PHF20 collaborates with PARP1 to promote stemness and aggressiveness of neuroblastoma cells through activation of SOX2 and OCT4

Abstract

The differentiation status of neuroblastoma (NB) strongly correlates with its clinical outcomes; however, the molecular mechanisms driving maintenance of stemness and differentiation remain poorly understood. Here, we show that plant homeodomain finger-containing protein 20 (PHF20) functions as a critical epigenetic regulator in sustaining stem cell-like phenotype of NB by using CRISPR/Cas9-based targeted knockout (KO) for high-throughput screening of gene function in NB cell differentiation. The expression of PHF20 in NB was significantly associated with high aggressiveness of the tumor and poor outcomes for NB patients. Deletion of PHF20 inhibited NB cell proliferation, invasive migration, and stem cell-like traits. Mechanistically, PHF20 interacts with poly(ADP-ribose) polymerase 1 (PARP1) and directly binds to promoter regions of octamer-binding transcription factor 4 (OCT4) and sex determining region Y-box 2 (SOX2) to modulate a histone mark associated with active transcription, trimethylation of lysine 4 on histone H3 protein subunit (H3K4me3). Overexpression of OCT4 and SOX2 restored growth and progression of PHF20 KO tumor cells. Consistently, OCT4 and SOX2 protein levels in clinical NB specimens were positively correlated with PHF20 expression. Our results establish PHF20 as a key driver of NB stem cell-like properties and aggressive behaviors, with implications for prognosis and therapy.

Figure 1

High-throughput screening of key regulators for NB differentiation using a CRISPR/Cas9 sgRNA library. (A) Bright-field microscopy of crystal violet staining of SH-SY5Y cells with and without RA treatment. Neurite outgrowth (arrows) began at Day 3 post-RA treatment. Scale bar, 50 μm. (B) The mRNA expression of SOX2, OCT4, NANOG, and NESTIN of SH-SY5Y cells at 0, 36, and 72 h post-RA treatment. (C) A schematic diagram of the sgRNA library screening system. (D) Heat maps generated from sgRNA library screening of SH-SY5Y cell differentiation analysis. (E) Western blot analysis of PHF20 expression in control cells by non-specific sgRNA and PHF20 KO SH-SY5Y cells by two different PHF20-specific sgRNAs. (F) Crystal violet staining in control cells and PHF20 KO SH-SY5Y cells. Dense neurite networks (arrows) were found in PHF20 KO SH-SY5Y cells. (G) The mRNA expression of SOX2, OCT4, and NANOG in control cells and PHF20 KO SH-SY5Y cells from two different sgRNAs. Data are plotted as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls using Student’s t-test.

Figure 2

PHF20 is highly expressed in NB and correlates with the poor outcome of NB patients. (A) Western blot analysis of PHF20 expression in nine NB cell lines and normal PBMCs. (B) IHC staining of PHF20 in NB of Grades 1–3 from patients and comparison with normal peripheral nervous tissue. (C) The statistical results showed the proportion of PHF20-positive cells in each group. (D) The association between PHF20 expression in NB and tumor-free survival time of selected patients was analyzed by Kaplan–Meier analysis in TCGA dataset. Scale bar, 50 μm. Data are plotted as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls using Student’s t-test.

Figure 3

PHF20 promotes proliferation of NB cells in vitro and in vivo. (A) Demonstration of ablation of PHF20 in NB PHF20 KO cells by western blotting analysis. PHF20 KO clones were generated with PHF20 sgRNA #1 and #2. (B) A total of 5000 wild-type (WT) and PHF20 KO SH-EP cells and 50000 WT and PHF20 KO SK-N-AS cells were plated in a 96-well plate using 200 μl medium. Cell viability was assayed using CellTiter-Glo®. (C) Representative xenografts excised from NSG mice. The number of mouse xenografts and tumor incidence in each group is noted on the right. (D) Growth of tumors following subcutaneous injection of PHF20 KO or control cells. (E) The tumor weight of subcutaneous xenografts formed by NB WT and PHF20 KO cells is illustrated. (F) Hematoxylin and eosin (H&E) staining and IHC staining of PHF20 and Ki-67, as well as the TUNEL assay of xenografts. (G) The statistical results showing the proportion of Ki-67-positive cells in each field and the proportion of apoptotic cells in the TUNEL assay. Scale bar, 50 μm. Data are plotted as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls using Student’s t-test.

Figure 4

PHF20 promotes migration and invasion of NB cells. (A) PHF20 KO and its control cells were subjected to transwell matrigel invasion assays. (B) Quantification of migrated cells through Matrigel for each cell line is shown. (C) Expression levels of Egfr, Wnt3a, Mycn, and Bmi1 were analyzed by quantitative real-time PCR (qPCR) in PHF20-deficient and control NB cells. (D) Expression levels of N-cadherin (N-cad), E-cadherin (E-cad), Vimentin, and Slug were analyzed by qPCR in PHF20-deficient and control NB cells. Scale bar, 50 μm. Data are plotted as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls using Student’s t-test.

Figure 5

PHF20 confers stem cell-like behavior to NB cells. (A) A tumor sphere formation assay was performed to assess the self-renewal capacity of WT and PHF20 KO cells. Five random wells were photographed. The sphere number was counted after 7 days. (B) A summary of the tumor incidence data for animals after subcutaneous injection of PHF20 KO or control cells is shown. (C) Expression levels of SOX2, OCT4, and NANOG were analyzed by qPCR in PHF20 KO and control NB cells. (D) Western blot analysis of SOX2, OCT4, and NANOG expression in PHF20 KO and control NB cells. β-actin served as a loading control. (E) IHC staining of SOX2, OCT4, and NANOG from xenografts. (F) The statistical results showing proportion of SOX2-, OCT4-, and NANOG-positive cells in each field. Scale bar, 50 μm. Data are plotted as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls using Student’s t-test.

Figure 6

PHF20 regulates SOX2 and OCT4 expression and H3K4 trimethylation by interacting with PARP1. (A) Analysis of PHF20 binding to the promoter regions of SOX2, OCT4, and NANOG in NB cells by ChIP–qPCR assay with PHF20-specific antibody. The data are presented as fold enrichment relative to input DNA. (B) ChIP–qPCR analysis of H3K4me3 and H3K27me3 of the SOX2, OCT4, and NANOG promoters in PHF20 KO and control cells. (C) Cell lysate of the PHF20 KO cells with ectopic flag-PHF20 expression cell lysate was subjected to immunoprecipitation with PHF20-specific antibody. The resolved proteins were subjected to Coomassie blue staining and excised for mass spectrometry. The top scored proteins that may interact with PHF20 were listed. (D) 293T cells were transfected with Flag-PHF20 or HA-tagged SSRP1 or PARP1. Cell extracts were immunoprecipitated with anti-Flag beads, followed by immunoblotting with anti-HA and anti-Flag antibodies. (E) Western blot analysis of PHF20, SOX2, OCT4, and NANOG expression in SSRP1 and PARP1 knockdown (KD) SH-EP cells using shRNA. Data are plotted as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls using Student’s t-test.

Figure 7

OCT4 and SOX2 play dominant roles in PHF20-induced stemness in NB cells. (A) Western blot analysis of SOX2 and OCT4 overexpressed either individually or in combination in PHF20 KO cells. (B) Representative xenografts excised from different groups of NSG mice are shown. Incidence was calculated by the number of tumors formed divided by total number of mice for each group. (C) The average tumor weight of varied groups. (D) H&E and IHC staining of PHF20, Ki-67, SOX2, OCT4, and NANOG expression in different groups. (E) IHC staining of SOX2 and OCT4 in NB tissue samples of Grades 1–3 from patients and in normal peripheral nervous tissue. Scale bar, 50 μm. Data are plotted as mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with controls using Student’s t-test.