIFT)—we assessed the predicted influence of the amino acid substitution 975aa R to H. Both analyses converged on a consensus: R975H is likely to be damaging to the structure and function of CHD4 (PolyPhen-2, Fig. 1E), with potential repercussions on CHD4 protein function (SIFT, Fig. 1F). We then compared the predicted structure of CHD4 wild type (WT) and CHD4 R975H using UCSF ChimeraX. In CHD4 WT, a salt bridge forms between the positively charged Nε in the side chain of 975R and the negatively charged Oε in the side chain of 1182E. The salt bridge could stabilize the α-helix structure of ATPase lobe 2. R975H disrupts the salt bridge and could cause instability of ATPase lobe 2 (Fig. 1G). In summary, our comprehensive analysis revealed a notable occurrence of CHD4 mutations in endometrial carcinoma, with particular emphasis on the recurrent CHD4 R975H mutation. Predictive analyses indicated that this mutation holds significance, potentially influencing the functional landscape of CHD4 in the context of endometrial cancer.CHD4 R975H promotes EC cell proliferation in vitro and tumor growth in vivoTo further investigate the impact of the CHD4 R975H mutation on endometrial cancer, we engineered stable cell lines expressing CHD4 WT, CHD4 R975H, or short hairpin RNA targeting CHD4 (shCHD4). Given prior observations by Li et al., suggesting an increase in cell invasion and sphere formation capacity with both CHD4 R975H and shCHD4 in the EFE184 and SKUT-2 endometrial cancer cell lines15, we opted for the Ishikawa cell line, which is derived from an Asian EC patient. Inducible promoters within the SB vector (for CHD4 WT and CHD4 R975H) and the lentiviral vector (for shCHD4) were activated by doxycycline. The overexpression and knockdown efficacy of CHD4 WT and shCHD4 were validated through qRT–PCR (Fig. 2A), while the expression of the CHD4 R975H mutant was confirmed by western blot analysis (Fig. 4B).Figure 2The CHD4 R975H mutation promotes a cancer stem cell (CSC)-like phenotype in endometrial cancer cells. (A) CHD4 expression levels in the constructed CHD4 WT (left) and shCHD4 (right) cell lines determined via qRT‒PCR. (B–F) Transwell, colony formation and MTT assays of the CHD4 R975H, CHD4 WT and shCHD4 cell lines. (B) Stained invasive cells in the transwell assay. (C) Quantification of invasive cells. (D) Stained colonies in the colony formation assay. (E) Quantification of colonies. (F) Absorbance of the chromogenic product formazan produced by live cells at 490 nm. The number of live cells is linearly related to the absorbance. (G) Tumor weight and volume in the mouse experiment.Subsequently, the CHD4 WT, CHD4 R975H, and shCHD4 cell lines were subjected to transwell, colony formation, and MTT assays. In transwell assay, CHD4 R975H demonstrated a proinvasive effect, in contrast with the inhibitory effects observed for CHD4 WT and shCHD4 (Fig. 2B,C). Colony formation assay revealed that CHD4 R975H promoted colony formation, while CHD4 WT and shCHD4 suppressed colony formation (Fig. 2D,E). Correspondingly, the MTT assay results indicated that CHD4 R975H enhanced cell proliferation, whereas CHD4 WT and shCHD4 repressed cell proliferation (Fig. 2F).To further investigate the oncogenic role of the CHD4 R975H mutation, we conducted an in vivo experiment using a mouse xenograft model. Equal numbers of CHD4 R975H cells, either pre-induced with doxycycline or untreated, were subcutaneously injected into the flanks of nude mice. Six weeks post-injection, the results revealed that the expression of CHD4 R975H led to a significant increase in both tumor weight and volume compared to the control group (Fig. 2G). These comprehensive findings revealed that CHD4 R975H augments the CSC-mediated invasive and proliferative capabilities of endometrial cancer cells. In stark contrast, CHD4 WT and shCHD4 manifested opposing effects, collectively revealing the dynamic influence of CHD4 mutations on endometrial cancer cellular behaviors.CHD4 R975H results in a cancerous transcriptome profile in EC cellsTo unravel the underlying mechanisms through which CHD4 R975H enhances tumorigenesis in endometrial cancer (EC) cells, we conducted RNA-Seq analysis of total RNA extracted from the CHD4 R975H cell line cultured with or without doxycycline (Fig. 3A,B). The ensuing gene set enrichment analysis (GSEA) yielded insightful outcomes. A summary of the GSEA gene sets, spanning KEGG legacy gene sets, hallmark gene sets, and GO molecular function gene sets, provided a comprehensive overview (Fig. 3C). KEGG gene set analysis revealed that CHD4 R975H induced the expression of genes related to cancer-associated pathways. Hallmark gene sets further illuminated the spectrum of pathway activation, encompassing TNF-α signaling via NF-κB, KRAS signaling, the P53 pathway, Myc targets, and mTORC1 signaling—pathways commonly implicated in cancer19,20,21,22,23. GO functional gene sets confirmed the heightened growth factor activity induced by CHD4 R975H, a critical factor known to play a pivotal role in immune suppression within the tumor microenvironment24.Figure 3The CHD4 R975H mutant induces a cancer-specific transcriptome change in endometrial cancer cells. (A) Heatmap of gene expression profile of RNA-Seq result. (B) DEG volcano plot of RNA-Seq result. Differentially expressed genes (DEGs) are identified as | log2 fold change |> 0.5, p < 0.05. (C) Summary of GSEA results based on KEGG legacy gene sets, hallmark gene sets and GO molecular function gene sets. (D) GSEA results for Myc targets, mTOR signaling, TGF-β signaling, KRAS signaling, the p53 pathway, TNF-α signaling via NF-κB, growth factor signaling and EGFR signaling.Individual gene set analyses for each pathway highlighted the robust activation induced by CHD4 R975H. Notably, enrichment of EGFR-associated gene sets revealed the activation of this driver of tumorigenesis25. Furthermore, consistent with the findings of Li et al., our GSEA confirmed the activation of TGF-β signaling by the CHD4 R975H mutant, a key contributor to endometrial tumorigenesis15 (Fig. 3D). Collectively, these results reveal that CHD4 R975H orchestrates the tumorigenic transcriptome in EC cells, unveiling the comprehensive landscape of activated pathways underlying its oncogenic influence.CHD4 R975H stimulates oncogenic pathways and promotes cancer stemnessTo corroborate the findings from our bioinformatics analysis, we conducted qRT–PCR assays to assess the expression levels of a set of genes in the CHD4 R975H, CHD4 WT, and shCHD4 cell lines. These genes, identified as upregulated by RNA-seq, encompass hallmarks of pathways implicated in our previous analyses. The pathways and their corresponding hallmark genes are detailed below: mTOR (SLC2A1, TRIB3, SLC7A5), KRAS (ALDH1A3, EPHB2, HBEGF), Myc (Myc), P53 (EPHA2, S100A10), TGF-β (PMEPA1, ITGB6, PDGFB), TNF-α signaling via NF-κB (CCND1), EGFR (SERPINE2, MICAL2), and growth factor (FGF18, MET). Additionally, SEMA3C, known for promoting cervical cancer growth through the activation of the p-ERK pathway26, was included due to its prominent variation in expression within the top 50 genes identified by RNA-seq analysis (Supplementary Table 4). The results unequivocally demonstrated a significant increase in the expression levels of all the aforementioned genes in the CHD4 R975H cell line. In contrast, limited upregulation was observed in the CHD4 WT and shCHD4 cell lines (Fig. 4A), suggesting that only CHD4 R975H effectively initiates the activation of tumorigenic pathways, while CHD4 WT and CHD4 depletion exert minimal influence on the oncogenic signaling network. The activation of mTOR and TGF-β signaling driven by CHD4 R975H was further validated through western blot analysis (Fig. 4B).Figure 4The CHD4 R975H mutant activates carcinogenic pathways and promotes the stemness of endometrial cancer cells. (A) Expression levels of marker genes in cancer-related pathways in the CHD4 R975H, CHD4 WT and shCHD4 cell lines measured by qRT‒PCR. (B) Expression levels of hallmark proteins involved in stemness (CD133), mTOR signaling (total S6K and pS6K) and TGF-βsignaling (β-catenin) in CHD4 R975H cells detected via western blotting. Original blots are presented in Supplementary Fig. 2. (C) Expression levels of stemness marker genes in the CHD4 R975H, CHD4 WT and shCHD4 cell lines measured by qRT‒PCR. (D) Flow cytometry analysis of CD133 staining in CHD4 R975H cell line. (E) Spheroids of Ishikawa and AN3-CA CHD4 R975H cell lines. (F) Number and diameter of spheroids.In alignment with the findings of Li et al., Zhang et al., and Pratheeshkumar et al. on the role of CHD4 in cancer stemness in patients with endometrial cancer and other malignancies15,27,28, we sought to elucidate the impact of CHD4 R975H on the stemness of endometrial cells. We assessed the expression levels of a set of stemness marker genes (KLF4, NANOG, OCT4) in the CHD4 R975H, CHD4 WT, and shCHD4 cell lines. The results revealed a substantial increase in the expression levels of all stemness markers in the CHD4 R975H cell line, whereas no significant upregulation was observed in either the CHD4 WT or shCHD4 cell lines (Fig. 4C). This pronounced increase in stemness markers strongly suggested that CHD4 R975H actively promotes stemness in endometrial cancer cells. Intriguingly, CHD4 WT exhibited a suppressive effect on the expression of NANOG and OCT4, indicating an inhibitory influence on the stemness of endometrial cancer cells. Furthermore, the expression of CD133, a well-established marker of cancer stem cells, was elevated in the CHD4 R975H cell line, consistent with the qPCR results (Fig. 4B). Flow cytometry analysis of CD133 expression in CHD4 R975H cells revealed that, compared to the control group, the CHD4 R975H mutation significantly increased the proportion of CD133-positive cells (Fig. 4D). To further validate the promotive effect of CHD4 R975H on stemness traits in EC, we conducted spheroid formation assays in Ishikawa and AN3-CA CHD4 R975H cell lines. The results showed that CHD4 R975H expression markedly enhanced the growth of EC spheroids compared to the control group (Figs. 4E,F). Together, these findings provide compelling evidence that the CHD4 R975H mutation stimulates stemness in EC.CHD4 R975H induces TAM polarization to M2-like macrophagesIn light of the intricate composition of the tumor microenvironment (TME), encompassing diverse molecular constituents such as the extracellular matrix, growth factors, cytokines, and both nonimmune and immune cells29, we conducted an immune infiltration analysis focusing on multiple immune cell subgroups in endometrial cancer (EC) patients with the R975H mutation, other CHD4 mutations, or without any CHD4 mutation. The results revealed a discernible disparity in the distribution of various immune cells among the aforementioned patient categories (Supplementary Fig. S1). Given the multifaceted roles of tumor-associated macrophages (TAMs) and their ability to polarize towards either M1-like or M2-like macrophages, with M2-like macrophages well-established for their protumorigenic function in fostering immune suppression30, our attention turned to scrutinizing variations in the abundance of M2 macrophages in EC patients with or without CHD4 mutations. The findings indicated a significant enrichment of M2 macrophages in the tumor tissue of patients with CHD4 mutations compared to those without, suggesting a potential association between CHD4 mutation and M2-like macrophage polarization (Fig. 5A).Figure 5The CHD4 R975H mutation drives M2-like polarization in tumor-associated macrophages. (A) Abundance of M2 macrophages in tumor tissue of EC patients with or without CHD4 mutation. (B) Differential expression analysis of CCL and CXCL gene sets in tumor tissue of patients above. (C) Expression levels of M2 macrophage marker genes in the THP-1 cell line cocultured with the CHD4 R975H, CHD4 WT and shCHD4 cell lines measured by qRT‒PCR. (D) Flow cytometry analysis of CD68 + CD163/CD206 staining in cocultured THP-1 cell line. (E) Immunohistochemistry analysis of CD163/CD206 in tumor tissue of nude mice used in the mouse xenograft model. (F) Quantification of positive area of immunohistochemistry assay.Recognizing the pivotal role of chemokines, secreted by cancer cells, as potent regulators influencing TAM polarization31, we further conducted a differential expression analysis of CCL and CXCL gene sets in EC patients with or without CHD4 mutations. The results unveiled upregulation of CCL4, CCL7, CCL8, CCL13, CXCL9, CXCL10, CXCL11, and CXCL13, coupled with downregulation of CCL14 in the tumor tissue of patients with CHD4 mutations in comparison to those without (Fig. 5B). This intriguing pattern aligns with the established functions of these chemokines: CCL7 activation promotes M2 macrophage enrichment in lung adenocarcinoma32; CCL14 induces M1 polarization and suppresses M2 polarization of TAMs in colon cancer33; in colorectal cancer, M2 macrophages release CXCL13, stimulating cancer metastasis34. The observed differential expression trends of these chemokines align cohesively with the heightened abundance of M2 macrophages in patients with CHD4 mutations. Collectively, these findings hint at the potential impact of CHD4 mutations within endometrial cancer cells on M2-like macrophages.To explore whether CHD4 R975H could induce TAM polarization toward M2-like macrophages, we collected conditioned media from the CHD4 R975H, CHD4 WT, and shCHD4 cell lines. Subsequently, we separately cocultured THP-1 cells with conditioned medium from the aforementioned cell lines for 2 days and assessed the expression levels of CD23, CCL18, and CCL22, which are markers of M2 macrophages35,36. The results unequivocally demonstrated that CHD4 R975H significantly increased the expression levels of CD23, CCL18, and CCL22, whereas CHD4 WT and shCHD4 exhibited no discernible effect on their expression (Fig. 5C). This compelling evidence substantiates that CHD4 R975H actively stimulates TAM polarization toward an M2-like macrophage phenotype, whereas CHD4 WT and shCHD4 exert no discernable influence on TAM polarization. To further substantiate the inducible effect of CHD4 R975H on TAM M2-like polarization, we analysed the expression of CD163 and CD206 in cocultured THP-1 cells, both well-defined markers of M2 macrophages. The results revealed that, compared to the control group, CHD4 R975H expression significantly increased the ratio of CD68/CD163 and CD68/CD206 double-positive cells (Fig. 5D). To further confirm that CHD4 R975H triggers TAM M2-like polarization in vivo, we harvested tumor tissue from nude mice used in the mouse xenograft model and conducted immunohistochemistry of CD163 and CD206. The results showed that, compared to the control group, tumor tissue with CHD4 R975H expression exhibited higher expression of both CD163 and CD206. This demonstrates that CHD4 R975H expression in EC cells induces the M2-like polarization of infiltrated TAMs in vivo (Fig. 5E,F). Overall, these findings clearly demonstrate that the CHD4 R975H mutant induces TAM M2-like polarization in EC.
