DNA damage-mediated FTO downregulation promotes CRPC progression by inhibiting FOXO3a via an m⁶A-dependent mechanism

Summary

Polyadenosine diphosphate-ribose polymerase inhibitors (PARPi) represent a promising novel treatment for castration-resistant prostate cancer (CRPC) with encouraging results. However, the combination targets in CRPC remain largely unexplored. N6-methyladenosine (m⁶A) has been shown to play a crucial role in cancer progression and DNA damage response. Here, we observed a higher overall level of m⁶A and a downregulation of Fat mass and obesity-associated protein (FTO), which correlated with unfavorable clinicopathological parameters in prostate cancer (PCa). Functionally, reduced FTO promotes PCa growth, while overexpression of FTO has the opposite effect. Mechanistically, FOXO3a was identified as the downstream target of FTO in PCa. FTO downregulates the expression of FOXO3a in an m⁶A-dependent manner, leading to the degradation of its mRNA. Importantly, DNA damage can degrade FTO through the ubiquitination pathway. Finally, we found that overexpression of FTO can enhance the effect of PARPi on PCa. Therefore, our findings may provide insight into novel therapeutic approaches for CRPC.

Graphical abstract

Highlights

• •Prostate cancer shows elevated m⁶A RNA methylation levels and decreased FTO expression
• •FTO performs a tumor-suppressive functional role in prostate cancer
• •FTO mediates FOXO3a mRNA degradation in an m⁶A-dependent way
• •DNA damage induces FTO protein degradation, and FTO potentiates PARPi synergistic effect

Molecular biology; Cell biology; Cancer

Introduction

Prostate cancer (PCa) is the most frequently diagnosed malignant tumor of the male genitourinary system.¹ Androgen deprivation therapy is the standard treatment for patients with advanced PCa. Second-generation androgen receptor inhibitors, such as enzalutamide, have significantly improved progression-free survival and overall survival (OS) in patients with metastatic, hormone-sensitive PCa.² However, the majority of patients with advanced PCa eventually develop castration-resistant prostate cancer (CRPC), which has a poor prognosis.^3,4 Polyadenosine diphosphate-ribose polymerase inhibitors (PARPi) represent a new form of treatment for patients with metastatic CRPC with relevant genomic alterations in homologous recombination repair (HRR) genes.^5,6 Unfortunately, only about 10% of patients with PCa harbor pathogenic mutations in these HRR genes, leaving the majority of patients unable to benefit from this promising treatment strategy.⁷ Further investigation into combination targets for PARPi may enhance efficacy through synergistic activity and potentially sensitize inherently PARPi-resistant tumors to PARPi therapy.

N6-methyladenosine (m⁶A) RNA modification is an essential mechanism of post-transcriptional regulation of gene expression. It represents the most predominant and conserved internal mRNA modification, accounting for over 80% of all types of RNA methylation modification. m⁶A regulators are categorized into three types: writers (m⁶A methyltransferases), erasers (demethylases), and readers (proteins that recognize m⁶A).⁸ The m⁶A modification process is dynamic and reversible, controlled by the actions of writers and erasers. There is substantial evidence suggesting the role of m⁶A modification in regulating various aspects of RNA biology, including RNA metabolism, folding and structure, nuclear processing, mRNA export, maturation, degradation, and translation.⁹ Increasingly, studies have highlighted the significance of RNA m⁶A modifications in tumor occurrence, development, immune response, and drug resistance.¹⁰ Although there have been limited reports on the function of m⁶A in CRPC, the underlying regulatory mechanisms are largely unknown.

In this study, we observed that UV-induced DNA damage diminishes Fat mass and obesity-associated protein (FTO) expression in PCa cells. We demonstrated the upregulation of m⁶A and downregulation of FTO expression in CRPC tumor tissues compared to adjacent normal tissues. Subsequently, we unveiled that FTO stabilizes FOXO3a mRNA level by removing m⁶A modifications, thereby suppressing the proliferation of CRPC cells. Notably, we found that PCa cells with high FTO expression can be selectively killed by PARPi. Overall, FTO holds promise as a novel biomarker for PARPi therapy in CRPC, validating its role in regulating CRPC progression and providing a potential biomarker for the therapeutic efficacy of PARPi in treating CRPC.

Results

The upregulation of m⁶A and downregulation of FTO correlate with unfavorable clinicopathological parameters in PCa

To investigate the role of m⁶A in CRPC, we performed RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) analyses on tumor and adjacent normal tissues obtained from three CRPC specimens (Figure 1A). Comprehensive visualization of RNA-seq and MeRIP-seq results is depicted in the circos plot (Figures 1B and S1A). MeRIP-seq revealed elevated m⁶A level in CRPC tissues, although the results did not reach statistical significance (Figure S1B). This result is consistent with previous reports analyzing The Cancer Genome Atlas (TCGA) database in PCa.^11,12 The Kyoto Encyclopedia Genes and Genomes (KEGG) analysis of these differentially expressed genes (DEGs) is presented in Figure 1C. Metagene analysis of MeRIP-seq data reveals enrichment of m⁶A sites near stop codons (Figure 1D). Additionally, motif analysis comparing CRPC tumor and adjacent normal tissues was conducted and depicted in Figure 1E.

Figure 1.

The upregulation of m⁶A and downregulation of FTO correlate with unfavorable clinicopathological parameters in PCa

(A) Flow chart of MeRIP and RNA-seq for detecting tumor and adjacent tissues in 3 pairs of CRPC tissues.

(B) Overall display of 3 CRPC RNA-seq and MeRIP-seq data in the circos figure.

(C) KEGG analysis revealed the pathways associated with the DEGs identified via RNA-seq.

(D) Metagene analysis revealed an m⁶A distribution pattern in the tumor and adjacent tissues of patients with CRPC, as determined by MeRIP-seq.

(E) The m⁶A motif and sequence enriched in tumor and adjacent tissues of CRPC.

(F) Heatmap representing the differential expression of m⁶A-related genes in 3 pairs of CRPC and adjacent tissues.

(G and H) Representative images (G) and Immunohistochemical Reactive Score (IRS) and statistical results (H) of FTO immunohistochemistry analysis of PCa and adjacent tissues from the paired groups.

(I) OS of patients with PCa with high and low FTO mRNA level determined via Kaplan-Meier survival curve analysis based on TCGA dataset. Error bars represented mean ± SD. ∗∗∗p < 0.001.

We then assessed the expression of m⁶A methyltransferase (METTL3, METTL14, METTL16, RBM15, and WTAP), m⁶A readers (HNRNPA2B1, YTHDC1/2, and YTHDF1/2/3), and demethylase (FTO and ALKBH5) in paired PCa tissues and adjacent tissues. The FTO demethylase was found to be downregulated in all PCa tissues (Figure 1F). We utilized data analysis from EMBL-EBI (www.ebi.ac.uk) on the gene expression profiles of 14 patients with PCa as reported in the previous study, which is consistent with our findings (Figure S1C).¹³ To further confirm the relationship between FTO expression and clinicopathological parameters, we conducted an immunohistochemical (IHC) staining assay using a PCa tissue microarray. The analysis revealed that the FTO protein level was significantly downregulated in tumor compared to adjacent tissues (Figures 1G and 1H). Moreover, analysis of TCGA database showed that patients with PCa with higher FTO expression exhibited longer disease-free survival than those with lower FTO expression (Figure 1I), indicating that low FTO levels were associated with an adverse prognosis. Previous reports have shown that patients with PCa with elevated m⁶A levels have poorer OS compared to those with lower m⁶A levels.¹¹ Taken together, these findings suggest that the downregulation of FTO expression in CRPC affects prognosis by mediating m⁶A modification.

Silencing FTO promotes PCa proliferation in vitro

To explore the role of FTO in PCa, we utilized lentivirus-based short hairpin RNA to knock down FTO expression in PC3 and DU145 PCa cell lines and investigated its biological function. The successful knockdown of FTO mRNA and protein was confirmed by qPCR and western blotting, respectively (Figures 2A and 2B). Subsequently, we performed a CCK8 experiment on PC3 and DU145 cells with FTO knockdown, which demonstrated that FTO knockdown significantly enhanced cell proliferation (Figure 2C). We further confirmed the inhibitory effect of FTO knockdown on cell proliferation through a colony formation assay, where the colony formation rates of FTO knockdown PC3 and DU145 cells were higher compared to shNegative Control (shNC) cells (Figure 2D). Moreover, FTO knockdown significantly inhibited apoptosis of DU145 cells following treatment with oxaliplatin (Figure 2E).

Figure 2.

Silencing FTO promotes PCa proliferation in vitro

(A and B) Examination of FTO expression level in PC3 and DU145 cells transfected with LV-shFTO via qRT-PCR (A) and western blotting (B).

(C) Determination of the absorbance at 450 nm of PC3 and DU145 cells transfected with LV-shFTO via a CCK8 experiment.

(D) Colony formation assay of PC3 or DU145 cells with FTO knockdown.

(E) Effects of FTO knockdown on apoptosis after DMSO or oxaliplatin (10 μM) treatment in DU145 cells via flow cytometry analysis. Error bars represented mean ± SD. ∗∗∗p < 0.001.

To evaluate the function of FTO in vivo, we utilized a nude mouse subcutaneous xenograft model. DU145 cells with FTO knockdown exhibited a significantly higher tumor growth rate and increased tumor weight (Figures S2A–S2D). Histological examination of Ki-67 revealed that subcutaneous tumors derived from FTO knockdown DU145 cells exhibited a higher cell proliferation index (Figure S2E). Overall, these results indicated that reduced FTO promotes PCa growth.

FTO overexpression inhibits PCa proliferation both in vivo and in vitro

Next, we established the stable FTO overexpression cell lines using DU145, PC3, and Lymph Node Carcinoma of the Prostate (LNCaP) cells. As expected, FTO mRNA and protein were successfully overexpressed (Figures 3A, 3B, S3A, and S3B). A CCK8 assay showed that FTO overexpression significantly inhibited cell growth (Figures 3C and S3C). We further validated that the overexpression of FTO markedly reduced the colony formation rates (Figure 3D). Therefore, we hypothesized that FTO may influence cell proliferation capacity by modulating the cell cycle. As anticipated, the overexpression of FTO in DU145 cells promoted cell-cycle arrest, specifically in the G0/G1 phase (Figure 3E). Apoptosis assay showed that FTO overexpression promotes apoptosis in PC3 and LNCaP cells (Figures 3F and S3D). Subsequently, we assessed the potential impact of FTO on several genes associated with the cell cycle and apoptosis. Western blot analysis revealed that FTO overexpression upregulated the expression of MLH1, BIM, and FasL, which are associated with the cell cycle and apoptosis (Figure 3G). Then, we subcutaneously implanted DU145 cells overexpressing FTO into nude mice. Unexpectedly, FTO overexpression exhibited powerful anticancer abilities in vivo, completely suppressing tumor formation in mice (Figure 3H). Overall, these results indicated that FTO overexpression inhibits PCa proliferation both in vivo and in vitro.

Figure 3.

FTO overexpression inhibits PCa proliferation and growth

(A and B) Examination of FTO expression level in PC3 and DU145 cells overexpressing FTO via qRT-PCR (A) and western blotting (B).

(C) Determination of absorbance at 450 nm of PC3 and DU145 cells overexpressing FTO via a CCK8 experiment.

(D) Examination of colony formation assay of PC3 and DU145 cells overexpressing FTO.

(E and F) Effects of FTO overexpression on cell cycle (E) or apoptosis (F) in PC3 cells via flow cytometry analysis.

(G) Examination of cell cycle or apoptosis relative protein expression level in PC3 or DU145 cells overexpression FTO via western blotting.

(H) Xenograft tumor experiments were carried out in nude mice with FTO overexpressing DU145 cells, and images show nude mice bearing tumors after being euthanized at the eighth week are shown. Error bars represented mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

FTO is positively correlated with FOXO3a expression

As an important member of the FOXO family, FOXO3a serves as a central transcription factor, playing a critical role in mediating multiple physiological and pathological processes, including cell proliferation, apoptosis, reactive oxygen species (ROS) response, and regulation of cell cycle and metabolism.^14,15 PI3K-AKT signaling phosphorylates FOXO3a and promotes its exit from the nucleus, allowing cells to pass through the G1-S phase and inhibiting apoptosis.¹⁶ Additionally, FOXO3a overexpression inhibits tumor growth in PCa.¹⁷ More importantly, the KEGG analysis revealed alterations in the FoxO signaling pathway and apoptosis in CRPC tissues compared to those in adjacent normal tissues (Figure 1C). Given previous reports suggesting an interaction between FTO and FOXO3a,¹⁸ we conducted an analysis of the PCa TCGA dataset. Our analysis revealed a significant positive correlation between the expression level of FTO and FOXO3a (Figure S4A). Although, the basal level of FTO and FOXO3a in PCa cells showed no significant correlation (Figure S4B). Next, we assessed FOXO3a mRNA expression level in PC3 and DU145 cells with either FTO knockdown or overexpression using quantitative reverse-transcription PCR (qRT-PCR) and western blot assays. As anticipated, the mRNA expression level of FOXO3a was upregulated in overexpressing FTO and downregulated in FTO knockdown cells (Figures 4A–4C). Moreover, the protein expression level of FOXO3a increased with FTO overexpression and decreased with FTO knockdown (Figures 4D–4F). We then investigated whether FOXO3a could counteract the effects of FTO depletion or overexpression on PCa cell proliferation. We generated DU145 cells with both FTO knockdown and FOXO3a overexpression. As expected, FOXO3a overexpression significantly rescued the promotion of PCa cell proliferation that resulted from FTO knockdown, as indicated by the CCK8 experiment (Figure 4G). Furthermore, we employed an immunohistochemistry assay to assess the expression of FOXO3a protein in a PCa tissue, which had also been utilized in Figure 1E. The IHC results revealed a notable downregulation of FOXO3a protein level in PCa tissues compared to adjacent tissues (Figures 4H and S4C). Furthermore, histological examination revealed that subcutaneous tumors derived from FTO-knockdown DU145 cells exhibited a higher expression of FOXO3a (Figure S2E). Taken together, these findings indicate that FTO inhibits the progression of PCa by modulating the expression of FOXO3a.

Figure 4.

FTO is positively correlated with FOXO3a expression

(A–C) Examination of FOXO3a mRNA expression level in DU145 or PC3 cells with FTO overexpression or knockdown via qRT-PCR.

(D–F) Examination of FOXO3a protein level in DU145 or PC3 cells with FTO overexpression or knockdown via western blotting.

(G) Determination of absorbance at 450 nm of FTO knockdown PC3 cells undergoing LV-FOXO3a overexpression transfection via a CCK8 experiment.

(H) Representative images of FOXO3a immunohistochemistry analysis in PCa and adjacent normal tissue. Error bars represented mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

FTO mediates FOXO3a mRNA degradation in an m⁶A-dependent way

FTO has been reported to play m⁶A-dependent roles in tumorigenesis and progression in several cancers.¹⁹ To confirm whether FOXO3a is a target of FTO-mediated m⁶A modification, we conducted m⁶A dot blot assays using PC3 and DU145 cell lines with either FTO knockdown or overexpression. The results demonstrated that the level of m⁶A modification decreased significantly when FTO was overexpressed and increased upon FTO knockdown (Figures 5A and 5B). To further explore the potential regulatory mechanism of FTO on FOXO3a, we initially compared the stability of FOXO3a mRNA between FTO stable knockdown cells and control cells using actinomycin D. Compared to the negative control, we observed a significant decrease in the mRNA decay rate of FOXO3a following FTO overexpression in DU145 cells, whereas the results were reversed in PC3 cells with FTO downregulation (Figures 5C and 5D). Then, we assessed the m⁶A modification of FOXO3a mRNA through MeRIP-seq in 3 CRPC tissues. The results indicated that m⁶A peaks showed higher enrichment in the tumor tissues compared to adjacent tissues in the indicated region of FOXO3a (Figure S4D). We further performed MeRIP-qPCR in FTO-downregulated PC3 cells. The m⁶A enrichment of FOXO3a was remarkably increased when FTO was knocked down (Figure 5E). Consistent with other research,²⁰ we demonstrated that FOXO3a is regulated by FTO in an m⁶A-dependent manner.

Figure 5.

FTO mediates FOXO3a mRNA degradation in an m⁶A-dependent way

(A and B) Examination of m⁶A level in PC3 or DU145 cells with FTO overexpression (A) or knockdown (B) via m⁶A dot blot assays.

(C and D) After actinomycin D (Act. D) treatment at the indicated time points, the RNA level of FOXO3a was determined after FTO overexpression (C) or knockdown (D).

(E) MeRIP-qPCR was performed to detect alterations in the m⁶A level of FOXO3a in FTO knockdown PC3 cells. Error bars represented mean ± SD. ∗∗p < 0.01; ∗∗∗p < 0.001.

FTO is degraded by DNA damage in PCa cells and potentiates the synergistic effect of PARPi

Considering that FOXO3a plays a role in mediating DNA damage and tumorigenesis and responds to various cellular stresses, such as UV irradiation and oxidative stress,¹⁵ we wondered whether DNA damage has any impact on the expression of FTO. Therefore, we exposed PC3 and DU145 cells to UV irradiation to trigger the DNA damage response. The expression level of the FTO protein progressively decreased over time following DNA damage (Figures 6A and 6B). Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) are known to be key kinases in DNA damage repair pathways²¹; we investigated whether these two kinases could also affect the expression of FTO. We found that the decrease in FTO protein expression after UVC treatment was inhibited by the ATM inhibitor but not the ATR inhibitor in PC3 cells (Figure 6C). FTO protein expression was decreased after cycloheximide (CHX) treatment in DU145 cells (Figure S5A). Meanwhile, we found that the downregulation of FTO expression induced by CHX could also be rescued by MG132 in both PC3 and DU145 cells (Figures S5B and S5C). This result indicates that the degradation of FTO is mediated by the proteasome pathway. Subsequently, PC3 cells were treated with the proteasomal inhibitor MG132, and it was observed that the reduction in FTO induced by UVC was rescued by MG132 (Figure 6D). Furthermore, our observations indicated an increase in FTO ubiquitination level following exposure to UVC, suggesting the involvement of the ubiquitin (Ub)-proteasome pathway in mediating FTO degradation (Figure 6E). These observations strongly suggest that DNA damage leads to a decrease in FTO level through the Ub-mediated pathway in PCa.

Figure 6.

FTO is degraded by DNA damage in PCa cells and potentiates the synergistic effect of PARPi

(A and B) Western blot analysis showing the protein level of FTO and γH2AX after UVC treatment.

(C) Western blot analysis of FTO level in PC3 cells treated with DMSO, an ATR inhibitor (10 μL), or an ATM inhibitor (5 μM) after UVC treatment for the indicated times.

(D) Western blot analysis of FTO level in PC3 cells treated with DMSO or MG132 (5 μM) for 12 h after UVC treatment.

(E) Immunoblotting was performed to detect ubiquitinated FTO using the immunoprecipitation assay on PC3 cells overexpressing FTO, which were transfected with an HA-tagged Ub plasmid and subsequently exposed to UVC conditions.

(F) Determination of absorbance of PC3 cells with FTO overexpression at 450 nm via a CCK8 experiment after olaparib treatment for indicated concentration.

(G and H) Determination of the absorbance at 450 nm of PC3 (J) or DU145 (K) cells with FTO knockdown via a CCK8 experiment after olaparib treatment for indicated concentration.

(I) The schematic diagram illustrates that UV irradiation leads to the downregulation of FTO in PCa, which in turn weakens FOXO3 RNA stability and inhibits FOXO3a protein translation, thereby suppressing CRPC cell apoptosis and promoting proliferation. Error bars represented mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Based on the crucial role of FTO in DNA damage, we hypothesized that the combination of FTO and olaparib may exhibit a synergistic anticancer effect. Therefore, we treated cell lines overexpressing or knocked down FTO with olaparib. The results demonstrated that cells overexpressing FTO exhibited increased sensitivity to olaparib, whereas cells with FTO knockdown showed resistance to olaparib (Figures 6F–6H and S3E).

In summary, FTO demethylates the mRNA of FOXO3a, enhancing its stability and expression, thereby promoting FOXO3a-mediated apoptosis of PCa and inhibiting proliferation. UV exposure promotes the degradation of FTO through the ubiquitination pathway, thereby accelerating the degradation of FOXO3a and promoting the proliferation of CRPC cells (Figure 6I). Our findings indicate that the expression level of FTO may serve as a molecular marker for the clinical use of PARPi in PCa and as a potential target for the development of combination therapies involving PARPi.

Discussion

As the first identified RNA demethylase, dysregulation of FTO is closely associated with obesity as well as energy homeostasis and plays an important role in various types of cancers.²² For example, in leukemia, FTO can regulate the stability of its target mRNA by removing m⁶A modifications, thereby promoting tumor stem cell self-renewal, reprogramming cancer immunity and metabolism, and promoting cancer cell proliferation.^23,24,25 FTO was identified as a prognostic factor in lung squamous cell carcinoma, promoting cell proliferation and invasion.²⁶ In glioblastoma, FTO exerts a pro-cancer effect.²⁷ FTO can also promote aerobic glycolysis in tumor cells by regulating PD-L1 expression and the expression of genes related to the glycolytic pathway, resulting in impaired T cell activation.²⁸ However, consistent with previous studies,^12,29,30 our study found that FTO is expressed at a low level in PCa tissues. Combined with functional characterization, this suggests that FTO serves as a tumor suppressor gene in regulating genesis and progression of CRPC. The divergent roles of FTO in various cancer types may constrain its potential as a therapeutic target. Further comprehensive research is essential to investigate this matter thoroughly.

The DNA damage repair system detects and repairs damaged DNA, playing a crucial role in maintaining and transmitting genetic information.³¹ The RNA demethylase ALKBH5 acts as a crucial regulator safeguarding cells against DNA damage and apoptosis induced by ROS-induced stress.³² METTL14 plays a pivotal role in global genome repair and the development of skin tumors induced by UVB radiation.³³ Here, we report that UV irradiation mainly causes a decrease in FTO protein expression in PCa cells through Ub-mediated proteasome-associated degradation. These findings suggest a novel potential mechanism for regulating FTO protein homeostasis and propose a novel m⁶A-dependent gene regulation model in the epigenetics of DNA damage. Previous research has shown that hypoxia can induce post-translational ubiquitination, resulting in FTO protein degradation.³⁴ Together, these findings suggest that RNA modifications can swiftly respond to stress conditions through the regulation of FTO proteostasis.

Previous research has indicated that FTO interacts with FOXO3a to enhance its nuclear translocation, leading to its suppression in gliomas.¹⁸ In our study, we found that FTO in PCa cells decreases the m⁶A modification of FOXO3a mRNA, thereby inhibiting FOXO3a mRNA degradation. The distinct effects of one specific molecule are highly intriguing. This phenomenon may be explained by tissue specificity differences in various organs, and further investigation is needed for deeper insights.

PARP is an essential factor in repairing DNA single-strand breaks by initiating a signaling cascade and recruiting the necessary repair machinery to the affected regions. PARPi have ushered in a new era of precision therapy, and the combination of olaparib with second-generation inhibitors can significantly enhance the OS of patients with CRPC.^35,36 Food and Drug Administration has approved olaparib with abiraterone and prednisone (or prednisolone) for breast cancer-mutated metastatic CRPC. Other study proposes novel genes, such as ARH3, contributing to PARPi resistance in CRPC.³⁷ Our study suggests that FTO may serve as a biomarker for olaparib therapy.

Taken together, our study reveals a tumor suppressor gene FTO in PCa progression. Mechanistically, we identified FTO enhances the mRNA stability of FOXO3a, which inhibits the proliferation of PCa. Furthermore, DNA damage regulates FTO by enhancing its ubiquitination and subsequent degradation. At last, FTO may serve as a potential therapeutic target in combination with PARPi in CRPC.

Limitations of the study

There are several limitations in this study. First, we identified elevated m⁶A levels in CRPC tissues using MeRIP-seq. However, the number of CRPC patient samples is limited because these patients typically have fewer opportunities for surgery. Additionally, MeRIP-seq requires a substantial amount of solid tissue to obtain sufficient RNA, which restricts its use in clinical detection. Second, the proteins involved in the ubiquitination regulation of FTO remain unidentified. Third, although we observed that elevated FTO enhances the synergistic effect of the PARPi olaparib in PCa cells, we lack direct clinical evidence linking FTO expression to the efficacy of PARPi in patients with PCa. Furthermore, since there is currently no known FTO activator, we do not have in vivo efficacy data on the combined application of FTO activation and PARPi, which would have significant clinical implications.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rabbit monoclonal anti-FTO	Abcam	Cat #ab124892; RRID: AB_10972698
Rabbit monoclonal anti-H2AX	ABclonal	Cat #AP0099; RRID: AB_2771168
Rabbit monoclonal anti-FOXO3	Cell Signaling Technology	Cat #12829S; RRID: AB_2636990
Rabbit monoclonal anti-m6A	Cell Signaling Technology	Cat #56593; RRID: AB_2799515
Biological samples
Prostate cancer tissue microarray	Outdo Biotech Company	Cat #HProA150PG02
BALB/c nude	SPF Biotechnology	N/A
NSG	SPF Biotechnology	N/A
Chemicals, peptides, and recombinant proteins
RPMI 1640 medium	Life Technologies	Cat #11875093
fetal bovine serum	Gibco	Cat #10270107
Penicillin-Streptomycin Solution	Vivacell	Cat #C3420-0100
Lipofectamine 2000	Thermo Fisher Scientific	Cat #11668019
Polybrene	Biosharp	Cat #BL628A
puromycin	TargetMol	Cat #T19978
Blasticidin S HCl	APExBIO	Cat #B4879
RNAiso Plus	TAKARA	Cat #9109
Actinomycin D	GLPBIO	Cat #GC16866-5
20×SSC	Solarbio	Cat #S1030
RNase inhibitor	Beyotime	Cat #R0102
proteases inhibitor	Beyotime	Cat #P1006
Proteinase K	Meilunbio	Cat #MA0006
RIPA lysis buffer	Beyotime	Cat #P0013B
4% paraformaldehyde	Beyotime	Cat #P0099-500mL
Olaparib	Beyotime	Cat #SC9118-5mg
Oxaliplatin	Aladdin	Cat #D109812-250mg
KU-55933 (ATM inhibitor)	Beyotime	Cat #SC9136-5mg
AZD6738 (ATR inhibitor)	TargetMol	Cat #T3338-5mg
Cycloheximide	Selleck	Cat #S7418-500mg
MG-132	Selleck	Cat #S2619-5mg
Critical commercial assays
StarScript III One-Step RT-PCR Kit	GenStar	Cat #A230-10
SYBR Green PCR Master Mix	Vazyme	Cat #Q311-02
CCK-8 Cell Counting Kit	Vazyme	Cat #A311-01
Annexin V-FITC Apoptosis Detection Kit	Beyotime	Cat #C1062
Cell Cycle and Apoptosis Analysis Kit	Beyotime	Cat #C1052
Deposited data
RNA-seq, meRIP-seq data	This paper	SRA: SRP498052
14 prostate cancer patients RNAseq data	EMBL-EBI	E-MTAB-567
Experimental models: Cell lines
Human: PC3	ATCC	CRL-1435 ™
Human: DU145	ATCC	HTB-81 ™
Human: LNCaP	ATCC	CRL-1740 ™
HEK293T	ATCC	CRL-3216™
Recombinant DNA
FTO-Flag and constructs	This paper	N/A
FOXO3a-Flag and constructs	This paper	N/A
Software and algorithms
Applied Biosystems 7500 Real-Time PCR System	Thermo Fisher	Cat #4397808
SYNERGY Microplate Reader	BioTek	https://imagej.nih.gov/ij
Clarity Western ECL Subetrate	Bio-Rad	https://www.flowjo.com/
Bio-Imaging System	Bio-Rad
GraphPad Prism 9	La Jolla	http://www.graphpad.com
ImageJ	ImageJ	https://imagej.nih.gov/ij
FlowJo	Tree Star software, inc.	https://www.flowjo.com/
RStudio (V 4.2.2)	R	https://posit.co/download/rstudio-desktop/

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dinglan Wu (wudinglan@163.com).

Materials availability

This study did not generate new unique reagents and all materials in this study are commercially available. The nucleotide sequences for plasmids construction in this study are provided in the Supplementary tables. Any additional analysis information for this work is available by request to the lead contact.

Data and code availability

This paper analyzes existing, publicly available data. The accession number for the datasets is listed in the key resources table. Raw RNAseq and metadata are deposited in the Gene Expression Omnibus database and are publicly available as of the date of publication. The accession number is listed in the key resources table. All original code is available in this paper’s supplemental information. Any additional information required to reanalyze the data reported in this paper is available upon request. Source data for other figures will also be provided upon request from the lead contact.

Experimental model and study participant details

Cell lines and cell culture

PC3, DU145 and LNCaP and HEK293T cell lines were obtained from ATCC (ATCC Cell Resource Center, Manassas, VA, USA). All the cells were cultured in RPMI 1640 medium (Life Technologies, USA) and HEK293T cells were grown in Dulbecco’s Modified Eagle media (DMEM) (Life Technologies, USA) with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Inc., USA) and 1% antibodies (penicillin/streptomycin). The cell lines were maintained in a humidified cell incubator with 5% CO₂ at 37°C.

Clinical samples

A tissue microarray containing 95 primary or localized prostate cancer tissues and 3 normal prostatic tissues was purchased from Shanghai Outdo Biotech Company (Number: HProA150PG02). CRPC tissue specimens used were surgical specimens from CRPC patients underwent cytoreductive radical prostatectomy. All studies were approved by the Ethics Committee of the Shenzhen Hospital of Southern Medical University, and informed consent was obtained from all patients (Ethical approval number for human participants: NYSZYYEC20210011).

Tumor xenografts in nude mice

For the in vivo experiment, 4-6 week-old male BALB/c and NSG mice were obtained from SPF (Beijing) Biotechnology Co. Ltd and held under specific pathogen-free conditions. The mice were kept in ventilated cages and allowed to acclimate for at least three days before any manipulation. All animal studies were approved by the Institutional Animal Care and Use Committee of Peking university Shenzhen Graduate School (Ethical approval number for animal participants: AP0044007).

Method details

Tissue specimens and tissue microarray analysis

Formalin-fixed paraffin-embedded (FFPE) sections of 5 mm thickness were prepared on charged glass slides. After deparaffinization and rehydration, heat-mediated antigen retrieval was carried out with 10 mM sodium citrate (pH = 6). Endogenous peroxidase activity was blocked by adding 3% hydrogen peroxide. The chips were incubated with diluted antibodies overnight at 4°C in a humidified chamber. Then, the tissue chips were incubated with an HRP-conjugated secondary antibody for 1h. Standard DAB staining was performed for chromogenic detection of the IHC targets. All tissues were assigned a score based on staining intensity in the epithelial compartment (1, no staining; 2, low positive; 3, positive; 4, high positive) and the percentage of positive cells (0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%)). The final stain score was calculated and analyzed.

RNA sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq)

Total RNA was extracted from tissue samples using TRIzol reagent (Invitrogen). Subsequently, RNA-seq and MeRIP-seq were performed simultaneously (Aksomics Inc., Shanghai, China). For RNA-seq, 1 μg of total RNA from tissue was subjected to poly(A) mRNA isolation using Dynabeads Oligo-dT. A cDNA library was constructed following the protocol for the TruSeq RNA Library Prep Kit v2 (Illumina). Ultimately, 2 × 150 bp paired-end (PE150) sequencing was performed on an Illumina NovaSeq 6000. For MeRIP-seq, polyadenylated RNAs were enriched from total RNA and fragmented into approximately 100-nucleotide-long oligonucleotides. Protein-A/G beads were incubated with anti-m ⁶A affinity purified antibody for 30 min at room temperature. The mixture was then immunoprecipitated by incubating with fragmented mRNA at 4°C for 2 h. High-throughput m⁶A sequencing was performed on the Illumina NovaSeq 6000 platform. The KAPA Stranded RNA-seq Kit was used to construct m⁶A-modified mRNA and input RNA sequencing libraries separately. Paired-end reads were harvested from an Illumina HiSeq 4000 sequencer, and the quality was controlled by Q30. After 3′ adapter-trimming and low-quality read removal by Cutadapt software (v1.9.3), the reads were aligned to the reference genome (UCSC MM10) with Hisat2 software (v2.0.4). Methylated sites on RNAs (peaks) were identified by MACS software. Nonmatching methylated sites on RNAs were identified by diffReps. These peaks identified by both software programs as overlapping with exons of mRNA were figured out identified and chosen by custom scripts. Image analysis and base calling were performed using the Solexa pipelinev1.8 (OffLine Base Caller software, v1.8). Sequencing quality was examined by FastQC software (v0.11.7). HISAT2 software (v2.1.0) was used to align immunoprecipitation and input trimmed reads to the Ensembl Rnor_6.0 genome sequences. The aligned reads were used for peak calling of the MeRIP regions by exomePeak (v2.13.2) with default settings. Statistically significant MeRIP enriched regions (peaks) were also identified by exomePeak for each separate m⁶A-immunoprecipitation/input sample pair. Identified MeRIP-enriched regions were annotated using the Ensembl Rnor_6.0 database. Alignment statistical analysis was employed to preserve valid sequences for the identification of subsequent significant differentially methylated regions. Hierarchical clustering, scatterplots, and volcano plots were conduced in the R environment for statistical computing and graphics. Mapped reads representing enriched m⁶A peaks of RNA fragments across the genome were visualized using the Integrated Genome Viewer (IGV v2.4.14, Broad Institute). The sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database under the accession code SRP498052.

Methylated RNA immunoprecipitation qRT-PCR (MeRIP-qPCR)

Total RNA was extracted from PC3 shNC or shFTO cells using RNAiso Plus (Takara), after which DNase was added to remove DNA. The m⁶A RNA enrichment kit (IVDSHOW, China) was used to perform MeRIP according to the manufacturer’s instructions. In brief, the RNA sample was incubated in an immune capture buffer containing affinity beads and bead-antibody complexes. Subsequently, the RNA sequence, encompassing both ends of the m⁶A region, was cleaved using an RNA cleavage enzyme mix. The enriched RNA was then released with Protein Digestion Solution, purified, and eluted. The purified methylated RNA and input mRNA were reverse-transcribed and analyzed by qPCR. Fold enrichment was determined as the ratio of m⁶A modified mRNA to input mRNA.

Plasmid construction and infection

Short hairpin RNAs (shRNAs) targeting FTO constructs, as well as the overexpression plasmids of FTO-Flag and FOXO3a-Flag, were purchased from Hitrobio (Beijing, China). The plasmid contained the full-length coding region of FTO/FOXO3a and was fused with flag tags in the N-terminal. The sequences of shRNAs for FTO and FTO-Flag/FOXO3a-Flag are listed in the Supplemental tables.

Plasmids, including transgenes and packaging plasmids, were co-transfected into HEK 293 T cells using Lipofectamine 2000 (11668019, Thermo Fisher Scientific Inc., USA) according to the manufacturer’s protocol. Viruses were collected after 48 h. When tumor cells reached a density of around 70%–80%, the collected viruses were transfected with an appropriate concentration of polybrene (BL628A, Biosharp, China). After 2 days, puromycin (T19978, TargetMol, China) or Blasticidin S HCl (B4879, APExBIO, China) was used to select the transfected cells.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

With reference to the product specification, total RNA was extracted from PC cells using TRIzol reagent (Invitrogen, Massachusetts, USA) and then subjected to reverse transcription into complementary DNA (cDNA) using the StarScript III One-Step RT-PCR Kit (GenStar, Beijing, China) following the manufacturer’s instructions. Subsequently, SYBR Green PCR Master Mix (Vazyme) and Applied Bio-systems 7500 Real-Time PCR System (Thermo Fisher, USA) were employed for qPCR assay. Three independent assays were performed to collect data and Actin was used as the endogenous control. All primers were synthesized by Bioengineering Co., Ltd. (Shanghai).

Cell counting kit-8 (CCK-8) assay

According to the product specification, the CCK-8 kit (Biosharp, China) was used to assess the proliferation abilities of PC3 and DU145 cells following the manufacturer’s instructions. In brief, PC3 and DU145 cells were inoculated into 96-well plates (2000 cells per well), and 10 μL of CCK-8 reagent was added to each well at the specified time points (0, 24, 48, 72, 96h) for 1 h incubation in the dark. Finally, the absorbance at a wavelength of 450nm was measured using an SYNERGY Microplate Reader (BioTek).

Nude mouse xenograft model

In the animal experiments, 12 nude mice were randomly divided into two groups. Subcutaneous tumor growth assays were performed using vector or FTO DU145 cells (5×10⁶ cells per mouse). The control group (n = 6) and the treatment group (n = 6) were injected with 100 μL of PBS supplemented with 100 μL of Matrigel (Yeassen). NSG mice (n = 7) were subjected to subcutaneous tumor growth experiments by injecting vector or FTO DU145 cells (5×10⁶) on the respective sides of their back. Tumor growth was monitored weekly by measuring tumor size from the outside of the mouse skin. The volume was calculated using the formula V = 1/2 × larger diameter × (smaller diameter)2. Eight weeks later, the tumors were harvested and weighed following standard processes approved by the institution. Tumor samples were paraffin-fixed and prepared for immunohistochemistry analysis.

Western blotting assay

Western blotting was performed as described in a previous study.³⁸ The antibodies used were as follows: FTO (1/1000, ab124892, Abcam, USA), H2AX (1/1000, AP0099, ABclonal, USA), FOXO3a (1/1000, 12829S, Cell Signaling Technology). The level of protein expression was detected by Clarity Western ECL Substrate (Bio-Rad, USA) using a Bio-Imaging System (Bio-Rad, USA).

Colony formation

Transfected PC3 and DU145 cells were incubated for an appropriate duration to allow colony formation. Subsequently, the colonies were fixed in 4% paraformaldehyde and then dyed with crystal violet. Each experiment was independently replicated three times.

Cell cycle analysis

Cells were harvested and stained with 400 μL of propidium iodide (PI). Cell cycle analysis was performed using a flow cytometer (ID7000 Spectral Cell Analyzer, SONY, Japan) according to the manufacturer’s instructions of the Cell Cycle and Apoptosis Analysis Kit (C1052M, Beyotime, China).

Cell apoptosis assay

The apoptotic cells were detected using the Cell Apoptosis Analysis Kit (C1062M, Beyotime). Cells were harvested and stained with PI and Annexin V-FITC and analyzed by flow cytometer (ID7000 Spectral Cell Analyzer, SONY, Japan).

Measurement of mRNA stability

FTO stable knockdown and overexpression cell lines, as well as control groups, were treated with the transcription inhibitor actinomycin D (2 μg/mL) for the indicated durations before cell collection. Total RNA was isolated using TRIzol, and RT-qPCR was performed to quantify the relative level of target mRNAs. The 0 h time point was used as an internal control.

RNA m⁶A dot blot

Total RNA was extracted using TRIzol reagent and mRNA isolation was performed with the BeyoMag mRNA Purification Kit using Magnetic Beads (R0071S, Beyotime) following the manufacturer’s instructions. Briefly, total RNA is mixed with BeyoMag Oligo (dT) 25 magnetic beads, the oligo(dT) sequences on the surface of the beads specifically bind to the poly(A) tails at the 3′ end of mRNA via base pairing. Subsequently, under the influence of an external magnetic field, the beads can be separated from the corresponding solution. After thorough washing to remove impurities, mRNA is eluted from the beads using elution buffer, resulting in obtaining high-purity intact mRNA. For dot blot analysis, mRNA was denatured with 2× RNA denaturation buffer at 65°C for 5 min and added to 20× SSC (S1030, Solarbio) before being immediately placed on ice. Next, the nylon membrane was activated with 10×SSC and completely dried before the RNA spotting. Subsequently, 1.5μL of denatured RNA was added to an activated nylon membrane and cross-linked with UV light for 6 min. Following cross-linking, the membrane was stained with methylene blue as a loading control. After washing with water three times, the membrane was blocked with 5% fat-free milk buffer for 1 h at room temperature and then incubated with the m⁶A antibody (56593, CST) overnight at 4°C. After washing three times, the membrane was exposed to horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The secondary antibody was washed with TBST (Tris-buffered saline with Tween) and the membranes were detected using the Bio-Rad ChemiDoc Touch Imaging System.

Quantification and statistical analysis

Statistical analysis was conducted using GraphPad Prism v. 9.00 (GraphPad Software, La Jolla, CA, USA). Spearman correlation analysis was employed to assess the relationship between FTO and FOXO3a. Two-tailed unpaired Student’s t test or nonparametric Mann-Whitney U test was utilized for comparing two groups and analysis of variance (ANOVA) was used to compare differences among multiple groups. Experiments were repeated at least three times and the data are presented as mean ± SD. A significance threshold of p < 0.05 was applied. Significance was set at p < 0.05 (∗), <0.01 (∗∗), or <0.001 (∗∗∗).

Published: July 14, 2024