ALKBH5 promotes ovarian cancer progression by activating Notch2 signaling

Abstract

Background

ALKBH5, one of the RNA N6-methyladenosine (m6A) demethyltransferases, has been suggested to be involved in the progression of several cancers. The aim of this study was to investigate clinical significance and biological functions of ALKBH5 in promoting ovarian cancer progression.

Results

We found a significant upregulation of ALKBH5 expression in ovarian cancer tissues compared with normal tissues. Correlation analyses indicated an association between heightened ALKBH5 expression and FIGO stage, as well as lymph node metastasis. Importantly, increased ALKBH5 expression indicated shorter progression-free survival and overall survival. Moreover, we found that hypoxia induced an increase in ALKBH5 expression in ovarian cancer via an HIF-1α-dependent mechanism. Loss-of-function assays demonstrated that ALKBH5 knockdown inhibited ovarian cancer cell progression both in vitro and in vivo. Furthermore, we found that knockdown of ALKBH5-meidated m6A demethylation decreased Notch2 mRNA stability and expression, resulting in the inhibition of cell proliferation, invasion and metastasis in OC cells.

Conclusion

In summary, our findings demonstrated that ALKBH5 promotes the progression of ovarian cancer by activating Notch2 signaling, and suggested that ALKBH5 functions as an oncogene and may serve as a prognostic biomarker and therapeutic target in ovarian cancer.

Introduction

Ovarian cancer (OC) remains one of the most lethal gynecological malignancies, ranking as the fifth leading cause of cancer-related mortality among female patients. Owing to a lack of early warning symptoms and effective screening methods, approximately 75% of cases are typically diagnosed at an advanced stage [1]. At present, chemotherapy stands as the primary treatment modality for OC [2, 3]. Although most patients respond to initial treatment, the prognosis remains poor. Early diagnosis and risk stratification will improve prognosis by allowing earlier disease intervention [4–6] Thus, there is an imperative need to explore the molecular mechanisms underlying OC progression to identify novel prognostic biomarkers and therapeutic targets for this disease.

N6-methyladenosine (m6A) is the most prevalent internal, reversible, and dynamic RNA modification, wielding regulatory control over stability, turnover, localization, and its translation of target mRNA [7, 8]. m6A modification is catalyzed by a methyltransferase complex comprising METTL3/14 and WTAP, while being erased by demethylases such as ALKBH5 and FTO. Furthermore, this modification is recognized by m6A reader proteins such as YTHDF1/2/3 and YTHDC1/2 [9, 10]. Recent studies have underscored the pivotal role of m6A modifications in tumorigenesis, tumor growth, metastasis, and drug resistance [11–13]. ALKBH5 is one of nine mammalian members of the AlkB family of Fe(II)⁻ and α-ketoglutarate-dependent dioxygenases and can demethylate the m6A modification in RNA to adenosine [14]. Functioning as a key demethylase in m6A modification, ALKBH5 has been implicated in the regulation of various biological and pathophysiological processes, including meiosis, gametogenesis, autophagy, and cancer progression [15, 16]. Recent studies also suggest that ALKBH5 exerts a significant influence on OC and may represent a potential therapeutic target [17–22].

In this study, we aimed to investigate ALKBH5 expression in OC and its association with clinical factors and outcomes. We found that the knockdown of ALKBH5 could inhibit JAG2/Notch2 signaling, resulting in the inhibition of cell proliferation, migration and invasion in OC cells. These findings indicated that ALKBH5 might serve as a promising prognostic biomarker and therapeutic target for OC.

Methods

Cell culture and reagents

The normal ovarian epithelial cell line IOSE-80 was purchased from the Bena Culture Collection (Kunshan, Jiangsu Province, China). The human ovarian cancer cell lines A2780, ES2, SKOV3, and OVCAR3 were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Rockville, MD, USA) and 1% penicillin/streptomycin (Beyotime, Shanghai, China) in a humidified incubator of 5% CO₂ at 37 °C. Cell lines were reauthenticated by Short Tandem Repeat (STR) analysis every 6 months after resuscitation in our laboratory. LW6 was purchased from Merck Millipore (Eschborn, Germany) and was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 25 mM.

Plasmid and lentiviral transfection

Two shRNAs targeting ALKBH5 (shALKBH5-1: 5’-AAAGGCTGTTGGCATCAATA-3’ and shALKBH5-2: 5’-CCACCCAGCTATGCTTCAGAT-3’), shRNA targeting YTHDF1 (shYTHDF1: 5’-GCACTGACTGGTGTCCTTT-3’), shRNA targeting YTHDF2 (shYTHDF2: 5’-CAAGGAAACAAAGTGCAAA-3’), shRNA targeting YTHDF3 (shYTHDF2: 5’-TAAGTCAAAGAAGACGTATTA-3’), and a negative control were purchased from Shanghai OBio Technology. Expression plasmid for pCMV6-Notch2 and pCMV6-XL5 empty plasmid was purchased from Origene (Rockville, MD). Cells were infected with lentiviruses for 48 h. Infected cells were selected with 2 µg/mL puromycin (Beyotime).

Cell Counting Kit-8 (CCK-8) assays

2 × 10⁴ cells were seeded in 100 µL of complete culture media in 96-well plates for various time periods, followed by addition of 10 µL of CCK-8 buffer (Beyotime) to each well and incubation at 37 °C for another 1 h. Then the absorbance at 450 nm was measured using a microplate reader (BioTek, Vermont, USA).

Colony formation

Cells were seeded in 6-well plates with culture medium with 10% FBS (1000 cells per well). After incubating the plates for 14–18 days, culture medium was taken off and the colonies were washed with phosphate buffered saline (PBS) (Beyotime) twice. The next step is the colonies were staining with 0.2% crystal violet for 30 min at room temperature. After washing 2 times with PBS, cell numbers were counted under the microscope.

Wound-healing assay

Cells were seeded into six-well plates at a density of 5 × 10⁵ cells per well and allowed to attain a confluence of 90%–95%. Subsequently, a linear scratch was created using a sterile pipette. Following this, the cells were washed with PBS and cultured in a serum-free medium. The wounded region was observed with a microscope, and images were taken at 0, and 24 h.

Transwell assay

Cells were resuspended at 4 × 10⁴ cells/well in serum–free medium. A total of 200 µL of the cell suspension was seeded in the Matrigel–coated chamber (354480, Corning), while a total of 750 µL of complete medium was added into the lower chamber. The cells were incubated for 24 h at 37 °C. Subsequently, the cells on the upper surface were removed, and those on the lower surface were fixed with methanol and then with 0.1% crystal violet. Invaded cells were imaged and counted.

Real-time quantitative RT-PCR

Total RNA was extracted from cells using the E.Z.N.A.^® HP Total RNA Kit (Omega Bio-tek, Doraville, GA, USA). Reverse transcription and real-time PCR were performed utilizing the PrimeScript^® RT Reagent Kit (TakaRa, Shiga, Japan) in combination with the SYBR Green PCR Master Mix (Applied Biosystems, Vilnius, Lithuania). The transcript levels were subsequently analyzed using the 2^−ΔΔCt method. The primer sequences were used as the following: GAPDH, forward 5ʹ-GTCTCCTCTGACTTCAACAGCG-3ʹ and reverse 5ʹ-ACCACCCTGTTGCTGTAGCCAA-3ʹ; ALKBH5, forward 5ʹ-CCCGAGGGCTTCGTCAACA-3ʹ and reverse 5ʹ-CGACACCCGAATAGGCTTGA-3ʹ; Notch1, forward 5ʹ-CGCTGACGGAGTACAAGTG-3ʹ and reverse 5ʹ-GTAGGAGCCGACCTCGTTG-3ʹ; Notch2, forward 5ʹ-CAACCGCAATGGAGGCTATG-3ʹ and reverse 5ʹ-GCGAAGGCACAATCATCAATGTT-3ʹ Notch3, forward 5ʹ-TGGCGACCTCACTTACGACT-3ʹ and reverse 5ʹ-CACTGGCAGTTATAGGTGTTGAC-3ʹ; Notch4, forward 5ʹ-TGTGAACGTGATGTCAACGAG-3ʹ and reverse 5ʹ-ACAGTCTGGGCCTATGAAACC-3ʹ; JAG1, forward 5ʹ-TGCTACAACCGTGCCAGTGACT-3ʹ and reverse 5ʹ-TCAGGTGTGTCGTTGGAAGCCA-3ʹ; JAG2, forward 5ʹ-TGGGACTGGGACAACGATAC-3ʹ and reverse 5ʹ-AGTGGCGCTGTAGTAGTTCTC-3ʹ.

Western blotting

Total protein was extracted from cells using RIPA lysis buffer (Thermo Scientific) in the presence of Halt Protease and Phosphatase Inhibitor Cocktail (Pierce Chemical, Dallas, Texas, USA). The protein concentration of lysates was measured using a BCA Protein Assay Kit (Thermo Scientific). Equivalent amounts of protein were separated by 8–12% SDS-PAGE, the proteins were then transferred to PVDF membranes. Blocked with 5% skim milk, the membranes were incubated with primary antibodies overnight at 4 °C and then the secondary antibodies for 1 h at room temperature. Bound antibodies were visualized by ECL reagents (Thermo Fisher).The primary antibodies included anti-β-Actin antibody (#3700, Cell Signaling Technology, Mass, USA), anti-GAPDH antibody (#2118, Cell Signaling Technology), anti-ALKBH5 antibody (#ab75359, Abcam), anti-HIF1α antibody (#36169, Cell Signaling Technology), anti-Notch2 antibody (#5732, Cell Signaling Technology), anti-JAG2 antibody (#3230, Cell Signaling Technology), anti-CCND1 antibody (#2978, Cell Signaling Technology), anti-c-myc antibody (#9402, Cell Signaling Technology), anti-Hes1 antibody (#11988, Cell Signaling Technology), anti-YTHDF2 antibody (#ab246514, Abcam).

RNA stability assay

Cells were seeded in 6-well plate. After 24 h, cells were treated with 1 mg/mL Actinomycin D (Sigma, #A9415) at indicated time points. Extracting RNA from these cell pellets were used for further qRT-PCR experiments.

RNA Immunoprecipitation (RIP)

The RIP assay was conducted by using RNA-binding protein kit (Millipore, MA, USA) following the manufacturer’s instructions. The Protein A/G magnetic beads were incubated with 10 µL of YTHDF2 antibody (#ab246514, Abcam, Mass, USA) for a minimum of 4 h at 4 °C. Subsequently, the cells were lysed using RIP buffer and subjected to overnight incubation with washed magnetic beads at 4 °C. RNA extraction was performed using Trizol reagent (Invitrogen, CA, USA), followed by determination of target gene expression through qRT-PCR.

m⁶A RNA Immunoprecipitation (RIP)-qPCR

Total RNA was extracted from cells under indicated treatment, and then treated with DNase (Sigma) to remove genomic DNA. After mRNA purification and fragmentation, the fragments were incubated with m⁶A primary antibody for immunoprecipitation using a Magna MeRIP™ m⁶A kit (#17–10,499, Merck Millipore, MA, USA). Enriched m⁶A modified mRNA was then detected by qRT-PCR.

Chromatin Immunoprecipitation (ChIP)

ChIP was performed by using EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s protocols. Briefly, cells were fixed with 1% formaldehyde and quenched in 125 mM glycine. Protein-DNA complexes were immunoprecipitated using anti-Hif1α antibody (#36169, Cell Signaling Technology) or control IgG. The ChIP DNA was isolated and amplified by PCR. The specific primers used for the amplification of Hif1α binding site in ALKBH5 promoter are: #1, 5’-GCCTTTGCTGACTGCTTCAA-3’ (forward) and 5’-CGCAAGGCTAAGGCTAAGCTA-3’ (reverse), #2, 5’-CACCAAGGAGGCTCGACG-3’ (forward) and 5’-GCGGACGTTTCACGAGTCAC-3’ (reverse), #3, 5’-TATGAGCGCACCCCTGTAGA-3’ (forward) and 5’-CTGAGGGGCTTCAAGGGAAC-3’ (reverse).

Animal experiments

For ovarian cancer xenograft models, ALKBH5-knockdown A2780 cells or control cells (5 × 10⁶) were injected subcutaneously into 6-week-old female BALB/c nude mice (Guangdong medical laboratory animal center). Tumor volume was measured and calculated using the formula: V = 1/2 × larger diameter × (smaller diameter)². At the end of the studies, the mice were sacrificed, and tumors were dissected for further IHC and immunofluorescence analysis. For ovarian cancer lung metastasis models, ALKBH5-knockdown A2780 cells or control cells (1 × 10⁶) were injected into the tail vein of 6-week-old female BALB/c nude mice (n = 6). At the end of the studies, mice were sacrificed. The lungs were fixed in 4% paraformaldehyde, paraffin–embedded and sliced. Lung sections were stained by hematoxylin and eosin (H&E). The number of metastatic colonies in the lungs was counted. All animal works were performed in accordance with protocols approved by the Animal Experimentation Ethics Committee of Guangzhou Medical University (G2023-260).

Tissue samples and patient data

The ethics approval for present study was obtained from the Ethics Committee of the Guangzhou Medical University (GYZL-2023-ST109). We collected 84 formalin-fixed paraffin-embedded ovarian tissues, including 8 normal ovarian tissues, 74 ovarian malignant tumor tissues from patients treated initially at the Affiliated Cancer Hospital of Guangzhou Medical University between 2018 and 2023. Relevant clinical parameter data were collected from the hospital medical record system and the definite histological diagnosis and grading came from the pathological reports. The clinical-stage was determined based on the International Federation of Gynecology and Obstetrics, 2009 (FIGO, 2009).

Immunohistochemistry (IHC) staining

Paraffin–embedded sections of clinical ovarian tissues, tumor tissues from mouse xenograft models were subjected to immunohistochemistry. Briefly, the sections were deparaffinized in xylene, rehydrated with graded alcohol, and microwaved in 10 mM sodium citrate (pH 6.0) for 20 min. Hydrogen peroxide (0.3%) was applied to block endogenous peroxide activity. After incubation with 10% normal goat serum, the sections were incubated with primary antibodies: anti-ALKBH5 (#ab75359, Abcam), anti-Ki67 (#9449, Cell Signaling Technology), anti-E-cadherin (#3195, Cell Signaling Technology), and anti-vimentin (#5741, Cell Signaling Technology), followed by incubation with horseradish peroxidase (HRP)-conjugated antibody. The sections were visualized by a DAB visualization kit (Maixin Bio, China), and then counterstained with hematoxylin. IHC staining was evaluated by two independent pathologists. IHC score was calculated based on both the extent and the intensity of staining. The staining extent was scored as 0, 0–5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100% according to the percentage of positively stained cells. The percentage and intensity scores were multiplied to obtain a total score (range, 0–12), and the tumors were finally determined as negative (−), score 0; lower expression (+), score ≤ 4; moderate expression (++), score 5–8; and high expression (+++), score ≥ 9. An optimal cutoff value was identified: specimens with the final scores ≥ 6 were defined as high expression and specimens with the final scores < 6 were defined as low expression. The cutoff value for ALKBH5 was chosen based on a measure of heterogeneity using the log-rank test statistical analysis with respect to overall survival. All patients were followed up yearly, with the last follow-up being conducted in September 2023. Of the 74 patients, none of patients were lost to follow-up and seventeen patients were alive at the final follow-up in September 2023.

Statistical analysis

Statistical analyses were conducted using GraphPad Prism 8.0 and SPSS 19.0 software. Group comparisons were analyzed using the t-test and the χ² test. Results are presented as the mean ± S.D. of multiple independent experiments. All the experiments were performed at least three times. Percent survival curves were obtained by the Kaplan-Meier method, and differences were compared by log-rank test. A threshold of P < 0.05 was considered statistically significant.

Results

Increased ALKBH5 expression predicts a poor prognosis in patients with OC

To investigate the association between ALKBH5 expression and OC, we examined ALKBH5 expression in 74 independent primary OC tissues and 8 normal ovarian tissues through immunohistochemistry. The results revealed a significant upregulation of ALKBH5 in OC tissues (Fig. 1A, B). Moreover, we investigated potential correlations between ALKBH5 expression and clinicopathological factors. We found significant correlations between ALKBH5 expression and FIGO stage, tumor differentiation, and lymph node metastasis (p < 0.05), while no significant correlations were found with age, pathological classification, tumor size, or CA125 levels (Table 1). Furthermore, we assessed the correlation between ALKBH5 expression and overall survival (OS) in the patients with OC. We found that patients with high ALKBH5 expression exhibited poorer OS compared to those with low ALKBH5 expression (Fig. 1C). In addition, TCGA database analysis also showed that patients with OC exhibiting elevated ALKBH5 expression had shorter progression-free survival (PFS) and OS compared to those with low ALKBH5 expression (Fig. 1D, E). Taken together, these results suggested that the upregulation of ALKBH5 might serve as an indicator of an unfavorable prognosis in patients with OC.

Fig. 1.

Increased ALKBH5 expression predicts a poor prognosis in patients with OC. A Representative images of ALKBH5 IHC staining in OC specimens. OC tissue sections were quantitatively scored, considering both the percentage of positive-stained cells and staining intensity. The resultant percentage and intensity scores were multiplied to obtain a total score (range: 0–12). Tumors were categorized as negative (−) with a score of 0, exhibiting lower expression (+) with a score of ≤ 4, demonstrating moderate expression (++) with a score of 5–8, and presenting high expression (+++) with scores of ≥ 9. B IHC staining scores for ALKBH5 in adjacent normal tissues and OC tissues. C Survival curves of patients with OC exhibiting low ALKBH5 expression compared to those exhibiting high ALKBH5 expression. D Kaplan–Meier OS curves (http://kmplot.com/analysis/) of patients with OC relative to varying expression levels of ALKBH5. E Kaplan–Meier PFS curves (http://kmplot.com/analysis/) of patients with OC relative to varying expression levels of ALKBH5

Table 1.

Correlation between the clinicopathological parameters and expression of ALKBH5

Parameters	Total	ALKBH5
		Low (n = 38)	High (n = 38)	P-value
Age (Years)
≤ 50	24	10	14	0.3236
> 50	52	28	24
Differentiation grade
G1/G2	32	20	12	0.0631
G3	44	18	26
FIGO stage
I/II	40	27	13	0.0013
III/IV	36	11	26
Lymph node metastasis
No	49	31	18	0.0059
Yes	27	9	20
CA125 (U/mL)
≤ 35	12	7	5	0.5292
> 35	64	31	33

ALKBH5 transcription is induced by HIF-1α in OC Cells

The expression of ALKBH5 was assessed in a panel of OC cell lines (A2780, ES-2, SKOV3, and OVCAR3) and normal ovarian epithelial cells (IOSE-80) via qRT-PCR and western blotting. We found a substantial increase in ALKBH5 expression in OC cells compared to normal ovarian epithelial cells (Fig. 2A, B). Furthermore, we investigated ALKBH5 expression in OC cells subjected to hypoxia. Two OC cell lines (SKOV3 and A2780) were exposed to 20% or 1% O₂, and subsequently, RNA and protein were extracted for qRT-PCR and western blotting, respectively. We found a significant increase in mRNA and protein levels of ALKBH5 in OC cells under hypoxic conditions (Fig. 2C, D). However, treatment with the selective HIF-1α inhibitor LW6 led to decreased ALKBH5 expression in OC cells subjected to hypoxia (Fig. 2E, F). ChIP-qPCR analysis further showed that HIF1α antibody enrichment with ALKBH5 ChIP#2 primers amplified ALKBH5 promoter DNA (motif: cacgtga), with the hypoxia-treated group yielding significantly more product than the negative control (NC) group (Fig. 2G-H), indicating the HIF1α binding the locus within this region. Moreover, analysis of the GEPIA database revealed that ALKBH5 was mostly positively correlated with HIF-1α in OC (Fig. 2I). Taken together, these results suggested that HIF-1α upregulated ALKBH5 expression in OC cells.

Fig. 2.

ALKBH5 transcription is induced by HIF-1α in OC cells. A, B The expression of ALKBH5 in a panel of OC lines and normal ovarian IOSE-80 cells was assessed by qRT-PCR (A) and western blotting (B). C-D A2780 or SKOV3 cells were cultured under normoxia (20% O₂) or hypoxia (1% O₂) conditions, and the expression of ALKBH5 was assessed by qRT-PCR (C) and western blotting (D). E-F A2780 or SKOV3 cells following treatment with 20 µM LW6 or vehicle were cultured under normoxia (20% O₂) or hypoxia (1% O₂) conditions for 12 h, and the expression of ALKBH5 was assessed by qRT-PCR (E) and by western blotting (F). G Putative Hif1α binding motifs in ALKBH5 promoter. H Enrichment of Hif1α in the ALKBH5 promoter was determined by the ChIP-qPCR assay. I Correlation between the mRNA expression of HIF-1α and ALKBH5 in the GEPIA database. (* P < 0.05, ** P < 0.01)

Knockdown of ALKBH5 inhibits OC cell proliferation, migration and invasion

Since ALKBH5 was found to be upregulated in OC tissues and cells, we subsequently investigated its biological role in OC progression. Two ALKBH5-targeted shRNAs were employed to suppress ALKBH5 expression in SKOV3 and A2780 cells (Fig. 3A, B). Colony formation assay showed a significant reduction in colony formation upon ALKBH5 knockdown (Fig. 3C, D). Moreover, CCK-8 assays revealed a significant decrease in the proliferation of OC cells following ALKBH5 knockdown (Fig. 3E).

Fig. 3.

Knockdown of ALKBH5 inhibits OC cell proliferation, migration and invasion. A-B A2780 or SKOV3 cells were stably transfected with ALKBH5 shRNA or control shRNA, and the expression of ALKBH5 was evaluated by qRT-PCR (A) and western blotting (B). C-D A2780 or SKOV3 cells were stably transfected with ALKBH5 shRNA or control shRNA, and cell proliferation was evaluated by colony formation assay. E A2780 or SKOV3 cells were stably transfected with ALKBH5 shRNA or control shRNA, and cell proliferation was evaluated by CCK-8 assay. F A2780 or SKOV3 cells were stably transfected with ALKBH5 shRNA or control shRNA, and cell migration ability was assessed by wound-healing assay. (G-H) A2780 or SKOV3 cells were stably transfected with ALKBH5 shRNA or control shRNA, and cell invasion ability was assessed by Transwell assay. (* P < 0.05, ** P < 0.01)

We further investigated whether ALKBH5 knockdown impacts the metastatic capacity of OC cells. Wound healing assays showed a significant reduction in cell migration in SKOV3 and A2780 cells following ALKBH5 knockdown compared to the controls (Fig. 3F). Moreover, transwell assays demonstrated a significant suppression of OC cell invasion upon ALKBH5 knockdown (Fig. 3G, H). Epithelial-mesenchymal transition (EMT) is related to cancer migration and invasion. Consistently, we found that knockdown of ALKBH5 significantly increased the expression levels of E-cadherin while decreased the expression levels of vimentin in SKOV3 and A2780 cells (Fig. 3B). These results suggested that knockdown of ALKBH5 suppressed OC cell proliferation, migration and invasion in OC cells.

Knockdown of ALKBH5 suppresses tumor growth and metastasis in vivo

We further assessed the effects of ALKBH5 knockdown on tumor growth and metastasis in vivo. ALKBH5-knockdown A2780 cells or control cells were implanted into the right flanks of nude mice by subcutaneous injection. The mean volumes of xenograft tumors generated from ALKBH5-knockdown A2780 cells were significantly smaller than those originating from control cells (n = 5 animals per group, P < 0.05) (Fig. 4A). The weight of excised tumors from ALKBH5-knockdown xenograft was significantly lower than control group (Fig. 4B-C). Immunohistochemical staining for Ki67 showed decreased expression in ALKBH5-knockdown tumors compared with control tumors (Fig. 4D). Moreover, tumors formed by ALKBH5-knockdown H2780 cells showed obviously increased in the expression of E-cadherin but decreased in the expression of vimentin (Fig. 4D). To further investigate the effect of ALKBH5 knockdown on tumor metastasis, we implanted ALKBH5-knockdown A2780 cells or control cells into nude mice through the lateral tail vein. We found that lung metastasis was apparent in mice injected with control cells. In contrast, few lung metastatic were detected in mice injected with ALKBH5-knockdown A2780 cells (Fig. 4E-F). Taken together, these results indicated that ALKBH5 has the ability to promote tumor growth and metastasis in vivo.

Fig. 4.

Knockdown of ALKBH5 inhibits OC growth and metastasis in vivo (A-C) A total of 1 × 10⁶ A2780/ALKBH5 shRNA or A2780/Control shRNA cells were inoculated subcutaneously into the female nude mice (N = 5 per group), (A) The tumor size was measured at indicated time intervals and calculated. The tumor volume was calculated using the formula: V = 1/2 × larger diameter × (smaller diameter)², and growth curves were plotted using average tumor volume within each experimental group at the set time points, (B) At the end of treatment, tumors were excised and imaged, (C) Tumor weights were measured. D Tumor tissues were fixed, sectioned, and placed on slides. Tumor specimens were subjected to IHC staining for ALKBH5, Ki67, E-cadherin and Vimentin. E-F A total of 2 × 10⁵ A2780/ALKBH5 shRNA or A2780/Control shRNA cells were tail vein–injected into nude mice, (E) Representative H&E images of lung tissues showing metastatic nodules, (F) Average number of lung metastases nodules in the indicated groups. (* P < 0.05, ** P < 0.01)

ALKBH5-mediated m6A modification increased Notch2 expression in OC cells

Notch signaling has been implicated in cancer initiation and progression across various cancer types, including OC [23, 24]. We further investigated the potential mechanisms through which ALKBH5 regulates Notch receptors (Notch 1–4) and Notch ligands (Jagged 1–2) in OC cells. We found a significant reduction in the mRNA expression of Notch2 in ALKBH5-knockdown SKOV3 and A2780 cells (Fig. 5A). In agreement with the mRNA expression of Notch2, specific knockdown of ALKBH5 significantly decreased Notch2 protein levels in SKOV3 and A2780 cells (Fig. 5B).To verify Notch2 as a potential target of ALKBH5 for m6A modification, we employed the SRAMP database (http://www.cuilab.cn/sramp) analysis and identified four GGACU motifs in the 3’-UTR of Notch2 mRNA (Fig. 5C). MeRIP-qPCR was then applied to confirm the ALKBH5-mediated m6A demethylation of Notch2 mRNA. The results showed that the m6A-specific antibody, but not the IgG control, significantly enriched Notch2 mRNA in ALKBH5-depletion SKOV3 and A2780 cells (Fig. 5D). To study the role of ALKBH5-induced m6A demethylation on the 3’-UTR of Notch2, we generated luciferase reporters containing either wild-type (WT) Notch2 3’-UTR or mutant 3’-UTR (GGACU to GGCCU). The luciferase assays showed that specific knockdown of ALKBH5 significantly inhibited Notch2 WT-3’-UTR reporter activity, but had little effect on the luciferase activity of Notch2 mutant-3’-UTR reporter (Fig. 5E), indicating that m6A methylation in the 3’-UTR was critically involved in ALKBH5-induced Notch2 expression. Recent studies have suggested the critical role of ALKBH5-mediated m6A modification in regulating mRNA stability. Indeed, RNA stability assays showed that specific knockdown of ALKBH5 expression significantly reduced Notch2 mRNA stability in SKOV3 and A2780 cells (Fig. 5F). Moreover, we found a positive correlation between ALKBH5 and Notch2 as well as JAG2 expression in OC (Fig. 5G). It has been reported that m6A readers YTHDF1/2/3 are responsible for m6A-mediated mRNA destabilization [25]. We found that knockdown YTHDF2, but not YTDHF1/3, significantly increased the mRNA expression of Notch2 (Fig. 5H). Specific knockdown of YTHDF2 also enhanced the stability of Notch2 mRNA in A2780 and SKOV3 cells (Fig. 5I). Moreover, we performed RNA immunoprecipitation (RIP)-qPCR assays and discovered that YTHDF2 strongly bound with Notch2 mRNA (Fig. 5J). We further conducted rescue assays to determine the importance of YTHDF2 in ALKBH5-induced Notch2 expression. Our studies showed that the inhibitory effects of ALKBH5 knockdown on Notch2 mRNA and protein expression were almost completely reversed by specific knockdown of YTHDF2 in both A2780 and SKOV3 cells (Fig. 5K-L). These data demonstrated that ALKBH5-mediated m6A demethylation maintained Notch2 mRNA stability and expression by abolishing YTHDF2-dependent mRNA degradation. Furthermore, we found that knockdown of ALKBH5 significantly decreased the expression of CCND1, Hes1 and MYC, which are downstream targets of the Notch pathway (Fig. 5M). Collectively, these results demonstrated the critical role of ALKBH5-mediated m6A modification in regulating Notch2 signaling in OC cells.

Fig. 5.

ALKBH5-mediated m6A modification increased Notch2 expression in OC cells. A A2780 and SKOV3 cells were stably transfected with ALKBH5 shRNA or control shRNA, and the mRNA levels of Notch1, Notch2, Notch3, Notch4, JAG1, and JAG2 were determined by qRT-PCR. B A2780 and SKOV3 cells were transfected with ALKBH5 shRNA or control shRNA, and the expression levels of Notch2 was measured by western blotting. C Schematic representation of positions of m6A motifs within GLUT4 mRNA. D A2780 and SKOV3 cells were stably transfected with ALKBH5 shRNA or control shRNA, the m6A modifications of Notch2 mRNA was measured by MeRIP-qPCR. E A2780 and SKOV3 cells were co-transfected with ALKBH5 shRNA or control shRNA and wild-type (WT) or mutant (Mt) pmirGLO-Notch2-3’-UTR reporter for 24 h, the relative luciferase activity was measured. F A2780 and SKOV3 cells stably transfected with ALKBH5 shRNA or control shRNA were treated with actinomycin D (2 µg/mL) at the indicated time points, and the expression of Notch2 mRNA was measured by qRT-PCR. G Correlation between the mRNA expression of ALKBH5 and Notch2 in OC, based on the GEPIA database. H A2780 cells were stably transfected with YTHDF1 shRNA, TYHDF2 shRNA, TYHDF3 shRNA or control shRNA, the expression of Notch2 mRNA was measured by qRT-PCR. I A2780 and SKOV3 cells stably transfected with YTHDF2 shRNA or control shRNA were treated with actinomycin D (2 µg/mL) at the indicated time points, and the expression of Notch2 mRNA was measured by qRT-PCR. J RIP-qPCR analysis showing the enrichment of Notch2 mRNA in A2780 cells using anti-IgG and anti-YTHDF2 antibody. K-L A2780 and SKOV3 cells were co-transfected with ALKBH5 shRNA and YTHDF2 shRNA, the expression of Notch2 mRNA was measured by qRT-PCR (K), the expression levels of Notch2, ALKBH5, and YTHDF2 were measured by western blotting (L). M A2780 and SKOV3 cells were transfected with ALKBH5 shRNA or control shRNA, the expression levels of CCND1, Hes1, and c-myc were measured by western blotting. (*P < 0.05, **P < 0.01)

ALKBH5 promotes OC cell proliferation, migration and invasion through activation of Notch2 signaling

To determine whether the role of Notch2 in ALKBH5-induces cell proliferation, migration and invasion, ALKBH5-knockdown A2780 cells were subjected to ectopic overexpression of Notch2. Western blot analysis confirmed that the levels of Notch2, which were decreased by ALKBH5 knockdown, were restored by co-transfection with Notch2 overexpression vector (Fig. 6A). Accordingly, overexpression of Notch2 increased the expression of CCND1, Hes1 and MYC in ALKBH5-knockdown A2780 cells (Fig. 6A). Functional experiments indicated that restoration of Notch2 promoted the proliferation and colony formation ability in ALKBH5-knockdown A2780 cells (Fig. 6B-D). Furthermore, overexpression of Notch2 also rescued the invasion of A2780 cells transfected with ALKBH5 shRNA (Fig. 6E-F). More, in vivo assay results showed that knockdown of ALKBH5 resulted in a significant decrease in tumor volumes and weight of A2780 xenograft, whereas overexpression of Notch2 reversed the effect of ALKBH5 shRNA and increased xenograft growth (Fig. 6G-I). In addition, overexpression of Notch2 showed an increased in Vimentin expression, but reduction in E-cadherin expression in ALKBH5-knockdown A2780 xenograft (Fig. 6J). Taken together, these results suggested that ALKBH5 promotes OC progression at least in part through activation of Notch2 signaling.

Fig. 6.

ALKBH5 promotes OC progression through activation of Notch2 signaling.(A) A2780 cells were co-transfected with ALKBH5 shRNA or control shRNA and pCMV or pCMV-Notch2, the protein levels of Notch2, CCND1, Hes1, and c-myc were measured by western blotting. B A2780 cells were co-transfected with ALKBH5 shRNA or control shRNA and pCMV or pCMV-Notch2, cell proliferation was evaluated by CCK8 assay. C-D A2780 cells were co-transfected with ALKBH5 shRNA or control shRNA and pCMV or pCMV-Notch2, cell proliferation was evaluated by colony formation assay. E-F A2780 cells were co-transfected with ALKBH5 shRNA or control shRNA and pCMV or pCMV-Notch2, cell invasion ability was assessed by Transwell assay. G-I A2780 cells transfected with ALKBH5 shRNA alone or ALKBH5 shRNA combined with pCMV-Notch2 were inoculated subcutaneously into the female nude mice (N = 6 per group), the tumor volume was measured on the indicated days (G), at the experimental endpoint, the tumors were dissected and imaged as indicated (H), Tumor weights were measured (I). J Tumor specimens were subjected to IHC staining for E-cadherin and Vimentin. (*P < 0.05, **P < 0.01)

Discussion

m⁶A is one of the most prevalent RNA modifications in eukaryotes [26]. Accumulating evidence indicates that dynamically balanced m6A modifications, which are regulated by m6A methyltransferases and demethylases, are closely associated with the development and progression of various malignancies [10]. The significance of ALKBH5, functioning as an m6A demethylase, in the regulation of cancer progression has been explored in previous studies [16]. A few studies have reported that ALKBH5 overexpression inhibits tumor development in pancreatic cancer [27, 28], gastric cancer [29], and esophageal cancer [30]. In contrast, ALKBH5 has been shown to promote cancer progression by mediating the m6A demethylation of mRNA in breast cancer [31, 32], colorectal cancer [33, 34], glioblastoma [35], and lung cancer [36]. These contrasting roles of ALKBH5 in cancer progression suggest that ALKBH5 might have different functions across various cancer types. In OC, ALKBH5 has been demonstrated to activate the NF-kB, FAK, and JAK2/STAT3 signaling pathways, suggesting a pivotal role for ALKBH5 in driving OC progression [17, 18, 37–39]. Consistent with these findings, our study demonstrated elevated ALKBH5 expression in OC tissues, which was correlated with tumor stage and histological grade. Notably, increased ALKBH5 expression was associated with significantly reduced OS and PFS in patients with OC, indicating ALKBH5 as a potential biomarker for OC. Moreover, knockdown of ALKBH5 inhibited OC cell proliferation and invasion. These findings underscore that heightened ALKBH5 levels can enhance the aggressive and proliferative potential of OC cells, thus positioning ALKBH5 as a promising therapeutic target for OC.

The dysregulation of Notch signaling has been reported in various types of human cancers, including colorectal, esophageal, prostate, and hepatocellular carcinoma [24]. In OC, approximately 23% of patients exhibit alterations in Notch signaling, which directly contribute to the development, progression, and occurrence of OC [23, 40]. Overexpression of Notch is associated with cell hyperproliferation, tumor metastasis, and poor survival in patients with OC. Consequently, targeting the Notch signaling pathway is considered an effective strategy for inhibiting OC [41, 42]. The carcinogenic function of Notch in OC may be mediated through JAG/Notch signaling activation [43]. Our findings contribute to a better understanding of the mechanisms underlying Notch1 functions in the progression of pancreatic cancer. In this study, we identified JAG2 and Notch2 as targets of ALKBH5. Consistent with our findings, Ding et al. suggested that the loss of ALKBH5 leads to an increase in the abundance of m6A RNA modification, consequently reducing the expression of several target genes, including the components of the Notch signaling pathway Jagged1 and Notch2 in thymocytes [44].

Hypoxia plays a pivotal role in the development and progression of OC [45]. Here, we observed a notable increase in ALKBH5 expression levels in OC cells subjected to hypoxic conditions. The hypoxia-induced upregulation of HIF-1α and HIF-2α transcriptional activity is critical for the regulation of numerous genes associated with cancer progression. Previous studies have demonstrated that HIF-1α enhances ALKBH5 expression by binding with its promoter, thereby promoting the transcription of ALKBH5 [32, 46]. Indeed, we found that treatment with the selective HIF-1α inhibitor LW6 reduces ALKBH5 expression in OC cells subjected to hypoxia. This outcome suggested that hypoxia indeed upregulates ALKBH5 expression in an HIF-1α-dependent manner.

In summary, our findings suggested that ALKBH5 could serve as a novel indicator of adverse prognosis in patients with OC and represents a promising target for the development of novel treatments.

Authors’ contributions

Y.L., C.P., and R.W. designed the study. H.Liu., Y.L, and Q.W. developed research methodology. H.Luo., Z.G., and Y.L. performed the experiments, collected and analyzed the experimental data. L.L, Q.W., and H.Liu. provided administrative, technical, and material support. H.Luo., C.P., and R.W. performed literature review, wrote and finalized the manuscript.

Funding

This work was supported by the Quanzhou science and Technology Project (2022NS075), the Natural science foundation of Guangdong province (2023A1515010097).

Data availability

The data for this study is available from the corresponding author.

Declarations

Ethics approval and consent to participate

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Guangzhou medical University.

Consent for publication

Not applicable.

Competing Interests

The authors declare no competing interests.