FTO-Dependent N⁶-Methyladenosine Modifications Inhibit Ovarian Cancer Stem Cell Self-Renewal by Blocking cAMP Signaling

Abstract

N⁶-Methyladenosine (m⁶A) is the most abundant modification of mammalian messenger RNAs (mRNA). RNA methylation fine tunes RNA stability and translation, altering cell fate. The fat mass- and obesity-associated protein (FTO) is an m⁶A demethylase with oncogenic properties in leukemia. Here we show that FTO expression is suppressed in ovarian tumors and cancer stem cells (CSC). FTO inhibited the self-renewal of ovarian CSC and suppressed tumorigenesis in vivo, both of which required FTO demethylase activity. Integrative RNA-sequencing and m⁶A mapping analysis revealed significant transcriptomic changes associated with FTO overexpression and m⁶A loss involving stem cell signaling, RNA transcription, and mRNA splicing pathways. By reducing m⁶A levels at the 3’UTR and the mRNA stability of two phosphodiesterase genes (PDE1C and PDE4B), FTO augmented second messenger 3’, 5’-cyclic adenosine monophosphate (cAMP) signaling and suppressed stemness features of ovarian cancer cells. Our results reveal a previously unappreciated tumor suppressor function of FTO in ovarian CSC mediated through inhibition of cAMP signaling.

INTRODUCTION

RNA modifications have been implicated in dysregulated gene expression under physiological and pathological conditions(1–3). N⁶-methyladenosine (m⁶A), the most prevalent internal modification in eukaryotic messenger RNA (mRNA), is distributed widely across the transcriptome and alters mRNA stability and translation efficiency(1, 2). The effects of m⁶A on mRNA fate rely on specific RNA binding proteins (“readers”)(3, 4), which mediate enhanced mRNA decay or stability, and/or enhanced mRNA translation(5–7). The deposition of m⁶A is catalyzed by methyltransferase like 3 (METTL3), 14 (METTL14) and Wilms Tumor 1 Associating Protein (WTAP) and is removed by RNA demethylases, the fat mass and obesity associated protein (FTO) and the alkylation repair homolog proteins 5 (ALKBH5)(2). The functions of RNA marks are context-dependent and remain incompletely elucidated.

The link between m⁶A and oncogenesis was first described in acute myelogenous leukemia (AML), where overexpression of methyltransferases leading to increased m⁶A deposition inhibited stem cell differentiation and promoted leukemogenesis(8–10). Mutations of key regulatory genes, METTL3, METTL14 and WTAP were found in AML and associated with poor clinical outcomes(8, 11, 12). The functions of epitranscriptomic marks regulating the fate of methylated transcripts in solid tumors are just emerging. In lung cancer, METTL3 enhances translation of EGFR and TAZ leading to increased proliferation(13). ALKBH5 was shown to stimulate the growth of glioblastoma cells by stabilizing nascent RNA transcripts for the transcription factor FOXM1(14). In contrast, reduced m⁶A methylation due to hotspot mutations in the METTL14 complex or due to decreased METTL3 expression, promoted endometrial carcinogenesis through an AKT-dependent mechanism(15). The role of the m⁶A regulatory machinery in ovarian cancer (OC) remains unexplored. A single previous report implicated m⁶A modifications in stabilization of the transcript for the receptor FZD10 in ovarian cancer cells resistant to PARP inhibitors(16), suggesting a potential role regulating DNA damage response mechanisms. Here we sought to determine the function of the demethylase FTO in OC.

FTO, a gene linked to a predisposition for childhood and adult obesity(17), regulates energy homeostasis by controlling food intake and fine tuning of nutrient sensing at the cellular level(18). FTO was the first recognized nucleic acid demethylase(19), physiologically targeting m⁶A residues in mRNA(20). RNA demethylation by FTO was detected in the midbrain of FTO-deficient mice and linked to altered dopaminergic transmission (21). FTO was found to be highly expressed in AML where it promoted cell transformation and proliferation through post-transcription regulation of ASB2, RARA, MYC and CEBPA(22, 23).

High-grade serous ovarian cancer (HGSOC) is initially highly responsive to platinum-based therapy and has a propensity for early metastasis (24). Cancer stem cells (CSCs) are characterized by the ability to self-renew, grow as spheres, differentiate and generate tumors and have been implicated in HGSOC initiation, metastasis, and recurrence after chemotherapy (25),(26). CD133 and ALDH have been recognized as robust ovarian CSCs selection markers for (27, 28) and were used in these experiments.

We report that FTO expression is suppressed in HGSOC compared to fallopian tube epithelium. FTO overexpression inhibited colony and sphere formation in vitro and tumor initiation in vivo. Mechanistically, FTO induced higher levels of the second messenger cAMP by inhibiting its hydrolysis mediated by PDE4B and PDE1C through reduction of m⁶A levels and stability of mRNA transcripts. Our findings point to a tumor-suppressor function of FTO in HGSOC.

Materials and Methods:

Cell culture:

SKOV3 and HEK293T cells were purchased from American Type Culture Collection (Rockville, MD). COV362 and OVCAR5 cells were provided by Dr. Kenneth Nephew (Indiana University). OVCAR5 and COV362 cells were maintained in high glucose (4.5 g/L) DMEM medium (Corning, Corning, NY) containing 10% FBS, 1% penicillin-streptomycin, and 0.1 × Non-Essential Amino Acid. SKOV3 and primary cells recovered from human malignant ascites associated with OC were cultured in 1:1 MCDB 105 and Medium 199 (Cellgro) supplemented with 10% FBS (Cellgro) and 100 units/mL penicillin and 100 ug/mL streptomycin.

Cell lines were tested to be pathogen and mycoplasma negative (Charles River Research Animal Diagnostic Services, Wilmington, MA) and periodically by using the Universal Mycoplasma Detection Kit (ATCC). Low passage number was used during experiments.

Human specimens:

Advanced high grade serous ovarian tumors or malignant ascites associated with OC were collected fresh under Northwestern University approved protocol IRB#: STU00202468; n=5), processed immediately through enzymatic and mechanical disassociation into single cell suspension and cultured under stem cell conditions, as previously described(29, 30) and used for experiments. Cryopreserved HGSOC specimens (n=10) and normal ovarian epithelial tissue (n=6) were obtained from the Indiana University Simon Cancer Center Tissue Bank repository. Human fallopian tube epithelial cells were provided by Dr. Theresa Woodruff of Northwestern University and were obtained from routine hysterectomies, from consenting patients, as previously described (31). Informed written consent was obtained for tissue collection, which was approved by the institutional IRB in accordance with the Declaration of Helsinki.

Plasmid construction:

FTO was PCR-amplified and sub-cloned into plenti-GIII-CMV-GFP-2A-puro lentivirus-based vector (Applied Biological Materials Inc., Richmond, BC) by using Nhel and EcoRV digestion. The wild type FTO and mutant FTO were cloned into lentivector-based pMIRNA1 (SBI, Mountain View, CA) using the following primers: forward 5’-AGAGCTCTAGAACCACCATGGATTACAAAGATGAC-3’ and reverse 5’-CTAAGATTGCGGCCGCCTAGGGTTTTGCTTCCAGAAGC-3’. The lentiviral vector based shRNAs targeting human PDE1C (shPDE1C-1, shPDE1C-2) and human PDE4B (shPDE4B-1, shPDE4B-2) and non-targeting control shRNA were purchased from Applied Biological Materials (Richmond, BC, Canada). The lentiviral vector based shRNAs targeting human FTO (shFTO-1, shFTO-2) and non-targeting control shRNA were purchased from Origene Technologies (Rockville, MD). Lentiviral particles were produced in 293T cells by co-transfecting the lentiviral construct and the packaging genes VSV-G and Pspax2. The viral particles were harvested 72 hours after transfection and added to cells in the presence of polybrene (8 μg/ml) for 48 hours.

RNA extraction, quantitative RT-PCR analysis, Colony-forming assay, and Western blotting methods are included in SM.

Sequences of primers for FTO, ALDH1A1, SOX2, NANOG, OCT4 are included in Supplementary Table S1.

m⁶A dot blot assay:

mRNA was isolated from total RNA by using the Dynabeads mRNA purification kit (Ambion) according to the manufacturer’s instructions and quantified by NanoDrop 2000 spectrophotometry. The assay was performed following a published protocol (www.bio-protocol.org/e2095) with some modifications. Briefly, the mRNA samples were loaded onto an Amersham Hybond-N+ membrane (GE Healthcare) and UV-crosslinked. The membranes were blocked with 5% nonfat dry milk and incubated with an anti-m⁶A antibody (Synaptic Systems) overnight at 4°C followed by incubation with HRP-conjugated goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology) for 1 hour at room temperature. The membrane was developed with a chemiluminescent substrate (Thermo Scientific) before imaging.

m⁶A RNA methylation quantification:

N6-methyladenosine RNA methylation was quantified by using the EpiQuik m⁶A RNA methylation quantification kit (Epigentek Group Inc., Farmingdale, NY). Briefly, 300 ng total RNA was bound to wells, incubated with capture antibody for 60 min, followed by detection antibody for 30 min, and enhancer solution for 30 min at room temperature. The reaction signal was read with an xMark microplate spectrophotometer (Biorad) at 450 nm.

Immunohistochemistry (IHC):

A tissue microarray (TMA) (OVC1021) containing 92 ovarian cancer specimens (45 serous, 2 clear cell, 27 endometrioid, 18 mucinous) was obtained from Pantomics Inc. (Fairfield, CA). Sections from 6 normal fallopian tubes were obtained from the Tissue Pathology Core of the Lurie Cancer Center (Chicago, IL). The FTO antibody (ab92821) was from Abcam Inc. (Cambridge, MA). Detailed methods are in SM. H-scores were calculated for each specimen based on the intensity (0–3+) and percentage of cells staining (0–100%), using the formula: intensity * percentage/100.

RNA-seq:

The RNA-seq libraries were prepared using the NEBNext Ultra II RNA library prep kit from Illumina (New England Biolabs Inc., Ipswich, MA) and mRNA isolated from 1 μg of total RNA. After first-strand cDNA synthesis, second-strand cDNA synthesis, end repair of cDNA library, dA-tailing of cDNA library, Adaptor ligation, and PCR enrichment, the RNA-seq libraries were checked by using a BioAnalyzer, and sequenced on Illumina NextSeq500 with single-end, 75-bp read length. Each group was sequenced in triplicate. Data analysis is described in Supplemental Methods. Reads of RNA-seq shown in Supplementary Table S2 and RNA-seq QC is included in Supplementary Figure S10.

MeRIP-seq:

To examine m⁶A modifications across the genome, the Magna MeRIP m6A kit (Millipore, Billerica, MA) was used. Briefly, 5 μg of mRNA was sheared to approximately 100 nt in length by metal-ion induced fragmentation, purified, and incubated with m⁶A antibody (Synaptic Systems) or mouse IgG (control) –conjugated magnetic protein A/G beads in 500 μl 1 × IP buffer supplemented with RNase inhibitors at 4°C overnight. Methylated RNAs were immunoprecipitated with beads, eluted by competition with free m⁶A, and recovered with the RNeasy kit (QIAGEN). One tenth of fragmented RNA was saved as input control. MeRIP libraries were prepared by using the NEBNext Ultra II RNA library prep kit for Illumina (New England Biolabs Inc., Ipswich, MA), starting from the first-strand cDNA synthesis step. The libraries were sequenced on Illumina NextSeq500 with single-end 75-bp read length. Sequencing was performed in triplicates for GFP and FTO samples, along with their respective controls, resulting in 12 raw fastq files of average read depth 30.6 million reads. Quality check and analysis are described in Supplemental Methods. The accession numbers for RNA-Seq and MeRIP-Seq data reported in this paper are GSE130349 and GSE130350, respectively. Reads of MeRIP-seq show in Supplementary Table S2 and MeRIP-seq QC is included in Supplementary Figure S11.

Gene-Specific m⁶A qPCR:

mRNA was purified from DNase I treated total RNA by using the Dynabeads® mRNA Purification Kit (Life technology). 5 μg mRNA was sheared to approximately 100 nt in length by metal-ion induced fragmentation. One tenth of fragmented RNA was saved as input control, and then the MeRIPed RNAs was obtained as described above. Relative enrichment of m⁶A was quantified by the Verso SYBR Green 1-step RT-qPCR (ThermoFisher Scientific) using the primers listed in Table S1. The relative mRNA expression in each sample was calculated by the value of Cq in m⁶A IP portion divided by the value of Cq from the input (CqIP/CqInput). Gene-specific m⁶A levels were controlled to the relative mRNA expression levels between FTO-overexpressing and control samples (ΔCqtarget/ΔCqcontrol).

RNA stability assay:

FTO and FTO-mut-overexpressing cell lines, shFTO cell lines and their control cell lines were treated with the transcription inhibitor actinomycin D (5 μg/ml) at 0, 3 and 6 hours before cell collection. Total RNA was isolated by using Trizol and purified with RNeasy kit (QIAGEN). RT-PCR quantified the relative levels of target mRNAs by using the Bio-Rad iTaq universal SYBR green system. The degradation rate of mRNA (Kdecay) was estimated by using the equation: In(C/C0)=-Kdecayt; where C0 is the concentration of mRNA at time 0 hour, before the decay starts, t represents the transcription inhibition time, while C is the mRNA concentration at the time t. The Kdecay can be derived from calculating the exponential decay fitting of C/C0 versus time t. The half-time (t1/2) representing C/C0=50%/100%=1/2 is calculated by using the following equation: ln(1/2)= -Kdecayt1/2. The mRNA half-life time value was estimated as t1/2= ln2/Kdecay.

Preprocessing and analysis of TCGA ovarian cancer exon-array database:

Raw Affymetrix Exon-array data for 569 high grade serous ovarian tumors and 8 control samples (fallopian tube epithelium) from TCGA and GTEx were processed with Multi-Mapping Bayesian Gene expression for Affymetrix whole-transcript arrays (MMBGX) to obtain gene- and isoform-level expression estimates. Ensembl database (version 56) was used as the reference genome. All expression estimates were normalized across the samples using locally weighted scatter plot smoothing (loess) algorithm. Gene-level expression between OC specimens and control samples were compared with the two-sample t-test with unequal variances and statistical significance was declared at p = 0.05(32, 33).

Spheroid Formation Assay:

One hundred OC cells, were seeded into 96-well ultra-low attachment plates (Corning, Tewkesbury, MA) in stem cell media (Stemcell technologies, Cambridge, MA) with 10% Mammocult proliferation supplements, (Stemcell technologies) 4μg/ml heparin (Stemcell technologies), 0.48μg/ml hydrocortisone (Stemcell technologies), and 1% penicillin-streptomycin solution (100⨯, Corning). Media was refreshed every 3 days. The numbers of spheroids per well were counted under an inverted microscope (5 fields per well) on day 14 of culture. Each experiment was performed in triplicate.

Extreme Limited Dilution Analysis (ELDA):

A serial of dilution of cells (5, 10, 50, 100, 500, 1000, 2000, and 5000) were FACS sorted directly into 96 well low-attachment plates and cultured in stem cell medium as described above for 14 days. Each dilution included 12 replicates. The numbers of spheroids per well were counted for each cell dilution. The CSC frequency for the two conditions was calculated by using the ELDA software (http://bioinf.wehi.edu.au/software/elda/). The same approach was applied to cells dissociated from xenografts using serial dilution numbers of 1250, 2500, 5000 and 10000 cells.

Aldefluor assay and Fluorescence-Activated Cell Sorting:

Dissociated OC single cells were centrifuged at 1,500 rpm for 5 min, resuspended in Aldefluor assay buffer (Stemcell Technologies) containing the ALDH substrate, bodipyaminoacetaldehyde (BAAA) at 1.5mM. After incubation for 45 min at 37 °C, cells were centrifuged at 1,500 rpm for 5 min at 4 °C, washed with cold Aldefluor assay buffer and incubated in the same buffer supplemented with 0.5% BSA and CD133/1-PE-Vio615 (2 μl/ million cells, Miltenyi Biotec, Auburn, CA) for 40 minutes on ice. The ALDH+/CD133+ population was gated using control cells incubated under identical conditions in the presence of the ALDH inhibitor (DEAB), and anti-mouse IgG1-PE-Vio615 (1μl/ million cells Miltenyi Biotec) isotype control. Flow cytometry and sorting used the FACSAria II flow cytometer (BD Biosciences, San Jose, CA) under sterile conditions.

In vivo xenograft experiments:

Animal studies were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of Northwestern University (# IS00003060). Female nude, athymic, BALB/c-nu/nu mice (6–7 weeks old; Harlan) were injected subcutaneously (s.c.) with serially diluted numbers (5,000, 10,000, or 20,000) of OVCAR5_GFP or OVCAR5_FTO cells mixed with Matrigel (Fisher Scientific). Mice were monitored bi-weekly and time to tumor initiation and tumor growth were recorded. Tumor length (l), width (w) and height (h) were measured with digital calipers and tumor volumes (v) were calculated as v = ½ × l × w × h. CSC frequency in single cell suspensions dissociated from harvested xenografts and statistical significance were determined by using the ELDA software (http://bioinf.wehi.edu.au/software/elda/).

Isolation of Tumor Cells used previously described methods (30, 34) and is described in SM. Cyclic AMP XP Assay: Cyclic AMP (cAMP) activity was measured with an AMP XP assay kit (Cell Signaling) according to the manufacturer’s instructions. In brief, 6 −10 × 10⁴ cells plated into 96-well-plates under standard cell culture conditions were lysed in 100 μl of 1 × lysis buffer for 15 minutes on ice. Thereafter, HRP-linked cAMP solution and cell lysates were incubated at room temperature for 3 hours on a plate shaker. Wells were washed 4 times before incubating in TMB substrate. The absorbance was measured at 450 nm and the percentage of cAMP activity was calculated as 100 × (1-A/A₀), where A was the sample’s absorbance and A₀ was the absorbance at minimal stimulation (no cAMP).

Statistical Analysis:

Data were analyzed and presented as means ± SD. Two-tailed Student’s t test compared means between two groups, and one-way ANOVA was used for multiple group comparisons; p < 0.05 was considered significant.

RESULTS

FTO is expressed at lower levels in OC cells, tumors, and in ovarian cancer stem cells (OCSCs):

To understand whether RNA methylation is involved in the process of ovarian cancer (OC) initiation or progression, we compared the expression levels of m⁶A writers and erasers between normal fallopian tube epithelium (FTE), normal ovarian epithelium and ovarian tumors. No significant differences were observed between HGSOC specimens (n=10), normal FTE (n=4) and normal ovarian tissue (n=5) for METTL3, METTL14, WTAP, or ALKBH5 (Figure 1A). In contrast, the mRNA expression levels of the demethylase FTO were significantly lower in ovarian tumors compared to FTE and normal ovary tissue (Figure 1A, p = 0.0485). An analysis of the TCGA Affymetrix Exon-array data including 569 high-grade serous and 8 control FTE specimens for the five genes revealed that FTO and ALKBH5, but not METTL3, METTl14 or WTAP, were significantly downregulated in HGSOC relative to FTE (p < 0.001; Figure 1B and Figure S1). Among the 569 tumor samples, 497 (87%) and 566 (99%) displayed downregulated FTO and ALKBH5 expression levels, respectively, relative to normal controls. Because FTO expression levels were found to be significantly suppressed in both the TCGA database and the in-house dataset, we focused on FTO for further studies. FTO protein expression was measured by immunohistochemistry (IHC) in OC specimens (n= 92; all histological subtypes) included in a tissue microarray and FT (n=6). FTO expression quantified as H score was down regulated in OC compared to FTE (p<0.05), for the serous, endometrioid, and mucinous groups (Figures 1C–D). Likewise, significantly lower FTO mRNA and protein expression levels were detected in OC cell lines compared to primary FTE cell cultures (Figures 1E–F, p < 0.01). Collectively, the data support that FTO expression is significantly downregulated in OC cells and tumors.

Figure 1: FTO is downregulated in OC cells and ovarian tumors.

(A) mRNA expression levels measured by qRT-PCR for the RNA methylation regulatory genes METTL3, METTL14, WTAP, FTO, and ALKBH5 between normal fallopian tube epithelium (FTE; n=4), ovarian epithelium (n=5) and human OC specimens (n=10). (B) Expression levels of FTO and ALKBH5 in HGSOC (n=569) compared to normal fallopian tube epithelium (FT; n=8) extracted from the TCGA database. (C) Representative FTO IHC staining in fallopian tube and OC specimens (magnification 100⨯). Scale bars, 200 μm. (D) H-scores compared FTO expression in fallopian tube (n=6) compared with endometrioid (n =27; p = 0.037), mucinous (n = 18; p = 0.019), serous (n=45, p = 0.036) and clear cell (n=2, NS) sub-types. (E) FTO mRNA expression levels in primary human fallopian tube epithelial cells (n=2; FEM121, FEM122) and OC cell lines (SKOV3, OV90, HEY, COV362, OVCAR3, OVCAR4, OVCAR5, and OVCAR8). (F) FTO protein expression levels were measured by Western blotting in human immortalized fallopian tube epithelial cells (FT; FT-190) and OC cells lines (SKOV3, OV90, HEY, COV362, OVCAR3, OVCAR4, OVCAR5, and OVCAR8). (G) FTO mRNA expression levels in ALDH⁺ and ALDH⁻ FACS sorted cells from OVCAR5 and COV362 cell lines. (H) FTO mRNA expression levels in ALDH⁺CD133⁺ (DP) and ALDH⁺ compared with ALDH⁻CD133⁻ (DN) FACS-sorted cells derived from HGSOC. Bars display mean fold changes +/− SD (n=5). (I) Quantification of m⁶A levels in OVCAR3, COV362, and OVCAR5 cells cultured as monolayers or spheroids. (J) M⁶A levels in FACS-sorted ALDH⁻ and ALDH⁺ cells from OC cell lines and from human tumors. Bars represent means ± SD. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant (p > 0.05).

Given the previously reported role of m⁶A modifications in the regulation of normal and leukemic stem cell populations(8, 9), FTO expression was measured in cell sub-populations derived from cell lines or human ovarian tumors. Cells characterized by high ALDH activity possess tumor initiating capacity, self-renewal ability, effectively form spheres, express “stemness” associated transcription factors, and are quiescent(27, 35),(36). FTO expression levels were significantly lower in the ALDH⁺ population compared to ALDH⁻ cells derived from OVCAR5 and COV362 cell lines (Figure 1G, p < 0.01). Similarly, FTO expression levels were lower in ALDH⁺ and ALDH⁺/CD133⁺ cells compared to the ALDH⁻/CD133⁻ cell populations derived from human HGSOC specimens (n=5; Figure 1H, p < 0.01). FACS-based separation of ALDH⁺/CD133⁺ cells is shown in Figure S2.

To determine the effects of FTO overexpression on m⁶A levels, an m⁶A RNA quantification assay was used. This assay requires >10⁵ cells and is not applicable to rare cell populations, such as CSCs, therefore, cultures were enriched under sphere conditions(29, 30). Global m⁶A mRNA levels were significantly increased in spheroids derived from OVCAR3, OVCAR5 and COV362 cells compared to monolayer cultures (Figure 1I, p < 0.05). Global m⁶A levels were significantly higher in ALDH⁺ vs. ALDH⁻ cells (Figure 1J, p < 0.05), FACS sorted from HGSOC cell lines or human tumors and propagated under non-differentiating culture conditions, as spheres. These results show increased global m⁶A RNA levels, accompanied by FTO downregulation, in OCSCs relative to non-CSCs.

FTO overexpression inhibits ovarian CSC proliferation/self-renewal and tumor-initiation capacity:

To investigate the functions of FTO in OCSCs, we developed stable cell lines overexpressing FTO. FTO mRNA and protein expression levels were increased in OVCAR5, COV362, and SKOV3 cells stably transduced with FTO-carrying lentivirus compared with vector control (Figures 2A–B and Figures S3A–C). Accordingly, the global m⁶A levels on mRNA were decreased in FTO-overexpressing OVCAR5, COV362, and SKOV3 cells (Figures S3D–E). Notably, m⁶A levels were significantly higher in OVCAR5-FTO cells grown under non-differentiating conditions as spheroids (OVCAR5-FTO-Sph) compared to monolayer cultures (Figure S3E, p<0.05), consistent with the concept that RNA methylation would be increased in cultures enriched with CSCs, associated with FTO downregulation.

Figure 2: FTO overexpression inhibits colony forming ability of OC cells and cancer stem cell characteristics.

(A-B) FTO overexpression in OVCAR5 cells (A) and COV362 cells (B) were verified by using qRT-PCR (mRNA) and western blot (protein). (C, D) Numbers of colonies generated by OVCAR5 (C) and COV362 cells (D) transduced with FTO or control vector (n=3). (E) Numbers of spheroids formed by OVCAR5 cells transduced with control or FTO during 14 days culture (n=3). (F) ALDHA1 mRNA expression levels in SKOV3 and OVCAR5 cells +/− FTO. (G) mRNA expression levels for the stemness-associated transcription factors OCT4, NANOG and SOX2 in OVCAR5 cells transduced with control or FTO; * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant (p > 0.05).

Colony-forming assays showed that FTO-overexpressing OVCAR5, COV362 and SKOV3 cells formed significantly fewer colonies compared to (GFP-expressing) control cells (Figure 2C–D, p < 0.01 and Figure S3F). Similarly, much fewer spheres were formed by FTO-overexpressing OVCAR5 vs. control cells (Figure 2E, p < 0.01). The morphology of spheres derived from FTO-overexpressing cells was less well structured, as such cells formed loose aggregates, without a peripheral ring, as compared to control cells, which formed classic spheres (Figure 2E). To further characterize the effects of FTO on stem cell characteristics, the percentage of ALDH⁺ cells and the expression of stemness associated transcription factors (NANOG, SOX2, and OCT4) were assessed. The percentage of ALDH⁺ cells was significantly lower in FTO-overexpressing OVCAR5 vs. control cells (Figure S3G, p < 0.05). Moreover, expression levels of ALDH1A1 (Figure 2F, p < 0.001) and of transcription factors (NANOG, SOX2, and OCT4) were significantly lower in FTO-overexpressing OVCAR5 and SKOV3 vs. control cells (Figure 2G, p < 0.05 and Figure S3H).

Lastly, the effects of FTO on cancer stemness were assessed through limiting dilution assays. In vitro, spheroid formation was measured from serially diluted FTO-overexpressing or control OVCAR5 cells (Table S3 and Figure S3I). Extreme limiting dilution analysis (ELDA) calculations(37) demonstrated that the frequency of CSCs was significantly decreased in FTO-overexpressing OVCAR5 cells (1 in 677.36) relative to the control cells (1 in 7.19; p = 1.99e⁻⁴³, Table S3). Tumor initiation capacity (TIC) was assessed by injecting subcutaneously (S.C.) serially diluted GFP- or FTO-overexpressing OVCAR5 cells (5,000, 10,000, and 20,000 cells) into female nude mice. Tumor size derived from FTO-expressing cells was significantly smaller at all dilutions compared to tumors derived from the control GFP-expressing cells (Figures 3A–C, p < 0.05). Time to tumor initiation was prolonged for FTO-overexpressing vs. vector-transduced OVCAR5 cells (41 days vs. 23 days, p < 0.05) (Figure S4A). ELDA calculations(37) indicated that the CSCs’ frequency was significantly lower in FTO-expressing OVCAR5 (1 in 119,931) vs. control cells (1 in 10,941; p = 0.00491; Figure 3D). Further, FACS demonstrated that the population of ALDH⁺ cells was significantly lower in tumors from mice xenografted with FTO-overexpressing OVCAR5 cells than those from control xenografts (Figure 3E–F, p < 0.01). Increased FTO expression in xenografts was verified by qRT-PCR (Figure 3G, p< 0.01). Expression levels of stemness-associated factors (ALDH1A1, NANOG, SOX2, and OCT4) were significantly lower in FTO-expressing tumor cells vs. control cells (Figures 3H; p < 0.05). Cells dissociated from FTO-overexpressing xenografts had diminished sphere forming ability and contained fewer CSCs compared to cells dissociated from control xenografts (Figure S4B–C, p = 4.08e⁻⁵). Overall, these data suggest that forced expression of FTO reduced the population of CSCs and inhibited their specific functions.

Figure 3: FTO inhibits tumor initiation capacity.

(A-C) Representative images (top) and tumor volumes (means ± SD, bottom) of subcutaneous (s.c.) xenografts formed by 5,000 (A), 10,000 (B), or 20,000 (C) OVCAR5_GFP and OVCAR5_FTO cells (n=3 or 5). (D) Numbers of mice harboring detectable tumors for each cell dilution. CSC frequency was calculated using the ELDA software (P = 0.00491). Plot indicates the log fraction of mice which did not form tumors versus the number of injected cells. (E) Side scatter shows the percentages of ALDH⁺ cells in single cell suspensions derived from OVCAR5-GFP or OVCAR5-FTO xenografts. (F) Percentages of ALDH⁺ cells from single cell suspensions derived from OVCAR5-GFP or OVCAR5-FTO xenografts (n=3). (G) qRT-PCR measured FTO mRNA expression levels in harvested OVCAR5-GFP and OVCAR5-FTO xenografts. (H) Fold changes (means ± SD) for ALDH1A1, NANOG, SOX2 and OCT4 mRNA levels in OVCAR5-FTO vs. OVCAR5-GFP-derived xenografts. * p < 0.05; ** p < 0.01; *** p < 0.001.

FTO knock-down promotes CSC self-renewal

Loss-of-function studies used OVCAR5 cells stably transduced with individual shRNAs targeting FTO (shFTO-1/2) or a control (nontargeting) shRNA (shCTL). The downregulation of FTO at both RNA and protein levels mediated by shFTO was confirmed (Figure 4A) along with higher levels of m⁶A measured in FTO-depleted compared to control cells (Figure 4B and Figure S5A). Depletion of FTO by shFTO-1 or shFTO-2 resulted in substantial upregulation of ALDH1A1 (Figure 4A, p < 0.001). Depletion of FTO by shFTO-2 potently induced the expression of all stem cell factors (SOX2, OCT4, and NANOG), while transduction of shFTO-1 only induced OCT4 expression (Figure 4C, p < 0.001). FTO knockdown resulted in significantly increased numbers of colonies (Figure 4D, p < 0.05) and spheres (Figure 4E; p < 0.05), relative to the control group. FTO knockdown led to an increased population of ALDH⁺ cells compared to vector-transduced cells (Figure 4F, p < 0.01). Tumor initiation capacity (TIC) also was assessed by injecting subcutaneously (S.C.) serially diluted shCTL or shFTO cells (500, 1,000, and 2,000 cells) into female nude mice. Time to tumor initiation was prolonged for shFTO vs. shCTL cells (9 days vs. 14 days, p = 0.0491; Figure S5B). ELDA calculations indicated that the CSCs frequency was higher in shFTO (1 in 298) vs. shCTL (1 in 1106; p = 0.0320; Figure S5C). Overall, depletion of FTO in OC cells caused an increased level of m⁶A, induced cancer cell proliferation as spheres, induced colony formation, and promoted a CSC phenotype.

Figure 4: FTO knockdown promotes ovarian cancer cell stemness.

(A) FTO and ALDHA1 mRNA expression levels in OVCAR5 cells transduced with non-targeting shRNA (shCTL) or shRNA targeting FTO (shFTO-1 and shFTO-2). Western blotting assessed FTO protein expression in shCTL, shFTO-1 and shFTO-2 transduced cells. (B) m⁶A levels were quantified in shCTL and shFTO transduced OVCAR5 cells by using the EpiQuik m⁶A RNA methylation kit. (C) mRNA expression levels of the stemness markers, NANOG, SOX2, and OCT4, were determined by qRT-PCR in FTO knockdown vs. control cells (n=3). (D) Colonies formed by shCTL, shFTO-1 and shFTO-2 transduced OVCAR5 cells (top panels) and average numbers of colonies formed by shCTL, shFTO-1 and shFTO-2 transduced OVCAR5 cells (bottom panels). (E) Average numbers of spheroids formed by shCTL, shFTO-1 and shFTO-2 transduced OVCAR5 cells (n=3). (F) FACS quantification of ALDH⁺ cells in shCTL and shFTO-2 transduced OVCAR5 cells (n=3). (G) FTO protein and mRNA expression levels after stable transduction of FTO and FTO-mutant (FTO-mut) in OVCAR5 cells. (H) Numbers of colonies generated from OVCAR5 cells stably transduced with vector control, FTO or FTO mutant. (I) Numbers of spheres generated from OVCAR5 cells stably transduced with vector control, FTO or FTO mutant. (J) qRT-PCR measured mRNA expression levels for NANOG, OCT4, and SOX2 in vector control, FTO or FTO mutant transduced OC cells. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

The demethylase function of FTO regulates cancer stemness phenotype

To determine whether the effects of FTO are due to its demethylase function, we used a mutant carrying two point mutations H²³¹A and D²³³A, which disrupt its enzymatic function(38). FTO and FTO-Mut were stably overexpressed in OVCAR5 cells (Figure 4G). To confirm the mutant’s loss of demethylase function, global m⁶A mRNA levels were measured by ELISA in total RNA from FTO and FTO-Mut transduced cells and found to significantly decreased in FTO-overexpressing OVCAR5 cells compared to control cells, but not altered in FTO-Mut transduced cells (Figure S5D). While wild-type FTO overexpression reduced colony (Figure 4H) and sphere formation (Figure 4I) compared to control, forced expression of the mutant did not significantly alter the phenotype. Similarly, OCT4 and SOX2 mRNA expression levels were downregulated in cells expressing wild-type FTO, but not in cells transduced with the FTO mutant (Figure 4J, p<0.05). Collectively, these data support that the demethylase activity of FTO is required for its suppressing function against the OCSC phenotype.

Signaling pathways affected by the FTO/m⁶A axis in OC cells

To decipher the mechanisms by which FTO-mediated m⁶A modifications altered stemness, RNA-sequencing (RNA-Seq) and methylated RNA immunoprecipitation-sequencing (MeRIP-seq) compared OVCAR5 cells overexpressing FTO or vector. A total of 2,406 significant differential m⁶A peaks were distributed in the 3’ UTR (18%), 5’UTR (7%), introns (29%), and coding sequence (CDS, 32%; Figure 5A). A similar pattern was observed for total m⁶A peaks (22,703 peaks; 18% were in the 3’UTR, 10% in 5’ UTR, 27% in intronic regions, and 28 % in CDS; Figure S6A). Motif analysis of the peaks region using DREME(39) showed that the common m⁶A motif GGAC was significantly enriched (Figure S6B; p-value of 3.5e-042). There were 4,550 differentially expressed genes observed between FTO and vector-transfected OC cells, as determined by RNA-seq (FDR < 0.05, p < 0.05). Integration of the differentially expressed (n=4,550 genes) and differentially m⁶A marked transcripts (n=1,272 RNA transcripts) yielded 731 overlapping genes (Figure 5B), of which 93.4% were demethylated in FTO-expressing cells (p < 0.05; fold-change ≥ 1.2). Heatmap analysis indicated a clear separation between the gene expression profiles of GFP- and FTO-expressing cells (Figure 5C). Among the 731 genes, 246 were hypo-methylated and down-regulated, 437 were hypo-methylated and up-regulated, 28 were hyper-methylated and down-regulated, and 20 were hyper-methylated and up-regulated (Figure 5D).

Figure 5: Genome wide m⁶A modifications and transcriptomic changes induced by FTO.

(A) The distribution of differential m⁶A peaks (n=2406) in the 3’UTR, 5’UTR, intron, non-coding, stop codon, intergenic, or CDS regions. (B) Venn diagram illustrates the numbers of genes with significant expression or m⁶A markings changes (FDR<0.05, p<0.05) as measured by RNA-seq and MeRIP-seq. (C) Heatmap obtained through unsupervised complete-linkage hierarchical clustering based on Euclidean distance and scaling across rows demonstrates differential gene expression between FTO- and vector-expressing OVCAR5 cells (n = 3). Differentially expressed and differentially m⁶A marked genes (p <0.05 and FDR < 0.05) were included (n = 731 genes). (D) Genes displaying a significant change in m⁶A marks and expression levels in FTO- vs. control OVCAR5 cells were grouped in 4 categories: hypomethylated/downregulated (n=246; lower left quadrant); hypomethylated/upregulated (n=437, lower right quadrant); hypermethylated/downregulated (n=28; upper left quadrant); hypermethylated/upregulated (n=20; upper right quadrant). (E) GSEA reveals significantly up-regulated (red) and down-regulated (blue) pathways (p ≤ 0.05, FDR ≤ 0.05) in FTO- vs. control- transduced OVCAR-5 cells. Enrichment Map and Auto Annotate applications in Cytoscape were used to construct the visual representation. Nodes represent different gene sets from the GSEA database and edges represent the similarity coefficients between two linked nodes (gene sets). This enrichment map was manually curated after removing un-informative individual gene sets. (F) Top downregulated or upregulated transcripts in FTO-overexpressing cells. Bars represent fold change, extracted from RNA-sequencing analysis. (G) Interaction network of relevant biological pathways affected by FTO overexpression using genes selected from the hypomethylated/downregulated group.

Gene Set Enrichment Analysis (GSEA) on genes pre-ranked by log-2 fold change and log p-values yielded “embryonic stem cells associated with H3K27me3” (Normalized Enrichment Score (NES) of −5.12, FDR < 0.0001; Figure S6C) as being enriched in GFP expressing cells and “downregulated hematopoietic stem cells” as being enriched in FTO expressing cells (NES =2.26, FDR=0.005; Figure S6C); supporting FTO’s role inhibiting stemness. Other gene sets represented in FTO cells vs. controls include “downregulated genes in metastasis” (NES=4.07) and “downregulated genes in neoplastic transformation” (NES 3.32; Figure S6C), supporting a tumor suppressor function. An enrichment map was built by using significant gene sets (p < 0.05 and FDR < 0.05). FTO overexpression led to up-regulated gene sets linked to immune response and RNA transcription pathways and down-regulated DNA repair pathways, stem cell signaling, and mRNA splicing (Figure 5E). The top hypomethylated and differentially expressed genes are included in Tables S4, S5, and S6 and include the adhesion molecule ICAM1, the phosphodiesterases PDE1C and PDE4B, the stem cell related factor Noggin, the signaling molecule TACSTD2, and others (Figure 5F). Focusing on genes related to signaling, selected from the “hypomethylated-downregulated” group, and analyzed by using Ingenuity Pathway Analysis, we identified gene networks related to signal transduction, cAMP signaling, RNA transcription and various cancer related pathways (Figure 5G and Figure S6D). Importantly, genes related to cAMP hydrolysis (PDE1C and PDE4B) and signaling were represented in these networks and studied further.

M⁶A-modified mRNAs regulate the expression of two phosphodiesterases

Among the top five differentially m⁶A modified and expressed genes, the phosphodiesterases 4B (PDE4B) and 1C (PDE1C) were hypomethylated and downregulated in FTO-overexpressing cells compared to controls (Table S4). The differentially m⁶A enriched regions were included in the 3’ UTR for PDE4B and spanned the last three exons of the PDE1C transcript, including the last exon with STOP codon and 3’UTR, and are shown in Figure 6A. Validation studies showed that FTO knockdown significantly up-regulated expression of both PDE4B and PDE1C in OVCAR5 cells (Figure 6B, p<0.01). Conversely, expression of PDE4B and PDE1C was significantly down-regulated upon forced expression of wild-type FTO (but less by the FTO mutant) in OVCAR5, COV362, and SKOV3 cells (Figure 6C, p<0.01; Figure S7A–B, p<0.05). Furthermore, expression levels of PDE4B and PDE1C were higher in ALDH⁺/CD133⁺ cells compared to the ALDH⁻/CD133⁻ cell populations derived from human HGSOC specimens (n=3; Figure 6D, p < 0.001). Significantly reduced m⁶A levels at the 3’ UTR of PDE4B and near the 3’UTR for PDE1C were validated in cells overexpressing wild-type FTO (but not in those overexpressing FTO mutant) by gene-specific m⁶A qRT-PCR using m⁶A immunoprecipitated total RNA (Figure 6E), confirming the findings of MeRIP-sequencing. To further demonstrate that m⁶A modifications at the 3’UTR of these transcripts alter mRNA stability, we treated cells overexpressing FTO or FTO-mutant with actinomycin D, a transcription inhibitor, and measured the half-lives of PDE1C and PDE4B transcripts. Indeed, wild-type FTO overexpression led to significantly shortened half-life of PDE1C (5 to 2.9h, Figure 6F) and PDE4B (7.87 to 2.87h, Figure 6G), while overexpression of FTO mutant did not significantly alter PDE1C (5 to 4.5h, Figure 6F) and only mildly affected PDE4B half-life (7.87 to 4.91h, Figure 6G). Collectively, these data suggest that FTO directly affects the mRNA stability of the two phosphodiesterase genes. Because recent studies have reported binding of the insulin growth factor 2 mRNA binding proteins 2 and 3 (IGF2BP 2/3) to m⁶A modified regions leading to enhanced mRNA stability(40), we further checked the possible binding sites of IGF2BP2 and IGF2BP3 within these differentially enriched regions, using RBPmap with maximum stringency (p-value < 0.001) and conservation filter. The bioinformatic tools detected possible binding sites in a 10,000 bp region: for PDE4B, five sites for IGF2BP2 and four for IGF2BP3 and for PDE1C, fifty-three sites for IGF2BP2 and forty-two for IGF2BP3 (average z-score >3; Figure S8).

Figure 6: PDE1C and PDE4B are FTO target genes in OC cells.

(A) Differences in m⁶A peaks distributions as detected by MeRIP-seq in the 3’UTR of PDE4B and the last 3 exons of PDE1C mRNA transcripts in control or FTO-transduced OVCAR5 cells. (B-C) mRNA expression levels of PDE4B and PDE1C in shCTL or shFTO transduced OVCAR5 cells (B) or in control, FTO, or FTO-mutant-transduced OVCAR5 cells (C). ** p < 0.01; *** p < 0.001; ns, not significant. (D) PDE4B and PDE1C mRNA expression levels in human HGSOC specimens derived ALDH/CD133 double positive (DP+) cells compared to ALDH/CD133 double negative cells (DN-). (***, p <0.001). (E) Gene-specific m⁶A PCR measured changes in m⁶A levels in the 3’UTR of PDE4B and associated with PDE1C in FTO, FTO-mutant, vs. control-transduced OVCAR5 cells. * p < 0.05; ** p < 0.01; ***, p <0.001; ns, not significant. (F-G) PDE1C (F) and PDE4B (G) mRNA transcript half-lives in OC cells transduced with control, FTO, or FTO-mutant.

FTO/m⁶A regulated phosphodiesterases are involved in maintaining stemness

To determine whether the two phosphodiesterase genes play a role in maintaining cancer stemness, we used shRNA mediated knockdown and small molecule inhibitors. Knockdown of either PDE1C or PDE4B (Figure 7A) downregulated the expression of stemness-associated factors (NANOG, OCT4 and SOX2; Figure 7B–C) and inhibited spheroid formation (Figure 7D) consistent with a stemness-inhibitory effect induced by FTO overexpression. Furthermore, PDE1C knockdown could at least in part reverse the effects of FTO knockdown on promoting expression of ALDH1A1, NANOG, OCT4 and SOX2 in OVCAR5 cells (Figures 7E–I), suggesting that these genes are critical targets of FTO and are required for maintaining the stemness phenotype in OC cells.

Figure 7: FTO-regulated PDE4B and PDE1C maintain the OC stemness phenotype.

(A) PDE1C and PDE4B mRNA levels in OVCAR5 cells transduced with control or PDE1C or PDE4B targeting shRNA. (B-C) NANOG, OCT4, and SOX2 mRNA levels in OVCAR5 cells transduced with control or PDE1C (B) or PDE4B (C) targeting shRNA. (D) Sphere formation derived from OVCAR5 cells transduced with control or PDE1C or PDE4B targeting shRNA. (E) PDE1C mRNA levels in sh-control or sh-FTO transduced OVCAR5 cells, subsequently transduced with control or PDE1C shRNA (2 sequences). (F-I) ALDH1A1 (F), NANOG (G), OCT4 (H) and SOX2 (I) mRNA levels in sh-control or sh-FTO transduced OVCAR5 cells, subsequently transduced with control or PDE1C shRNA. (J) cAMP activity in GFP or FTO transduced OVCAR5 cells. (K) Cyclic AMP activity was detected in rolipram (1μM) and vinpocetine (50μM) treated shCTL or shFTO stably transduced OVCAR5 cells. (L) Cyclic AMP activity was determined in ALDH⁺ cells compared with ALDH⁻ cells FACS-sorted from OVCAR5 and COV362 cells. * p< 0.05; ** p< 0.01; *** p< 0.001; ns, not significant (p > 0.05). (M) Proposed mechanism by which FTO regulates tumor initiation and ovarian cancer stem cells.

Similar observations were made by using small molecule inhibitors. Vinpocetine, an alkaloid derived from the Vinca plant, inhibits PDE1C, increasing levels of both cAMP and cGMP(41), which in turn, can activate protein kinase A (PKA) and alter transcription. Rolipram, inhibits cAMP hydrolysis by blocking PDE4B(42). Inhibition of both phosphodiesterases, especially of PDE1C, blocked spheroid formation (Figure S9A, p<0.05) and expression of stem cells markers, ALDH, OCT4, NANOG, and SOX2 (Figure S9B–C, p<0.05), supporting that FTO⊣PDE signaling plays a role in cancer stemness regulation. To demonstrate that the effects of phosphodiesterase inhibitors on stemness are dependent on FTO, we measured ALDH1A1 and SOX2 mRNA expression levels in response to the PDE1C and PDE4B inhibitors in stably transfected OVCAR5 cells with shRNA control or shRNA targeting FTO. The reduction in ALDH1A1 and SOX2 mRNA expression levels induced by the PDE1C inhibitor vinpocetine was partially rescued in sh-FTO compared to control cells (Figure S9D–E, p<0.001). Similarly, the effects of the PDE4B inhibitor rolipram on ALDH1 and SOX2 mRNA expression levels were partially rescued in sh-FTO compared to control cells (Figure S9F–G, p<0.05).

As the main function of phosphodiesterases is to regulate cAMP and cGMP balance though hydrolysis, we next measured whether modulation of FTO levels alters cAMP levels. Increased cAMP levels were found in FTO-overexpressing cells compared to control cells, consistent with downregulation of phosphodiesterase expression levels in FTO-overexpressing OC cells (Figure 7J, p <0.05). Furthermore, cAMP levels augmented by treatment with PDE1C and PDE4B inhibitors, were diminished in OC cells in which FTO was knocked down by shRNA (Figure 7K), further supporting the role of this demethylase regulating the cAMP balance. Lastly, we measured cAMP in CSCs vs. non-CSCs sorted from OC cell lines. Significantly decreased cAMP levels were found in ALDH⁺/CD133⁺ cells compared with ALDH⁻/CD133⁻ cells FACS-sorted from OVCAR5 and COV362 cell lines (Figure 7L, p<0.01), suggesting that cAMP signaling is significantly inhibited in CSCs. Collectively, these data support that m⁶A modified PDE1C and PDE4B regulate cancer stemness by modulating the intracellular cAMP balance, which is decreased in ovarian CSCs vs. non-CSCs, as proposed in the model shown in Figure 7M.

DISCUSSION

Our results define a previously unappreciated tumor-suppressor role of FTO in HGSOC, which is opposite to the tumor-promoting function of FTO previously reported in other types of cancers. Through both gain- and loss-of-function studies, we demonstrated that FTO is a tumor suppressor and inhibits the stemness features of ovarian CSCs. Through transcriptome-wide RNA-seq and m⁶A mapping, we identified two phosphodiesterase genes (PDE4B and PDE1C) as FTO target transcripts that regulate cAMP signaling and play a critical role in maintaining the stemness of ovarian CSCs. Our findings are highly significant, as they define for the first time a tumor-suppressor role for FTO in solid tumors and identify cAMP signaling as a key pathway modulated by m⁶A mRNA modifications in this context.

Alterations of m⁶A levels have been previously linked to abnormal cellular differentiation states in cancer (43). As a key regulatory enzyme for m⁶A modifications, studies of FTO have been initiated in cancer; with initial reports pointing to pro-tumorigenic functions in glioblastoma and leukemia. Overexpression of FTO was detected in brain tumors and an FTO inhibitor suppressed tumor formation and progression in glioblastoma models(44). FTO was also found to be highly expressed in AML, where it promoted leukemic cell growth and transformation by regulating m⁶A deposition at the 3’ UTR of Ankyrin Repeat and SOCS box protein 2 (ASB2) and the retinoic acid receptor α (RARA) involved in cell differentiation(22). R2-hydroxyglutarate (R-2HG), an oncometabolite which accumulates in leukemias or gliomas associated with mutant isocitrate dehydrogenases, exhibited anti-tumor activity by suppressing FTO and increasing global levels of m⁶A(23). The R-2HG-FTO axis contributed to enrichment in m⁶A peaks in the 5’UTR and CDS regions of MYC and CEBPA transcripts, suppressing their expression and function, and exerting anti-leukemogenic effects(23). In contrast, our results show that overexpression of FTO associated with decreased m⁶A levels suppressed tumorigenicity and sphere forming capacity of ovarian CSCs, while FTO depletion increased tumorigenicity and stemness characteristics, providing compelling evidence for a tumor suppressor function. Importantly, a mutant FTO construct, lacking the demethylase function, did not alter the stemness phenotype, suggesting that the role of FTO in ovarian CSCs relies on its m⁶A demethylase activity.

The context-dependent function of FTO may be accounted for by tissue-specific targets regulating unique circuitries in distinct cancers. We show that FTO had significant effects upon the transcriptome of OC cells with over 700 transcripts being significantly deregulated as a consequence of m⁶A modifications. Pathways related to stem cell signaling, RNA transcription, mRNA splicing, and DNA repair were significantly altered by overexpression of FTO. Among potential targets, two phosphodiesterases, PDE4B and PDE1C, were hypomethylated and downregulated in response to FTO over-expression. The mRNA half-life of these two transcripts was significantly altered by FTO over-expression, but not by the mutant construct, supporting the direct effect of this demethylase on PDE4B and PDE1C. M⁶A methylation is typically recognized by reader proteins, which guide the subsequent processing of mRNAs. The classical pathway involves YT521-B homology domain containing proteins (YTHDFs), which recognize m⁶A modifications and direct mRNAs towards decay, resulting in decreased gene expression(45). Another mechanism involves alterations in the local structure of RNA, resulting in alternative splicing(46). Emerging data indicate that mRNAs with reduced m⁶A alterations could be downregulated due to increased instability(47). The two targets described here PDE1C and PDE4B fall into this category, being downregulated in response to decreased m⁶A mRNA modifications. A recently proposed mechanism accounting for this phenomenon involves IGF2BP1–3, which recognize m⁶A modified targets and stabilize them, leading to increased gene expression(40).

This is the first report linking FTO to regulation of cellular processes mediated by the second messenger cAMP. Interestingly, recent studies have associated cAMP and cGMP signaling with maintenance of CSCs in breast cancer models and inhibitors of PDE were shown to deplete ALDH⁺ populations(48). The main effector of cAMP is protein kinase A (PKA) which phosphorylates several metabolic enzymes and the cAMP-response element-binding protein CREB(49). Here we show that cAMP levels were significantly downregulated in ovarian CSCs vs. non-CSCs and that FTO overexpression induced increased cAMP levels, while its knockdown decreased them. Interestingly, PKA activation induced by cAMP was shown to cause epigenetic reprogramming leading to differentiation of tumor initiating mammary cells(50), further supporting the significance of this pathway to inhibiting stemness. In all, our findings identify the cAMP pathway as a new FTO target in CSCs, associated with tumor inhibiting functions, and provide key new information on the role of m⁶A modifications regulated by FTO in the fine tuning of cellular fate in HGSOC.

SIGNIFICANCE.

A new tumor suppressor function of the RNA demethylase FTO implicates m⁶A RNA modifications in the regulation of cyclic AMP signaling involved in stemness and tumor initiation.