Abstract
Gastric cancer (GC) is characterized with differentiation disorders, the precise mechanisms of which remain unknown. Our previous study showed that PHF10 exhibits oncogenic properties in GC, with its histological presentation indicating a potential role in the modulation of differentiation disorders in GC. This study reveals a significant upregulation of PHF10 in GC tissues, showing a negative correlation with differentiation level. PHF10 was found to impede the differentiation of GC cells while promoting their stemness properties. This was attributed to the formation of a positive feedback loop between PHF10 and E2F1, resulting in dysregulated expression levels in GC. Additionally, PHF10 was found to mediate the transcriptional repression of the target gene DUSP5 in GC cells through the assembly of the SWI/SNF complex, leading to an elevation in pERK1/2 levels. In GC tissues, a negative association was noted between the expression of E2F1 or PHF10 and DUSP5, whereas a positive correlation was observed between the expression of E2F1 or PHF10 and pERK1/2. Additional rescue experiments confirmed that the inhibitory effect on differentiation of GC cells by PHF10 is dependent on the DUSP5-pERK1/2 axis. The signaling cascade involving E2F1-PHF10-DUSP5-pERK1/2 was identified as an important player in regulating differentiation and stemness in GC cells. PHF10 emerges as a promising target for differentiation induction therapy in GC.
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Introduction
Gastric cancer (GC) is a malignancy associated with a high incidence of morbidity and mortality [1]. The characteristic of differentiation disorders is a significant feature of GC [2, 3]. The Corea pathway outlines the progression of intestinal GC from normal gastric mucosa through stages of chronic superficial gastritis, chronic atrophic gastritis, intestinal epithelial metaplasia, dysplasia, and ultimately carcinogenesis [4, 5]. Within the context of intestinal epithelial metaplasia, gastric epithelial cells undergo dedifferentiation to acquire characteristics of gastric or intestinal stem cells/progenitor cells, followed by trans-differentiation into metaplastic cells exhibiting an intestinal epithelial phenotype. If gastric or intestinal stem cells/progenitor cells acquire key oncogenic driving mutations, it can result in the development of GC. Research has indicated that abnormal expression of CDX1 is a significant factor in the development of gastric epithelium differentiation disorders and carcinogenesis [6]. Upregulation of CDX1 can be facilitated by the transcription of stem cell-related reprogramming factors KLF5 and SALL4, leading to intestinal metaplasia of gastric epithelium through the Wnt pathway and contributing to the progression of GC [7]. The etiology of differentiation disorders remains poorly understood, necessitating further investigation into the intricate molecular mechanisms that underlie dysdifferentiation. This research is crucial for advancing differentiation induction therapy for GC.
In our previous work, the identification of plant homeodomain finger protein 10 (PHF10) through SEREX analysis revealed its close association with GC [8]. PHF10 shares homology with the Drosophila transcription co-activator SAYP, possessing a SAY domain in its N-terminal region and two PHD domains in its C-terminal region [9]. The PHD (plant homeodomain) is a highly conserved zinc finger domain found in eukaryotes that serves as a characteristic domain for a variety of transcription factors (TFs) [10, 11]. This domain allows TFs to interact with histone H3, specifically H3K4, and modulate the transcription of target genes through epigenetic modifications [12, 13]. Our prior research demonstrated that PHF10 enhances the G1/S transition and inhibits apoptosis in GC cells by suppressing the transcription of Caspase-3 [14], suggesting its oncogenic role in GC. Additionally, the expression of PHF10 was notably elevated in poorly differentiated GC compared to well-differentiated GC [15]. The findings suggest that PHF10 may have a crucial role in the regulation of differentiation disorders in GC.
The SWI/SNF (switch/sucrose non-fermentable) chromatin remodeling complex is known to have significant implications in gene expression, regulation, particularly in processes such as cell maintenance and differentiation in cancer stem cells [16, 17]. This complex comprises Brahma-related gene 1 (BRG1)-associated factor (BAF) and polybromo-associated BRG1-associated factor (PBAF), which is composed of a catalytic subunit with ATPase activity, conserved core regulatory subunits, and variable subunits [18]. The SWI/SNF complex consists of the catalytic subunit BRG1 or BRM, along with core regulatory subunits SNF5, BAF155, and BAF170 [19]. Recent high throughput sequencing studies have revealed frequent mutations in SWI/SNF complex members such as BRG1, BRM, ARID1A, and ARID1B in GC, suggesting their involvement in the pathogenesis of GC [20, 21]. Additionally, PHF10 has been identified as a transcriptional regulator within the PBAF complex, but its potential role in the formation of the SWI/SNF complex in GC tumorigenesis remains unclear [9, 22].
Cell differentiation disorder is the key step of carcinogenesis. The study focuses on investigating the role of PHF10 in GC cell differentiation disorders, specifically examining its expression patterns during the transition from normal gastric mucosa to GC. Tissue samples were used to verify PHF10 expression patterns and evaluate the correlation between PHF10 levels and clinical pathological parameters, including the degrees of GC differentiation and prognosis. Further, a detailed examination of PHF10 in GC cell differentiation was conducted. With the emergence of additional insights into the molecular mechanisms involved, PHF10 is increasingly viewed as a promising target for guiding differentiation induction therapy in GC.
Materials and methods
Cell culture
The GC cell lines MKN28, MKN45, SGC7901, HS-746T, and AGS were procured from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. MKN28 originates from highly differentiated human gastric adenocarcinoma, while SGC7901 originates from poorly differentiated human gastric adenocarcinoma. The GC cell lines BGC-823 and CRL-5822 were purchased from American Type Culture Collection (ATCC). The immortalized cell line GES-1, derived from human gastric mucosal epithelium, was maintained in our laboratory. All cell lines were cultured in DMEM supplemented with 10% FBS, 100 μ/mL streptomycin, and 100 μ/mL penicillin in a cell incubator with 5% CO2 at 37 °C.
RNA isolation and quantitative real‑time PCR (qRT-PCR) analysis
Total RNA from cells was extracted utilizing the Trizol reagent (Invitrogen, USA), followed by reverse transcription employing Superscript IV reverse transcriptase (Invitrogen). A SYBR Green reagent (Applied Biosystems, USA) was used to perform qRT-PCR. The mRNA expression levels were normalized to GAPDH. Primer sequences are provided in Table S1.
Western blot (WB)
Proteins extracted from tissues or cells were subjected to SDS-PAGE for separation and subsequently transferred to PVDF membranes. The primary antibodies utilized in this study included PHF10 (GeneTex, USA, GTX116314), E2F1 (Santa Cruz, USA, sc-193), DUSP5 (Santa Cruz, sc-393801), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756), BRM (Abcam, USA, ab15597), ERK1/2 (Cell Signaling, USA, CST9926), and pERK1/2 (Cell Signaling, CST9910). GAPDH served as the internal control for normalization purposes.
Immunohistochemistry staining (IHC)
IHC was conducted in adherence to established procedures, utilizing primary antibodies targeting PHF10 (GeneTex, GTX116314), E2F1 (Santa Cruz, sc-193), DUSP5 (Santa Cruz, sc-393801), ATP4B (Santa Cruz, sc-376393), PGC (Santa Cruz, sc-5815), and a biotinylated swine anti-rabbit secondary antibody (Dako, Denmark). The staining intensity of each specimen was assessed based on color intensity (0: no staining; 1: weak staining; 2: moderate staining; 3: strong staining) and the range of positive staining cells (1: 0–25%; 2: 26–50%; 3: 51–75%; 4: 76–100%). The IHC score was calculated by multiplying the staining intensity score by the staining cell range score. Expression levels were categorized as low if the score was 6 or below, and high if the score exceeded 6.
Lentivirus transduction
The full-length cDNA of each gene and their respective controls were amplified and subcloned into the pFLAG-CMV4 plasmid, then packaged as lentivirus with labels assigned as Flag-PHF10, PHF10-vector, E2F1, E2F1-vector, DUSP5, and DUSP5-vector. Short hairpin RNAs (shRNA) specific to the mRNA or negative control shRNA were subcloned into a plasmid and packaged as lentivirus, labeled as shPHF10, shNC, E2F1-sh1, E2F1-sh2, E2F1-sh3, and E2F1-sh4. For lentivirus transfection, cells were seeded at 60% confluency in 6-well plates. Lentivirus particles containing shPHF10, PHF10, E2F1-sh1/2/3/4, E2F1, DUSP5, and their respective controls were diluted to the appropriate concentration in OptiMEM supplemented with 5 μg/mL polybrene. Positive cells were subsequently selected using puromycin (Sigma-Aldrich). The shRNA target sequences utilized were as follows: shPHF10, 5′-CAGCATTGCGCAGTGATGA-3′; shPHF10-NC, 5′-CATCATGGCGTAGTGGTGA-3′. E2F1-sh1, 5′-CCTCTTCGACTGTGACTTT-3′; E2F1-sh2, 5′-AGAGCAAACAAGGCCCGAT-3′; E2F1-sh3, 5′-TGGACTCTTCGGAGAACTT-3′; E2F1-sh4, 5′-CTATGAGACCTCACTGAAT-3′; E2F1-shNC, 5′-TTCTCCGAACGTGTCACGT-3′.
Transfection
Transfection were conducted using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Cells were collected 72 h post transfection for qRT-PCR or WB analysis. DUSP5 siRNA and negative control siRNA-NC were procured from Origene (SR301299).
Transmission electron microscope
Cells were initially fixed with 2.5% glutaraldehyde at 4 °C for 2 h, followed by a replacement with 0.2 M sucrose, and subsequent fixation with 1% OsO4. The cells were then rinsed, dehydrated with ethanol, and placed in EMBed 618 (propylene oxide) in a desiccator. Subsequently, the cells were embedded in beam capsules, baked in an oven at 60 °C, sectioned to a thickness of 0.5 µm, and collected on grids. These grids were stained with uranyl acetate and lead citrate for 5 min before imaging the cells using a Philips CM-120 transmission electron microscope.
Sphere formation
Cells were dissociated using Accutase and subsequently re-suspended in sphere formation medium composed of Neural Basal medium supplemented with B-27, EGF (20 ng/mL), bFGF (10 ng/mL) and heparin (5 mg/mL). The cells were then seeded onto ultralow attachment 6-well plates (Corning, USA) at varying concentrations (SGC7901, 5000 cells/well; MKN28, 10,000 cells/well; GES-1, 20,000 cells/well). Primary spheres were observed after 7 days, dissociated into single-cell suspension, seeded at individual cell density. Secondary spheres were quantified after another 7 days. The sphere formation medium was refreshed every other day, and the spheroids in suspension culture were monitored and counted under microscope.
Chromatin immunoprecipitation (ChIP) assay
The chromatin immunoprecipitation Kit (Cat.17-371, Millipore) was utilized for the ChIP assay in accordance with the manufacturer’s instructions. GC cells were cultured to 90% confluence and subsequently cross-linked with 1% formaldehyde for 10 min prior to undergoing cell lysis on ice. The cross-linked DNA fragments were sonicated to an average size of ~200 bp. Immunoprecipitation was performed using specific antibodies or control IgG. Following elution of protein/DNA complexes and reversal of cross-links, the DNA was purified and analyzed by qRT-PCR. The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756). The primer for PHF10 promoter is 5′-TTGGGCTGACGTGAGAAG-3′ (forward) and 5′-ACCGCCATGGCCGTC-3′ (reverse). The primer for DUSP5 promoter is 5′-GCAAATCTCCGACGTCCTCT-3′ (forward) and 5′-GATCCGGCCTTCAGCTTCA-3′ (reverse).
Luciferase assay
The E2F1 binding site in the PHF10 promoter is situated at –159 ~ –149 bp. Constructs of the wild-type PHF10 promoter (PHF10-WT) (–2000 ~ –1 bp), a truncated PHF10 promoter lacking the E2F1 binding site (PHF10-△), and a mutated PHF10 promoter with altered E2F1 binding site (PHF10-MUT) were cloned into the luciferase reporter vector pGL4. The binding site for the PHF10-SWI/SNF complex in the DUSP5 promoter is located at –460 ~ –300 bp. Constructs of the wild-type DUSP5 promoter (DUSP5 WT) (–2000 ~ –1 bp), a DUSP5 promoter lacking the PHF10-SWI/SNF complex binding site (DUSP5-△), and a DUSP5 promoter with a mutated PHF10-SWI/SNF complex binding site (DUSP5-MUT) were also prepared. Co-transfection of cells with the reporter construct and the internal control vector (pRL-TK) was performed for the reporter assay. Luciferase activity was measured using the dual-luciferase reporter assay system (Promega, USA).
Immunofluorescence assay
Cells were fixed in 4% paraformaldehyde for 30 min and washed with PBS for 3 times. Subsequently, the cells were incubated with Triton for 15 min, washed with PBS for 3 times, and then sealed with serum at 37 °C for 30 min. Following the removal of BSA, the cells were incubated with the primary antibody at 4 °C overnight. After washing three times with PBS, the cells were incubated with a fluor secondary antibody for 1 h. Afterwards, they were washed with PBS for 3 times, and incubated with 1 μg/mL DAPI for 5 min. Finally, the cells were sealed with ProLongTM Gold anti-quenching agent, and images were captured using a fluorescence microscope.
Co-immunoprecipitation (Co-IP) assay
Cell lysates were pre-cleared with control agarose resin through rotation at 4 °C for 1 h. Following this, the lysates underwent immunoprecipitation using suitable antibodies or corresponding IgG control through rotation at 4 °C overnight. The beads were then washed with elution buffer and boiled for 5 min the following morning. The proteins that were bound were separated using SDS-PAGE and subsequently analyzed through WB with corresponding antibodies.
Bioinformatic analysis
The bioinformatic analyses were conducted using the R4.2.1 software. Visualization of GO, KEGG, and GSEA data was achieved through the ggplot2 package. The promoter region of PHF10 was retrieved from NCBI, specifically from –2000 to –1 bp. The prediction of TFs was performed by integrating data from PROMO (https://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) and ChIP-seq datasets. Binding motifs were identified using JASPAR.
Statistical analysis
The results were presented as mean ± SD and statistical analyses were conducted using SAS version 9.4, employing the student’s t-test, chi-squared test, or Fisher’s exact test. All statistical tests were two-tailed, with differences deemed statistically significant at a significance level of P < 0.05.
Results
PHF10 involves in GC cell differentiation
In our prior investigation involving the IHC staining of 190 cases of GC tissues [15], it was observed that the level of PHF10 was elevated in poorly differentiated cases compared to well-differentiated cases. This finding suggests a negative correlation between PHF10 expression levels and the degree of differentiation in GC (Fig. 1A). We conducted a comprehensive analysis of PHF10 levels in a total of 190 samples, consisting of 90 paired non-cancerous gastric tissues. Within this sample set, 61 samples exhibited normal gastric mucosa, 12 showed signs of chronic gastritis, and 17 displayed intestinal metaplasia. Our findings indicate a gradual increase in PHF10 levels across the progression from normal gastric mucosa to chronic gastritis, intestinal metaplasia, and ultimately GC development, as illustrated in Fig. 1B. Utilizing RNA sequencing data from TCGA-STAD, we identified 68 differentially expressed genes (DEGs, |Fold Change | >2.0) between groups with high and low PHF10 expression levels. Applying GO and KEGG analysis, the DEGs were observed to be predominantly enriched in processes related to epidermis development and epidermal cell differentiation (Fig. 1C). Furthermore, pathways associated with protein digestion and gastric acid secretion were also identified (Fig. 1D). Additionally, GSEA revealed that cellular functions linked to epithelium differentiation and development were enriched in GC patients exhibiting low levels of PHF10, while the MAPK signaling pathway was activated in the high PHF10 group (Fig. 1E). These results suggest that PHF10 may play a significant role in the differentiation of GC.
A Comparison of PHF10 IHC scores across various differentiation levels of GC (n = 190). B A histogram illustration of PHF10 positivity in normal gastric mucosa, chronic gastritis, intestinal metaplasia and GC tissues. C Results from GO analysis. Genes of the epidermis development and epidermal cell differentiation were mostly enriched. D Results from KEGG enrichment analysis. Genes of the protein digestion, gastric acid secretion and IL-17 signaling pathway were enriched. E GSEA analysis revealed dysregulated pathways associated with PHF10 expression. The GSEA analysis of GO between the high and low PHF10 expression samples revealed significant difference in the enrichment of cell differentiation pathways, while the GSEA analysis of KEGG pathway enrichment showed MAPK signaling pathway activation. F WB validation of PHF10 knockdown SGC7901, PHF10 overexpression MKN28 and GES-1 cells. G QRT-PCR detection of gastric epithelial maturation and differentiation markers following PHF10 knockdown in SGC7901 cells. H The expression levels of gastric epithelial stem progenitor cells and GC stem cell markers in SGC7901-shPHF10 cells. I The expression patterns of markers for gastric epithelial maturation and differentiation following the upregulation of PHF10 in MKN28 cells. J The expression levels of CD44, CE133, Villin 1 and SOX9 in MKN28-PHF10 cells. K The expression of markers for gastric epithelial maturation and differentiation in GES-PHF10 cells. L The expression levels of gastric epithelial stem progenitor cells and GC stem cell markers in GES-1 cells overexpressing shPHF10. *P < 0.05.
PHF10 inhibits gastric epithelium differentiation and promotes gastric cell stemness
To investigate the impact of PHF10 on GC cell differentiation, we initially assessed the mRNA levels of PHF10 in seven GC cell lines (MKN28, MKN45, CRL-5822, HS-746T, SGC7901, BGC-823, and AGS) as well as the gastric epithelial immortalized cell line GES-1. Our findings revealed a significantly elevated expression of PHF10 in GC cell lines compared to GES-1 (Fig. S1A). Subsequently, we included SGC7901 (derived from poorly differentiated human GC with relatively high PHF10 expression), MKN28 (derived from well-differentiated human GC with relatively low PHF10 expression), and the normal gastric epithelial cell line GES-1 in our study (Fig. S1C). Additionally, we generated one PHF10 knockdown cell line (SGC7901-shPHF10 and corresponding control) and two PHF10 overexpression cell lines (MKN28-PHF10, GES-1-PHF10, and corresponding controls) for further investigation (Figs. 1F and S1D–F). The expression of gastric epithelium maturation and differentiation markers (ATP4A and ATP4B for parietal cells, MIST1 and Pepsinogen I for chief cells, NEUROG3 and GAST for gastric neuroendocrine cells, TFF1, TFF2, MUC5AC, MUC6, and GKN1 for gastric mucus cells) was analyzed, along with markers associated with gastric epithelium dedifferentiation (GC stem cell markers CD44 and CD133, gastric epithelial stem cell marker Villin 1, gastric epithelial progenitor cell marker SOX9) in PHF10 knockdown or overexpression groups. Our study revealed that sustained suppression of PHF10 in SGC7901 cells led to upregulation of markers associated with gastric epithelial maturation and differentiation (Fig. 1G), while concurrently downregulating markers indicative of gastric epithelial stem/progenitor cells and GC stem cells (Fig. 1H). Conversely, persistent overexpression of PHF10 in MKN28 cells and GES-1 cells resulted in the downregulation of markers associated with gastric epithelial maturation and differentiation (Fig. 1I, K), and the upregulation of markers indicative of gastric epithelial stem/progenitor cells and GC stem cells (Fig. 1J, L). These findings suggest that PHF10 may play a role in impeding the maturation and differentiation processes within the gastric epithelium.
Given the dynamic nature of cell differentiation, we proceeded to conduct transient transfection experiments to observe changes in differentiation markers following upregulation of PHF10 over a 96-h period. The results showed that the expression levels of ATP4B, Pepsinogen I, GAST, and TFF1 (representing distinct mature gastric epithelial cell types) exhibited an inverse relationship with PHF10 levels (Figs. 2A–D and S2A), whereas CD44 and SOX9 displayed a positive correlation with PHF10 expression (Fig. 2E, F). These results provide additional evidence supporting the suppressive impact of PHF10 on the maturation and differentiation of gastric epithelial cells.
A Parietal cell marker ATP4B. B Main cell marker Pepsinogen I. C Gastric neuroendocrine cell marker GAST. D Gastric mucus cell marker TFF1. E GC stem cell marker CD44. F Gastric progenitor cell marker SOX9. *P < 0.05; **P < 0.01; ***P < 0.001.
PHF10 inhibits subcellular morphology and enhances sphere formation ability in GC cells
Transmission electron microscopy detection was utilized to examine the impact of PHF10 on subcellular morphology and structure during GC cell differentiation. The negative control, SGC7901 cells, displayed characteristics typical of malignant tumors, including variations in microvilli thickness, length, and quantity, uneven distribution of microvilli on the cell surface, well-developed rough endoplasmic reticulum, sparse mitochondrial cristae, limited Golgi complex and lysosomes in the cytoplasm, varying depths of pits on the nuclear membrane, well-developed euchromatin, and a homogeneous fine-grained cytoplasm in the nucleus (Fig. 3A, B). In comparison to SGC7901 cells, PHF10 knockdown cells exhibited distinct subcellular morphology, characterized by a decreased presence of microvilli on the cell surface and shallower pits in the nuclear membrane (Fig. 3C), diminished rough endoplasmic reticulum, and the presence of high electron density lysosomes in the cytoplasm (Fig. 3D), increased mitochondrial cristae with defined structure (Fig. 3E), the development of a Golgi complex associated with normal secretion function (Fig. 3F), demonstrating a differentiated subcellular morphological structure. Simultaneously, the presence of degenerative cells displaying vacuolized cytoplasm (Fig. 3G) and apoptotic cells (Fig. 3H) was observed. However, there was no significant disparity in morphology between MKN28 control cells and those overexpressing PHF10 (Fig. S2B and S2C).
A SGC7901 cells exhibited numerous microvilli on the cell surface and pits on the nuclear membrane (×15,000). B SGC7901 cells manifested a significant presence of rough endoplasmic reticulum in the cytoplasm (white arrow), limited mitochondrial cristae (black arrow) (×60,000). C SGC7901-shPHF10 cells exhibited a decrease in microvilli and pits on the nuclear membrane (×10,000). D SGC7901-shPHF10 cells presented a notable quantity of lysosomes with high electron density (white arrow) (×60,000). E The cytoplasm of SGC7901-shPHF10 cell exhibited an increase in mitochondrial cristae (white arrow) (×60,000). F Cells with PHF10 knockdown displayed alterations in the Golgi complex (white arrow) (×30,000). G Cytoplasmic vacuolization was observed in SGC7901-shPHF10 cells (×30,000). H Apoptotic cells were identified in SGC7901-shPHF10 cells (×15,000). I–K The sphere formation capacity of SGC-7901, MKN28 and GES-1 cells manipulated by PHF10 was evaluated by measuring the number and diameter of primary and secondary spheres separately. *P < 0.05; **P < 0.01; ***P < 0.001.
To assess the impact of PHF10 on GC stemness, sphere formation assays were conducted using SGC7901-shPHF10, MKN28-PHF10, GES-1-PHF10, and their respective control cells. The results indicated that SGC7901 cells formed spheres with less compact structures (Fig. 3I), whereas MKN28 and GES-1 cells formed denser spheres (Fig. 3J, K). Following the knockdown of PHF10, a significant decrease in both the number and diameter of primary and secondary spheres formed in SGC7901 cells was observed (Fig. 3I). Conversely, overexpression of PHF10 in MKN28 cells led to a significant increase in both the number and diameter of spheres formed (Fig. 3J). In GES-1-PHF10 cells, there was an increase in the number of primary and secondary spheres formed, although there was no significant change in sphere diameter (Fig. 3K). These findings indicate that PHF10 may enhance the sphere formation capacity of both GC and GES-1 cells.
PHF10 inhibits GC cell differentiation in vivo
The study examined the differentiation markers present in implanted tumor tissue of SGC7901-shPHF10, MKN28-PHF10, and their respective control groups. The results indicated that PHF10 suppressed the expression of markers associated with gastric epithelium maturation and differentiation, such as ATP4B, Pepsinogen I, GAST, TFF1, TFF2, and GKN1, while simultaneously upregulating the expression of CD44, VIL-1, and SOX9, markers associated with gastric cancer stem cells, gastric epithelial stem cells, and gastric epithelial progenitor cells, respectively (Fig. 4A, B). These findings were was consistent with the results of our in vitro experiments.
A The expression of differentiation markers in mice xenografts was compared between the SGC7901-shPHF10 and control groups. B The levels of various markers were assessed in the MKN28-PHF10 and control groups using qRT-PCR. C The morphology of xenografts in the SGC7901-NC and SGC7901-shPHF10 groups was examined through HE staining (×200, ×400). Nuclei were represented in blue, while cytoplasm was represented in red. Tumor cells in the SGC7901-shPHF10 group exhibited a more organized arrangement and formed gastric gland tubular structures compared to those in the SGC7901-NC group. D The morphology of xenografts in the MKN28-vector and MKN28-PHF10 groups was examined (×200, ×400). Nuclei were represented in blue, while cytoplasm was represented in red. Tumor cells in xenografts formed by MKN28-PHF10 and MKN28-vector exhibited diffuse and irregular distribution, lacking the formation of glandular-like structures. E–G IHC staining was performed on SGC7901-shPHF10 cells within the glandular tubular structure of SGC7901-shPHF10 xenografts (×200). Nuclei were represented in blue, ATP4B was represented in yellow in panel (E), PGC was represented in yellow in panel (F), and panel (G) showed negative expression of Adiponectin. H The expression level of E2F1 mRNA in normal and GC tissues. I The co-expression of E2F1 and PHF10 in GC tissue. J The analysis of PHF10 and E2F1 mRNA levels in SGC7901 cells following knockdown of E2F1 or PHF10. K The examination of PHF10 and E2F1 mRNA levels in MKN28 cells overexpressing E2F1 or PHF10. L The examination of PHF10 and E2F1 protein levels in SGC7901 cells after knockdown of E2F1 or PHF10. M The assessment of PHF10 and E2F1 protein levels in MKN28 cells following overexpression of E2F1 or PHF10. N, O ChIP-qPCR experiments in SGC7901 (N) and MKN28-E2F1 (O) cells. P Dual luciferase reporter assay in MKN28-E2F1 and MKN28-E2F1 vector cells. *P < 0.05; **P < 0.01; ***P < 0.001.
Furthermore, HE staining was conducted on the mice xenografts from our previous study [15], revealing that the tumor cells in the SGC7901 control group exhibited a diffuse distribution and irregular arrangement. Conversely, in the SGC7901-shPHF10 group, the tumor cells were neatly arranged, forming gastric gland tubular structures (Fig. 4C). Additionally, the tumor cells in the xenografts generated by MKN28-PHF10 and MKN28-vector displayed diffuse and irregular distribution, lacking the ability to form glandular-like structures (Fig. 4D). Concurrently, it was observed that SGC7901-shPHF10 cells exhibited gastric gland-like structures with notable vacuolar alterations under HE staining. To mitigate potential confounding factors stemming from adipocytes in nude mice, the presence of gastric epithelium markers ATP4B and PGC was confirmed (Fig. 4E, F), while the mouse adipocyte marker Adiponectin was not detected (Fig. 4G). These findings collectively suggest that PHF10 may contribute to the suppression of gastric cancer cell differentiation in an in vivo setting.
E2F1-PHF10 positive feedback promotes dysregulation of PHF10 expression in GC
It has been previously demonstrated that PHF10 is notably upregulated in GC [14], however, the specific mechanism underlying this dysregulation remains unclear. While gene mutations are recognized as a significant factor in the dysregulation of oncogenes or tumor suppressors in GC, such as ARID1A and BRM loss [23, 24], the frequency of mutations in PHF10 in GC is notably low [25]. This suggests that dysregulation of PHF10 may occur at the epigenetic level, with upstream TFs disorder being a common contributing factor. The promoter sequence of PHF10 was obtained from NCBI (–2000 ~ –1 bp) and analyzed using the online transcription factor prediction database PROMO and ChIP-seq datasets. Two TFs, E2F1 and PAX5, were identified as potential binders to the PHF10 promoter. The binding motif was further analyzed using JASPAR (Fig. S3D). Given E2F1’s known role in promoting the transition from G1 to S phase, and our previous research indicating PHF10’s involvement in the G1 phase of the cell cycle [14, 26]. E2F1 was selected as a candidate TF for further investigation.
The study assessed the expression levels of E2F1 mRNA in cell lines and 30 pairs of GC tissues, using the same specimens for PHF10 mRNA detection. Results indicated that akin to PHF10, E2F1 exhibited significantly higher expression in GC cells compared to GES-1, with SGC7901 showing the highest expression and MKN28 displaying relatively lower levels (Fig. S1B). Furthermore, the expression of E2F1 in GC tissue was notably elevated compared to paired normal tissue, showing statistical significance (P < 0.05, Fig. 4H). Additionally, the frequency of E2F1 mutations in GC was found to be minimal, suggesting that mutations are not a primary factor contributing to E2F1 dysregulation (Fig. S3A, B). Furthermore, a significant positive correlation was observed between the expression levels of E2F1 and PHF10 (r2 = 0.3760, P = 0.0003, Fig. 4I). These findings suggest a potential association between E2F1 and PHF10 expression in GC.
Subsequently, SGC7901-shE2F1 and MKN28-E2F1 cells were generated. RT-PCR analysis revealed a decrease in PHF10 mRNA levels upon E2F1 knockdown in SGC7901 cells (Fig. 4J), whereas overexpression of E2F1 in MKN28 cells led to a significant upregulation of PHF10 mRNA (Fig. 4K). Consistent results were observed at the protein level, supporting the notion that E2F1 may positively modulate PHF10 expression (Fig. 4L, M). Furthermore, an analysis of E2F1 levels following manipulation of PHF10 revealed an unexpected finding: PHF10 positively influenced E2F1 protein expression, while showing no significant impact on E2F1 mRNA levels (Fig. 4J–M). These results suggest the possibility of PHF10 regulating E2F1 expression at the post-transcriptional level. Consequently, a positive feedback regulatory mechanism between E2F1 and PHF10 in GC cells is hypothesized.
In order to determine the direct interaction between E2F1 and PHF10, ChIP-qPCR and dual luciferase reporter assays were applied. ChIP-qPCR analysis revealed successful amplification of the PHF10 promoter region in E2F1 antibody immunoprecipitated DNA (Fig. 4N, O). The dual luciferase reporter assay demonstrated a significant increase in luciferase activity when wild-type PHF10 promoter was transfected into MKN28-E2F1 vector cells (P < 0.01) and MKN28-E2F1 cells (P < 0.001) compared to control. Conversely, transfection with truncated or mutated E2F1 binding sites (–159 ~ –149 bp) resulted in a rapid decline in luciferase activity (P < 0.05), although it remained slightly elevated compared to the control. The luciferase levels in MKN28-E2F1 cells were found to be higher compared to those in vector cells, indicating a positive correlation between luciferase intensity in MKN28 cells transfected with wild-type PHF10 promoter and E2F1 expression levels (P < 0.05, Fig. 4P). These findings indicate that PHF10 is a direct target of E2F1.
Overall, E2F1 directly targets PHF10, leading to the activation of PHF10 transcription. This, in turn, results in the upregulation of E2F1 protein levels, highlighting the importance of the positive feedback regulatory loop between E2F1 and PHF10 in the dysregulation of GC.
PHF10 participates in the formation of SWI/SNF complex in GC cells
In order to investigate the involvement of PHF10 in the assembly of the SWI/SNF complex in GC cells, immunofluorescence experiments were conducted on PHF10 and the conserved catalytic subunits of the SWI/SNF complex in SGC7901 and MKN28 cells. As BRM expression is absent in GC cells (Fig. S4A), immunofluorescence analysis of PHF10 and BRG1 was carried out. The results revealed the colocalization of PHF10 and BRG1 in the nucleus of both cell lines (Fig. 5A, B). Subsequently, Co-IP were performed on SGC7901-shPHF10, MKN28-PHF10, and their respective control cells using antibodies against PHF10, BRG1, SNF5 and BAF155. Our research findings demonstrate that PHF10, BRG1, SNF5, and BAF155 proteins can be successfully immunoprecipitated regardless of the specific antibody used, suggesting their presence within the same protein complex (Fig. 5C–J). The silencing of BRM proteins in GC cells resulted in minimal precipitation. Additionally, the levels of BRG1, SNF5, and BAF155 exhibited a positive correlation with PHF10 and other proteins within the complex (Fig. 5C–J). To further elucidate this observation, we conducted qRT-PCR and WB analyses to investigate the expression of BRG1, SNF5, and BAF155 following PHF10 overexpression or downregulation. The results showed that the downregulation of PHF10 does not impact the expression of BRG1, SNF5 and BAF155 (Fig. S4A and S4B). This suggests that the observed phenomenon may be attributed to the significant role of PHF10 in maintaining the stability of the SWI/SNF complex in GC. Consequently, it can be concluded from these results that PHF10 is involved in the assembly of the SWI/SNF complex in GC.
A, B The nuclear co-localization of PHF10 and BRG1 was observed in SGC7901 cells (A) and MKN28 cells (B). C–F Co-IP assays were performed using antibodies against PHF10 (C), BRG1 (D), BAF155 (E) and SNF5 (F) in SGC7901-shPHF10 and control cells. G–J Co-IP experiments for PHF10 (G), BRG1 (H), BAF155 (I) and SNF5 (J) were conducted in MKN28-PHF10 and control cells.
PHF10 directly targets DUSP5 through SWI/SNF complex
The above findings revealed that PHF10 is a significant factor in cell differentiation in GC, yet its precise molecular mechanism remains unknown. Given its involvement in the chromatin remodeling complex SWI/SNF, it is hypothesized that transcriptional regulation mediated by this complex may be crucial for PHF10’s role in regulating differentiation in GC. The absence of PHF10 in the TF database complicates the direct prediction of its downstream targets. Hence, the ChIP-Seq data of BRG1, SNF5, BAF155, and BAF170 were integrated and analyzed [27], with a focus on identifying targets associated with cell differentiation that exhibited elevated scores. The investigation revealed that DUSP5, a suppressor of the MAPK (ERK1/2) pathway, possesses four binding sites situated within the –460 ~ –300 bp region of the promoter, which are bound by the aforementioned molecules. Consequently, it is plausible that DUSP5 serves as a downstream target for PHF10 in GC cells, potentially influencing the regulation of cell differentiation.
To verify this, we conducted an analysis of E2F1, PHF10, and DUSP5 mRNA levels following their individual or combined manipulation. Our findings indicate that E2F1 and PHF10 have a negative regulatory effect on DUSP5 expression in SGC7901 and MKN28 cells, whereas DUSP5 does not impact the expression of E2F1 and PHF10 (Fig. 6A–F). Furthermore, the knockdown of PHF10 reversed the decrease in DUSP5 expression caused by DUSP5 siRNA, while the overexpression of PHF10 partially reversed the increase in DUSP5 expression induced by overexpressing DUSP5 (Fig. 6A–F). Subsequently, the phosphorylation level of ERK1/2 exhibited a corresponding decrease or increase in conjunction with the decrease or increase of DUSP5 (Fig. 6G–I), aligning with the established role of DUSP5 as a bispecific phosphatase involved in the dephosphorylation of the active phosphate group of pERK1/2 [28].
A–C Detection of E2F1, PHF10 and DUSP5 expression in SGC7901 cells by qRT-PCR. D–F The mRNA levels of E2F1, PHF10 and DUSP5 in MKN28 cells were also assessed. G, H WB analysis was performed to determine the levels of DUSP5, ERK1/2 and pERK1/2 levels in SGC7901 cells. I The levels of E2F1, PHF10, DUSP5, ERK1/2 and pERK1/2 levels in MKN28 cells were evaluated. J ChIP-qPCR was carried out using antibodies against PHF10, BRG1, BAF155 and SNF5 in SGC7901 cells, with IgG antibodies serving as a negative control. The purified DNA after precipitation was subsequently amplified using the same set of primers for PCR analysis. K The promoter region of DUSP5 was amplified in MKN28-PHF10 cells using antibodies against PHF10, BRG1, BAF155 and SNF5. L A dual luciferase reporter assay was conducted in both MKN28-PHF10-Vector and MKN28-PHF10 cells.
To validate DUSP5 as a direct target of the PHF10-SWI/SNF complex, ChIP-qPCR analysis was performed on SGC7901 and MKN28-PHF10 cells. The findings demonstrated that BRG1, BAF155, and SNF5 effectively amplified the promoter region of DUSP5 using a common primer following immunoprecipitation with PHF10 (Fig. 6J, K), suggesting that key components of the SWI/SNF complex and PHF10 are capable of binding to the same region of the DUSP5 promoter. Additionally, the results of a dual luciferase reporter assay indicated a significant decrease in fluorescence following transfection of the wild-type DUSP5 promoter in both MKN28-PHF10-vector and MKN28-PHF10 cells. In comparison to the wild-type group, the groups transfected with truncated or mutated forms at the binding site of PHF10, exhibited partially reversed fluorescence, albeit still slightly lower than the control group. Additionally, the fluorescence intensity of MKN28-PHF10 cells surpassed that of the control cells, suggesting a positive correlation between fluorescence intensity and PHF10 expression (Fig. 6L). These findings suggest that DUSP5 serves as a direct target for the PHF10-SWI/SNF complex.
Co-expression of E2F1-PHF10-DUSP5-pERK1/2 occurs in GC tissue
To validate the observed correlation between the expression of E2F1, PHF10, DUSP5, and pERK1/2 in vitro, we conducted qRT-PCR, IHC, and WB analyses on tissue samples from GC patients. Our findings revealed a negative correlation between the mRNA expression of E2F1 or PHF10 and DUSP5 (E2F1 vs DUSP5, r2 = 0.5318, P = 0.0091; PHF10 vs DUSP5, r2 = 0.7345, P = 0.0011, Fig. 7A). Additionally, the protein levels of E2F1 or PHF10 were positively associated with pERK1/2, but negatively associated with DUSP5 (Fig. 7B, C). These results suggeat that the E2F1-PHF10-DUSP5-pERK1/2 pathway is indeed present in GC tissue.
A The relationship between E2F1 or PHF10 and DUSP5, was examined in 30 pairs of GC tissues through qRT-PCR analysis. B IHC staining was performed for E2F1, PHF10 and DUSP5 in two patient samples. Patient 1 exhibited poorly differentiated and diffuse GC, while patient 2 had well differentiated and intestinal GC. C WB analysis was conducted to measure the levels of E2F1, PHF10, DUSP5 and pERK1/2 in the aforementioned patient samples. D The expression levels of gastric epithelium differentiation markers (ATP4B, PG I, GAST, TFF1, CD44 and SOX9) were detected by qRT-PCR in SGC7901 cells. E The mRNA levels of gastric epithelium differentiation markers were assessed in MKN28 cells. F, G Sphere formation was quantified in SGC7901 cells (F) and MKN28 cells (G). H A schematic illustrating the mechanism of PHF10-mediated dysdifferentiation in GC cells.
PHF10 mediates differentiation disorders in GC through E2F1-PHF10-DUSP5-pERK1/2 pathway
In order to provide additional validation of the role of the E2F1-PHF10-DUSP5-pERK1/2 axis in mediating the differentiation barrier effect of GC cells, rescue experiments were conducted. These experiments involved overexpressing E2F1 or knocking down DUSP5 in SGC7901-shPHF10 cells, as well as knocking down E2F1 or DUSP5 in MKN28-PHF10 cells, to determine if the phenotypes mediated by PHF10 in cell differentiation could be reversed. The findings indicated that the upregulation of gastric epithelium maturation and differentiation markers ATP4B, PG I, GAST, and TFF1, as well as the downregulation of dedifferentiation markers CD44 and SOX9 in SGC7901-shPHF10 cells were partially or completely reversed by E2F1 overexpression or DUSP5 knockdown (Fig. 7D). Conversely, in MKN28-PHF10 cells, the downregulation of differentiation markers and upregulation of dedifferentiation markers were reversed by E2F1 knockdown or DUSP5 overexpression (Fig. 7E). Meanwhile, the overexpression of E2F1 or the downregulation of DUSP5 in SGC7901-shPHF10 cells resulted in a decrease in sphere formation ability (Fig. 7F). Similarly, the downregulation of E2F1 or the upregulation of DUSP5 in MKN28-PHF10 led to an increase in sphere formation ability (Fig. 7G). These findings suggest that PHF10 may play a role in the disruption of GC cell differentiation through the E2F1-PHF10-DUSP5-pERK1/2 pathway.
Discussion
The dysdifferentiation mechanism of GC remains poorly understood, despite being a common manifestation of differentiation disorders [29]. This study elucidates a crucial molecular mechanism underlying GC cell differentiation disorder involving the dysregulation of PHF10. This dysregulation is closely linked to patient prognosis in GC, as it inhibits cell differentiation through the E2F1-PHF10-DUSP5-pERK1/2 pathway, ultimately facilitating the progression of GC.
PHF10, functioning as a TF, is crucial in mediating the differentiation of neural stem/progenitor cells into mature neurons [30, 31]. The potential association of PHF10 with malignant tumor differentiation disorders remains uncertain due to insufficient evidence. This study aimed to investigate the expression of PHF10 in GC tissues and paired non-cancerous tissues, revealing a negative correlation between PHF10 expression and the degree of differentiation. Furthermore, our findings indicate a gradual increase in PHF10 levels from normal gastric mucosa to various stages of GC progression. Consequently, a strong positive association between the development, advancement, and differentiation abnormalities of GC was demonstrated. Additionally, our research indicated that PHF10 suppressed the differentiation of gastric epithelial cells and enhanced the stemness of gastric cells both in vitro and in vivo. Moreover, our findings suggested that PHF10’s role in inhibiting GC cell differentiation was closely linked to the SWI/SNF chromatin remodeling complex. Nevertheless, the necessity of the SWI/SNF complex for PHF10 to regulate differentiation requires validation through the silencing of key components within the complex in future investigations.
E2F Transcription Factor 1 (E2F1), a member of the E2F TF family, is known to play a pivotal role in various biological processes, such as cell cycle regulation, apoptosis, and maintenance of stemness [32,33,34]. This study aimed to explore the dysregulation mechanism of PHF10 expression in GC, and our findings suggest that E2F1 may act as an upstream TF of PHF10 based on bioinformatics predictions. Our analysis of GC tissue samples revealed a significant upregulation of E2F1, which exhibited co-expression with PHF10. Moreover, the direct targeting of PHF10 by E2F1 was validated through ChIP-qPCR and dual luciferase assay. Additionally, our findings demonstrate that PHF10 reciprocally regulates the expression of E2F1 at the protein level, indicating a positive feedback loop between the two proteins. This regulatory mechanism may provide insight into the concurrent dysregulation of E2F1 and PHF10 in GC.
Dual Specificity Phosphatase 5 (DUSP5) is a member of the dual-specificity MAPK phosphatases (MKPs or DUSPs) family [35], known for their ability to hydrolyze active phosphorylation groups on serine and threonine residues of phosphorylated MAPK (ERK1/2, JNK and P38) simultaneously, thereby reverting the corresponding MAPK protein to its inactive state [36]. Consequently, DUSPs play a crucial role as negative regulators of the MAPK pathway. Previous research has indicated that DUSP5 is frequently subject to epigenetic silencing in GC due to high levels of methylation in its promoter region [37, 38]. The suppression of DUSP5 expression was found to be closely associated with the unfavorable prognosis of GC patients. Through experiments conducted on GC cell lines, it was observed that the overexpression of DUSP5 led to a decrease in pERK1/2 levels, resulting in the inhibition of cell proliferation and the promotion of G1 phase arrest. Conversely, the knockdown of DUSP5 resulted in an increase in pERK1/2 levels, leading to enhanced cell proliferation [37]. This study further elucidated that the overexpression of PHF10 facilitated the direct transcriptional repression of DUSP5 by participating in the assembly of the SWI/SNF complex, thereby representing an additional crucial mechanism contributing to the silencing of DUSP5 expression in GC. Our research identified DUSP5 as a crucial target for PHF10 in the regulation of differentiation disorders in GC cells.
Through our findings, we have uncovered the significance of the signaling pathway E2F1-PHF10-DUSP5-pERK1/2 in the differentiation of GC cells (Fig. 7H). E2F1 functions as a TF that directly enhances the transcriptional expression of PHF10. Consequently, PHF10 also elevates the protein levels of E2F1. This positive feedback mechanism results in the aberrant overexpression of E2F1 and PHF10 in GC. PHF10, a crucial component of the SWI/SNF complex, was found to play a significant role in maintaining the stability of the complex. Additionally, PHF10 was observed to interact with the DUSP5 promoter, resulting in the transcriptional repression of DUSP5. This repression led to a reduction in DUSP5 activity, specifically in the hydrolysis of pERK1/2 active phosphate groups. Consequently, the levels of activated pERK1/2 increased, thereby activating the MAPK pathway and influencing cellular function regulation.
Conclusions
The findings of this study demonstrate a significant upregulation of PHF10 in GC tissue, which is inversely associated with the level of differentiation. PHF10 was found to suppress the differentiation of GC cells while enhancing their stemness properties. Additionally, PHF10 was shown to engage in a positive feedback loop with E2F1, resulting in dysregulated expression in GC. Furthermore, PHF10 was found to transcriptionally inhibit the target gene DUSP5 in GC cells by forming the SWI/SNF complex, leading to an elevation in pERK1/2 levels. In conclusion, the E2F1-PHF10-DUSP5-pERK1/2 axis represents a significant pathway in the development of differentiation disorders in GC cells, highlighting PHF10 as a promising target for differentiation induction therapy in GC.
Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.
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Acknowledgements
We thank Prof. Qinlong Gu for his good advice and all the participants of the Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.
Funding
This study was supported by grants from National Natural Science Foundation of China (No. 82072605).
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ZF, BY and ZY conceived the study. ZF and WY designed and performed the experiments. ZF and WY analyzed the data. JL provided the needed reagents. ZF and BY wrote the manuscript. MY and BL contributed to discussion and revised the manuscript. All the authors approved the final version of the manuscript.
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The study was approved by the Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, and conducted in accordance with the Helsinki Declaration of 1975. Written informed consents were obtained from all patients. The experimental procedures with animals were approved by the animal ethics committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, and all experiments were performed following the guidelines for ethical review of animal welfare of laboratory animals, designated by the Ministry of Science and Technology of China (2018).
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Fan, Z., Yan, W., Li, J. et al. PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis. Cancer Gene Ther (2024). https://doi.org/10.1038/s41417-024-00820-5
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DOI: https://doi.org/10.1038/s41417-024-00820-5
