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      Overactivation of histone deacetylases and EZH2 in Wilms tumorigenesis

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          Abstract

          Wilms tumor (WT) is the most common childhood kidney cancer. Although WT is largely curable, current treatments fail in up to 15 percent of patients. Moreover, survivors suffer from the complications and late effects of the aggressive treatments. Thus, there is a critical need to improve our understanding of tumorigenesis to develop novel therapies to reduce the treatment burden while maintaining excellent survival rates. WT is believed to arise from the immature kidney cells, nephron progenitor cells (NPCs), which have failed to differentiate properly. Previous studies revealed that Wilms cells share a transcriptional and epigenetic landscape with normal renal stem cells. 1 Although several studies have shown the positive associations between WT in children and embryonic exposure to adverse environments, the underlying mechanisms remain unknown. Altered epigenetics is central to oncogenesis in many pediatric cancers. The critical contribution of epigenetic dysregulation to pediatric tumors provides a compelling rationale for the therapeutic potential of epigenetic drugs. Histone deacetylases (HDACs) and Enhancer of Zeste Homolog 2 (EZH2, a histone H3K27 methyltransferase), have been demonstrated to play a critical role in self-renewal and differentiation of mouse NPCs. 2 , 3 In addition, altered expression and mutations of HDACs and EZH2 have been linked to many human cancers, including WT. Thus, they are among the most promising therapeutic targets for cancer treatment. We reasoned that WT would result from the unrestrained proliferation of progenitor cells due to overactive HDAC1/2 (HDAC1 and HDAC2), and EZH2. We tested this hypothesis by analyzing a clinical specimen received from the left kidney tumor of an 11-year-old male patient diagnosed with WT and four other human WT specimens. We have the approval from Tulane Human Research Protection Office & Institutional Review Boards (Study number: 2019–623) to study WT specimens. The tumor (9.5 cm × 8 cm × 7.5 cm) is classified as favorable for the histology of indeterminate cell tumors. As shown in Figure 1A, the tumor exhibits a biphasic pattern with significant blastemal component admixed with stromal component. No significant epithelial component is present. The blastema represents the undifferentiated and malignant component, consisting of small round blue cells with overlapping nuclei and brisk mitotic activity. The blastemal component shows somewhat a basaloid growth pattern. The stromal component is also prominent and includes hypercellular undifferentiated mesenchymal cells. Figure 1 Overactivation of histone deacetylases and EZH2 in Wilms tumor and cell proliferation. (A) Histological morphology of Wilms tumor (right two panels) and the adjacent normal tissues (left panel), revealing the tumor's biphasic pattern with significant blastemal component admixed with the stromal component. Scale bar = 100 μm. (B) Immunostaining revealed the significantly elevated expression and activity of HDAC1 and HDAC2 in Wilms tumor compared with adjacent normal tissues. NCAM, a critical cell surface marker of a putative WT stem cell line, was also highly expressed in the tumor. Scale bar = 100 μm. (C) Higher expression of SIX1 and SIX2 in Wilms tumor. PCNA staining also demonstrated the dramatically increased cell proliferation of tumor tissue. Scale bar = 100 μm. (D) Loss of H3K27Ac and gain of H3K27Me2/3 in Wilms tumor compared with adjacent normal tissues. Scale bar = 100 μm. (E) Western blotting showed the HDAC1/2 knockdown downregulated SIX1/2 expression in HEK293 cells. The number below the protein band is the relative band intensity normalized with the band intensity of β-actin. (F) Merck 60 and Tazemetostat demonstrated synergistic growth-suppressive effect in G401 kidney cancer cells. Cells were plated in 96-well plates and treated with Tazemetostat or Merck60 alone or both at the ratio of 1:4 for 72 h. Cell viability was determined using an MTT assay. The data represent the mean of three independent experiments ± standard error of the mean. The presented CI values indicate the interaction of Merck60 with Tazemetostat when the combined treatment inhibits cell growth by 50% or 60%, compared to the control for their treatment alone. Fig. 1 Phosphorylation of HDAC1 and HDAC2 was well characterized biochemically and positively correlated with the deacetylase activity of HDAC1/2. Immunofluorescence (IF) demonstrated dramatically higher levels of HDAC1, p-HDAC1 (phosphorylated at Ser 421, 423), HDAC2, and p-HDAC2 (phosphorylated at Ser 394) (Fig. 1B1, 3, 5, 7) in the tumor tissues compared with adjacent normal tissues (Fig. 1B2, 4, 6, 8), strongly suggesting a role of the overactive HDAC1/2 in Wilms tumorigenesis. Neural cell adhesion molecule (NCAM, a cell surface marker of the WT stem cell) was also highly expressed in the tumor (Fig. 1B3), indicating the presence of undifferentiated and proliferating WT blastemal cells. Of note, DBA-staining is a sensitive indicator of the morphogenetic activity of the collecting duct system. The lack of DBA-staining (Fig. 1B7) revealed the developmental deficiency of kidney tumor tissue. Consistent with a higher level of HDAC1/2 (Fig. 1B), histone H3 acetylation on Lysine 27 (H3K27Ac) was markedly reduced in the tumor specimen (Fig. 1D1) compared with adjacent normal tissues (Fig. 1D2). We also detected higher H3 histone tri-methylation on Lysine 27 (H3K27Me3) in the tumor (Fig. 1D3), suggesting the overactivation of EZH2. EZH2 was reported to be highly expressed in WT and has been associated with WT progression. We also examined the level of the sine oculis (SIX) family members in these WT tissues. The SIX family of transcription factors are key regulators for developmental processes and tumorigenesis. Members of this family control gene expression to promote cell proliferation and govern cell differentiation. SIX1/2 are the master transcription factors especially expressed in the human nephron progenitors and play a critical role in balancing the self-renewal and differentiation of progenitor cells. 4 Persistent SIX2 in human WT cells shifts the balance from a differentiation path toward a cell proliferation pathway. SIX1 is predominantly expressed in blastemal cells of WT and is identified as a candidate maker for blastemal. As predicted, we detected higher levels of SIX1 and SIX2. Cumulative evidence suggests that SIX1 and SIX2 drive the proliferation of the metanephric mesenchyme of developing human kidneys and have an oncogenic function in certain tumors. Consistently, IF for PCNA showed dramatically increased cell proliferation in tumor tissue (Fig. 1C5, 6). E-cadherin is a tumor suppressor protein, and the loss of its expression in association with the epithelial–mesenchymal transition (EMT) occurs frequently during tumorigenesis. Interestingly, E-cadherin was drastically reduced in the tumor compared to its adjacent normal tissues (Fig. 1C7, 8). To study the regulation of SIX1/2 expression by HDAC1/2, we did HDAC1/2 knockdown in human embryonic kidney 293 (HEK 293) cells. The cells were transfected with siRNA-HDAC1 and/or siRNA-HDAC2, and HDAC1/2, SIX1/2 level was evaluated by WB analysis and quantified against the endogenous β-actin level. As a result (Fig. 1E), the knockdown of HDAC1/2 together significantly downregulated the expression of SIX1 and slightly downregulated the expression of SIX2, indicating the positive regulation of SIX1/SIX2 by HDAC1/2. Of note, in humans, SIX1 is the direct target of SIX2. 4 The more marked downregulation of SIX1 may partially result from impaired SIX2 activity after HDAC1/2 knockdown. A previous study showed that SIX1 and SIX2, which encode transcription factors with non-redundant roles in renal development, are responsible for Wilms tumorigenesis. 5 Furthermore, SIX1 and SIX2 are associated with the high-risk blastemal subtype and with the presence of undifferentiated blastema in WT. Our study in the mouse model also revealed that the deletion of Hdac1/2 resulted in the deletion of the progenitor pool and downregulation of key progenitor genes that are mutated in WT, such as Six 2 and Wt1. 2 We also demonstrated that Ezh2 is the dominant H3K27 methyltransferase in Six2+ NPCs and is required for NPC proliferation. 3 Based on these findings, we reason that SIX1/2 upregulation by HDAC1/2 and EZH2 overactivation may play an important role in Wilms tumorigenesis. Studies have demonstrated that EZH2 interacts with HDAC1/2 and that HDACs are required for the transcriptional repression by EZH2. Concomitant inhibition of HDACs and EZH2 has proven highly synergistic and very potent for the treatment of many types of human cancers. To develop a potentially effective epigenetic therapy for WT, we examined the effects of co-treatment with benzamide-based HDAC1/2-selective inhibitor Merck 60 and FDA-approved EZH2 inhibitor Tazemetostat in G401 cells. The line was utilized for studies of chromosomal changes in WT and formerly classified as a WT cell line. Due to a change in the classification, the cell line was found to be more appropriately classified as derived from a rhabdoid tumor of the kidney. Our result showed that the co-treatment synergistically suppresses the proliferation of G401 kidney cancer cells (Fig. 1F). In summary, our studies show the elevated expression and activity of HDAC1/2 and EZH2 in WT samples, positive regulation of SIX1/SIX2 by HDAC1/2 during tumorigenesis, and synergic effect of HDAC1/2 and EZH2 inhibition in suppressing tumor cell proliferation. Thus, specifically co-targeting HDAC1/2 and EZH2 may provide a promising therapeutic approach to treat WT and other kidney cancer with low toxicity and low side effects. Epigenetic therapies have been proven to be effective in well-defined clinical contexts. The availability of epigenetic drugs will facilitate the translation of this research into promising therapies. Author contributions Hongbing Liu designed the study, performed the experiments and data analysis, and wrote the manuscript. Chao Hui Chen and Nguyen Yen Nhi Ngo performed the experiments and data analysis. Alun Wang and Samir El-Dahr performed data analysis. All authors approved the final version of the manuscript. Conflict of interests All authors declare no competing interests. Funding This study was partly supported by the 10.13039/100000968 American Heart Association (No. 17SDG33660072) and the Louisiana Board of Regents Support Fund (No. LEQSF-RD-A-18) to Dr. Hongbing Liu.

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          Most cited references5

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          Recurrent DGCR8, DROSHA, and SIX homeodomain mutations in favorable histology Wilms tumors.

          We report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations. Significantly decreased expression of mature Let-7a and the miR-200 family (responsible for mesenchymal-to-epithelial transition) in miRNAPG mutant tumors is associated with an undifferentiated blastemal histology. The combination of SIX and miRNAPG mutations in the same tumor is associated with evidence of RAS activation and a higher rate of relapse and death.
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            Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators.

            Nephron endowment is determined by the self-renewal and induction of a nephron progenitor pool established at the onset of kidney development. In the mouse, the related transcriptional regulators Six1 and Six2 play non-overlapping roles in nephron progenitors. Transient Six1 activity prefigures, and is essential for, active nephrogenesis. By contrast, Six2 maintains later progenitor self-renewal from the onset of nephrogenesis. We compared the regulatory actions of Six2 in mouse and human nephron progenitors by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Surprisingly, SIX1 was identified as a SIX2 target unique to the human nephron progenitors. Furthermore, RNA-seq and immunostaining revealed overlapping SIX1 and SIX2 activity in 16 week human fetal nephron progenitors. Comparative bioinformatic analysis of human SIX1 and SIX2 ChIP-seq showed each factor targeted a similar set of cis-regulatory modules binding an identical target recognition motif. In contrast to the mouse where Six2 binds its own enhancers but does not interact with DNA around Six1, both human SIX1 and SIX2 bind homologous SIX2 enhancers and putative enhancers positioned around SIX1. Transgenic analysis of a putative human SIX1 enhancer in the mouse revealed a transient, mouse-like, pre-nephrogenic, Six1 regulatory pattern. Together, these data demonstrate a divergence in SIX-factor regulation between mouse and human nephron progenitors. In the human, an auto/cross-regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis. By contrast, the mouse establishes only an auto-regulatory Six2 loop. These data suggest differential SIX-factor regulation might have contributed to species differences in nephron progenitor programs such as the duration of nephrogenesis and the final nephron count.
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              Histone deacetylases 1 and 2 regulate the transcriptional programs of nephron progenitors and renal vesicles

              Nephron progenitor cells (NPCs) are Six2-positive metanephric mesenchyme cells, which undergo self-renewal and differentiation to give rise to nephrons until the end of nephrogenesis. Histone deacetylases (HDACs) are a group of epigenetic regulators that control cell fate, but their role in balancing NPC renewal and differentiation is unknown. Here, we report that NPC-specific deletion of Hdac1 and Hdac2 genes in mice results in early postnatal lethality owing to renal hypodysplasia and loss of NPCs. HDAC1/2 interact with the NPC renewal regulators Six2, Osr1 and Sall1, and are co-bound along with Six2 on the Six2 enhancer. Although the mutant NPCs differentiate into renal vesicles (RVs), Hdac1/2 mutant kidneys lack nascent nephrons or mature glomeruli, a phenocopy of Lhx1 mutants. Transcriptional profiling and network analysis identified disrupted expression of Lhx1 and its downstream genes, Dll1 and Hnf1a/4a , as key mediators of the renal phenotype. Finally, although HDAC1/2-deficient NPCs and RVs overexpress hyperacetylated p53, Trp53 deletion failed to rescue the renal dysgenesis. We conclude that the epigenetic regulators HDAC1 and HDAC2 control nephrogenesis via interactions with the transcriptional programs of nephron progenitors and renal vesicles. Summary: Histone deacetylases 1 and 2 perform redundant, sequential and essential roles in the balance of nephron progenitor cell self-renewal and differentiation, as well as in progression of nephrogenesis.
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                Author and article information

                Contributors
                Journal
                Genes Dis
                Genes Dis
                Genes & Diseases
                Chongqing Medical University
                2352-4820
                2352-3042
                28 November 2022
                September 2023
                28 November 2022
                : 10
                : 5
                : 1783-1786
                Affiliations
                [a ]Department of Pediatrics, Tulane University School of Medicine, New Orleans, LA 70112, USA
                [b ]Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA
                Author notes
                []Corresponding author. Tel.: +504 988-6244 hliu8@ 123456tulane.edu
                Article
                S2352-3042(22)00298-7
                10.1016/j.gendis.2022.10.026
                10363625
                72ca9c02-f1ac-499c-9a41-8032d1311477
                © 2022 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 15 June 2022
                : 24 October 2022
                : 31 October 2022
                Categories
                Rapid Communication

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