Introduction
Lymphoma is a common hematological malignant tumor that originates in the lymph nodes or lymphoid tissue, which could be classcially divided into Hodgkin’s Lymphoma (HL) and Non-Hodgkin’s Lymphoma (NHL) [
1,
2]. The authoritative epidemiology data indicates that the 2022 estimated new cases of lymphoma would be 89,010, resulting in approximately 21,170 death in the United States [
3]. Of note, among all pathological subtypes of NHL, Diffuse large B cell lymphoma (DLBCL) ranks the first type and occupies nearly 40% of all diagnosed cases [
4]. Like the other subtypes of lymphoid malignancies, DLBCL exihibits various heterogeneous features in regard to morphology, biological aggressiveness, as well as clinical presentation [
5,
6]. The rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) is an acknowledged standard strategy for DLBCL, alleviating burden of more two-thirds of cases [
7]. However, nearly 30% of DLBCL patients still have unsatisfactory effects and suffer from recurrent attacks due to ambiguous symptoms and unclear pathogenesis [
8]. As reported, three scoring systems intergrating multiple clinical characteristics have been commonly adopted to determine prognosis of patients for 20 years, like the International Prognostic Index (IPI), revised IPI (R-IPI), and National Comprehensive Cancer Network IPI (NCCN-IPI) [
9‐
11]. Nevertheless, the NCCN-IPI score is not completely effective to predict the clinical outcomes of DLBCL, implicating the existed tumor heterogeneity [
12]. In recent years, high-throughput sequencing methods have identified different molecular subtypes of DLBCL, like germinal center B-cell-like (GCB) DLBCL and activated B-cell-like (ABC) DLBCL [
13]. Differing from GC-like subtype, ABC-like DLBCL correlates with a worse prognosis, which is characterized by constitutively activated NF-kB signaling [
5]. As a result, further investigations on molecular mechanisms of DLBCL is of great importance to optimize prognostic hazard stratification and endow novel therapeutical targets.
In recent decades, numerous researches indicate that chromatin remodelers and histone modifiers are reported to exert essential roles in multiple tumorigenesis and aggressiveness, including DLBCL [
14‐
16]. Of note, as a highly conserved histone chaperone heterotrimer and containing p48, p60, and p150 subunits (CHAF1A), chromatin assembly factor-1 (CAF-1) participates in various biological events, especially responsible for chromatin structure restoration upon DNA repair [
17]. As the key component of CAF-1, CHAF1A cooperates with other factors, like heterochromatin protein 1 (HP1), to modulate DNA mismatch repair, DNA replication process, as well as epigenetic regulation of related genes. Besides, CHAF1A could also regulate H3K9 trimethylation that influences expressions of key target genes related with proliferation, survival, and differentiation [
18]. For instance, CHAF1A participates in a complex with methyl CpG DNA binding domain protein 1 (MDB1) and histone methyl transferase SETDB1. Intensive documents have implicated that CHAF1A is abnormally regulated or expressed in various malignancies, including neuroblastoma, prostate cancer, breast cancer, as well as hepatocellular carcinoma (HCC) [
17,
19,
20]. Furthermore, dysregulation of CHAF1A correlates tightly with genomic instability and leads to high morbid risks of leukemia, lymphoma, or other solid tumors. However, no studies are currently reported to elucidate the underlying associations between CHAF1A and lymphoma, especially the DLBCL. Meanwhile, the potential mechanisms that contribute to abnormal CHAF1A expressions are still unidentified.
As one of the adaptor proteins of the CUL3–RBX1 E3 ubiquitin ligase complex (CRL3), Speckle-Type Poz Protein (SPOP) could selectively recruit substrates via its.
N-terminal MATH domain [
21]. Next, the BTB and BACK domains of SPOP are mainly responsible for promoting oligomerization and interaction with CUL3. A list of substrates of SPOP were identified that are oncoproteins in multiple tumors, including BRD4, AR, GLI, SRC-3, Caprin1, and PD-L1 [
22,
23]. Besides, SPOP could also mediate the nondegradative ubiquitination of p62 at residue K420 within the UBA domain to attenuate the Nrf2-mediated transcriptional activation of antioxidant genes [
24]. SPOP was regarded to be a tumor suppressor in many cancers, like prostate cancer, colorectal cancer, breast cancer, and endometrial cancer (PMID: 31,771,591; 31,772,275; 31,911,863). In contrast, the tumorigenic activity of SPOP in renal cell carcinoma occurs via the ubiquitination and degradation of many factors of cellular proliferation and apoptosis, like PTEN, ERK phosphatases, Daxx, and the Hedgehog pathway transcription factor Gli2 [
25,
26]. These findings supported that SPOP may have double-faced and distinct roles in different tumors. However, little researches were available to uncover the associations between SPOP and lymphoma. Although Xiaofeng Jin et al. have found that CRL3–SPOP ubiquitin ligase complex suppresses the growth of diffuse large B-cell lymphoma by negatively regulating the MyD88/NF-κB signaling, whether SPOP plays essential functions in hematologic malignancies still remains undefinited [
27]. Apart from NF-κB signaling, whether SPOP regulates other biological pathways involved in aggressiveness of DLBCL is an interesting project to be thoroughly elucidated.
In this study, we found CHAF1A is highly expressed in DLBCL and contributes to malignant proliferation and growth. SPOP functions as a negative regulator of CHAF1A via interacting with and inducing the degradative ubiquitination of CHAF1A. Down-regulated or DLBCL-associated SPOP mutations contribute to CHAF1A accumulations, thereby enhancing tumor autophagy of DLBCL in a TFEB-dependent manner. Therefore, our study provided a novel link between SPOP/CHAF1A axis and tumor autophagy of DLBCL, acting as the basis for finding novel epigenetic targets for DLBCL treatment.
Methods and materials
Cell culture
Human lymphoblastoid B cell (GM12878), human renal epithelial cells (293 T) and DLBCL cells (OCI-LY7, DB, U2932, and FARAGE) were purchased from American Type Culture Collection (ATCC, USA). The 293 T cells were maintained in DMEM with 10%(v/v) FBS. The GM12878 cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640; Gibco) supplemented with 15% fetal bovine serum (FBS; Gibco) and 1% penicillin–streptomycin (Gibco) at 37℃ with 5% CO2.
Collection of DLBCL tissues and immunohistochemistry (IHC) assays
Eighty paired B cell lymphoma tissues and adjacent normal tissues were obtained from the Shengjing Hospital of China Medical University. This study was reviewed and approved by the Shengjing Hospital of China Medical University. All participants had signed informed consent. When we obtained the samples, the tissues were frozen at − 80 °C. No patients had received chemotherapy or radiotherapy before surgery. For the IHC assay, DLBCL tissues from patients were taken to paraffin imbedding and cut, and stained by hematoxylin. 4 μm thick sections of tissues were sliced. After deparaffinization and rehydration, samples were blocked from endogenous peroxidases with 3% solution of hydrogen peroxide. Following this, IHC staining was performed using the specific primary antibodies against CHAF1A according to standard protocols. After 1 × PBS rinses for 15 min, tissue sections were incubated with the rabbit anti-goat biotinylated secondary antibody, and then followed by incubation with strept avidin-horseradish peroxidase complex (SABC) and stained with 3,3′-diaminobenzidine tetrachlorhydrate dihydrate (DAB). Sections were counterstained with hematoxylin. The staining results were evaluated by two independent observers.
Quantitative real-time PCR (RT-qPCR)
Total RNA was isolated from cells using the TRIzol reagent (Tiangen), and cDNA was reversed-transcribed using the Superscript RT kit (TOYOBO) following the manufacturer’s instructions. PCR amplification was performed using the SYBR Green PCR master mix Kit (TOYOBO). All quantitations were normalized to the level of endogenous control GAPDH. The primers of genes in this study were listed as the following: CHAF1A: F: 5′-GATGCTGCGGAGGTCCAA-3′; R: 5′-ATACGTCACCCCTGCTCTCA-3′; TFEB: F: 5′-CAAGCTCAGGCTGGGAGC-3′; R: 5′-GTATTGATGGCCGGGGTGG-3′.
First of all, the DLBCL cells (U2932, FARAGE) were seeded into 96-well plates with the concentration of 1 × 103 cells/well. All cells were incubated for 0, 24, 48, 72, and 96 h. Next, the cells were subjected to the addition of cell counting kit 8 (CCK-8; Dojindo, Tokyo, Japan) and incubated for 2 h. The optical density was measured at 450 nm. For colony formation assay, indicated cells in 6-well plates (5 × 102 cells/well) were cultured for two weeks. Then, the cells were subjected to the fixation using methanol and then stained using crystal violet (SigmaAldrich). The number of colonies containing more than 50 cells was counted manually.
Transwell assays
Cell migration was determined by Transwell (Costar) migration assay. DLBCL cells (U2932, FARAGE) were precultured in serumfree medium for 48 h. For migration assay, 3 × 104 cells were seeded in serum-free medium in the upper chamber, and the lower chamber was filled with RPMI1640 containing 5% FBS. After 48 h, the non-migrating cells on the upper chambers were carefully removed with a cotton swab, and migrated cells underside of the filter stained and counted in nine different fields.
In vivo ubiquitination assays
The 293 T cells were transfected with HA-Ub and other indicated constructs. After 36 h transfection, cells were lysed in 1% SDS buffer (Tris pH 7.5, 0.5 mM EDTA, 1 mM DTT) and boiled for 10 min. For immunoprecipitation, the cell lysates were diluted tenfold in Tris–HCl buffer and incubated with anti-FLAG M2 agarose beads for 4 h at 4 °C. The bound beads are then washed four times with BC100 buffer (20 mM Tris–Cl, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 20% glycerol) containing 0.2% Triton X-100. The protein was eluted with 3 × FLAG peptide for 2 h at 4 °C. The ubiquitinated form of CHAF1A was detected by Western blot using the anti-HA antibody.
Western blotting
After 48 h transfection, cells were lysed by RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor PMSF (Wuhan Boster Biological Technology, Ltd, Wuhan, China). Protein concentration was detected with bicinchoninic acid (BCA) method. Separated proteins were denatured at 95 °C for 5 min. Equal amount of protein (20 μg) was added into each well in 12% SDSPAGE, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Afterwards, PVDF membranes were blocked in 5% skim milk for 1 h at room temperature, following the incubation with specific primary antibodies at 4 °C overnight. PVDF membranes were then sealed with secondary antibody for 1 h at 37 °C. The protein signals were visualized with ECL solution (Millipore) and scanned by QUANTITY ONE software (Bio-Rad, Hercules, CA, USA). All antibodies used in this study were listed as the following: anti-SPOP (Abcam, ab192233); anti-CHAF1A (Cell signaling Technology, CST#5480); anti-HA (Abcam, ab9110); anti-p62 (Cell signaling Technology, CST#23,214); anti-Beclin-1 (Cell signaling Technology, CST#4122); anti-β-actin (Cell signaling Technology, CST#41,470); anti-FLAG (Cell signaling Technology, CST#14,793).
Chromatin immunoprecipitation (ChIP)
ChIP assay was conducted by the application of SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 (Cell Signaling Technology, USA) in accordance with the manufacturer's instructions. Antibodies against CHAF1A was applied to mmunoprecipitate the crosslinked protein-DNA complex, with anti-IgG as negative control. The immunoprecipitated DNA underwent purification and was analyzed by RTqPCR with primers specific for the predicted binding sites on the promoter of TFEB.
Luciferase reporter assay
The pmirGLO dual-luciferase vector (Promega, Madison, WI, USA) containing TFEB sequence was cotransfected with CHAF1A plasmids into 293 T cells. The luciferase reporter assay was conducted in shCtrl and shCHAF1A#1/2 cells. The TFEB promoter was sub-cloned into the pGL3-basic vector (Promega), then co-transfected into 293 T cells with EV or FLAG-CHAF1A, individually. Luciferase activities were explored via DualLuciferase Reporter Assay System (Promega).
Transmission electron microscopy (TEM)
DLBCL cells were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide at 4 °C and immersed in spur resin after dehydration. The cells were then stained with 4% uranyl acetate and lead citrate. Finally, images were captured using a TEM (Hitachi, Ltd).
In vivo tumor model
The 6-week-old beige female mice with severe combined immunodeficiency (SCID) were obtained from the SLAC Laboratory Animal Co., Ltd. and reared in a pathogen-free environment. All mice were randomly divided into two groups. As previously illustrated, DLBCL cells (either transfected with CHAF1A-OE vectors or empty control vectors) were subcutaneously injected to SCID-beige mice to establish xenograft models (n = 8 per group). At the end of 5 weeks, mice were killed and in vivo tumors were dissected and weighed. The ethics approval number for animal assays is 20210014-R.
Confocal microscopy
The sections were fixed and permeabilized with 0.3% Triton (Solarbio Life Sciences) for 10 min. After blocking non-specific binding using 5% BSA, sections were incubated with LC3 (1:100, Cat No. 14600–1-AP, Proteintech Group, Inc). After transfecting RFP-GFP-LC3 adenovirus for 48 h, DLBCL cells were fixed, and autophagosome dots were photographed and counted using a laser scanning confocal microscope.
Statistical analysis
Data were revealed as mean ± SD. Variance analyses were implemented via Student’s t test or one-way ANOVA. Pearson Correlation Coefficient was utilized for verifying significance of the correlation among SPOP, CHAF1A and TFEB expression. The P < 0.05 was considered statistically significant. Statistical analyses were conducted employing SPSS 22.0 (IBM, Armonk, NY, USA). All experiments were repeated in independent experiments at least three times.
Discussion
Intensive studies have highlighted that dysregulation of the epigenome is an essential factor during the pathogenesis of malignancies, including DLBCL [
30]. As is well documented, EZH2 encodes the catalytic subunit of Polycomb repressive complex 2 (PRC2) and participates in restricting gene expression via regulating methylation of histone H3 on lysine 27 (H3K27) [
31]. EZH2 is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. High-throughput sequencing have detected nealy 20% Tyr641 mutations across EZH2 in GCB-DLBCL samples [
32]. DLBCLs harbouring EZH2
Y641 mutations exhibit notably higher enrichment of trimethylated histones (H3K27me3) modifications versus DLBCLs with wild type EZH2, implicating the elevated PRC2 activity. Besides, nearly 30% of DLBCLs have the histone monomethyltransferase KMT2D mutations. Most of mutations are nonsense or frameshift mutations that could contribute to loss-of-function of KMT2D [
33,
34]. KMT2D deficiency could lead to the decrease of H3K4 methylation, contributing to altered expressions of genes related to JAK-STAT signaling, cell cycle, as well as apoptosis. Furthermore, CREBBP and EP300 mutations are often mutually exclusive in DLBCLs, correlating with tumor recurrence and inferior clinical outcomes [
35,
36]. These two epigenetic regulators belong to KAT3 family members of histone acetyltransferases, depending on H3K27 acetylation to function as transcriptional co-activators. Therefore, aberrant chromatin-modifying genes with mutations or abnormal expressions could potentiate lymphomagenesis via altering dysregulation of chromatin signatures. In this study, we focused on the epigenetic roles of CHAF1A in DLBCLs. Bioinformatic analysis and IHC assays consistently showed that CHAF1A expresses highly in DLBCL than normal tissues. Kaplan–Meier survival curves also indicated that DLBCLs with high CHAF1A expressions had worse OS outcomes relative to those with low CHAF1A. We further compared the differential expression levels of CHAF1A in multiple DLBCL cell lines. We utilized the lentiviruses to knock down CHAF1A expressions and found that targeting CHAF1A could significantly suppress cell growth, colony formation, and migration abilities. Meanwhile, ectopic expression of CHAF1A could restore the impaired malignant features, implicating that CHAF1A is indispensable for DLBCs aggresiveness. The subcutaneous mice model further suggested that CHAF1A could promote in vivo tumor growth, as revealed by larger tumor volumes and tumor weight. Mechanistically, we observed that E3 ligase enzyme SPOP could interact with CHAF1A and promote CHAF1A ubiquitination and ubiquitin-dependent proteasomal degradation. Aberrant DLBCL-associated SPOP mutations, like F102I or D104H, are deficient in mediating CHAF1A degradations. Low or mutated SPOP could result in accumulated CHAF1A proteins, thereby enhancing cell malignant aggressiveness. SPOP deficiency mainly depended on CHAF1A to accelerate in vivo DLBCL tumor growth. To further uncover the downstream pathways that are required for SPOP/CHAF1A axis, we found that CHAF1A mainly activated TFEB expressions. CHAF1A directly binds to the promoter region of TFEB to enhance the regional activity, as evidenced by the active chromatin indicator of H3K27ac. Subsequent analysis further revealed that CHAF1A could enhance the transcriptional activity of TFEB to promote its downstream targets, including CLEAR, CTSA, CTSD, MCOLN1, LAMP2, or ARSB. Thereby, SPOP/CHAF1A axis could restrict the expression levels of autophagy and lysosomal biogenesis-related genes. Based on the basic theory, we proposed that targeting TFEB-dependent autophagy process is effective to suppress DLBCLs with aberrant SPOP/CHAF1A axis.
Autophagy is an evolutionarily conserved physiological process during which the cellular components are recycled by lysosomes for subsequent degradation [
37,
38]. This process could be induced by nutrient- and energy-limiting conditions, imbalance of reactive oxygen species (ROS), as well as endoplasmic reticulum (ER) stress [
39]. Multiple aspects of biological processes are modulated by autophagy, such as cell proliferation, immune regulations, neurodegenerative disorders, or metabolic deseases [
40]. In oncology, autophagy was regarded to possess essential roles to mediate resistance to targeted treatment and immunotherapy [
41,
42]. As autophagy may have multi-faceted roles in cancer and is partially elucidated nowadays, development of agents that target autophagy is promosing to treat cancer [
43,
44]. During the early stage of tumorigenesis, autophagy mainly suppress the accumulations of oncogenic p62 proteins, cell injury, and inflammation [
45]. Therefore, autophagy may exert tumor-suppressive roles in restricting proliferation, invasion, or distal metastasis at the early stages. From the another aspect, autophagy can maintain functional mitochondria to reduce DNA damages or ROS stress, thereby enhancing the survival capacity and resistance of tumors against environmental factors. Multiple factors have contributed to the roles of autophagy on the aggressiveness of the cancers, like cancer stages, genetic phenotypes, as well as tumor microenvironment. As a master regulator of lysosomal biogenesis and autophagy, TFEB correlates tightly with multiple physiological and pathological events [
46,
47]. TFEB mainly localizes to the cytoplasm under the normal situations. However, nutrient starvation could enhance TFEB nuclear translocation to exert its transcriptional activity. Previous studies have indicated that TFEB-driven autophagy is indespensible for TGF-β-induced pancreatic tumorigenesis, promoting tumor cells migration ability and in vivo metastasis [
48]. Besides, TFEB could elevate PD-L1 expressions to promote immune evasion resistance to mTOR inhibition in renal cell carcinoma, highlighting the significant relationships between TFEB and immunotherapy [
49]. Given that little was reported about the roles of TFEB in DLBCL, we found that CHAF1A mainly depends on activated TFEB to drive tumor progression. The TFEB-dependent autophagy is required for the oncogenic roles of CHAF1A in DLBCLs.
Although intensive researches have comprehensively described the roles of aberrant SPOP mutations or expressions on kidney cancer, prostate cancer and endometrial cancer, the specific roles of SPOP on hematologic tumors are still unclear [
25,
50]. SPOP is observed to mutate in a subset of lymphoid malignancies, like DLBCLs. The lymphoid malignancies-associated SPOP mutants failed to bind to MyD88 and further restrict NF-κB activation, thus enhancing DLBCL progression. Xiaofeng Jin et al. have indicated that SPOP is a tumor suppressor in DLBCL and defective mutations in the SPOP–MyD88 binding interface may contribute to aberrant MyD88/NF-κB activation [
27]. In line with the above findings, we identified that SPOP was downregulated and habours defective mutations in DLBCL, contributing to CHAF1A accumulations. Apart from MyD88/NF-κB axis, we also proposed that CHAF1A/TFEB may be the another bypass that triggers DLBCL aggressiveness induced by SPOP deficiency. In addition, Qing Shi et al. have found that cytoplasmic SPOP could bind and induce the non-degradative ubiquitination of p62, thereby decreasing p62 puncta formation and suppressing p62-dependent autophagy [
24]. Apart from the identified p62/SQSTM1-dependent pathway, our study also indicated that SPOP could employ CHAF1A/TFEB axis to inhibit TFEB-dependent autophagy. Therefore, our study not only supplied novel insights on the roles of SPOP/CHAF1A axis in DLBCL tumorigenesis, but linked the mechanistic associations between SPOP and TFEB-dependent autophagy.
However, there are still some unclear problems that deserve further improvements. Firstly, the DLBCL patients samples are limited and it may take a long time to collect enough eligible patients to assess the clinical significance of SPOP/CHAF1A/TFEB axis in DLBCLs. Meanwhile, apart from SPOP-mediated ubiquitination mechanisms, we speculated that there may exist other upstream signals and epigenetic events that govern high CHAF1A expressions, including methylation, N6-methyladenosine (m6A) modifications, as well as phosphorylation at specific residues. Last of all, the in vivo efficacy of targeting TFEB on DLBCL may need more pre-clinical models to figure out.
In conclusion, our research, for the first time, highlighted the SPOP/CHAF1A ubiquitination crosstalk in the pathogenesis of DLBCL. Aberrantly high CHAF1A expressions potentiate the aggressiveness of DLBCL and indicate a inferior prognosis for patients. Deficient SPOP contributes to accumulated CHAF1A proteins, thereby sustaining tumor autophagy via induction of TFEB. Targeting TFEB is effective for DLBCL with aberrant SPOP/CHAF1A axis. These findings systematically elucidated the biological roles of SPOP/CHAF1A/TFEB pathway, endowing novel therapeutic strategies in DLBCL.
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