CHAF1B promotes the progression of lung squamous-cell carcinoma by inhibiting SETD7 expression

Abstract

The p60 subunit of the chromatin assembly factor-1 complex, that is, chromatin assembly factor-1 subunit B (CHAF1B), is a histone H3/H4 chaperone crucial for the transcriptional regulation of cell differentiation and self-renewal. CHAF1B is overexpressed in several cancers and may represent a potential target for cancer therapy. However, its expression and clinical significance in lung squamous-cell carcinoma (LUSC) remain unclear. In this study, we performed weighted gene correlation network analysis to analyze the Gene Expression Omnibus GSE68793 LUSC dataset and identified CHAF1B as one of the most important driver gene candidates. Immunohistochemical analysis of 126 LUSC tumor samples and 80 adjacent normal lung tissues showed the marked upregulation of CHAF1B in tumor tissues and the negative association of its expression level with patient survival outcomes. Silencing of CHAF1B suppressed LUSC proliferation in vitro and LUSC tumor growth in vivo. Furthermore, bulk RNA sequencing of CHAF1B knockdown cells indicated SET domain containing 7 (SETD7) as a significant CHAF1B target gene. In addition, CHAF1B competitively binds to the SETD7 promoter region and represses its transcription. Altogether, these results imply that CHAF1B plays a vital role in LUSC tumorigenesis and may represent a potential molecular target for this deadly disease.

Keywords

CHAF1B / cell cycle / lung squamous cell carcinoma / SETD7 / WGCNA

1 Introduction

Lung cancer was the leading cause of cancer-related mortality globally in 2022, with an estimated 2.5 million new cases and 1.76 million deaths worldwide. In China alone, approximately 1.06 million new cases and 733 300 deaths have been recorded in 2022 [1,2]. Lung cancer falls under two major histological types: small-cell lung cancer (SCLC), which represents 15% of lung cancers, and non-small cell lung cancer (NSCLC), which accounts 85% of lung cancers. NSCLC can be further classified into lung adenocarcinoma (LUAD; 40% of NSCLC), lung squamous-cell carcinoma (LUSC; 30%), and large-cell lung carcinoma (LCLC; 15%) [3]. LUSC develops in the proximal region of the lungs and originates from the basal cells of bronchi. This condition is distinguished by its distinct squamous morphology and high genomic tumor mutational burden [4]. Targeted therapies, such as inhibitors of epidermal growth factor receptor and immune checkpoint inhibitors (ICIs), including those targeting programmed cell death 1 and programmed cell death-ligand 1 proteins, have considerably prolonged the overall survival of patients suffering from LUAD [5–7]. However, the effectiveness of ICIs covers only 20% of LUSCs, and no targeted therapy is available because of limited driver genes and biomarkers [8]. Currently, the 5-year survival rate of patients with LUSC is less than 15% [9,10]; therefore, new molecular indicators and treatment targets must be developed for the effective management of LUSC.

The p60 subunit of the chromatin assembly factor 1 (CAF-1) complex, known as the seven-tryptophan-aspartate-repeat-containing protein CAF-1 subunit B (CHAF1B), mediates the interaction between the antisilencing function 1A histone chaperone/histone H3/histone H4 and CHAF1A within the CAF-1 complex [11]. CHAF1B is a central facilitator delivering newly synthesized histones to DNA replication forks for chromatin assembly during the S phase of the cell cycle; and it also regulates nucleotide excision repair following DNA damage [12]. Under physiologic conditions, CHAF1B plays a critical role in preventing the mislocalization of death domain-associated protein-dependent centromere protein A and chromosomal instability [13]. In addition, CHAF1B acts as an oncoprotein and shows a high expression in solid tumors [14–17] and hematopoietic malignancies [18–20]. In acute myeloid leukemia (AML), the effects of CHAF1B are linked to AML blast self-renewal capacity and their undifferentiated state [11]. CHAF1B functions downstream of miR-3619-5p and circular RNA circ_0026134 in NSCLC [21]. In patients with LUAD, CHAF1B serves as an E3 ubiquitin ligase that induces the degradation of the nuclear receptor corepressor 2, promotes cisplatin resistance [22], enhances cell proliferation, and exhibits an inverse correlation with a poor prognosis [23]. However, the role of CHAF1B in LUSC has rarely been reported.

In this study, we aimed to construct LUSC-related gene networks and identify the central nodes using the Gene Expression Omnibus GSE68793 LUSC data set and weighted gene co-expression network analysis (WGCNA) [24]. The top three enriched pathways in LUSC comprised the cell cycle, DNA replication, and base excision repair pathways, and CHAF1B was one of the most considerably altered genes. CHAF1B was highly expressed in LUSC tumor samples and negatively associated with patient prognosis. The knockdown of CHAF1B elevated the expression of SET domain containing 7 (SETD7) and inhibited LUSC cell proliferation in vitro and in vivo. This study revealed that CHAF1B may have an important role in carcinogenesis and serve as a potential molecular target for LUSC treatment.

2 Materials and methods

2.1 Patient samples

This study received approval from the Institutional Review Boards of the Cancer Hospital, Chinese Academy of Medical Sciences, and Peking Union Medical College (approval No. NCC2020A190). A minimum of two independent pathologists verified the diagnostic assessment of patients with LUSC. Microarray slides of LUSC tissue (product Nos. HLug-Squ150Sur-02 and HLugS180Su02) were obtained from Shanghai Outdo Biotech, China. Written informed consent was obtained from all patients. Tab.1 shows the comprehensive pathological details of these samples.

2.2 Cell culture

LUSC (H520, H1703, and H226) and LCLC (H1299) cell lines were obtained from the American Tissue Culture Collection (ATCC, USA) and grown in Dulbecco’s modified Eagle medium or Roswell Park Memorial Institute 1640 with 10% fetal bovine serum (Gibco, USA). Cells were cultured in a climate-controlled environment to maintain optimal growth conditions.

2.3 Cell viability assay

H520 and H1299 cells were transfected with siCHAF1B or a negative control siRNA (siNC) (GenePharma) using Lipofectamine 3000 transfection reagent. Table S1 lists the corresponding the siRNA sequences. The cells were harvested, reseeded at an appropriate density, and cultured for various time intervals. Cell proliferation was assessed using a 1:10 dilution of Cell Counting Kit-8 (CCK-8) solution in the culture medium, with a subsequent 1 h incubation. Cell viability was assessed through measurement of the optical density (OD) of each well at 450 nm. Relative cell viability was determined using the formula: (OD_exp– OD_blank)/ (OD_control– OD_blank) × 100%, where OD_exp and OD_control denote the absorbance values of the experimentally treated and control cells, respectively.

2.4 Colony formation assay

H520 and H1299 cells were transfected with siNC or siCHAF1B for 48 h using Lipofectamine 3000 reagent. After harvest, the cells were plated at an appropriate density. After 7–14 days, the cells were fixed in methanol, washed twice with phosphate buffered saline (PBS), and stained with 0.005% crystal violet (Sigma-Aldrich, USA). Colonies with more than 50 cells were identified and quantified.

2.5 Cell cycle

H520, H1703, H226, and H1299 cell lines were transfected with siNC or siCHAF1B using Lipofectamine 3000 reagent for 72 h. After transfection, the cells were fixed in 70% ethanol at −20 °C, washed twice with PBS, and treated with ribonuclease A. Subsequently, the cells were stained with propidium iodide for DNA labeling. Cell distribution across different cell cycle phases was assessed using a FACSCalibur flow cytometer (Becton Dickinson, USA).

2.6 RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)

RNA was isolated using TRIzol (Invitrogen, USA) and reverse transcribed into cDNA using HiFiScript RT SuperMix (Vazyme, China). qRT-PCR was conducted using the SYBR qPCR Master Mix (Vazyme, China) and the reference gene GAPDH as a control. Gene expression was calculated using the 2^-ΔΔCT method. Table S1 provides the primer sequences.

2.7 Immunoprecipitation (IP) and Western blot

The cells were lysed on ice using an IP buffer and high-speed centrifugation, and the supernatants were incubated with antibodies (Table S1) overnight. Protein A/G agarose was added, and the beads were washed and boiled in the loading buffer. The input and IP samples were subjected to Western blot analysis. Table S1 lists the specific antibodies used.

2.8 Lentivirus-mediated transfection

To construct the shCHAF1B-expressing vector, we inserted shCHAF1B sequences into the pSIH1-H1-Puro shRNA vector backbone, which was then co-transfected with psPAX2 and pMD2.G into HEK293T cells. Then, lentiviral supernatants were collected from the HEK293T cells. CHAF1B-stable knockdown cells were established by infecting H520 and H1299 cells with lentiviral supernatants in the presence of polybrene (Yeasen Biotechnology, China), followed by puromycin selection. Table S1 lists the shRNA primers used.

2.9 Immunohistochemical analysis

Deparaffinization of 5 µm-thick paraffin-embedded LUSC tissue sections with xylene was conducted, followed by rehydration in a series of alcohols. The tissue sections were subjected to a heat-induced epitope retrieval step in a citrate buffer solution and blocked with 5% bovine serum albumin. Next, the sections were incubated with primary antibodies overnight, followed by a 90 min incubation with horseradish peroxidase-conjugated secondary antibodies (Table S1). 3,3′-Diaminobenzidine was used as a chromogenic reagent. The immunoreactivity score (IRS) was determined through separate scoring of the staining intensity (SI, 0–3 points) and the proportion of positively stained cells (RP, 0–4 points) in the immunohistochemical sections. In addition, composite IRS (0–12 points) was obtained using the formula SI × RP.

2.10 Chromatin immunoprecipitation

Cells cultured in a 10 cm dish were crosslinked with 1% formaldehyde, and the reaction was neutralized with glycine. Sonication was performed to fragment the chromatin associated with proteins into smaller pieces via. Protein–DNA complexes were immunoprecipitated using an anti-CHAF1B antibody, with normal rabbit IgG serving as the negative control. Table S1 lists the antibodies used in this study. Next, Protein A + G Agarose/Salmon Sperm DNA was mixed into the solution at 4 °C. After IP, DNA was decrosslinked, and proteins were removed via digestion using proteinase K. DNA was subsequently collected and prepared for PCR. Table S1 contains the primer sequences used in this work.

2.11 Animal studies

The animal experiments were approved by the relevant ethics committee of the Chinese Academy of Medical Sciences Cancer Institute and Hospital (NCC2020A190). Five-week-old, female nonobese diabetic/ShiLtJGpt-Prkdc^em26Cd52Il2rg^em26Cd22/Gpt mice (NCG; GemPharmatech, China) were maintained in a specific pathogen-free environment. H520-NC and H520-shCHAF1B cells (2 × 10⁶) were subcutaneously inoculated into the mice (n = 7 per group). Tumor size measurement was conducted every four days, with volumes calculated as 1/2 × length × width². Once tumors reached 2 cm or the mice displayed symptoms of morbidity, the tumors were excised, photographed, and subjected to subsequent experimental analyses.

2.12 Online data availability

The gene expression dataset for LUSC (GSE68793) was obtained from the website of NCBI Series GSE68793. The TIMER database was used in the evaluation of expression of CHAF1B in tissues. Survival prognoses were analyzed using the Kaplan–Meier plotter, and the enriched pathways correlated with CHAF1B were assessed using the LinkedOmics database.

2.13 Statistical analysis

Each experiment was conducted in triplicate at least. Mono-methyltransferase data are expressed as mean values ± standard deviation (SD). Statistical evaluations were conducted using SPSS (version 23.0) and GraphPad Prism (version 8.0) software. The statistical methods applied to the data included two-tailed Student’s t, chi-square, and log-rank tests. P < 0.05 was considered statistically significant.

3 Results

3.1 Identification of CHAF1B as a key gene in LUSC

We analyzed the altered genes in the LUSC public data set (GSE68793), which comprised 134 tumor samples. The top 5000 altered genes were used to create joint-expression modules via WGCNA [24,25], and 24 modules were identified based on a single-step approach for module construction and identification (Fig. S1A). Fig. S1B shows the results of module–trait association analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed on the most significant module (black) to determine the top three most enriched pathways, namely, the cell cycle, DNA replication, and base excision repair pathways (Fig.1). To better identify the pivotal genes in this module, we evaluated protein–protein interactions and depicted them using Cytoscape with the MCODE plugin [26,27]. In general, 50 significant hub genes in the circle were identified as crucial targets among differentially expressed genes (DEGs) in LUSC (Fig.1). Comparison of these hub genes with the 648 cell cycle pathway genes [28] identified 27 candidates, including CDC45, TTK, and CHAF1B (Fig.1). Among these genes, CDC45 [29] and TTK [30] regulate the proliferation of LUSC and LUAD (Table S2). However, the significance of CHAF1B, an important cell cycle regulatory protein [31], has been understudied in LUSC. Therefore, CHAF1B was selected for further investigation.

The CHAF1B levels in cancerous and adjacent healthy tissue were assessed using the TIMER database [32–34], and the findings indicate the marked increase in the CHAF1B mRNA level in tumor samples of bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), colon adenocarcinoma (COAD), esophageal carcinoma (ESCC), head-and-neck carcinoma (HNSC), liver hepatocellular carcinoma (LIHC), LUAD, LUSC, prostate adenocarcinoma (PRAD), rectal adenocarcinoma (READ), stomach adenocarcinoma (STAD), thyroid cancer (THCA), and uterine corpus endometrial cancinoma (UCEC), compared with their counterpart normal tissue (Fig.1). Kaplan–Meier plotter database was used to assess the association between CHAF1B mRNA expression and overall survival of patients suffering from NSCLC [35,36]. We discovered that elevated CHAF1B expression was linked to poor prognosis of patients with NSCLC (Fig.1).

3.2 Validation of CHAF1B in LUSC tissue samples

To further validate the significance of CHAF1B in LUSC, we analyzed 126 LUSC samples and 80 adjacent normal lung tissues from tissue microarrays via immunohistochemistry. Among these patients, 119 (94.4%) were male, 7 (5.56%) were female, 55 (43.7%) were aged ≤ 60 years, and 71 (53.6%) were > 60 years of age (Tab.1). However, no data were available on the smoking status of these 126 patients, although most male patients with LUSC are smokers. CHAF1B was mainly localized in the nuclear compartment but was rarely detected in the cytoplasmic fraction (Fig.2 and 2B). A higher CHAF1B IRS was observed in tumor tissues than in adjacent normal lung tissues (Fig.2). In tumor tissues, CHAF1B expression fell under four categories based on SI: negative, weak, moderate, and strong (Fig.2). The median IRS value served as a threshold, with IRS values above it being classified as high, and those at or below classified as low. CHAF1B expression exhibited an inverse association with overall survival (Fig.2), which is consistent with the findings of the LUSC data set analysis presented in Fig.1. These data suggest that CHAF1B serves as an oncoprotein in LUSC progression.

3.3 CHAF1B promotes LUSC cell proliferation

We explored the effects of CHAF1B on LUSC proliferation through transfection of shCHAF1B siRNA or lentivirus into H520, H1703, H226, and H1299 cells, followed by cell proliferation analysis using CCK8 assay. CHAF1B knockdown considerably suppressed the proliferation of these cell lines (Fig.3–3D, S2A, and S2B). Correspondingly, CHAF1B overexpression promoted the growth of H1703 and H226 cells after transfection with CHAF1B plasmids (Fig.3 and 3F). Colony formation experiments demonstrated the reduced colony formation in H520 and H1299 cells after CHAF1B knockdown (Fig.3 and 3H). The effect of CHAF1B on the cell cycle was further determined, and we observed that siCHAF1B-transfected cells were arrested in the S phase (Fig.3). These findings suggest the crucial role of CHAF1B in LUSC growth.

3.4 SETD7 serves as a potential downstream target of CHAF1B

To gain insights into the downstream targets of CHAF1B in promoting LUSC, we conducted bulk RNA sequencing on siNC- and siCHAF1B-transfected H520 and H1299 cells and evaluated the genes with altered expression (increased and decreased) (Fig. S3A). Compared with the siNC groups, 109 DEGs were found in siCHAF1B-transfected H1299 and H520 cells (Fig.4). Notably, eight DEGs, including the SETD7 (also termed SET7/9, and KMT7), hyaluronan binding protein 4 (HABP4), tropomyosin 4 (TPM4), caspase 7 (CASP7), dehydrogenase/reductase 2 (DHRS2), regulator of solute carriers 1 (RSC1A1), neuroplastin (NPTN), and heme oxygenase 1 (HMOX1), were selected for further analysis because their fold changes (log₂) were > 1 or < −1 in both cell lines (Fig.4). To assess the mRNA levels of these genes after CHAF1B knockdown in H520 and H1299 cells, we conducted qRT-PCR, and the findings reveal the significant upregulation of SETD7 in H520 and H1299 cells (Fig.4).

SETD7 is a histone lysine methyltransferase containing seven structural domains responsible for the methylation of catalytic SETs [37]. SETD7 mediates KRAS methylation and subsequent degradation and attenuates the RAS/mitogen-activated protein kinase/extracellular-signal-regulated kinase signaling cascade; thus, it acts as a tumor suppressor in NSCLC [38]. We hypothesized that CHAF1B promotes LUSC progression by inhibiting the tumor suppressor SETD7. To test this possibility, we performed Western blot analysis, and the findings reveal the elevated SETD7 levels following CHAF1B knockdown (Fig.4 and S3B). By contrast, CHAF1B overexpression inhibited SETD7 expression in H1703 cells (Fig.4). Notably, CHAF1B protein levels exhibited no substantial change after SETD7 knockdown, which indicates that SETD7 did not regulate CHAF1B expression (Fig.4). Using the NSCLC data sets in Kaplan–Meier plotter [35,36], we demonstrated the linkage of elevated levels of SETD7 to improved patient outcomes (Fig.4). In addition, cell viability was increased during SETD7 knockdown, and co-knockdown of SETD7 and CHAF1B rescued H1299 and H520 cell proliferation compared with CHAF1B-silenced cells (Fig.4 and 4I). These data suggest a critical role for SETD7 in the CHAF1B pathway. The co-IP assay revealed no direct association between CHAF1B and SETD7 at the protein level (Fig.4). Chromatin IP experiments using PCR primer pairs for segments of the SETD7 promoter region (Table S1) unveiled that CHAF1B bound SETD7 at the regions of primer pairs 3, 4, 5, 8, 16, and 17 (Fig.4). These results prove the function of CHAF1B as a cotranscriptional regulator of SETD7 expression at the transcription level.

3.5 CHAF1B is required for tumor growth in mice

To evaluate the effect of CHAF1B on tumorigenicity in vivo, we subcutaneously injected NCG mice with control or CHAF1B knockdown H520 cells. The findings demonstrate that stable depletion of CHAF1B markedly inhibited tumor growth compared with that in the siNC group (Fig.5). Mice in the H520-shCHAF1B group displayed substantially lower tumor volumes and weights than those in the H520-shNC group (Fig.5 and 5C). Western blot analysis was conducted to detect the CHAF1B and SETD7 protein levels in harvested tumor tissues, and the results demonstrate the significant upregulation of SETD7 upon CHAF1B knockdown (Fig.5). Immunohistochemical analysis revealed the downregulation of CHAF1B and Ki67 in H520-shCHAF1B mice compared with those in the H520-shNC group (Fig.5). These findings imply the important role of in LUSC tumor development in vivo.

4 Discussion

CHAF1B was initially identified as part of the CAF-1 complex, which is a conserved three-component complex comprising RBBP4, CHAF1A, and CHAF1B; it facilitates histone H3–H4 deposition during the S phase of the cell cycle [39,40]. Physiologically, CHAF1B assembles chromatin during the S phase of DNA synthesis in the cell cycle [12]. During embryonic development, enhanced CHAF1B expression may contribute to a high cellular replication rate [41]. CHAF1B expression shows a positive association with the proliferation marker Ki67 in several solid tumors [14]. In AML, CHAF1B overexpression promotes leukemia, whereas the suppression of CHAF1B activity substantially inhibits leukemogenesis in vivo without impairing normal hematopoiesis [11]. In addition, CHAF1B silencing results in cell accumulation in the S phase during DNA replication due to chromatin assembly defects, which leads to programmed cell death [41]. However, the role of CHAF1B in LUSC remains unclear. This study showed the overexpression of CHAF1B in LUSC and the negative correlation of its expression level with the clinical outcomes of patients with LUSC. Stable and transient knockdown of CHAF1B notably suppressed LUSC cell proliferation and arrested cell cycle in the S phase. Consistently, in vivo experiments confirmed that the stable knockdown of CHAF1B in LUSC cells tremendously inhibited tumor growth. These data indicate the role of CHAF1B as an oncoprotein in LUSC.

CHAF1B is a key epigenetic regulator at the transcriptional level and associated with cell state and self-renewal. This molecule can bind to various gene enhancers and promoters, such as CCAAT enhancer binding protein alpha, which leads to the inhibition of these genes through the displacement of transcription factors [11]. To identify the target genes of CHAF1B in LUSC, we conducted bulk RNA-seq in CHAF1B silenced cells and observed that the expressions of eight candidate genes were markedly altered, with SETD7 being the most considerably upregulated gene. We further confirmed that CHAF1B bound SETD7 at its promoter and suppressed its expression, and knockdown of CHAF1B resulted in extensive upregulation of SETD7. SETD7 stabilizes the tumor suppressor p53 and facilitates its nuclear translocation to augment p53 transactivation through its lysine methylation at residue 372 [42]. These data suggest that CHAF1B promotes LUSC tumorigenesis, likely through the downregulation of SETD7 expression, and thus inhibits the SETD7-mediated p53 cascade.

SETD7 contains 366 amino acids and comprises three motifs in the membrane occupation–recognition nexus. This mono-methyltransferase can modify various substrates, including p53, KRAS, and Rb1 [37,38,43,44]. SETD7 is involved in several pathological processes, including abnormal development, aberrant tissue regeneration, and neurodegeneration. It functions as a tumor promoter in hepatocellular carcinoma [45] and displays a direct association with drug resistance and cancer relapse. However, SETD7 is also a tumor suppressor in KRAS-driven NSCLC [38]. Herein, we showed that SETD7 knockdown contributed to enhanced NSCLC cell proliferation and that a high SETD7 expression correlated with good survival outcomes in patients. This finding indicates that SETD7 may be a tumor suppressor in LUSC. To date, only a few molecules, such as long non-coding RNAs, including small nucleolar RNA host gene 15 (SNHG15) and miR-153-3p, have been shown to regulate SETD7 expression [46]. Here, we report for the first time that CHAF1B suppresses SETD7 expression by binding to its promoter and inhibiting its transcription, and these findings provide the potential mechanisms for controlling SETD7 expression.

In conclusion, CHAF1B expression is elevated in patients with LUSC and inversely correlates with survival outcomes. Ectopic CHAF1B expression promoted LUSC proliferation in vitro and in animal models. RNA-seq revealed that SETD7 is a downstream target of CHA1B, and CHAF1B suppressed SETD7 mRNA levels, possibly through competitive binding to SETD7’s promoter site and interference with transcription factor promoter occupancy. This study suggests that CHAF1B may serve as an oncoprotein in LUSC tumorigenesis and that CHAF1B-targeted therapy may provide a potential therapeutic approach for LUSC.

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