Abstract

Background

   The tumor suppressor FBW7 is the substrate recognition component of the
   SCF E3-ubiquitin ligase complex that mediates proteolytic degradation
   of various oncogenic proteins. However, the role of FBW7 in ovarian
   cancer progression remains inadequately understood.

Methods

   IP-MASS, co-IP, immunohistochemistry, and western blotting were used to
   identify the potential substrate of FBW7 in ovarian cancer. The
   biological effects of FBW7 were investigated using in vitro and in vivo
   models. LC/MS was used to detect the m^6A levels in ovarian cancer
   tissues. MeRIP-Seq and RNA-Seq were used to assess the downstream
   targets of YTHDF2.

Results

   We unveil that FBW7 is markedly down-regulated in ovarian cancer
   tissues and its high expression is associated with favorable prognosis
   and elevated m^6A modification levels. Consistently, ectopic FBW7
   inhibits ovarian cancer cell survival and proliferation in vitro and in
   vivo, while ablation of FBW7 empowers propagation of ovarian cancer
   cells. In addition, the m^6A reader protein, YTHDF2, is identified as a
   novel substrate for FBW7. FBW7 counteracts the tumor-promoting effect
   of YTHDF2 by inducing proteasomal degradation of the latter in ovarian
   cancer. Furthermore, YTHDF2 globally regulates the turnover of
   m^6A-modified mRNAs, including the pro-apoptotic gene BMF.

Conclusions

   Our study has demonstrated that FBW7 suppresses tumor growth and
   progression via antagonizing YTHDF2-mediated BMF mRNA decay in ovarian
   cancer.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12943-021-01340-8.

   Keywords: FBW7, YTHDF2, BMF, N6-methyladenosine, Ubiquitination

Background

   Epithelial ovarian cancer (EOC) is one of the most common causes of
   cancer-related mortality in women worldwide [[39]1]. Despite the
   advances in treatments with surgery and targeted chemotherapy [[40]2],
   the prognosis has only marginally improved because of the relapse and
   chemotherapy resistance [[41]3]. Therefore, thorough understanding of
   the pathogenic mechanisms of EOC is of great importance to develop new
   treatment strategies and ameliorate the overall prognosis of the EOC
   patients.

   F-box and WD repeat domain-containing 7 (FBW7), also known as FBXW7 or
   hCDC4, is encoded by the FBXW7 gene residing at chromosome 4q31 which
   is deleted in ~ 30% of human cancers [[42]4]. It belongs to the F-box
   protein family and acts as the substrate recognition component of the
   Skp1-Cullin-F-box (SCF) ubiquitin ligase complex. FBW7 selectively
   mediates ubiquitination and proteasomal degradation of oncogenic
   proteins, such as c-Myc, Cyclin E, c-Jun, Notch1, mTOR [[43]5], and is
   therefore regarded as a tumor suppressor. FBW7 was also shown to be
   transcriptionally induced by and synergize with p53 in maintaining
   genomic stability and suppressing carcinogenesis [[44]6–[45]8]. Several
   genetic models have elegantly demonstrated FBW7’s tumor inhibitory
   activity in vivo [[46]9]. Although loss of two alleles of Fbxw7 causes
   embryonic lethality of mice because of severe abnormalities in vascular
   development [[47]10], heterozygous inactivation or conditional knockout
   of this gene leads to profound chromosomal instability, upregulation of
   its targeting oncoproteins and increased tumor burden [[48]7, [49]8,
   [50]11–[51]14]. Also, FBW7 is able to facilitate nonhomologous
   end-joining (NHEJ) repair through the lysine 63-linked
   polyubiquitylation, but without degradation, of XRCC4 [[52]15].
   Consistent with its role as a tumor suppressor, the mutation of the
   FBXW7 gene was found in around 6% of all human cancers [[53]4], which
   not only abrogates the tumor suppressive activity of FBW7, but also
   endows these mutants with the oncogenic function [[54]13, [55]16].
   Additionally, FBW7 was found to be ubiquitously downregulated in human
   cancers which is related to unfavorable prognosis [[56]17]. The
   downregulation of FBW7 in tumors might be associated with the frequent
   mutation of the TP53 gene, and low FBW7 expression level along with
   TP53 mutation predicts distinctively poor prognosis in gastric cancer
   [[57]18]. In ovarian cancer, it was found that the frequency of the
   FBXW7 gene mutation is approximately 2.5 ~ 8.3% [[58]19, [59]20] and
   the downregulation of FBW7 expression can be attributed to the TP53
   gene mutation, particularly, in the serous carcinoma samples with the
   TP53 mutation frequency of ~ 95% [[60]21–[61]23]. However, the role of
   FBW7 in the progression of ovarian cancer remains incompletely
   understood.

   To unravel this question, we performed a set of bioinformatics and
   experimental analyses to dissect the role of FBW7 in ovarian cancer. In
   addition, a co-immunoprecipitation assay combined with mass
   spectrometry analysis was conducted and the human YTH domain family 2
   (YTHDF2), a N^6-methyladenosine (m^6A) binding protein, was identified
   as an interacting protein as well as a substrate of FBW7. YTHDF2 is an
   m^6A reader that selectively binds to the m^6A consensus motif and
   promotes mRNA decay [[62]24, [63]25]. The YTHDF2 encoding gene was
   found to be overexpressed and responsible for development of acute
   myeloid leukemia (AML) [[64]26, [65]27], whereas it turned into a tumor
   suppressor by targeting EGFR, IL11 and SERPINE2 mRNAs for degradation
   in hepatocellular carcinoma [[66]28, [67]29]. Very recently, YTHDF2 was
   found to be degraded by the E3 ubiquitin ligase complex consisting of
   Cullin 1 (CUL1), Cullin 4A (CUL4A), damaged DNA-binding protein 1
   (DDB1), and S-phase kinase-associated protein 2 (SKP2) [[68]30]. We
   reveal here that overexpression of YTHDF2 prompts ovarian cancer cell
   propagation by globally modulating mRNA turnover through m^6A
   modification, which is associated with worse overall survival in
   patients. Furthermore, FBW7 interacts with and promotes proteolytic
   degradation of YTHDF2, thereby stabilizing the pro-apoptotic BMF mRNA
   and evoking apoptosis of ovarian cancer cells. Collectively, our study
   as detailed below demonstrates the role of the FBW7-YTHDF2-BMF cascade
   in suppression of ovarian cancer.

Methods

The human specimens and cell lines

   Totally 120 Chinese patients diagnosed with high-grade serous ovarian
   carcinoma and 10 Chinese patients with ovarian cyst were involved in
   this study. All patients had surgical resection at Fudan University
   Shanghai Cancer Center (FUSCC). The tissue samples were collected
   immediately after surgery and stored in preservation buffer at − 80 °C.
   Informed consent was obtained from all patients, and the use of
   clinical samples in this study was approved by the ethics committee of
   FUSCC. Ovarian cancer cell lines used in this study, SKOV3, OVCA420,
   OVCA429 and OVCAR8 were obtained from ATCC.

Establishment of stable cell lines

   The specific shRNAs targeting FBXW7 or YTHDF2 (Supplementary Table
   S[69]1) were cloned into the lentiviral vector pLKO.1 (Sigma-Aldrich,
   St louis, MO, USA). The specific plasmid overexpressing FBW7 or YTHDF2
   was generated by inserting the full-length cDNA amplified by PCR into
   the lentiviral vector pCDH (Sigma-Aldrich). The 293 T cells were
   transfected with pCDH or pLKO.1 carrying the specific sequences, along
   with the packaging plasmids, psPAX2 and pMD2.G. The virus particles
   were generated and collected 48 h post-transfection and used to infect
   ovarian cancer cell lines which were then selected by puromycin
   (Sigma-Aldrich) for 3 days. Knockdown or overexpression efficiency was
   confirmed at both mRNA and protein levels.

Quantitative reverse transcription and quantitative RT-PCR (RT-qPCR)

   Total RNA was extracted from ovarian cancer samples or cell lines by
   using TRIzol reagent (Life Technologies, Waltham, MA, USA).
   Complementary DNA (cDNA) synthesis was performed with PrimeScript RT
   reagent Kit (Takara, Japan) using ~ 1 μg RNA each sample. RT-qPCR
   reactions were conducted using TB Green Premix (Takara, Japan). The
   primers for FBXW7, YTHDF2, BMF, KCNK3, MYBL2, LEMD1, SIRPD, LY6K,
   SEMA3C, C1QTNF1, ZDHHC14 and MOCS3 are listed in Supplementary Table
   S[70]1.

Immunoblotting (IB)

   Cells were harvested and lysed with RIPA buffer. Proteins were
   extracted and loaded in SDS-PAGE, and transferred onto PVDF membrane
   (Millipore, Billerica, MA, USA). After blocking with Bovine Serum
   Albumin (Beyotime, China) and sequential incubation with the primary
   and secondary antibodies, the blots were detected using the ECL
   detection kit (Millipore). The anti-FBW7 antibody was purchased from
   Bethyl Laboratories (Montgomery, TX, USA), and the anti-YTHDF2 and
   anti-GAPDH antibodies were purchased from Proteintech (Wuhan, Hubei,
   China).

Co-Immunoprecipitation (co-IP)

   Cells were harvested and lysed with mild RIPA buffer directly on plate
   for 30 min. Meanwhile, 50 μl dynabeads protein G (Life Technologies)
   were incubated with 3 μg antibody at room temperature for 1 h. Then mix
   the protein lysate with the beads-antibody complex and incubate
   overnight at 4 °C. Beads were washed three times with lysis buffer.
   Bound proteins and 10% inputs were detected by IB.

Immunofluorescence staining (IF)

   2 × 104 HeyA8 or OVCAR8 cells were seeded on coverslips in a 24-well
   dish and further incubated for 12 h. Cells were fixed with 4%
   paraformaldehyde for 15 min, then incubated with 0.5% Triton X-100 in
   PBS for 15 min, and washed three times in PBS. After blocking in 1% BSA
   for 30 min, the cells were incubated with rabbit anti-YTHDF2 antibody
   Proteintech (Wuhan, Hubei, China) and mouse anti-FBW7 antibody (Santa
   Cruz Biotechnology, CA, USA) overnight at 4 °C. After washing several
   times in PBS, the coverslips were incubated with anti-mouse secondary
   antibody conjugated with Alexa 488 and Cy3-conjugated anti-rabbit
   secondary antibody for 30 min. After washing several times in PBS, the
   Slides were mounted and sealed with DAPI Staining Solution. The images
   were acquired using a confocal microscope (Leica, Wetzlar, Germany).

Immunohistochemistry (IHC)

   Immunohistochemistry was performed using a primary antibody against
   YTHDF2 antibody Proteintech (Wuhan, Hubei, China) and FBW7 antibody
   (Bethyl Laboratories, Montgomery, TX, USA). The immunostaining results
   were examined by two researchers independently. Immunohistochemical
   evaluation was based on the intensity and percentage of membranous and
   cytoplasmic reactivity. A 10% expression threshold was defined as
   positive. Then, no positive cells, less than 10% of positive cells,
   10–50% of positive cells, more than 51% of positive cells were defined
   as negative, weak, moderate, strong expression respectively. Tumors
   with moderate and strong staining were regarded as high expression.

In vivo ubiquitination assay

   SKOV3 cells were transfected with different combinations of plasmids
   encoding FBW7, YTHDF2 and His-Ub. After 48 h, cells were harvested and
   divided into two parts, one for IB and the other for the ubiquitination
   assay. Briefly, cell pellets were lysed with buffer I (8 M urea, 0.1 M
   Na2HPO4/ NaH2PO4 (pH 8.0), 10 mM Tris-HCl (pH 8.0), 10 mM
   β-mercaptoethanol, 5 mM Imidazole) and incubated with Ni-NTA beads
   (Takara, Japan) for 6 h at room temperature. Beads were washed twice
   with buffer I, and twice with buffer II (8 M urea, 0.1 M
   Na2HPO4/NaH2PO4 (pH 6.3), 10 mM Tris-HCl (pH 6.3), 10 mM
   β-mercaptoethanol). The bound protein complex was eluted and analyzed
   by IB.

In vitro cell proliferation, colony formation, and anchorage-independent cell
growth assays

   For the cell proliferation assay, 2000 cells were seeded into 96-well
   plates, cell viability was assessed for 5 consecutive days by the Cell
   Counting Kit-8 (CCK-8) (Dojindo, Japan). For the colony formation
   assay, 1000 cells were seeded into 6-well plates for 2 weeks, and
   colonies were stained with crystal violet and counted. For the
   anchorage-independent cell growth assay, 20,000 cells were seeded into
   6-well plates coated with soft agar for 4 weeks, and the growing
   colonies were counted under the microscope. The colony formation assay
   was used to evaluate the long-term cell growth on a solid surface,
   while the anchorage-independent cell growth assay was employed to
   assess the ability of transformed cells to grow independently of a
   solid surface.

In vivo mouse xenograft study

   In vivo experiments were all conducted using 5-week old Female BALB/c
   nude mice (Shanghai SLAC Laboratory Animal Co., Ltd). 5 × 10^5 cells
   were suspended in 100 μl PBS and then were inoculated subcutaneously.
   After the tumors were formed, the tumor volumes were measured every 3
   days. Mice were sacrificed when the tumor diameter reached about 15 cm,
   and the tumors were excised and weighted. Tumor volume was calculated
   based on the formula: volume = length × width^2 × 0.5. Animal
   experiments were all performed according to the Control of Department
   of Laboratory Animal Science in Shanghai Medical College of Fudan
   University and the animal ethic principles.

RNA m^6A quantification by LC-MS/MS

   RNA m^6A quantification by LC-MS/MS was conducted as described
   previously [[71]31]. Briefly, total RNAs were isolated using TRIzol
   reagent (Life Technologies). 200 ng mRNA was incubated with nuclease P1
   (Sigma-Aldrich) in 20 μl buffer containing 25 mM NaCl, 2.5 mM ZnCl[2]
   at 37 °C for 2 h, then added 2.2 μl NH[4]HCO[3] (1 M) and alkaline
   phophatase (Sigma-Aldrich), and incubated at 37 °C for 2 h. Following
   centrifugation at 13000 rpm for 10 min at 4 °C, 10 μl of the solution
   was analyzed by LC-MS/MS at Mass Spectrometry Application Research
   Center of the Institutes of Biomedical Sciences in Fudan University.
   The expression level of m^6A was dichotomized for OS before the
   log-rank test according to optimal cutoff values calculated by the
   “surv_cutpoint” function of the “survminer” R package.

Analysis of BMF mRNA stability

   Cells were incubated with actinomycin D (5 μg/ml) for 0 h, 3 h or 6
   hoursfollowed by RNA extraction. The half-life of BMF mRNA was analyzed
   by quantitative RT-PCR. For the 3-deazaadenosine (DAA) treatment assay,
   cells were incubated with 50uM DAA followed by RNA extraction. The
   level of BMF mRNA was analyzed by quantitative RT-PCR.

Library preparing and RNA sequencing

   Transcriptome sequencing was performed by Cloud-Seq Biotech (Shanghai,
   China). RNA libraries were constructed by adopting the NEBNext® Ultra™
   II Directional RNA Library Prep Kit (New England Biolabs, Inc., MA,
   USA). Library sequencing was performed on the illumina Hiseq instrument
   with 150 bp paired end reads. After harvesting the Paired-end reads and
   quality controlled by Q30 and cutadapt software (v1.9.3), the high
   quality clean reads were aligned to the reference genome (UCSC hg19)
   with the hisat2 software (v2.0.4). Then, the gene level FPKM as the
   expression profiles of mRNA was analyzed by the Ensembl gtf gene
   annotation file and the cuffdiff software (part of cufflinks), the fold
   change and p-value were calculated according to FPKM, and the
   differentially expressed mRNAs with statistical significance were
   obtained. GO and Pathway enrichment analysis were performed according
   to the differentially expressed mRNAs.

m^6A-RNA immunoprecipitation (MeRIP) assay and m^6A sequencing

   Polyadenylated RNAs were prepared and sonicated into fragments of
   100–200 nt. A small portion of the RNA fragments was saved as input
   samples. MeRIP was performed as previously described [[72]32]. Briefly,
   4 μg fragmented RNAs were incubated with 2 μg anti-m^6A antibody
   (Synaptic Systems, Goettingen, Germany) in 1 x IP buffer (10 mM
   TrisHCl, pH 7.4, 150 mM NaCl, 0.1% NP-40) for 2 h at 4 °C. The m^6A-IP
   complex was then incubated with Dynabeads protein A (Life Technologies)
   for 2 h at 4 °C. The bound RNAs were washed and eluted through
   competition with N6-methyladenosine (Santa Cruz Biotechnology, CA, USA)
   and then purified by the RNA cleanup kit (Zymo Research, CA, USA). The
   NEBNext® Ultra II Directional RNA Library Prep Kit (New England
   Biolabs, MA, USA) was used for library construction. Library sequencing
   was performed on an illumina Hiseq instrument with 150 bp paired-end
   reads by Cloudseq Biotech Inc. (Shanghai, China).

Statistical analysis

   Statistical analysis was conducted by the SPSS software (IBM) and
   GraphPad Prism software (version 6). The differences between two groups
   or more than two groups were compared using Student’s t-test. Survival
   analyses were conducted by Kaplan-Meier curve and log-rank test. The
   linear regression test was used to analyze the genes expression
   correlation. p < 0.05 was regarded as statistically significant. All
   the data are showed as mean ± SD.

Results

Downregulation of FBW7 is associated with unfavorable prognosis and decreased
m^6A modification levels in human ovarian cancer

   Since a paucity of evidence had been found on the role of FBW7 in
   ovarian cancer, we first determined its clinical implication by
   comparing the expression of FBW7 in both cancerous and non-cancerous
   ovarian tissues. FBW7 was markedly downregulated in ovarian cancer
   tissues (Fig. [73]1a, b and Supplementary Fig. [74]1A). Consistently,
   FBW7 was also found to be significantly reduced in the TCGA RNA-Seq
   dataset of 586 ovarian cancer samples (Supplementary Figure [75]1B).
   Next, the FBW7 expression in the ovarian cancer tissue microarrays,
   containing 120 tumor samples, was analyzed by immunohistochemistry
   (IHC) staining and as thus the ovarian cancer specimens could be
   categorized into two groups with low or high expression level of FBW7
   (Fig. [76]1c and d). We then explored the relationship between FBW7
   expression and the clinicopathological features of ovarian cancer.
   However, no significant correlation was observed between FBW7
   expression and age, tumor stage, ascites, lymph node metastasis or
   menopause (Supplementary Table S[77]2; P > 0.05). The Kaplan-Meier
   survival analysis showed that high level of FBW7 is significantly
   associated with favorable overall survival in ovarian cancer (Fig.
   [78]1e, P = 0.04), which is consistent with the result from the TCGA
   ovarian cancer cohort (Supplementary Fig. [79]1C). Additionally, high
   level of FBW7 was moderately, but not significantly, correlated with
   better progress-free survival (PFS) (Fig. [80]1f), probably due to the
   limited sample size. Therefore, these observations indicate that FBW7
   is a favorable prognostic marker that may suppress ovarian
   tumorigenesis and progression.

Fig. 1.

   [81]Fig. 1
   [82]Open in a new tab

   Frequent downregulation of FBW7 in human epithelial ovarian cancer. a
   Expression of FBW7 in cancerous and normal ovarian tissues detected by
   IB. b Expression of FBW7 in cancerous and normal ovarian tissues
   analyzed by RT-qPCR. c-d Expression of FBW7 by IHC staining of human
   ovarian cancer tissue arrays. Scale bar, 200 μm. e FBW7 downregulation
   is significantly associated with shorter overall survival in epithelial
   ovarian cancer patients. f No significant correlation is observed
   between FBW7 expression and progression-free survival. g Quantification
   of m^6A levels in 13 normal ovarian specimens and 60 ovarian cancer
   tissues classified into FBW7 high expression and low expression groups.
   h Higher level of m^6A levels is significantly associated with better
   overall survival in epithelial ovarian cancer patients. m^6A high:
   m^6A ≥ 1.04%, m^6A low: m^6A < 1.04%. Results were presented as
   mean ± SD of three independent experiments. *P < 0.05 or **P < 0.01
   indicates a significant difference between the indicated groups

   FBW7 was a typical tumor suppressor, mainly involved in the process of
   ubiquitylation and degradation of many onco-proteins.
   N^6-methyladenosine (m^6A) has emerged as an abundant reversible
   modification, which has been reported to play critical roles in
   physiological and pathological processes. However, the regulation of
   the m^6A modification system remains elusive. Here we measured m^6A
   levels in 60 human ovarian cancer tissue samples. According to the
   optimal cutoff point calculated by “survminer” R package, samples with
   a m^6A expression no more than 0.01 were considered as the m^6A low
   expression population, while the rest was considered as the m^6A high
   expression population. Interestingly, m6A levels were elevated in
   patients with tumors expressing higher levels of FBW7 (Fig. [83]1g),
   and better overall survival was significantly correlated with higher
   levels of m^6A (Fig. [84]1h), which suggested FBW7 might be an upstream
   regulator for the m^6A modification system.

FBW7 inhibits ovarian cancer cell propagation as a tumor suppressor

   To investigate the biological function of FBW7 in ovarian cancer, we
   generated FBW7-stably overexpressing SKOV3 and OVCAR429 cell lines,
   both of which sustain low expression level of endogenous FBW7
   (Fig. [85]2a, Supplementary Fig. [86]2). Overexpression of FBW7
   significantly suppressed ovarian cancer cell proliferation (Fig.
   [87]2b), colony-forming ability (Fig. [88]2c), and
   anchorage-independent cell growth (Fig. [89]2d). The tumor inhibitory
   activity could be attributed to the induction of apoptosis, as ectopic
   FBW7 strikingly augmented the Annexin V-positive cell population (Fig.
   [90]2e). Furthermore, we established a xenograft mouse model to test if
   FBW7 suppresses ovarian tumor growth in vivo. In accordance with the
   cell-based results, ectopic FBW7 markedly repressed tumor growth in
   mice (Fig. [91]2f), as reflected by the significant inhibition of the
   tumor volume and weight when compared to the control group (Fig. [92]2g
   and h). In addition, we also generated FBW7-depleted HeyA8 and OVCAR8
   ovarian cancer cell lines using two independent shRNAs (Supplementary
   Fig. [93]3A). Consistently, ablation of FBW7 significantly promoted
   ovarian cancer cell proliferation (Supplementary Fig. [94]3B),
   colony-forming ability (Supplementary Fig. [95]3C), and
   anchorage-independent cell growth (Supplementary Fig. [96]3D). Taken
   together, these results demonstrate that FBW7 plays a tumor suppressive
   role in ovarian cancer.

Fig. 2.

   [97]Fig. 2
   [98]Open in a new tab

   Overexpression of FBW7 significantly suppresses ovarian cancer cell
   proliferation and growth in vitro and in vivo. a Stable overexpression
   of FBW7 in SKOV3 and OVCAR429 cells. The expression of FBW7 is verified
   at both mRNA and protein levels. b Overexpression of FBW7 significantly
   reduces the proliferation rate of SKOV3 and OVCAR429 cells. c
   Overexpression of FBW7 impairs the colony-forming ability of ovarian
   cancer cells. d Overexpression of FBW7 suppresses anchorage-independent
   growth ovarian cancer cells. e Overexpression of FBW7 promotes ovarian
   cancer cell apoptosis. f Overexpression of FBW7 in SKOV3 cells markedly
   suppresses ovarian tumor growth in the xenograft mice. g The size of
   tumors formed in the xenograft mice is monitored every 3 days. h
   Overexpression of FBW7 significantly reduces weight of tumors in the
   xenograft mice. Results were presented as mean ± SD of three
   independent experiments. *P < 0.05 or **P < 0.01 indicates a
   significant difference between the indicated groups

FBW7 induces ubiquitination and proteasomal degradation of YTHDF2

   To decipher the molecular mechanism behind FBW7-mediated regulation of
   the m^6A modification system, we conducted a co-IP assay using the
   anti-FBW7 antibody coupled with mass spectrometry analysis to identify
   the FBW7-interacting proteins in the FBW7-stably overexpressing SKOV3
   cells (Supplementary Fig. [99]4A-C, Supplementary table [100]3). This
   screening led to identification of YTHDF2 as one of the FBW7-binding
   proteins. To validate this interaction, we performed the reciprocal
   co-IP assays and indeed verified that FBW7 binds to YTHDF2 in SKOV3
   cancer cells (Fig. [101]3a). The immunofluorescence (IF) staining
   suggested that the interaction may occur in the cytoplasm (Fig.
   [102]3b). Since FBW7 is a component of the SCF E3-ubiquitin ligase, we
   sought to determine if FBW7 regulates YTHDF2 protein stability through
   their physical interaction. By introducing the FBW7-encoding plasmid
   into SKOV3 and OVCAR429 cells, we found that the protein level of
   YTHDF2 dramatically declined upon FBW7 overexpression, while this
   reduction is completely restored by treatment of cells with the
   proteasome inhibitor MG132 (Fig. [103]3c). Additionally, overexpression
   of FBW7 shortened the half-life of YTHDF2 protein as indicated by the
   cycloheximide-chase experiment (Fig. [104]3d). In line with these
   results, we also showed that FBW7 drastically enhances YTHDF2
   ubiquitination (Fig. [105]3e), which was validated by another set of
   ubiquitination assays using the anti-YTHDF2 antibody (Fig. [106]3f).
   Therefore, these findings demonstrate that FBW7 prompts
   ubiquitination-mediated proteolytic degradation of YTHDF2 in ovarian
   cancer to regulate the m^6A modification system.

Fig. 3.

   [107]Fig. 3
   [108]Open in a new tab

   FBW7 binds to and mediates the proteolytic degradation of YTHDF2 in
   ovarian cancer. a FBW7 interacts with YTHDF2 by the reciprocal co-IP
   assays in SKOV3 cancer cells. b The IF staining indicates the
   co-localization of FBW7 and YTHDF2 in the cytoplasm. c Ectopic
   expression of FBW7 reduces the YTHDF2 protein levels in SKOV3 and
   OVCAR429 cells, which can be blocked by the proteasome inhibitor MG132
   (20 μM). d The half-life of YTHDF2 is shortened upon FBW7
   overexpression. The cells were treated with 100 mg/ml of cycloheximide
   (CHX) and harvested at different time points as indicated. The
   quantification of YTHDF2 expression is shown in the lower panel.
   Overexpression of FBW7 shortened the half-life of YTHDF2 from 7.0 h to
   3.3 h. e FBW7 promotes ubiquitination of YTHDF2 in 293 T cells. Cells
   were transfected with combinations of plasmids encoding YTHDF2, FBW7 or
   His-Ub as indicated, and treated with MG132 (20 μM) or DMSO for 6 h
   before harvested for in vivo ubiquitination assay. Bound and input
   proteins were detected by IB assays with the indicated antibodies. f
   The ubiquitination of YTHDF2 is mediated by FBW7. Cells were
   transfected with combinations of plasmids encoding YTHDF2, FBW7 or
   His-Ub as indicated, and treated with MG132 (20 μM) or DMSO for 6 h
   before harvested for the IP assays using the anti-YTHDF2 antibody.
   Results were presented as mean ± SD of three independent experiments.
   *P < 0.05 or **P < 0.01 indicates a significant difference between the
   indicated groups

YTHDF2 is negatively correlated with FBW7 expression and prognosis of ovarian
cancer

   As YTHDF2 is a substrate of the tumor suppressor FBW7 for degradation,
   we inquired if the inverse correlation between the two proteins also
   exists in primary ovarian cancer tissues. First, we analyzed the
   expression of YTHDF2 in the cancerous and non-cancerous ovarian
   tissues, and found that YTHDF2 is frequently elevated in ovarian cancer
   (Fig. [109]4a). Consistently, YTHDF2 was significantly upregulated in
   ovarian cancer samples versus normal ovarian tissues in the TCGA
   database (Supplementary Fig. [110]5A). We then examined YTHDF2
   expression using the ovarian cancer tissue microarrays as mentioned
   above (Fig. [111]1c) and indeed found a negative correlation between
   the expression of YTHDF2 and FBW7 (Fig. [112]4b and c), which is in
   agreement with the results illustrated in Fig. [113]3c-f. According to
   the expression level of YTHDF2, these tumor specimens were categorized
   into two groups (Fig. [114]4d) and the clinicopathological features of
   each group were analyzed. However, no significant correlation was
   observed between YTHDF2 expression and age, tumor stage, ascites or
   lymph node metastasis (Supplementary table [115]4, Supplementary table
   [116]5; P > 0.05). The Kaplan-Meier survival analysis displayed that
   increased expression of YTHDF2 is significantly correlated with worse
   overall survival (OS) in ovarian cancer (Fig. [117]4e, P = 0.0013),
   which is in line with the result from the TCGA ovarian cancer cohort
   (Supplementary Fig. [118]5B). Nevertheless, YTHDF2 is not an
   appropriate prognostic marker for the progress-free survival (PFS)
   (Fig. [119]4f). And the OS of the FBW7 high expression /YTHDF2 low
   expression group was better than that of the BW7 low expression /YTHDF2
   high expression group (Supplementary Fig. [120]13). In multivariate
   analysis, YTHDF2 expression was adverse independent prognosticators for
   OS (Supplementary table [121]6). Thus, these observations strongly
   suggest that YTHDF2 may promote survival and growth of ovarian cancer,
   which is addressed as follows.

Fig. 4.

   [122]Fig. 4
   [123]Open in a new tab

   Upregulation of YTHDF2 is negatively associated with FBW7 expression
   and predicts unfavorable prognosis. a Expression of YTHDF2 in cancerous
   and non-cancerous ovarian tissues detected by RT-qPCR. b-c IHC staining
   of human the ovarian cancer tissue arrays shows that YTHDF2 expression
   is negatively correlated with FBW7 expression. d Expression of YTHDF2
   by IHC staining of the human ovarian cancer tissue arrays. Scale bar,
   200 μm. e YTHDF2 upregulation is significantly associated with better
   overall survival in ovarian cancer patients. f No significant
   correlation is observed between YTHDF2 expression and progression-free
   survival. * P < 0.05

YTHDF2 endorses ovarian cancer development as an oncogenic protein

   Although YTHDF2 was found to be involved in the development of several
   cancers [[124]26–[125]29, [126]33], it remains largely elusive if the
   protein is required for ovarian cancer cell survival and growth. To
   test this speculation, the YTHDF2-depleted SKOV3 and OVCAR420 cell
   lines were generated using two independent shRNAs (Fig. [127]5a). We
   showed that ablation of YTHDF2 significantly prohibits proliferation
   (Fig. [128]5b), colony-forming ability (Fig. [129]5c), and
   anchorage-independent growth (Fig. [130]5d) of the two ovarian cancer
   cell lines. Also, the flow cytometry analysis indicated that knockdown
   of YTHDF2 drastically triggers ovarian cancer cell apoptosis (Fig.
   [131]5e). Moreover, the xenograft mouse model revealed that YTHDF2
   depletion markedly hampers ovarian tumor growth in vivo (Fig. [132]5f)
   leading to significant reduction of the tumor volume and weight (Fig.
   [133]5g and h). And Knockdown of YTHDF2 significantly restores cell
   proliferation and colony-forming ability induced by FBW7 depletion
   (Fig. [134]5i-k). Thus, these findings indicate that YTHDF2 is
   essential for ovarian cancer cell propagation. To confirm these
   results, we also evaluated the biological function of ectopic YTHDF2 in
   ovarian cancer cell lines. By examining the expression of YTHDF2 in
   multiple ovarian cancer cell lines, we generated YTHDF2-stably
   overexpressing lineages using OVCAR433 and OVCAR8 cells that sustain
   relatively low expression of endogenous YTHDF2 (Supplementary Fig.
   [135]6 and [136]7A). As expected, overexpression of YTHDF2
   significantly boosted cell proliferation (Supplementary Fig. [137]7B),
   colony-forming ability (Supplementary Fig. [138]7C), and
   anchorage-independent growth (Supplementary Fig. [139]7D).
   Collectively, we demonstrate that YTHDF2 may drive ovarian cancer
   development, and that FBW7 restrains ovarian carcinogenesis by inducing
   proteolytic degradation and thus compromising the oncogenic activity of
   YTHDF2.

Fig. 5.

   [140]Fig. 5
   [141]Open in a new tab

   Ablation of YTHDF2 significantly suppresses ovarian cancer cell
   proliferation and growth in vitro and in vivo. a Stable expression of
   shRNAs against YTHDF2 in SKOV3 and OVCAR420 cells. The gene knockdown
   efficiency is verified at both mRNA and protein levels. b YTHDF2
   depletion significantly reduces the proliferation rate of SKOV3 and
   OVCAR420 cells. c YTHDF2 depletion impairs the colony-forming ability
   of ovarian cancer cells. d YTHDF2 depletion suppresses
   anchorage-independent growth of ovarian cancer cells. e YTHDF2
   depletion promotes ovarian cancer cell apoptosis. f YTHDF2 depletion in
   SKOV3 cells markedly suppresses ovarian tumor growth in the xenograft
   mice. g The size of tumors formed in the xenograft mice is monitored
   every 3 days. (H) YTHDF2 depletion significantly reduces weight of
   tumors in the xenograft mice. ** P < 0.01. Results were presented as
   mean ± SD of three independent experiments. *P < 0.05 or **P < 0.01
   indicates a significant difference between the indicated groups (i, j,
   k).

Identification of BMF as an effector of the FBW7-YTHDF2 cascade

   Since YTHDF2 was shown to prompt the decay of the m^6A-modified
   transcripts [[142]24], we determined if FBW7 regulates cellular m^6A
   enrichment through inhibition of YTHDF2. By employing a LC-MS/MS assay
   that allows quantification of m^6A level in cells, we showed that
   ablation of YTHDF2 significantly increases m^6A abundance in the
   ovarian cancer cell lines, OVCAR8 and HEY (Fig. [143]6a), which is in
   accordance with a previous study performed in HeLa cells [[144]24].
   Remarkably, overexpression of FBW7 led to significant elevation of m^6A
   level in SKOV3 and OVCAR429 cell lines (Fig. [145]6b), which
   phenocopies the effect of YTHDF2 depletion. In addition, we assessed
   m^6A enrichment in ovarian cancer tissues with high or low expression
   level of FBW7, which again verified that FBW7 expression is positively
   associated with m^6A level in tumors (Fig. [146]1g). Of note, we also
   ascertained that FBW7-mediated regulation of m^6A abundance is
   dependent on YTHDF2, as evidenced by the fact that knockdown of YTHDF2
   dramatically neutralized the effect of FBW7 depletion on m^6A level
   (Fig. [147]6c).

Fig. 6.

   [148]Fig. 6
   [149]Open in a new tab

   Regulation of m^6A-modified mRNA stability by the FBW7-YTHDF2 cascade.
   a Induction of the mRNA m^6A levels upon YTHDF2 depletion in OVCAR8and
   HEY cells. The RNA m^6A quantification was conducted by LC-MS/MS.
   [[150]31] The YTHDF2 depletion efficiency was shown in Supplementary
   Fig. [151]9A. b Induction of the mRNA m^6A levels upon FBW7
   overexpression in SKOV3 and OVCAR429 cells. The FBW7 overexpression
   efficiency was shown in Supplementary Fig. [152]9B. c Decline of the
   mRNA m^6A level caused by FBW7 depletion can be rescued by knockdown of
   YTHDF2. The YTHDF2 depletion and FBW7 overexpression efficiency was
   shown in Supplementary Fig. [153]9C. d RNA-sequencing analysis reveals
   a distinct gene expression patterns in response to YTHDF2 depletion in
   SKOV3 cells. e Distribution of m^6A modification in mRNA transcripts.
   The m^6A signal is mostly enriched in the coding sequences. f The m^6A
   consensus sequence motif is identified in ovarian cancer cells. g
   Integration of the RNA-Sequencing and m^6A-Sequencing results revealed
   25 genes with 39 peaks are consistently elevated upon YTHDF2 knockdown,
   which are displayed in Supplementary table [154]4 (h). The m^6A
   modification site of BMF mRNA was in the 3′-UTR region. i Knockdown of
   YTHDF2 increases BMF mRNA level in SKOV3 cells. j Knockdown of YTHDF2
   prolongs the half-life of BMF mRNA. Cells were treated with actinomycin
   D (5 μg/ml) for 3 or 6 h followed by RT-qPCR analysis. k The mRNA level
   of BMF is elevated upon the treatment of 50 μM 3-deazaadenosine (DAA)
   in SKOV3 cells. l The interaction between YTHDF2 and BMF pre-mRNA was
   detected by the RIP assay. m The mRNA level of BMF is significantly
   elevated in SKOV3 cells stably overexpressing FBW7. n The relative m6A
   level of BMF pre-mRNA was detected by MeRIP-qPCR in SKOV3 cells. o The
   FBW7-mediated regulation of BMF is dependent on YTHDF2 demonstrated by
   qRT-PCR detection. p YTHDF2 is inversely correlated with BMF expression
   in 64 epithelial ovarian cancer tissues. q FBW7 is positively
   correlated with BMF expression in 64 epithelial ovarian cancer tissues.
   r The working model for the regulation of m^6A-dependent turnover of
   BMF mRNA by the FBW7-YTHDF2 cascade. Results were presented as
   mean ± SD of three independent experiments. *P < 0.05 or **P < 0.01
   indicates a significant difference between the indicated groups

   Next, we examined if YTHDF2 orchestrates gene expression in ovarian
   cancer by conducting an RNA-sequencing analysis (Fig. [155]6d).
   Ablation of YTHDF2 led to upregulation of 348 genes and downregulation
   of 320 genes in SKOV3 cells. We also performed the genome-wide mapping
   of the m^6A modification in control and YTHDF2-depleted SKOV3 cell
   lines through a MeRIP (m^6A)-sequencing assay. The results indicated
   that the m^6A peaks mainly locate in the CDS regions (42.3%), then the
   stop codon sites (32.9%), start codon sites (14.5%), 5′ UTRs (7.7%) and
   3′ UTRs (2.6%) (Fig. [156]6e). The m^6A consensus sequence identified
   in SKOV3 cells was GGAC [U/A] (Fig. [157]6f) that is consistent with
   previous studies [[158]24, [159]34], indicating that YTHDF2 may also
   bind to m^6A in ovarian cancer. By integrating the RNA-Sequencing and
   m^6A-Sequencing results, we found that 25 genes with 39 peaks are
   consistently elevated upon YTHDF2 knockdown (Fig. [160]6g and
   Supplementary table [161]4). Among these dysregulated genes, BMF was
   considered an interesting target given that it encodes a pro-apoptotic
   BH3-only Bcl2 family protein required for inhibiting pro-survival Bcl2
   members and triggering apoptosis [[162]35] and that ablation of YTHDF2
   exhibited higher peak enrichment compared to the control group (Fig.
   [163]6h). We further testified that ablation of YTHDF2 significantly
   increases BMF mRNA expression level (Fig. [164]6i) and prolongs its
   half-life (Fig. [165]6j), which validates BMF mRNA as a target of
   YTHDF2 for degradation. Since the m^6A modification is indispensable
   for YTHDF2-induced mRNA decay, we determined if inhibition of m^6A
   stabilizes BMF mRNA. As shown in Fig. [166]6k, treatment of SKOV3 cells
   with the global methylation inhibitor, 3-deazaadenosine (DAA),
   significantly elevated the mRNA level of BMF, suggesting that
   methylation is critical for degradation of BMF mRNA (Fig. [167]6k). The
   interaction between YTHDF2 and BMF was verified by the RIP assay (Fig.
   [168]6l). And overexpression of YTHDF2 significantly reduces the
   expression of BMF (Supplementary Fig. [169]8A). The effect of YTHDF2 on
   cell proliferation and apoptosis can be reversed by the restoration of
   BMF expression (Supplementary Fig. [170]8B-E). Moreover, overexpression
   of FBW7, like knockdown of YTHDF2, strikingly induced BMF mRNA level
   (Fig. [171]6m), which is owing to YTHDF2 degradation by ectopic FBW7
   (Fig. [172]3c-f). Further, the overexpression of FBW7 or knockdown of
   YTHDF2 increased the levels of m6A modification of BMF pre-mRNA
   respectively, compared with modification levels of control vectors in
   SKOV3 cells (Fig. [173]6n).

   We also showed that the FBW7-mediated regulation of BMF is dependent of
   YTHDF2 (Fig. [174]6o and Supplementary Fig. [175]12,). Importantly and
   in line with these in vitro results, we also revealed that the
   expression of BMF is inversely correlated with YTHDF2 expression (Fig.
   [176]6p), whereas is positively associated with FBW7 expression (Fig.
   [177]6q, Supplementary Fig. [178]10), in ovarian cancer tissues. And
   the mRNA levels of BMF were elevated in patients with tumors expressing
   higher levels of FBW7, while YTHDF2 mRNA levels demonstrate no
   significant difference (Supplementary Fig. [179]11). Collectively, our
   results clearly demonstrate that FBW7 provokes the expression of
   pro-apoptotic BMF through an m^6A-dependent mechanism by interacting
   with and degrading the m^6A reader YTHDF2 in ovarian cancer (Fig.
   [180]6r).

Discussion

   The tumor suppressor FBW7 maintains genomic stability and prevents
   tumor growth by degrading multiple oncoproteins in a variety of human
   cancers. Here, we have unveiled that the m^6A-binding protein YTHDF2
   acts as a tumor promoter bolstering ovarian cancer cell propagation and
   the proteolytic degradation of YTHDF2 induced by FBW7 is required for
   the tumor inhibitory activity of the latter. Mechanistically, FBW7
   enhances stabilization of the pro-apoptotic BMF mRNA by abating
   YTHDF2-mediated m^6A-dependent mRNA decay. Therefore, our study depicts
   the role of the FBW7-YTHDF2-BMF cascade in the development of ovarian
   cancer.

   Since a seminal study suggested that FBW7, the human homologue of
   Drosophila archipelago, may be involved in ovarian carcinogenesis by
   regulating Cyclin E expression [[181]36], the expression pattern,
   mutation frequency, and clinical relevance of FBW7 in ovarian cancer
   has been evaluated [[182]19–[183]21]. In the present study, we have
   revealed that FBW7 is downregulated in ovarian carcinomas compared to
   non-cancerous ovarian tissues (Fig. [184]1a-b, Supplementary Fig.
   [185]1A-B) and that lower expression level of FBW7 is associated with
   unfavorable prognosis (Fig. [186]1e-f, Supplementary Fig. [187]1C),
   which is consistent with the previous study [[188]21]. These
   observations were further attested on the grounds of the fact that
   ectopic FBW7 impedes, while ablation of FBW7 fosters, ovarian cancer
   cell survival and growth (Fig. [189]2, Supplementary Fig. [190]3). It
   was found that FBW7 prompts ubiquitination and degradation of MCL1,
   while lack of FBW7 elevates MCL1 expression leading to resistance to
   antitubulin therapy in ovarian cancer [[191]37]. Also,
   Melanoma-associated antigen A1 (MAGEA1) was shown to suppress cell
   proliferation and migration probably through FBW7-mediated NICD1
   degradation in ovarian cancer [[192]38]. These findings definitely
   underscored the importance of FBW7 in ovarian cancer. Although a few
   substrates of FBW7 has been reported as mentioned above, our study
   uncovers the m^6A binding protein YTHDF2 as an additional substrate of
   FBW7 through a co-IP-MS analysis (Fig. [193]3a-b, Supplementary Fig.
   [194]4), and elaborates that FBW7 enhances ubiquitination and
   proteolytic degradation of YTHDF2 (Fig. [195]3c-f), which presents a
   novel mechanism behind FBW7-mediated tumor suppression.

   YTHDF2 is an m^6A reader that promotes rapid decay of m^6A-modified
   transcripts [[196]24]. Several studies indicate that YTHDF2 can perform
   either as an oncogenic protein or as a tumor suppressor relying on the
   context of different cancers [[197]26–[198]29] or by regulating
   different mRNA targets [[199]39]. Remarkably, we have provided the
   first line of evidence demonstrating that YTHDF2 is a tumor promoter in
   ovarian cancer, because ablation of YTHDF2 dampens cell proliferation
   and enhances apoptosis (Fig. [200]5), which recapitulates the tumor
   phenotype in vitro and in vivo caused by FBW7 overexpression (Fig.
   [201]2). The role of YTHDF2 in propelling ovarian cancer development is
   also verified by the clinical observation that YTHDF2 is upregulated in
   ovarian cancer samples and negatively associated with prognosis (Fig.
   [202]4a, e, Supplementary Fig. [203]5). Together with the findings that
   FBW7 induces YTHDF2 degradation in ovarian cancer cells (Fig.
   [204]3c-f) and inversely correlates with the expression of the latter
   in ovarian cancer tissues (Fig. [205]4b-c), we have convincingly
   demonstrated that FBW7 suppresses ovarian carcinogenesis and
   progression by antagonizing the oncogenic activity of YTHDF2.

   It has been shown that YTHDF2 targets a plethora of mRNAs for
   degradation in cancers, such as, TNFR2, c-Myc and CEBPA in leukemia
   [[206]26, [207]27], EGFR, IL11, SERPINE2 and SOCS2 in liver cancer
   [[208]28, [209]29, [210]39], and PD-1, CXCR4, and SOX10 in melanoma
   [[211]33]. In our attempt to identify YTHDF2-targeting mRNAs in ovarian
   cancer, the RNA-sequencing and m^6A-sequencing analyses were conducted,
   leading to the identification of a number of genes that could be
   regulated by YTHDF2 in an m^6A-dependent manner (Fig. [212]6g and
   Supplementary table [213]7). The pro-apoptotic BMF was further
   investigated as a key effector of the FBW7-YTHDF2 cascade in ovarian
   cancer, because BMF was found to play critical roles in restraining
   malignancies [[214]40] and maintaining the restricted number of
   primordial follicles and germ cells in the ovary by triggering
   apoptosis [[215]41]. Through the m^6A-sequencing experiment, we found
   that knockdown of YTHDF2 increases the m^6A accumulation in the 5′ UTR
   of BMF (Fig. [216]6h), suggesting that YTHDF2 depletion may elevate the
   level of this m^6A-modified mRNA. Indeed, our further results indicate
   that knockdown of YTHDF2 increases the expression of BMF by enhancing
   the stability of its mRNA (Fig. [217]6j-k). Most importantly, FBW7
   elevates BMF mRNA level and is positively associated with BMF
   expression in primary ovarian cancer tissues (Fig. [218]6q). These
   results mechanistically support the biological function of the
   FBW7-YTHDF2 cascade in triggering apoptosis and inhibiting growth of
   ovarian carcinoma. In addition, several other potentially interesting
   targets of YTHDF2 in ovarian cancer have been identified, such as the
   putative tumor suppressors TRERF1, ZDHHC14, DIS3L, Vps18, NOD1, and
   SLC9A8. These proteins were found to be involved in the regulation of
   cell cycle arrest [[219]42], caspase-dependent apoptosis [[220]43],
   microRNA stability [[221]44], ER signaling pathway [[222]45], and
   inflammation-associated tumorigenesis [[223]46, [224]47], respectively.
   Altogether, these findings profoundly demonstrate the role of the
   FBW7-YTHDF2 axis in restricting ovarian cancer by derepressing various
   tumor suppressors.

   Given that ovarian cancer sustains the highest frequency of TP53 gene
   mutation which leads to the downregulation of FBW7 [[225]6, [226]7,
   [227]21], our study indicates a potential interplay between mutant p53
   and the FBW7-YTHDF2 axis, and suggests an alternative working model for
   the “gain-of-function” of mutant p53 through the regulation of
   m^6A-dependent RNA turnover [[228]48]. Interestingly, a recent study
   showed that mutant p53-R273H impairs BMF mRNA expression in breast and
   colon cancer cell lines [[229]49], which is in agreement with and could
   be explained by our proposed model. Hence, it is worthwhile
   investigating in future if mutant p53 controls gene expression via
   regulation of the FBW7-YTHDF2 axis in ovarian cancer.

Conclusions

   In conclusion, our study explicitly demonstrates an unappreciated
   FBW7-YTHDF2-BMF axis in ovarian cancer. FBW7 interacts with and
   degrades the oncogenic YTHDF2, consequently leading to stabilization of
   m^6A-modified mRNAs, including the pro-apoptotic gene BMF, and
   impairment of ovarian cancer cell survival and proliferation. Our
   findings also unveil the clinical significance of the axis, which would
   be informative for future development of anticancer therapies.

Supplementary Information

   [230]12943_2021_1340_MOESM1_ESM.tif^ (9.5MB, tif)

   Additional file 1: Figure S1. (A) Expression of FBW7 in cancerous and
   normal ovarian tissues detected by IB, which is a supplement to Fig.
   [231]1a. (B) FBW7 is downregulated in the TCGA ovarian cancer cohort.
   (C) High expression level of FBW7 is significantly associated with
   better overall survival (OS) in the TCGA ovarian cancer cohort.
   [232]12943_2021_1340_MOESM2_ESM.jpg^ (100.2KB, jpg)

   Additional file 2: Figure S2. FBW7 expression in various human ovarian
   cancer cell lines.
   [233]12943_2021_1340_MOESM3_ESM.jpg^ (433.1KB, jpg)

   Additional file 3: Figure S3. Knockdown of FBW7 significantly promotes
   proliferation, colony formation, and anchorage-independent growth of
   ovarian cancer cells. (A) Stable expression of shRNAs against FBW7 in
   HeyA8 and OVCAR8 cells. The gene knockdown efficiency is validated at
   both mRNA and protein levels. (B) Knockdown of FBW7 significantly
   increases the proliferation rate of HeyA8 and OVCAR8 cells. (C)
   Knockdown of FBW7 enhances colony-forming ability of ovarian cancer
   cells. (D) Knockdown of FBW7 boosts anchorage-independent growth of
   ovarian cancer cells. Results were presented as mean ± SD of three
   independent experiments. *P < 0.05 or **P < 0.01 indicates a
   significant difference between the indicated groups.
   [234]12943_2021_1340_MOESM4_ESM.jpg^ (255.3KB, jpg)

   Additional file 4: Figure S4. (A-B) Validation of FBW7 accumulation in
   the immunoprecipitates by IB (A) and silver staining (B). (C) Mass
   spectrometry analysis of the immunoprecipitates with IgG or the
   anti-FBW7 antibody. Results were presented as mean ± SD of three
   independent experiments.
   [235]12943_2021_1340_MOESM5_ESM.jpg^ (123.5KB, jpg)

   Additional file 5: Figure S5. (A) YTHDF2 is upregulated in the TCGA
   ovarian cancer cohort. (B) High expression level of YTHDF2 is
   significantly associated with poor overall survival (OS) in the TCGA
   ovarian cancer cohort.
   [236]12943_2021_1340_MOESM6_ESM.jpg^ (106.1KB, jpg)

   Additional file 6: Figure S6. YTHDF2 expression in various human
   ovarian cancer cell lines.
   [237]12943_2021_1340_MOESM7_ESM.jpg^ (441.2KB, jpg)

   Additional file 7: Figure S7. Overexpression of YTHDF2 significantly
   promotes proliferation, colony formation, and anchorage-independent
   growth of ovarian cancer cells. (A) Stable overexpression of YTHDF2 in
   OVCA433 and OVCAR8 cells. The expression of YTHDF2 is validated at both
   mRNA and protein levels. (B) Overexpression of YTHDF2 significantly
   increases the proliferation rate of OVCA433 and OVCAR8 cells. (C)
   Overexpression of YTHDF2 enhances colony-forming ability of ovarian
   cancer cells. (D) Overexpression of YTHDF2 boosts anchorage-independent
   growth of ovarian cancer cells. Results were presented as mean ± SD of
   three independent experiments. *P < 0.05 or **P < 0.01 indicates a
   significant difference between the indicated groups.
   [238]12943_2021_1340_MOESM8_ESM.tif^ (16.3MB, tif)

   Additional file 8: Figure S8. (A) Overexpression of YTHDF2
   significantly reduces the expression of BMF. (B) The effect of YTHDF2
   on cell proliferation and colony-forming ability can be reversed by the
   restoration of BMF expression. (C) The BMF overexpression in SKOV3 and
   OVCAR429 cells increases apoptosis; (D) The YTHDF2 overexpression in
   the FBW7 OE stable cells attenuates the apoptosis caused by FBW7
   overexpression; (E) Knockdown of BMF in the YTHDF2 shRNA-stable cells
   reduced the apoptosis caused by the knockdown of BMF.
   [239]12943_2021_1340_MOESM9_ESM.tif^ (7.6MB, tif)

   Additional file 9: Figure S9. Efficiency of gene knockdown and
   overexpression was validated. (A) Knockdown of YTHDF2 was validated in
   OVCAR8 and HEY cells. (B) Overexpression of FBW7 was validated in SKOV3
   and OVCAR429 cells. (C) Knockdown of FBW7 and YTHDF2 was validated in
   OVCAR8 cells.
   [240]12943_2021_1340_MOESM10_ESM.tif^ (5.4MB, tif)

   Additional file 10: Figure S10. IHC staining of BMF in human the
   ovarian cancer tissue and normal ovary tissue.
   [241]12943_2021_1340_MOESM11_ESM.jpg^ (177.8KB, jpg)

   Additional file 11: Figure S11. The YTHDF2 and BMF mRNA level in FBW7
   high and low group.
   [242]12943_2021_1340_MOESM12_ESM.jpg^ (185.9KB, jpg)

   Additional file 12: Figure S12. The FBW7-mediated regulation of BMF is
   dependent on YTHDF2 demonstrated by WB detection.
   [243]12943_2021_1340_MOESM13_ESM.jpg^ (211.5KB, jpg)

   Additional file 13: Figure S13. (A) The OS curves of patients with
   different expression levels of FBW7 and YTHDF2. (B) The PFS curves of
   patients with different expression levels of FBW7 and YTHDF2. OS,
   overall survival; PFS, progress free survival.
   [244]12943_2021_1340_MOESM14_ESM.docx^ (81.5KB, docx)

   Additional file 14: Table S1. shRNA sequences and primer sequences for
   RT-qPCR.
   [245]12943_2021_1340_MOESM15_ESM.docx^ (19.3KB, docx)

   Additional file 15: Table S2. The relationship between FBW7 expression
   and the clinicopathological features of EOC.
   [246]12943_2021_1340_MOESM16_ESM.xlsx^ (243.8KB, xlsx)

   Additional file 16: Table S3. The list of differentially identified
   proteins by the co-IP-mass spectrometry analysis.
   [247]12943_2021_1340_MOESM17_ESM.docx^ (20.2KB, docx)

   Additional file 17: Table S4. The relationship between YTHDF2
   expression and the clinicopathological features of EOC.
   [248]12943_2021_1340_MOESM18_ESM.docx^ (57.4KB, docx)

   Additional file 18: Table S5. Correlations between the expression of
   FBW7/YTHDF2 and clinicopathological characteristics.
   [249]12943_2021_1340_MOESM19_ESM.docx^ (61.1KB, docx)

   Additional file 19: Table S6. Multivariate analysis of variables
   associated with PFS and OS.
   [250]12943_2021_1340_MOESM20_ESM.xlsx^ (70.8KB, xlsx)

   Additional file 20: Table S7. The list of 25 genes identified by
   integrating the RNA-sequencing and m6A-sequencing results.

Acknowledgements