Introduction
Triple-negative breast cancer (TNBC) is characterized by the lack of three key molecular signatures: estrogen receptor (ER) expression, progesterone receptor (PR) expression, and HER2 overexpression; it comprises approximately 15% of all invasive breast cancers [
1]. Given the lack of well-defined molecular targets, TNBC has the worst prognosis and fewest treatment options among all breast cancer types. TNBCs also contain a high proportion of breast cancer stem cells (BCSCs) and exhibit chemoresistance [
2]. Therefore, the exploration of novel and effective molecular targets to prevent and ultimately cure TNBC remains a critical unmet need.
It is well known that a small population of tumor cells with stem cell-like properties (stemness)—called cancer stem cells (CSCs)—exist within tumors. These CSCs are capable of self-renewal, pluripotent differentiation, metastatic dissemination, and therapeutic resistance. Based on cell surface markers and stem cell-like features, CSCs have been isolated and identified from numerous solid malignancies, including breast cancer [
3]. Accumulating evidence indicates that CSCs are responsible for drug resistance, metastasis, and tumor recurrence. In TNBC, BCSCs are commonly characterized by different markers, such as CD44/CD24, ALDH1, CD133, and EPCAM [
4]. In addition, the maintenance of BCSC stemness is typically driven by aberrant activation of transcription factors, such as OCT4, SOX2, and BMI1, together with aberrant activation of key signaling pathways, including Wnt, Notch, Hedgehog, and Hippo [
5]. Therefore, targeting BCSCs in TNBC may be a crucial strategy to achieve effective treatment.
F-box and WD repeat domain-containing 7 (FBXW7), also known as CDC4, is a substrate recognition component of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase. Given that most FBXW7 substrates are well-characterized oncoproteins, FBXW7 may serve as a putative tumor suppressor [
6]. Consistent with this notion, genomic deletion or mutation of FBXW7 has frequently been reported in various cancers, with a cancer-wide average frequency of 6%. Notably, almost half of the mutations are missense mutations in key arginine residues (Arg 465 and Arg 479) within the substrate recognition site [
7]. In our previous study, we showed that FBXW7 inhibits EMT and chemoresistance in NSCLC by regulating the ubiquitination and degradation of Snail [
8]. Subsequently, similar observations have been reported for other malignancies [
9]. To date, FBXW7 has been reported to regulate cell proliferation, cell migration, cell cycle arrest and apoptosis. However, its function and mechanism in regulating CSCs, especially BCSCs, have not yet been elucidated in TNBC.
Epigenetic regulators that mediate chromatin DNA or histone modifications may be involved in regulating BC heterogeneity, plasticity and tumorigenesis. Identifying the transacting factors and relevant pathways could help to reveal potential therapeutic targets for antitumor therapy. One such epigenetic factor, chromodomain-helicase-DNA-binding protein 4 (CHD4), is a core ATPase subunit of the nucleosome remodeling and deacetylase (NuRD) complex and contributes to protecting genome integrity by controlling the cell cycle and repairing DNA damage [
10]. CHD4 is also known to be involved in regulating transcriptional events during tumorigenesis and malignant progression [
11]. It has been confirmed to be closely associated with cancer stemness in hepatocellular carcinoma [
12], papillary thyroid carcinoma [
13] and endometrial cancer [
14]. For instance, CHD4 depletion and CHD4 mutations promote endometrial cancer stemness by activating TGF-beta signaling [
14]. Interestingly, CHD4 could enhance the metastatic ability and drug resistance of TNBC cells and is also a prognostic marker for TNBC patients [
15]. However, whether CHD4 plays an important role in mediating the stemness characteristics of BCSCs in TNBC is still poorly investigated.
Herein, we presented data highlighting a favorable prognostic factor—FBXW7—in TNBC patients and demonstrated that it could be a stemness marker of TNBC in vivo and in vitro. We also found that CHD4 is a potential downstream target of FBXW7, and the enhanced FBXW7 expression significantly suppresses the stemness properties of TNBC cells by facilitating ubiquitin-mediated degradation of CHD4 protein and then affecting the Wnt/β-Catenin pathway. Together, these findings show that FBXW7 physically interacts with CHD4 and mediates its ubiquitination, which highlights that targeting CHD4 may be a promising therapeutic strategy for eradicating BCSCs to overcome tumor relapse, metastasis and drug resistance in TNBC.
Methods and materials
Immunohistochemistry (IHC) staining
Human tissue microarray slides containing a total of 80 pairs of TNBC tumor tissues and matched adjacent tissues were purchased from Superbiotek, Inc. (BRC1601, Shanghai, China). Paraffin-embedded tumors were sliced into 6 μm thick sections. The tissue array slides were subjected to standard IHC using protein-specific antibodies as indicated: anti-FBXW7 antibody (1:300, Cat#ab105752, Abcam, USA) and anti-CHD4 (1:200, Cat#ab105752, Abcam, USA). The detailed clinicopathological characteristics are described in Table
1.
Table 1
The relationship between the expression of FBXW7 and the clinicopathological characteristics of TNBC patients
Age | 0.822572 |
> 50 years | 37 | 18 | 19 | |
≤ 50 years | 43 | 22 | 21 | |
Tumor size (cm) | 0.498962 |
> 2 | 43 | 24 | 21 | |
≤ 2 | 37 | 16 | 19 | |
TNM | 0.807006 |
I–II | 44 | 27 | 27 | |
III–IV | 26 | 13 | 13 | |
Lymph node | 0.073278 |
Negative | 42 | 25 | 17 | |
Positive | 38 | 15 | 23 | |
Survival status | 0.00022 |
Live | 50 | 17 | 33 | |
Death | 30 | 23 | 7 | |
Cell culture
Human mammary epithelial cells (MCF-10A), two non-TNBC cell lines (MCF-7 and T47D), and three TNBC cell lines (HCC1937, BT-549 and MD-MBA-231) were purchased from the Cell Bank of the Chinese Scientific Academy. MCF-10A cells were cultured in mammary epithelial cell growth medium (MEGM; Bulletkit, Lonza). MCF-7 cells were cultured in minimum essential medium (HyClone, Logan, UT, USA). BT549, T47D, and HCC1937 cells were cultured in RPMI-1640 medium (HyClone, Logan, UT, USA). MD-MBA-231 cells were cultured in DMEM. All of the above-listed media were supplemented with 10% fetal bovine serum (FBS) (BI, Beit-HaEmek, Israel) and 1% penicillin/streptomycin (Beyotime, Shanghai, China). For the mammosphere formation assay, cells were cultured in mammosphere medium containing DMEM/F12 supplemented with 5 mg/ml insulin (Sigma, St. Louis, USA), 2% B27 (Invitrogen, Carlsbad, CA, USA), 20 ng/ml EGF (Invitrogen, Carlsbad, CA, USA) and 20 ng/ml bFGF (Invitrogen, Carlsbad, CA, USA). All cells were cultured in a humidified incubator with an atmosphere of 5% CO2 at 37 °C.
Chemical reagents
MSAB (10 μM, MCE, HY-120697) was used to inhibit Wnt/β-catenin signaling. LiCl (10 mM, MCE, HY-W094474) was used to activate Wnt/β-catenin signaling. Cycloheximide (10 μg/ml, MCE, HY-12320) was used as a protein synthesis inhibitor. MG132 (MCE, 20 μM, HY-13259) was used as a proteasomal degradation inhibitor.
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from cells using TRIzol reagent (Beyotime, Shanghai, China) as previously described. Reverse transcription and qRT‒PCR were performed according to methods described previously. Following the standard protocol of the PrimeScript™ RT Reagent Kit (Takara, Dalian, China), reverse transcription was conducted to generate cDNA. qRT‒PCR analysis was carried out using SYBR-Green PCR master mix (Takara, Dalian, China). Finally, relative mRNA levels of FBXW7 were calculated with normalization to the reference gene GAPDH mRNA by using the 2
−ΔΔCq method. The sequences of primers used for qRT‒PCR analysis are listed in Table
2.
Table 2
Human primer sequences used for qRT-PCR
FBXW7 | CACTCAAAGTGTGGAATGCAGAGAC | GCATCTCGAGAACCGCTAACAA |
CHD4 | GGTTTTGGTTCCAAGCGTAA | CTCCTCCTCGCCTTTCTTTT |
GAPDH | CGGAGTCAACGGATTTGGTCGTAT | AGCCTTCTCCATGGTGAAGAC |
Western blot analysis and immunoprecipitation
Total protein was extracted from the treated cells by using radioimmunoprecipitation (RIPA) lysis buffer. The extracted total protein was then used in subsequent Western blot experiments as described previously. Sample proteins were separated by electrophoresis on 10% sodium-dodecyl sulfate polyacrylamide gels (SDS‒PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. The PVDF membrane was then incubated with primary antibodies overnight at 4 ℃. Antibodies used were anti-Flag antibody (1:1000, Cat#F1804, Sigma), anti-HA antibody (1:1000, Cat#H3663, Sigma), anti-FBXW7 antibody (1:1000, Cat#ab105752, Abcam, USA), anti-CHD4 antibody (1:1000, Cat#ab105752, Abcam, USA), anti-E-cadherin antibody (1:1000, Cat#3195, CST, USA), anti-N-cadherin antibody (1:1000, Cat #13116, CST, USA), anti-Snail1 antibody (1:1000, Cat #3879, CST, USA), anti-Vimentin antibody (1:1000, Cat #5741, CST, USA), anti-SOX2 antibody (1:1000, Cat#ab92494, Abcam, USA), anti-SOX2 antibody (1:1000, Cat#ab92494, Abcam, USA), anti-OCT4 antibody (1:1000, Cat# ab200834, Abcam, USA), anti-NANOG antibody (1:1000, Cat# ab109250, Abcam, USA), anti-EpCAM antibody (1:1000, Cat# ab213500, Abcam, USA), anti-P84 antibody (1:1000, Cat# ab54370, Abcam, USA) and anti-GAPDH antibody (1:1000, Cat#AC026, ABclonal, USA). Subsequently, the PVDF membrane was incubated with horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature. The secondary antibodies for Western blotting were goat anti-mouse IgG (1:5000, Cat#A0216, Beyotime, China) or goat anti-rabbit IgG (1:5000, Cat#A0208, Beyotime, China). Finally, the bands were visualized with an ECL detection system (GE Healthcare). For immunoprecipitation experiments, cells were harvested and lysed using ice-cold NP-40 lysis buffer (Beyotime, Shanghai, China) with protease inhibitors on ice for 30 min. The supernatant of each sample was incubated with 1 μg of anti-FBXW7 or anti-CHD4 at 4 °C overnight. A nonspecific anti-human IgG antibody was used as the negative control. After gentle rocking at 4 °C overnight, protein A/G agarose (#sc-2003, Santa Cruz Biotechnology, USA) was added to the incubated mix and maintained for 4 h at 4 °C to precipitate the antibody-protein complexes. The beads were subsequently collected and washed with IP buffer to collect the supernatant for subsequent Western blot analysis.
Transfection and infection experiments and plasmids
Specific small hairpin RNAs (shRNAs) targeting FBXW7 and negative control shRNA (sh-NC) were purchased from Gene Pharma Company (Shanghai, China) and cloned and inserted into the pLKO.1-GFP vector to obtain PLKO‐shFBXW7. Full-length human HA-tagged FBXW7 and ΔFbox deletion HA-tagged FBXW7 were cloned and inserted into the lentiviral vector pCDH (System Biosciences, Palo Alto, CA). Full-length FLAG-tagged CHD4 and ΔTPTPS internal deletion FLAG-tagged CHD4 were inserted into pHAGE-3 × FLAG plasmids to generate tagged proteins. In brief, for all virus production, lentiviral vectors and packaging constructs were transfected into 293FT cells by using Lipofectamine 3000 Transfection Reagent (Invitrogen). Supernatants containing lentiviruses were collected at 48–72 h after transfection and then used to infect TNBC cell lines. After 24 h of infection, the infected cells were screened with 2.5 μg/ml puromycin for one week, and the surviving cells were frozen and stored in liquid nitrogen for subsequent experiments. All target sequences for the shRNAs are given in Table
3.
Table 3
The shRNA target sequences for indicted genes
FBXW7 | AACACAAAGCUGGUGUGUGCA |
CHD4 | GCGGCAGTTCTTTGTGAAATG |
Flow cytometry assay
Cells were collected and washed twice with permeabilization wash buffer and were then resuspended in flow cytometry staining buffer (Bio-Rad), and cell numbers were counted. The dissociated cells were then stained with APC-conjugated anti-CD44 antibody (1:50, Cat# 559942, BD Pharmingen) and FITC-conjugated anti-CD24 antibody (1:50, Cat# 555427, BD Pharmingen) for 30 min at 4 °C. After washing twice in flow cytometry buffer, the cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences). For the ALDEFLUOR Assay, cells were harvested by trypsinization, washed in PBS, labeled with Aldefluor Reagent (StemCell Technologies, Grenoble, France) and incubated at 37 °C for 45 min. Finally, all samples were analyzed by a FACS machine (BD Calibur).
TNBC cells were dissociated into single cells and seeded in 6-well ultralow attachment plates (Corning, NY, USA) at a density of 1000 cells/well in serum-free stem cell-conditioned medium as described above. After culturing for 7–21 days, spheres with diameters > 50 μm were counted by using ImageJ software.
Ubiquitination and cycloheximide chase assay
For the in vitro ubiquitination assay. HEK293 cells were grown in a 10 cm dish until they reached 80–90% confluency and then were cotransfected with the indicated plasmids. After 48 h of transfection, the cells were treated with the proteasome inhibitor MG-132 (50 μg/ml) for 6 h before harvesting. Then, total protein was extracted from these treated cells and subjected to immunoprecipitation to detect CHD4 ubiquitination following the same protocol used for Co-IP. For the cycloheximide-chase degradation assay, the transfected cells were grown to a density of approximately 80% and exposed to 10 μg/ml cycloheximide for different incubation times. At the indicated time points, the treated cells were then lysed and used to extract proteins for subsequent Western blot assays as previously described.
Cell cycle and apoptosis analysis
For cell cycle analysis, cells (1 × 107) were harvested, washed with PBS and fixed in 70% ethanol at 4 °C overnight. After washing three times with PBS, the cells were incubated with 5 μl of RNase A for 1 h and stained with 10 μl of propidium iodide (PI) for 30 min at room temperature in the dark. FSC data were analyzed by using a FACSCalibur flow cytometer (BD). For the apoptosis assay, cells were harvested and double stained with Annexin V and propidium iodide (PI) using an Annexin V-FITC/PI Apoptosis Detection Kit (BestBio, Shanghai, China). Fluorescence was measured using a BD FACScan flow cytometer. FSC data were analyzed using Cell Quest software (BD Biosciences).
For the clonogenicity assays, TNBC cells were seeded in a 6-well plate (100 cells/well). After incubation for 2 weeks, colonies were washed with cold 1 × PBS, fixed with 4% paraformaldehyde for 5 min, and stained with 0.1 ml of 0.5% crystal violet for 10 min (Millipore Sigma). After fixation, colonies were washed and air-dried, and the colony numbers were counted with ImageJ software. For the migration assay, approximately 1 × 104 TNBC cells were cultured in the upper chamber containing 200 μl serum-free DMEM, and the lower chamber was filled with 600 μl DMEM containing 10% FBS. After 48 h of incubation, the nonmigrating cells on the upper surface of each filter were carefully removed with a cotton swab, and migrated cells adhering to the bottom were stained with 0.5% crystal violet for 20 min and manually counted in five nonoverlapping fields.
GST pull-down assay
The full-length cDNA sequence of human CHD4 was obtained by PCR using primers containing restriction enzyme sites for Eco RI and XhoI sites. The CHD4 cDNA was then cloned and inserted into the pGEX-5X-1 glutathione S-transferase (GST) fusion plasmid, which encodes a CHD4-GST fusion protein. The pGEX-5X-1-CHD4 vector was then transformed into competent Escherichia coli DH5α for expression. The recombinant GST fusion construct was amplified in bacterial cells, and the GST-CHD4 fusion protein from bacterial cells was purified by using GST-binding agarose resin as previously described. The GST protein was used as a negative control. The eluates were analyzed by Western blot with the indicated antibodies.
TOP/FOP luciferase reporter assay
For the TOP/FOP-Flash assay. shRNA-expressing cells were transfected with TOP flash or FOP flash reporter plasmids together with CMV-Renilla plasmid. The Renilla reniformis luciferase reporter was selected as an internal control. After 48 h of transfection, the relative luciferase activity was measured by using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity.
Immunofluorescence staining (IF)
Two TNBC cell lines were seeded on coverslips in a 24-well dish at 5 × 104 cells/well and further incubated for 24 h. Afterward, the cells were washed three times with cold PBS, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature, and washed three times in PBS. After blocking in 1% BSA for 30 min, the cells were incubated overnight at 4 °C with anti-CHD4 antibody and anti-FBW7 antibody. Subsequently, following several washes in PBS, the coverslips were treated with Alexa 488-conjugated anti-mouse secondary antibody as well as Cy3-conjugated anti-rabbit secondary antibody for a duration of 30 min. Upon repeated washing in PBS, the slides were mounted and sealed using DAPI Staining Solution. Confocal microscopy was used to acquire the images.
Tumor xenograft experiments
For xenograft models, BALB/c-nu mice aged between 4 and 6 weeks were randomly divided into four distinct groups. They were then subcutaneously injected with various cells as indicated at a concentration of 5 × 106 cells/200 μl (n = 4 per group). Tumor volume was measured once a week: Volume = 1/2 (Length × Width 2). The mice were sacrificed after 6 weeks postinjection, and the weight of the xenograft tumors was recorded. Subsequently, the tumors were fixed in 4% formaldehyde and analyzed by IHC staining with the indicated antibodies. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Zhengzhou University.
Statistical analysis
All cell experiments were carried out three times, and each group included triplicate samples. Quantitative data are presented as the mean ± SD, and qualitative data are presented as percentages. All statistical analyses were performed using SPSS 17.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 9 (GraphPad Prism, San Diego, CA, USA). Comparisons between the two groups were performed via Student’s t test, and multiple-group comparisons were performed via ANOVA. A p < 0.05 was considered to indicate statistical significance. GSEA was performed by using GSEA JAVA software (
http://software.broadinstitute.org/gsea/index.jsp).
Discussion
Despite the application of multimodal treatments, TNBC patients still have the worst prognosis of all subtypes of breast cancer. Previous studies have proposed that breast cancer stem cells (BCSCs) are enriched in TNBC [
17]. BCSCs are a small subpopulation of cells in breast cancer tissues with self-renewal ability, multilineage differentiation potential and high tumorigenicity. Due to these biological properties, BCSCs are often enriched in residual cancer tissue after breast cancer treatment with radiotherapy and chemotherapy [
18]. The resistance of BCSCs to conventional anticancer treatments has been considered responsible for recurrence and metastasis. There is substantial evidence to suggest that complete tumor eradication depends on the complete elimination of CSCs, but effectively identifying and targeting CSCs is not simple in cancer therapy. Thus, the precise molecular mechanisms underlying BCSC regulation in TNBC remain largely unknown.
FBXW7 mutations have been frequently observed in various cancer types. FBXW7 primarily targets oncoproteins for degradation and is thus widely recognized as a classic tumor suppressor gene. Previous studies on breast cancer have shown that the mutation frequency of FBXW7 in TNBC is considerably higher than that in ER receptor-positive BC [
19]. It has also been observed that FBXW7 plays a role in ubiquitinating and degrading EglN2, thereby inhibiting TNBC tumorigenesis [
20]. Additionally, a separate article reported that the circRNA produced by the FBXW7 gene hinders the malignant progression of TNBC cells through the ceRNA mechanism [
21]. Until now, only a few studies have explored the role of FBXW7 in the regulation of CSCs in several cancers, such as colorectal CSCs [
22], lung CSCs [
23], and live CSCs [
24]. However, the precise function and mechanism of FBXW7 in the CSC-like properties of TNBC cells remain largely unclear. Hence, this study mainly focused on the stemness of TNBC. In this study, we applied IHC staining to a tissue microarray and revealed that FBXW7 has markedly lower expression in tumor samples. Furthermore, clinical analysis revealed that downregulation of FBXW7 is associated with a poor prognosis in TNBC patients. This study aimed to characterize the functional role of FBXW7 in the stemness of TNBC. As expected, we confirmed that the level of FBXW7 was lower in tumor spheres than in adherent cells. The following functional experiments further validated that FBXW7 suppresses cell proliferation, migration, invasion, self-renewal, and tumorigenicity in TNBC. Thus, we speculated that FBXW7 functions as an inhibitory factor and regulates the stemness of TNBC. These findings may lay the foundation for further unraveling the molecular mechanism of FBXW7 in regulating CSCs in TNBC. To further elucidate the functional targets of FBXW7, we immunoprecipitated epitope-tagged FBXW7 and identified coprecipitating proteins using mass spectrometry. After intersecting these IP-MS proteins with multiple databases, CHD4 was finally screened out as a potential substrate protein for subsequent experiments. In vitro experiments further confirmed that CHD4 physically binds to FBXW7 and that its protein stability can be regulated by FBXW7-mediated ubiquitination and degradation. CHD4 plays a key role in regulating diverse cellular functions under physiological and pathological conditions, especially in carcinogenesis. Accumulating evidence has revealed that CHD4 is highly expressed in TNBC tissues and significantly positively correlated with tumor metastasis status, tumor recurrence, and poor prognosis [
15]. More recently, CHD4 was found to mediate EMT in TNBC cells, and silencing CHD4 expression in these cells increased drug sensitivity to cisplatin and PARP1 inhibitors. However, it remains unclear whether CHD4 regulates the stemness of TNBC cells. Interestingly, the regulation of cancer stemness by CHD4 in different malignant tumors is also controversial. Loss of CHD4 function promotes endometrial cancer stemness by activating the TGF-β pathway [
14] while suppressing stemness maintenance in papillary thyroid carcinoma [
13]. In our study, we used GSEA to find that stemness-related gene sets were significantly enriched in TNBC with high CHD4 gene expression. Correlation analysis also showed that CHD4 was significantly positively correlated with multiple stemness-related genes. Flow cytometry analysis further confirmed that CHD4 maintains the proportion of BCSCs in TNBC cells and partially reverses the inhibitory effect of FBXW7 on BCSCs. These results indicate that FBXW7-mediated degradation of CHD4 is a novel regulatory mechanism of cancer stemness in TNBC.
Wnt/β-catenin signaling is an evolutionarily conserved signaling pathway that is mainly responsible for embryonic development and tissue homeostasis [
25]. Growing evidence indicates that Wnt/β-catenin signaling plays a crucial role in maintaining stemness in TNBC [
26]. Strikingly, an early study mentioned that CHD4 promoted the nuclear accumulation of β-catenin in ovarian cancer [
27]. Consistent with the above reports, we also found that CHD4 was significantly positively correlated with β-catenin in TNBC tissues and promoted β-catenin nuclear accumulation in TNBC cells. Nuclear β-catenin accumulation is a hallmark of Wnt/β-catenin pathway activation. On the other hand, some studies have demonstrated that FBXW7 can directly ubiquitinate and degrade β-catenin to decrease the activity of the Wnt pathway [
28]. Moreover, our study confirmed that activating or inhibiting the Wnt pathway can inhibit the effects of FBXW7 and CHD4 on the proportion of BCSCs in TNBC cells. Therefore, we speculate that the FBXW7-CHD4-Wnt/β-catenin signaling axis plays a key role in regulating the stemness of TNBC.
Although the observations are interesting, there are still some limitations and shortcomings to this study. First, we found that GSK-3β knockdown can partially reverse the inhibitory effect of FBXW7 on the CHD4 protein, but we did not further analyze whether GSK phosphorylates CHD4 and clarify the phosphorylation site. Drug resistance is considered a characteristic of tumor-initiating stem cells. Considering that CHD4 knockdown sensitized TNBC cells to cisplatin and PARP inhibitors, it is necessary to further analyze the relationship between the “FBXW7-CHD4-Wnt/β-catenin” signaling axis and chemotherapy resistance in subsequent studies. Given that CHD4 serves as an epigenetic regulator, in future studies, we will further explore whether FBXW7 can also affect epigenetic modifications by degrading CHD4.
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