1、 Accumulation of fumarate induced IL6/JAK/STAT3 pathway activation in FH-knockdown cells
We successfully downregulated FH expression using si-FH-01 and si-FH-02 (Fig. 1A) and performed lentiviral transfection of ACHN and HK-2 cells with si-FH-01. We quantified the fumarate concentration in the intracellular and extracellular compartments of ACHN-shFH and HK-2-shFH cells, revealing that fumarate attains millimolar levels in both cellular compartments (Fig. 1B). Transcriptome sequencing revealed a remarkable elevation in the mRNA levels of STAT3 and IL-6R in ACHN-shFH cells (Fig. 1C). The expression levels of IL-6R, STAT3 and p-STAT3 significantly increased after the downregulation of FH in ACHN-shFH and HK-2-shFH cells (Fig. 1D, E). Rescue assays revealed that IL-6R and STAT3 levels decreased when FH was re-expressed in FH-knockdown cells (Fig. 1F, G). The expression level of FH was significantly downregulated in cancerous tissues, while the levels of IL-6R, total STAT3 (T-STAT3) and p-STAT3 were markedly increased in two paired FH-RCC samples (Fig. 1H, I).
Defective FH expression leads to the inhibition of the tricarboxylic acid cycle and accumulation of fumarate at millimolar concentrations 7,8. ACHN and HK-2 cells were stimulated with a gradient of monomethyl fumarate (MMF) for one weeks. The expression of IL-6R, STAT3 and p-STAT3 was significantly upregulated in ACHN and HK-2 cells upon stimulation with 0.1-1.0 mmol/L MMF, as evidenced by RT‒PCR and Western blot analysis (Fig. 1J). These findings suggested that the upregulation of IL-6R and STAT3 expression in FH-knockdown cells is driven by intracellular fumarate accumulation.
2. Knockdown of KDM4C or FH resulted in the upregulation of H3K36me3 expression and activation of the IL6/JAK/STAT3 pathway
To investigate the mechanism underlying the increased expression of IL-6R and STAT3 in FH-knockdown ACHN and HK-2 cells, we conducted bioinformatics analysis to identify relevant demethylation/acetylation regions in the promoters of IL-6R and STAT3 and designed primers for the potential target regions accordingly. DNA protein complexes were pulled down in ACHN-shFH and HK-2-shFH cells using histone antibodies. ChIP assay results indicated a significant increase in histone H3K36me3 levels across all four regions of the IL-6R gene promoter in ACHN-shFH and HK-2-shFH cells, as detected by RT-PCR and qPCR (Fig. 2A and Supplementary Fig. 1). Furthermore, a notable increase in H3K36me3 levels was observed in region B of the STAT3 gene promoter (Fig. 2A and Supplementary Fig. 1). We also assessed the levels of H3K4me3, H3K9me3, H3K27me3, and H3K36me3 after rescuing FH expression. After the decrease in FH expression, the level of H3K36me3 increased the most significantly, and rescuing FH expression decreased the abundance of H3K36me3 (Fig. 2B left). Subsequently, we quantified the level of H3K36me3 in fresh cancerous tissues from FH-RCC patients, revealing a significantly elevated abundance of H3K36me3 in cancerous tissues (Fig. 2B). Moreover, the Western blot results demonstrated that the downregulation of FH or the stimulation of ACHN and HK-2 cells with MMF leads to a significant increase in the abundance of H3K36me3 (Fig. 2B right). These results indicate that fumarate accumulation in FH-knockdown ACHN and HK-2 cells leads to an elevated level of H3K36me3 within the promoter region of the IL-6R and STAT3 genes, thereby promoting the transcriptional activation of the IL-6R and STAT3.
The histone demethylase KDM4, a member of the aKGDDs, is primarily involved in the regulation of histone H3K9me3 and H3K36me3 demethylation 29. We used siRNAs to knock down KDM4A, B, C, and D in ACHN and HK-2 cells (Fig. 2C). The level of H3K9me3 significantly increased upon downregulating KDM4A, whereas downregulating KDM4C expression led to significant elevations in the levels of H3K36me3, IL-6R, STAT3, and p-STAT3 (Fig. 2C, D).
We treated ACHN and HK-2 cells with a KDM4C inhibitor (1.2 µM) for 24–48 hours, followed by Western blotting and qPCR analysis. The results revealed a significant increase in the expression levels of H3K36me3, IL-6R, T-STAT3 and p-STAT3, upon inhibition of KDM4C activity (Fig. 2E, F). Therefore, these findings suggested that the observed significant increase in histone H3K36me3 levels in FH-knockdown cells, which activates IL-6R and STAT3 transcription, may be attributed to fumarate-mediated inhibition of KDM4C.
3、 Fumarate inhibits KDM4C-mediated demethylation of H3K36me3
The metalloprotein KDM4C, a member of the aKGDDs, relies on α-KG as a cofactor for its biological activity 30. Both fumarate stimulation and the inhibition of KDM4C resulted in increased H3K36me3 levels in ACHN and HK-2 cells. Given the structural resemblance between fumarate and α-KG, we postulated that fumarate competitively binds to α-KG, thereby inhibiting KDM4C activity. To test this hypothesis, we conducted in vitro histone demethylation experiments.
We transfected 293T cells with the overexpression plasmid Plv3-CMV-KDM4C to induce the expression of the fusion protein Flag-KDM4C (Fig. 3A). Subsequently, we performed purification using anti-Flag magnetic beads followed by concentration and further purification of the Flag-KDM4C fusion protein utilizing 100 kDa ultrafiltration tubes. The purity assessment through silver staining demonstrated that the purity exceeded 90% (Fig. 3B). Additionally, Western blot analysis confirmed that the purified protein corresponded specifically to KDM4C fused with Flag (Fig. 3C). The histone demethylase KDM4C has two enzyme active domains, JmjN and JmjC (Fig. 3C), which specifically bind to cofactors, i.e., iron ions and α-KG. The purified Flag-KDM4C fusion protein and H3K36me3 peptide (ab1785, Abcam) were added to histone demethylation buffer at 37°C for 2 hours, and then western blotting was used to detect demethylation. After the addition of varying molar concentrations of Flag-KDM4C to the histone demethylation buffer, a gradual decrease in the level of H3K36me3 was observed with increasing KDM4C concentration (Fig. 3D). However, when the KDM4C concentration reached 48 nM, there was no significant further reduction in the H3K36me3 level. Therefore, we selected a KDM4C concentration of 48 nM for our fumarate-inhibited demethylation experiment. We supplemented the histone demethylation buffer with a concentration gradient of MMF (0.1–1.2 mM) and observed a significant increase in H3K36me3 levels upon the addition of MMF at concentrations exceeding 0.1 mM (Fig. 3E). Our results showed that the demethylation of H3K36me3 by KDM4C was significantly inhibited upon treatment with millimolar concentrations of fumarate.
4、Inhibition of KDM4C activity led to increased expression of PD-L1 and the chemokine CXCL10
The level of p-STAT3 was significantly elevated in FH-knockdown cells, and as a transcription factor, p-STAT3 could enhance the expression of PD-L1 31. We conducted immunohistochemical staining of tissue sections from 107 patients diagnosed with FH-RCC to assess the expression of PD-L1. Our findings revealed that PD-L1 positivity was observed in 68.2% (73/107) of the FH-RCC patients, with a tumor proportion score (TPS) ≥ 1%. Moreover, strong PD-L1 expression (TPS > 50%) was detected in 37% (27/73) of these individuals (Fig. 4A).
The results from our previous studies demonstrated that FH-RCC exhibits a high level of immune infiltration, particularly with abundant CD4 + T and CD8 + T cells in the tumor stroma, indicating potential activation of immune signals within the tumor microenvironment (Fig. 4A). The chemokines CXCL9, 10, and 11 are capable of recruiting various immune cells expressing CXCR3 receptors on their cell membrane, including CD4 + T, CD8 + T, and Treg cells 32,33. Therefore, we investigated the expression of the CXCL10 in FH-RCC tissues and FH-knockdown cells. The results revealed higher expression level of CXCL10 in cancerous tissue and FH-knockdown cells compared to than in control groups (Fig. 4B-E), accompanied by a significant increase in PD-L1 expression (Fig. 4B-E).
To further validate the aforementioned experimental findings, ACHN and HK-2 cells were treated with a KDM4C inhibitor to assess changes in the PD-L1 and CXCL10 expression levels. qPCR and Western blot analysis revealed a significant increase in the levels of PD-L1 and CXCL10 following the inhibition of KDM4C activity (Fig. 4F, G). The suppression of KDM4C expression resulted in an increase in histone H3K36me3 levels within the promoter region of the CXCL10 gene, thereby facilitating its transcriptional activation 34. These findings are consistent with our own experimental observations.
5、 Upregulation of KDM4C in FH-knockdown cells decreased H3K36me3 levels
We assessed KDM4A, B, C, and D expression in FH-knockdown cells and noted a significant increase in KDM4C expression (Fig. 5A, B). Moreover, significantly higher KDM4C mRNA levels were observed in fresh tumor tissues from FH-RCC patients than in adjacent tissues (Fig. 5A). Furthermore, the targeted inhibition of KDM4C activity using a specific inhibitor led to the substantial upregulation of KDM4C mRNA expression in both HK-2 and ACHN cells (Fig. 5A). These results indicate that the suppression of KDM4C function triggers an induced feedback mechanism resulting in increased expression. We overexpressed either KDM4C or STAT3 in ACHN and HK-2 cells. qPCR and Western blot results showed that upregulating KDM4C expression significantly reduced STAT3 levels, while the substantial upregulation of STAT3 expression markedly increased KDM4C levels (Fig. 5C, D). Our data revealed a reciprocal regulatory loop between KDM4C and STAT3. KDM4C overexpression resulted in the significant downregulation of IL-6R, T-STAT3, p-STAT3 and PD-L1 expression in ACHN and HK-2 cells. Conversely, the overexpression of STAT3 had no significant effect on IL-6R expression but significantly upregulated p-STAT3 and PD-L1 expression (Fig. 5C, D). These results indicated that the downstream regulation of PD-L1 by p-STAT3 is independent of the IL-6R protein level.
We also overexpressed KDM4C in ACHN-shFH and HK-2-shFH cells. qPCR and Western blot analysis revealed a significant decrease in the levels of H3K36me3, IL-6R, T-STAT3, p-STAT3, PD-L1, and CXCL10 upon the upregulation of KDM4C expression in ACHN-shFH and HK-2-shFH cells (Fig. 5E, F). Meanwhile, we also overexpressed the mutant inactive form of KDM4C (H190A/E192A) in ACHN-shFH and HK-2-shFH cells. The qPCR and Western blot analysis indicated that there were no significant changes in the expression levels of H3K36me3, IL-6R, STAT3, PD-L1, and CXCL10 in FH-knockdown cells (Fig. 5E, F). Our findings suggested that the activation of the IL-6/JAK/STAT3 signaling pathway and the upregulation of PD-L1 and CXCL10 expression in FH-knockdown cells are not dependent on reduced FH expression or fumarate accumulation but rather result from the inhibition of KDM4C activity. This effect can be counteracted by upregulating KDM4C expression in FH-knockdown cells.
6、Cell proliferation and migration experiments and validation of the cell-derived xenograft (CDX) model
We investigated the impact of downregulated FH expression on the proliferation and migration of ACHN and HK-2 cells using CCK8 (Fig. 6A), scratch and transwell assays (Fig. 6C, D). Our findings revealed that while the reduced expression of FH did not significantly affect cell proliferation (Fig. 6A), it notably enhanced cell migration and invasion (Fig. 6C, D). CDX models were generated by subcutaneously injecting ACHN-shFH and ACHN-shNC cells into nude mice (Fig. 6B). There was no statistically significant difference in tissue size between the CDX-shFH and CDX-shNC groups, suggesting that the downregulation of FH expression did not exert a substantial impact on the proliferation rate of tumor cells (Fig. 6B). As seen in the magnified image in the upper right corner of Fig. 6E, lymph node metastasis was observed in 20% (1/5) of the nude mice in the CDX-shFH group. Additionally, the magnified image in the upper right corner depicts ACHN-shFH tumor cells, while the enlarged image in the lower left corner shows PAX8 immunohistochemical staining of metastatic ACHN-shFH cells, revealing positive nuclear staining (Fig. 6E). We observed that upregulation of KDM4C in HK-2-shFH and ACHN-shFH cells led to a reversal of IL6/JAK/STAT3 signaling pathway activation. Consequently, we induced overexpression of KDM4C in HK-2-shFH and ACHN-shFH cells and conducted scratch and transwell assays. The findings demonstrated that upregulation of KDM4C expression in HK-2-shFH and ACHN-shFH cells reduce cell migration and invasion (Fig. 6C, D). The above results have shown that fumarate significantly enhances the cell migration and invasion by inhibiting the activity of KDM4C. Furthermore, elevating the expression of KDM4C markedly reduces the migration and invasion of FH-knockdown cells.
Our findings revealed that FH protein expression was downregulated in CDX-shFH group, while 2SC protein expression was elevated (Fig. 6F). Compared with those in CDX-shNC group, the percentages of cells positive for STAT3, IL-6R, PD-L1 and CXCL10 were greater in CDX-shFH group, but there was no significant difference in the Ki67 index (Fig. 6F). q-PCR and Western blot results showed that the expression of the FH in the CDX-shFH group was significantly lower than that in the CDX-shNC group, while the levels of H3K36me3, IL-6R, T-STAT3, p-STAT3, PD-L1, and CXCL10 were markedly higher (Fig. 6G). The results of the previous cell experiments were validated through immunohistochemistry, Western blot and q-PCR analyses in CDX models.