The 6-methyladenine erasers ALKBH5 and FTO influence chemotherapy efficiency in bladder cancer cell lines

Abstract

Background

Bladder cancer (BLCA) is a prevalent and life-threatening condition that significantly impacts patients’ quality of life while imposing substantial financial costs on healthcare systems. Advancing our knowledge of the mechanisms underlying tumor development is crucial for improving treatment outcomes. Emerging studies emphasize the critical role of the RNA modification 6-methyladenine (m⁶A) and its associated proteins, methyltransferase-like 3 (METTL3), Vir-like m⁶A methyltransferase associated (VIRMA) (writers), Alkb homolog 5 (ALKBH5) and fat mass and obesity associated protein (FTO) (erasers), in maintaining m⁶A homeostasis. Dysregulation of these enzymes leads to aberrant m⁶A methylation, a hallmark of various cancers, including BLCA. Furthermore, m⁶A modifications influence cisplatin sensitivity, a key drug in muscle-invasive bladder cancer (MIBC) treatment. With this background, we investigated the combined effects of ALKBH5 and FTO knock-down in bladder tumor cell lines.

Methods

We first investigated the expression of METTL3, VIRMA, ALKBH5 and FTO in BLCA tissues and human bladder tumor cell lines from urinary cancer cells by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Simultaneous knock-down of the expression of the erasers was then performed to explore their consequences in bladder cells. We then conducted cisplatin and mitomycin (MMC) treatment in knock-down cells to decipher the effect of their reduction. The cell viability was evaluated with cell counting kit-8 (CCK-8) assay after the two treatment regimes.

Results

Lower expression of ALKBH5 and FTO was identified in BLCA tissue and bladder tumor cell lines. Notably, this trend was consistent across both low-grade and high-grade tissue samples. Furthermore, lower expression levels of ALKBH5 and FTO were observed in tumor cell lines derived from both men and women compared to the non-tumorigenic SV-HUC1 cell line. In contrast, both tissue and cell line data revealed an increased expression tendency of the m⁶A writers METTL3 and VIRMA. Additionally, knock-down of the two m⁶A erasers was found to enhance tolerance to cisplatin and MMC treatment, resulting in increased resistance to cell death.

Conclusions

Our findings reveal that ALKBH5 and FTO are down-regulated in BLCA and their knock-down confers resistance to cisplatin and MMC in vitro. This suggests that m⁶A erasers play a critical role in modulating chemotherapy sensitivity, potentially serving as biomarkers or therapeutic targets for enhancing treatment efficacy in BLCA.

Highlight box.

Key findings

• Alkb homolog 5 (ALKBH5) and fat mass and obesity associated protein (FTO), the RNA modification 6-methyladenine (m⁶A) erasers, are significantly downregulated in bladder cancer (BLCA), correlating with increased resistance to cisplatin and mitomycin (MMC) chemotherapy. These findings highlight the potential of m⁶A modulation as a therapeutic strategy to enhance treatment efficacy in BLCA.

What is known and what is new?

• BLCA is a common malignancy with poor outcomes, significantly affecting quality of life and healthcare costs. RNA modification m⁶A and its regulatory proteins, including the writers METTL3 and VIRMA and erasers ALKBH5 and FTO, are crucial for maintaining m⁶A homeostasis. Dysregulation of m⁶A has been implicated in cancer progression and chemoresistance, with m⁶A modifications influencing sensitivity to cisplatin, a key chemotherapeutic agent in BLCA treatment.

• This study demonstrates for the first time that ALKBH5 and FTO are downregulated in both BLCA tissues and cell lines. Furthermore, knock-down of these m⁶A erasers increases resistance to cisplatin and MMC, suggesting a novel role for m⁶A erasers in modulating chemotherapy sensitivity.

What is the implication, and what should change now?

• These findings provide new insights into BLCA biology and identify potential therapeutic targets for overcoming drug resistance.

Introduction

Bladder cancer (BLCA) is one of the most frequently occurring malignancy affecting the urinary tract and one of the most common types of cancer in Norway (1-3). The combination of high relapse frequency and treatment resistance makes BLCA one of the costliest cancers to treat, with a severe impact on life quality. The recurrence rate for non-muscle invasive bladder cancer (NMIBC) can be significantly high. Depending on risk factors, 31% to 78% of individuals may experience a recurrence or develop secondary BLCA within five years of treatment (4-6). BLCA is divided into NMIBC which represents 75% of BLCA, and muscle-invasive bladder cancer (MIBC). NMIBC refers to cancer cells confined to the urothelial layer of the bladder, while MIBC refers to tumors that invade the deeper muscular layer, including the detrusor muscle. Transurethral resection of bladder tumor (TURBT) is the standard initial procedure for both diagnosis and treatment of BLCA, including NMIBC and MIBC. While TURBT alone is typically used for NMIBC, MIBC patients often receive neoadjuvant chemotherapy before definitive surgery. For those who are not suitable candidates for radical surgery, a combination of radiation and chemotherapy is generally recommended as an alternative treatment approach. Importantly, chemotherapy for BLCA depends on the patient’s health, tumor stage, and several other factors (7-9), Cisplatin is typically used as systemic chemotherapy in MIBC patients, whereas mitomycin (MMC) is commonly administered intravesical for NMIBC. Both agents introduce DNA damage and lead to cell apoptosis in proliferating cells (10,11). Recent comparative analyses of international guidelines on BLCA management, particularly upper tract urothelial carcinoma (UTUC), emphasize the heterogeneity in diagnostic and treatment approaches across different medical associations [American Urological Association (AUA), European Association of Urology (EAU), National Comprehensive Cancer Network (NCCN)]. These variations highlight the need for standardized protocols to improve patient outcomes and treatment efficacy (12). BLCA has relatively few effective treatment modalities and resistance against standard chemotherapies causes significant mortality (13-15). However, immunotherapy has been a transformative breakthrough in BLCA treatment, offering patients an effective option that significantly improves outcomes and quality of life (16). Despite these advances, gaining deeper insights into the molecular mechanisms that regulate BLCA remains crucial for developing more targeted and personalized treatment strategies.

The discovery of the dynamic and reversible nature of some RNA modifications has led to a renewed focus on them and recent data gives optimism for attacking the balance of RNA modifications in cancer treatment. More than 100 internal modifications have been identified for the various RNA species (17,18), with the dynamically regulated N6-methyladenosine (m⁶A) on mRNA being the most studied (19). Epitranscriptomics, or “RNA epigenetics”, is now used as a collective name to describe all modifications of RNA. The enzymes responsible for the dynamics of mRNA modifications are plentiful and some of them are deregulated in cancer and other diseases (20,21). These epitranscriptomic regulators play a central role in regulating mRNA activity and stability and have key roles in cancer initiation or progression (22). However, their roles seem to depend on the cancer stage and type (23,24). The writers (METTL3, VIRMA, METTL14) deposit the m⁶A marks on mRNA whereas erasers (ALKBH5 and FTO) are demethylases, and change m⁶A into a normal adenosine by oxidative demethylation (Figure 1) (18).

Figure 1.

Schematic representation of m⁶A mRNA methylation and its regulators. (A) Illustration depicting the role of writers and erasers in mRNA regulation, where writers add the m⁶A modification, and erasers remove it. (B) m⁶A is a modification of the adenosine base at the nitrogen-6 position. RNA methylation is dynamic and are under the control of regulators which can methylate (writers: METTL3, VIRMA, METTL14) or demethylate (erasers: ALKBH5, FTO). ALKBH5, Alkb homolog 5; FTO, fat mass and obesity associated protein; m⁶A, 6-methyladenine; METTL3, methyltransferase-like 3; METTL14, methyltransferase-like 14; VIRMA, Vir-like m⁶A methyltransferase associated.

Until very recently, little was known about the implication of m⁶A and m⁶A regulators in BLCA development or progression. In 2018, an in-silico study based on TCGA analysis highlighted the significant gap in knowledge regarding urological tumors (25) and suggested a potential regulatory role for epitranscriptomic modifiers. Starting in 2019, several studies have shown that the deregulation of these modifiers contributes to BLCA progression (26-28). Recent studies have highlighted the critical role of m⁶A modifications in the progression of BLCA. The m⁶A RNA methylation landscape differs significantly between early-stage (NMIBC) and advanced-stage (MIBC), influencing RNA stability, alternative splicing, and tumor behavior (29). In early-stage BLCA, m⁶A modifications are relatively balanced, with controlled RNA methylation maintaining a stable gene expression environment. The activity of m6A writers (METTL3, METTL14) and erasers (ALKBH5, FTO) is regulated, preventing excessive oncogenic activation. In contrast, MIBC exhibits extensive m⁶A dysregulation with low ALKBH5 expression linked to a poorer prognosis in BLCA patient (30). While it is now evident that m⁶A erasers play a role in tumorigenesis and function as oncogenes in many cancers (31,32), research on the specific expression and impact of key erasers like FTO and ALKBH5 in BLCA remains limited (33-35). Further investigation is essential to understand their contributions fully and explore potential therapeutic opportunities.

Lately, new evidence displayed key roles of m⁶A erasers in BLCA drug resistance (30,36). Drug resistance is a major challenge encountered in many cancers (37,38). Thus, deepen knowledge in drug resistance will indeed help to understand chemo-resistance. ALKBH5 has been shown to decrease the cisplatin sensitivity in BLCA cell lines (30). Similarly, FTO induces cells resistance in cells treated with cisplatin (36). However, the effect of combined ALKBH5 and FTO deficiencies in BLCA has not yet been studied. Furthermore, knowing that MMC is a frequently used drug in NMIBC (39-41), whereas cisplatin is mainly used for MIBC (10,42); we have investigated the role of the m⁶A erasers following treatment with these drugs. We assessed the double knock-down of ALKBH5 and FTO to examine the combined role of m⁶A erasers in BLCA. We thus aimed to understand (I) how the dual knock-down affects BLCA cells, and (II) whether it acts as a drug resistor following cisplatin and MMC treatment. The dual knock-down approach allows us to explore potential compensatory mechanisms between these two m⁶A erasers, which have not been well characterized in the context of BLCA chemotherapy resistance in contrast to single knock-down (30,36).

The present study shows that the two erasers, ALKBH5 and FTO are downregulated in tumor tissue and BLCA cell lines. This downregulation is linked to the development of resistance to critical chemotherapy agents such as cisplatin and MMC. These findings uncover a previously unrecognized role of ALKBH5 and FTO in mediating drug resistance, offering new insights into the mechanisms of cancer therapy resistance. By highlighting the pivotal function of these erasers, our results pave the way for innovative therapeutic strategies targeting m⁶A regulation to overcome resistance and improve BLCA treatment outcome. We present this article in accordance with the MDAR reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-25-7/rc).

Methods

Biopsy and tissue sample processing

Fourteen urinary biopsies were obtained by cystoscopy from male and female patients diagnosed with BLCA. Donors are from age 50 to 80 with a median age of 69 (Table 1). This includes eleven men and three women with a mean age of 71 and 70 years, respectively (Table 1). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Regional Ethics Committee South-Eastern Norway Regional Health Authority (No. #2017/2170) and informed consent was obtained from all individual participants. During donor biopsies, healthy and tumor tissues were obtained from Bærum and Drammen hospital (Vestre Viken Hospital Trust) between the years 2018-2021. Healthy tissues from the patient were carefully extracted and examined, revealing a histologically normal appearance. We used only tissue from donors without known pathology or recidivist. Immediately after biopsy, tissues were preserved in RNA later stabilization reagent (Ambion, Massachusets, USA; #AM7020) and kept at −20 ℃ until further experimentation.

Table 1. Donor biopsy information, characteristics, and habits information.

Biopsy number	Gender	Age (years)	Tumor status	Stadium biopsy	Grade
1	M	76	Recidive	pT2 + CIS	HG, G3
2	M	76	Primary	pTa	LG, G1
3	M	80	Primary	pTa	HG, G2
4	W	79	Recidive	pTa	LG, G2
5	M	69	Primary	pTa	HG, G2
6	M	74	Primary	pTa	HG, G2
7	M	50	Primary	pT1 + CIS	HG, G3
8	M	72	Recidive	pTa	LG, G1
9	M	66	Primary	pTa	LG, G2
10	M	74	Primary	pT1	HG, G2
11	M	69	Primary	pTa	LG, G1
12	W	80	Primary	pTa	HG, G3
13	M	74	Primary	pTa	LG, G2
14	W	51	Primary	pTa	LG, G1

pT2, clinical term for “muscle invasive bladder cancer”; pTa and pT1, clinical term for “non-muscle invasive bladder cancer”. CIS, carcinoma in situ; HG, high grade; LG, low grade; M, man; W, women.

RNA extraction and cDNA generation

Total RNA extraction was performed using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany; #74136). Tissues are first disrupted and homogenized using the MP FastPRep-24 tissue and Cell homogenizer. Total RNA quantity was assessed using Nanodrop One/OneC (Fisher Scientific, NanoDrop™ One Microvolume UV-Vis Spectrophotometer), whereas quality was evaluated with the 2100 bioanalyzer system (Agilent, Santa Clara, USA) according the manufacturer’s protocol. mRNA extraction was performed using RNeasy Pure mRNA Bead Kit (Qiagen, #180244) with magnetic rack following the manufacturer’s recommendation. We used the Superscript IV Vilo MM with ezDNase (Invitrogen, Gotenborg, Sweden; #11766050), for cDNA synthesis, and 1 µg of cDNA was synthetized in accordance with the manufacturer’s protocol. Due to variability in RNA integrity and yield across samples, not all 14 tumor cases could be used for every experiment. Only samples with sufficient high-quality RNA were included in specific analyses to ensure reliable and reproducible results.

Real-time quantitative polymerase chain reaction (PCR)

Expression levels of genes regulating the m⁶A level in mRNAs was measured using a real-time PCR. The oligonucleotides sequences used are in Table S1. qRT-PCR was assessed using the StepOne Plus Real-Time PCR system (Applied Biosystems, Thermo Fisher Scientific, USA). A total of 2 ng of mRNA was used, and each sample was analyzed in triplicate. Normal tissues were used as calibrator for tissue sample, and the SV-HUC cell line for cell line experiments. GAPDH was used as endogenous control. RT-qPCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, #A25742). The PCR cycle was as follow: 95 ℃ incubation for 2 minutes followed by 40 cycles of 0.05 seconds at 95 ℃, 60 seconds at 58 ℃ for hybridization, and 40 seconds at 72 ℃ for elongation. As described in (43,44), our analysis was based on the cycle threshold (Ct) and the 2^−ΔΔCt method.

Cell culture

Human bladder tumor cell lines are from urinary cancer cells (LGC standards, ATCC). Cell lines were cultured in the appropriate medium (Table S2) complemented with 10% Fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life) at 37 ℃, 5% CO₂.

Drug treatment

Cisplatin (Selleckchem, Houston, USA, S1166) and mitomycin C from Streptomyces caespitosus (Merck Life Sciences, Saint-Louis, USA) were used as a drug treatment. Cells were treated with cisplatin for 48 hours at a concentration between 0–35 µM, whereas mitomycin C was used for 24 hours at a concentration and 0–50 µg/mL.

Transfection for ALKBH5 and FTO knock-down

To knock-down both ALKBH5 and FTO, we used the Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen, #13778150) 10 nM of ALKBH5 siRNA (Dharmacon, Cambridge, United Kingdom; #L-0-004281-02) and FTO siRNA (Ambion #4392420) were transfected in cell lines. Briefly, lipofectamine and siRNa were added to an appropriate volume of OptiMEM (Gibco, Waltham, USA, 31985070) without antibiotic, for 20 min. The mix was added to the cells for 2 hours and then removed to be replaced by a fresh and appropriate media according to the cell line (Table S2). After 48 hours, cells were harvested for subsequent analysis. Un-transfected cells and cells transfected with empty vectors were used as control and non-targeting control. Knock-down efficiency was confirmed by RT-qPCR.

Cell proliferation assay

Cell proliferation assay was assessed by Cell Couting Kit-8 (Abcam, #ab228554). Cells were seeded in 96-wells at a density of 10 000 cells per well. Cells were subsequently transfected with the ALKBH5 and FTO siRNA for 48 hours, and/ or treated with drug. WST-8 solution was then added to the 96 wells for 2 hours and absorbance value at 460 nm was measured using the Victor Nivo Microplate Reader (Perkin Elmer).

RNA sequencing

RNA from BLCA tissue was isolated and extracted from three different patients with RNAzol (Molecular Research Center, #MR-RN190-500) according to the manufacturer’s instructions. RNA quality and quantity was performed with RNA ScreenTape and TapeStation 4150 (Agilent). Samples were then sent to the Norwegian Sequencing Centre where the cDNA library preparation was performed followed by RNA sequencing (Illumina sequencing).

LC-MS/MS analysis of m⁶A ratio

RNA from tissue samples were extracted using the mirVana Isolation kit (Thermo Fisher) followed by gene read (Qiagen) to keep only pure mRNA. mRNA quality and quantity were assessed by Nanodrop and RNA ScreenTape. The Liquid chromatography with Tandem Mass Spectrometry LC-MS/MS was performed as previously reported (45). The ratio of m⁶A/A was calculated based on the calibrator (healthy tissue: NT) compared to the tumor tissue (TT). A total of 5 independent tissues were analyzed.

Statistical analysis

Control group (normal tissue or normal cell line) were used to normalize the data. Each experiment was performed in triplicate except for the RNA sequencing. Data are presented with mean ± standard error of the mean (SEM). The “N” designates the number of tissues or cell lines used in each experiment whereas the “n” indicates the number of repeated experiments or measures. Student t-test was performed and a P value <0.05 was considered statistically significant.

Results

ALKBH5 and FTO are significantly down-regulated in vivo and in vitro in BLCA

First, we investigated whether some of the well-known mRNA m⁶A regulators could be deregulated in BLCA. We analyzed the expression level of m⁶A writers and erasers by a RT-qPCR experiment on BLCA tissues (Figure 2A,2B and Figure S1A,S1B). We compared BLCA tissue to normal bladder tissue taken from the same donor. Both ALKBH5 and FTO are strikingly downregulated in carcinoma tissue (Figure 2A,2B). Regarding erasers, ALKBH5 is down-regulated by a factor of 1.6 in TT compared to NT (Figure 2A) whereas FTO is down-regulated by a factor of 2 (Figure 2B). These results are in accordance with previous studies presenting the downregulation of FTO and ALBKH5, promoting proliferation, migration, and BLCA progression (30,33,36). Furthermore, we found an in vivo upregulation of VIRMA and METTL3 that are part of the m⁶A methyltransferase complex (Figure S1). These findings align with recent studies highlighting the impact of METTL3 upregulation in promoting BLCA progression, as well as its role in driving tumor growth and invasion through the AFF4/NF-κB/MYC signaling network (26,46). The writers allow the deposition of m⁶A marks on mRNA while erasers influence its removal (Figure 1), thus, an opposing effect of writers and erasers could be anticipated. Furthermore, we have looked at the m⁶A level using LC-MS/MS analysis. Our results revealed elevated m⁶A level in tumor tissue when compared to normal tissue from the same patient (Figure 2C). These data are in line with elevated level of writers (Figure S1), lower erasers level (Figure 2A,2B) and previous studies (25-28,36). To substantiate these findings, we performed RNA sequencing (Figure 2D). Results showed a tendency of down-regulation of mRNA encoding the erasers, despite noticeable inter-individual variations (donor 2, Figure 2D). Yet, these results confirm the RT-qPCR results regarding ALKBH5 and FTO down-regulation.

Figure 2.

ALKBH5 and FTO are downregulated in tumor tissues and is correlated with m⁶A elevated level. (A) The ALKBH5 expression level was performed using the RT-qPCR technique, (B) the same as for FTO. Data information: GAPDH was used as endogenous control, and the adjacent healthy tissue (NT) was used as calibrator for each donor. NT is set as 1 in each replicate and compared to TT from each donor. RT-qPCR were performed in duplicate with three replicates for each run and donor. N=8, n=3. The 2^−ΔΔCt method was used for relative quantification (**, P≤0.005; ****, P≤0.0001 Student t-test). (C) Quantification of m⁶A marks on mRNA measured by LC-MS/MS assay. N=5, colored based paired. Data are presented as mean ± SEM. *, P≤0.05. (D) RNA sequencing exhibits ALKBH5 and FTO expression for 3 donors. ALKBH5, Alkb homolog 5; FTO, fat mass and obesity associated protein; ALKBH5, Alkb homolog 5; FTO, fat mass and obesity associated protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NT, normal tissue; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SEM, standard error of the mean.

We then examined whether the down-regulation pattern found in vivo could be similar in vitro. Using five different BLCA cell lines and one bladder cell line (Table S2), we analyzed the mRNA expression level of ALKBH5 and FTO (Figure 3). ALKBH5 was reduced by a factor of 2.08 in BLCA cell lines compared to nSV-HUC (Figure 3A) while FTO expression was reduced by a factor of 1.51 in BLCA cell lines compared to SV-HUC (control) (Figure 3B). These results corroborate with in vivo results as we have found 1.6-fold reduction in ALKBH5 and 2-fold reduction in FTO expression (Figure 2A,2B). The data obtained in cell lines confirm tissue experiment and are highly relevant as we pursue the study using cell lines.

Figure 3.

ALKBH5 and FTO mRNA expression in vitro. RT-qPCR analysis showing the expression of ALKBH5 (A) and FTO (B) in bladder cell line (SV-HUC) and bladder cancer cell line (Table S2). GAPDH was used as endogenous control and the SV-HUC as calibrator. Five different cell lines of bladder cancer cell line were used (n=5) with three replicate (n=3). Results are represented as the mean ± SEM. *, P≤0.05; ****, P≤0.0001. P value based on two-tailed paired student’s t-test from three independent experiments. ALKBH5, Alkb homolog 5; CCK-8, cell counting kit-8; FTO, fat mass and obesity associated protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SEM, standard error of the mean.

Double transfection is efficient and does not affect the cell viability

Several studies have explored the consequences of FTO knock-down in BLCA (33,35,36). They found a correlation between FTO and BLCA development, progression, proliferation, and migration (23-26). Additionally, Guo et al. revealed the crucial importance of FTO expression in BLCA and concluded that FTO downregulation has an oncogenic effect. Additionally, they used an FTO inhibitor and revealed the resistance of cells after cisplatin and FTO inhibitor treatment (47). Yu and colleagues have demonstrated that ALKBH5 knock-down protect against cisplatin-induced apoptosis. Here, we have studied the effect of double knock-down of the m⁶A erasers (30).

Simultaneous knock-down of ALKBH5 and FTO in two selected cell lines: 5637 and SW780, both isolated from the urinary bladder (Figure 4A,4B) were achieved. These cancer cell lines were compared with SV-HUC cells. Figure 4A shows the efficiency of ALKBH5 knock-down whereas the Figure 4B displays FTO knock-down. We found a 7 times reduction of ALKBH5 in SW780 and a 2.5 times reduction of ALKBH5 in 5637. Similar results were obtained for FTO knock-down (Figure 4B).

Figure 4.

Efficiency of double knock-down and viability. Relative expression level of ALKBH5 (A) and FTO (B) knock-down in three different cell lines (SV-HUC, SW780 and 5637) using que RT-qPCR techniques. Control (Ctrl) means non-transfected cells and is set as 1 in each cell line. (C) CCK-8 assay displayed cell viability after non-transfected cells (ctrl), empty vector transfection, and double knock-down. The experiment was performed 3 times for each cell lines (n=3). Data are presented as mean ± SEM. ALKBH5, Alkb homolog 5; CCK-8, cell counting kit-8; FTO, fat mass and obesity associated protein; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SEM, standard error of the mean.

Because of the known effect of transfection for cell viability (48), we examined cell-viability after ALKBH5 and FTO double transfection. Our results demonstrate that the double transfection does not affect the cell viability. Indeed, SV-HUC, SW780 or 5637 cells have all around 100% viability after the double transfection (Figure 4C). These data indicate that cells are healthy, and that cell viability will not affect results of further experiments.

Simultaneous transfection protects cells against cisplatin and MMC cytotoxicity

To explore the role of the double ALKBH5/FTO deficiencies in cisplatin toxicity, cells were simultaneously transfected with siRNA against these two m⁶A erasers. They were then subjected to different toxic cisplatin doses for 48 h (Figure 5). We used the cell counting kit-8 (CCK-8) assay to explore the cell viability after double knock-down of FTO and ALKBH5. We appreciate that cisplatin greatly decreases cells viability (Figure 5). In fact, we found 70% of cell death after 20 µM of cisplatin in SW780 and 5637 cells. Yet, when cells are double knock-down, the cisplatin toxicity drastically decrease with only 27% and 58% of cell death in SW780 and 5637 cells respectively (Figure 5).

Figure 5.

Cisplatin treatment after double knock-down. Differences in viability levels in simultaneous knock-down of ALKBH5 and FTO compared to cisplatin treatment. (A) SW780 and 5637 cells (B) were treated with different cisplatin concentrations (μM) in control cells (non-transfected cell line, bladder cancer cell line: SW780 and 5637) and simultaneous knock-down (bladder cancer cell line: SW780 and 5637). The cell viability was investigated by CCK-8 assay. The experiment was performed in triplicate (n=3) and analyzed by student t-test. Data are presented as mean ± SEM. *, P≤0.05; **, P≤0.005; ***, P≤0.001. ALKBH5, Alkb homolog 5; CCK-8, cell counting kit-8; FTO, fat mass and obesity associated protein; SEM, standard error of the mean.

We then investigated the effect of double knock-down after MMC treatment. MMC is one of the most used intervention for NMIBC (39). Doses from 0 to 50 µg/mL were used for 24 h (Figure 6). Results show a linear relation of cell viability to higher doses of MMC in the two cell lines (SW780 and 5637 cells) (Figure 6A,6B). When cells are ALKBH5 and FTO depleted by siRNA, the results depict a clear tendency. Indeed, when both SW780 and 5637 cells are treated with high MMC doses, cells are less sensitive to MMC treatment. We found 59% cell survival in the control group without ALKBH5/FTO knock-down whereas the viability following ALKBH5/FTO knock-down is 76% for SW780 cells (Figure 6A). Regarding 5637 cells, we found 40% versus 61% of cell viability in control group and knock-down group respectively (Figure 6B). These data suggest that combined ALKBH5/FTO knock-down increases the MMC tolerance.

Figure 6.

Mitomycin sensitivity of cells is increase upon simultaneous erasers knock-down. SW780 (A) and 5637 cells (B) were subjected to MMC doses from 0 to 50 μg/mL. The cells without any knock-down serve as the control group in this experiment (ctrl) and siRNA correspond to the simultaneous knock-down of ALKBH5 and FTO. Data information: n=3, student t-test. Data are presented as mean ± SEM. *, P≤0.05; **, P≤0.005; ***, P≤0.001. ALKBH5, Alkb homolog 5; FTO, fat mass and obesity associated protein; MMC, mitomycin; SEM, standard error of the mean.

Discussion

In this study, we investigated the impact of dual knock-down of the m⁶A erasers ALKBH5 and FTO on drug resistance in BLCA. In this paper, we focused on epigenetic regulation and chemotherapy resistance in BLCA. However, we are aware that other factors are important in the chemotherapy response such as the urinary microbiome. Indeed, the urinary microbiome (urobiome) has emerged as a potential factor in BLCA pathogenesis. Some bacterial species may contribute to chronic inflammation, immune suppression, and epigenetic alterations, all of which create a microenvironment favorable to cancer development (49).

While the aberrant expression of METTL3 has been extensively studied across various cancers (50), the role of m⁶A erasers has received relatively little attention. Recent years have seen a growing interest in understanding the role of FTO in BLCA, with several studies highlighting its aberrant levels in BLCA cell lines (33,35,51), but only one notable study has investigated ALKBH5 where they explored the function of ALKBH5 (30). This paper reveals that its knock-down promotes BLCA cell proliferation and reduces cisplatin sensitivity. Conversely, ALKBH5 overexpression was shown to reverse these effects, emphasizing its potential role in modulating chemoresistance and tumor progression (30). Similarly, FTO mRNA is downregulated in BLCA and its downregulation results in the promotion of proliferation and migration while cisplatin cytotoxicity is reduced with FTO downregulation (36). Crucially, none of these studies have addressed the combined effects of ALKBH5 and FTO knock-down, leaving a significant gap in understanding their interplay in driving drug resistance and cancer progression. This study addresses this critical knowledge gap, providing valuable insights into their collective role in BLCA by examining epigenetic regulation.

Erasers are weakly expressed in BLCA

Our results demonstrated lower expression of the two m⁶A erasers ALKBH5 and FTO (Figures 2A,2B,3) while proteins in the writer complex depicted elevated expression (Figure S1). Our data are in accordance with previous studies showing the relationship between METTL3 up-regulation and BLCA progression (26,46). In this study, Cheng and colleagues uncovered target genes of METTL3 and its oncogenic role through its methyltransferase activity. METTL3 is one of the most studied m⁶A regulator and its role in cancer is now well established with promising small-molecule inhibitor with chemotherapeutic potential being developed (52). ALKBH5 seems to have versatile roles that depends on the cancer type, with either oncogenic or tumor suppressor abilities (47). For instance, the ALKBH5 level is low in pancreatic cancer and ALKBH5 prevents progression of pancreatic cancer when overexpressed (47). On the other hand, elevated expression of ALKBH5 has been shown to stimulate the progression of colon cancer (53). Regarding FTO, its up regulation in breast cancer control cell migration and invasion, suggesting a role as an oncogene (54). However, in colorectal cancer, the level of FTO is low facilitating metastasis (55).

In our study, we found a 1.6-fold reduction in ALKBH5 and a 2-fold reduction in FTO expression in BLCA tissues (Figure 2A,2B). Admirably, the ALKBH5 level in cell lines is 2.08 less important in bladder cell lines compared to normal bladder cells whereas the FTO level is 1.51 lower in bladder cell lines. Interestingly, studies on FTO expression in BLCA have reported conflicting findings. While some studies suggest a downregulation of FTO (26,30,33), others report its upregulation in BLCA tissues (35,51). These discrepancies could arise due to several factors, including patient heterogeneity, tumor subtypes, and differences in study design. Future studies incorporating larger patient cohorts and standardized methodologies are needed to clarify the precise role of FTO in BLCA. Indeed, a recent study has demonstrated that the differences in epitranscriptomic regulation can be related to age (56). Furthermore, in our study, we found more expression of FTO in women compared to men and SV-HUC cells (Figure S2). On the contrary, we found no obvious grade-related differences of the erasers. Yet, they might be more downregulated in high-grade bladder tumor tissues (Figure S3). These data are yet interesting as a lower expression of ALKBH5 is correlated with a less favorable prognosis when compared to high ALKBH5 tumors (30). Finally, a study has identified distinct gene signatures of MIBC and NMIBC patients (57). Thus, BLCA is a versatile disease, and it has previously been shown that BLCA has a high degree of mutational heterogeneity (58). Overall, these studies call attention to the importance of BLCA treatment to be patient-dependent regarding epigenetic and epitranscriptomic variation as well as potential mutations. Altogether, our data and previous studies suggest that erasers are downregulated in urological cancer, that the erasers level might be gender dependent and that the same pattern is found in tissue and bladder cell lines.

Double knock-down of m⁶A erasers protect cells against drugs cytotoxicity

In our study, we aimed to elucidate the role of simultaneous knock-down of the two known m⁶A erasers. Thus, we performed a double knock-down of ALKBH5 and FTO (Figure 4A,4B). We confirmed that cell viability was not affected by transfection when treated with control siRNA, empty vector or double knock-down of FTO and ALKBH5 (Figure 4C). We have chosen to use 5637 and SW780, two cell lines regularly used in BLCA related studies (59,60). By performing the double knock-down, we could then explore the underlying consequences in drug treatment.

It has been recently established that METTL3 plays a role in radiotherapy resistance for glioblastoma cells and that METTL3 could be a target for glioblastoma therapy (61). As writers and erasers are intimately linked by m⁶A regulation, we could suspect that erasers might also influence chemotherapy resistance. Recent studies point in this direction, displaying a regulation and modulation of therapy response by ALKBH5 in tumor microenvironment and glioma stem cells (62,63). In addition, recent studies in BLCA have demonstrated the result of individual deregulation of ALKBH5 or FTO in cisplatin cytotoxicity (30,36). Thus, based on the recent data regarding cisplatin sensitivity, we have investigated the consequences of simultaneous knock-down in BLCA cells after cisplatin or MMC treatment. These two drugs are the most used in BLCA, and they are known to induce cell death. First, we identified an expected dose-toxicity with increasing doses of cisplatin and MMC (Figures 5A,5B,6A,6B). Furthermore, BLCA cells subjected to cisplatin treatment (considered as control cells) are more sensitive than cells subjected to cisplatin following ALKBH5 and FTO knock-down. Indeed, after 20 µM of cisplatin doses, whereas only 29% of control cells are alive, 73% and 42% of SW780 and 5637 cells remained viable (Figure 4). Regarding MMC, the double ALKBH5 and FTO knock-down led to 73% and 54% of survival after treatment with 30 µg/mL of MMC versus 61% and 28% in SW780 and 5637 for control cells. Recent studies have demonstrated that ALKBH5 enhances chemotherapy sensitivity in BLCA by regulating m6A-dependent pathways (30,64). Specifically, ALKBH5 inhibits tumor cell proliferation and sensitizes BLCA cells to cisplatin by targeting CK2α-mediated glycolysis (30). The loss of ALKBH5 leads to increased glycolytic activity, promoting chemoresistance and tumor progression. These findings highlight ALKBH5 as a potential therapeutic target, suggesting that restoring its function could improve chemotherapy efficacy in BLCA.

Until now, only MIBC patients are treated with systemic chemotherapy, the reason for this is the quite severe side effects of the drug used. However, a recent study shows that recidivist NMIBC patients that evolve in MIBC might have worse clinical prognosis and outcome (65). Cancers present thousands of deregulated genes or pathways (66,67). Among them, systemic inflammation plays a key role in BLCA progression, with inflammatory markers predicting oncological outcomes. The Systemic Immune-Inflammation Index (SII) has emerged as a promising biomarker, linked to tumor aggressiveness, recurrence, and survival. High preoperative SII correlates with increased nodal invasion and poorer recurrence-free and overall survival after radical cystectomy. As a non-invasive, cost-effective marker, SII could improve risk stratification, aiding in personalized treatment and identifying high-risk patients for intensified therapy or closer monitoring (68).

In our study, we focused on m⁶A erasers because they are poorly understood in BLCA. We found deregulation of m⁶A erasers to be similar in BLCA biopsies and BLCA cell lines. Our study’s small patient sample (N=14) may limit generalizability, but the cell line model supports our findings, warranting further validation in larger cohorts. Thus, using cell lines for upstream molecular analysis might be a helpful tool for further experiments.

Conclusions

In summary, our data reveal a critical role of m⁶A erasers in BLCA cell lines. Although our results are preliminary, there is no doubt that m⁶A proteins, including the erasers, should be evaluated as therapeutic targets in BLCA. Our results showed deregulation of ALKBH5 and FTO and highlight their potential role in cisplatin and MMC resistance.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Regional Ethics Committee South-Eastern Norway Regional Health Authority (No. #2017/2170) and informed consent was obtained from all individual participants.

Data Sharing Statement

Available at https://atm.amegroups.com/article/view/10.21037/atm-25-7/dss

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DOI: 10.21037/atm-25-7