The m6A demethylases FTO and ALKBH5 aggravate the malignant progression of nasopharyngeal carcinoma by coregulating ARHGAP35

Abstract

N⁶-methyladenosine (m⁶A) is an RNA modification that can be removed by demethylases [fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5)], which regulate gene expression and cell function. We show that m⁶A levels and m6A demethylase levels are altered in nasopharyngeal carcinoma (NPC) tissues vs. normal tissues. High FTO and ALKBH5 predict a poor prognosis in NPC patients. Silencing FTO and ALKBH5 inhibited the malignant behavior of patient-derived NPC cells in a short time. However, as time progressed, the inhibitory effect of FTO or ALKBH5 was weakened, and the cosilencing of FTO and ALKBH5 maintained a better inhibitory effect. Combined transcriptome and m⁶A-seq analysis revealed a downstream target gene that was jointly regulated by FTO and ALKBH5 in NPC, and ARHGAP35 was chosen to do further study. The synergistic silencing of FTO and ALKBH5 increased the methylation level on the mRNA CDS of a new transcription factor (ARHGAP35) and positively regulate the protein coding capacity and mRNA stability of ARHGAP35, thus leading to increased expression of ARHGAP35 and inhibition of the malignant phenotype of tumor cells. Our study revealed that the growth and metastasis of NPC can be stably inhibited through synergistic silencing of the demethylases FTO and ALKBH5, which play a positive role in the treatment of NPC by regulating the downstream transcript ARHGAP35 and increasing its m⁶A level.

Fig. 1. Expression and clinical significance of m⁶A and its demethylase in NPC.

A The representative images of m⁶A dot blot detection showed the overall abundance of m⁶A in NPC patients and normal human tissues. (Methylene blue staining: as the control group. NC: normal people, NPC: NPC patients, n = 3) B The content of m⁶A in total RNA of NPC patients and normal people (the same as A). C IHC staining of representative FTO and ALKBH5 of normal tissue, scale: 100 μm. D IHC staining of representative FTO and ALKBH5 of NPC tissue microarray, scale: 100 μm. E, F The expression of FTO and ALKBH5 in different clinical stages were statistically compared. Single factor analysis of variance. G Spearman correlation analysis of FTO score and ALKBH5 score. H–J Kaplan-Meier analysis then used log-rank test to compare the overall survival rate. All experiments were performed with 3 independent repetitions. All figures show the average ± SEM of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2. Low expression of FTO or ALKBH5 synergistically inhibits the progression of NPC in vivo.

A–F The stably silenced cells of shFTO, shALKBH5, shFTO+shALKBH5 or control group of CNE2, C666-1 cells were transplanted subcutaneously into nude mice (n = 5 for each group). On the 16th day, the subcutaneous tumor volume was displayed. The graft volume was measured every 4 days after the tumor was transplanted subcutaneously in nude mice, and the final volume of the removed graft was measured. Single factor analysis of variance **p < 0.01, ****p < 0.0001, one-way ANOVA. G–J immunohistochemistry (IHC) was used to analyze the expression of Ki67 and Cleaned Caspase3 among tumor groups. Scale: 50 μm. (****p < 0.001, one-way ANOVA) K–N The visual and quantitative analysis of lung metastasis in BALB/c mice. by one-way ANOVA. (*p < 0.05, one-way ANOVA). O, P Representative image of lung tissue stained with HE. Scale: 100 μm.

Fig. 3. Characteristics of m⁶A modification and gene expression changes in FTO and ALKBH5 deficient cells.

A There is a top-level consistent motif with m⁶A seq peak in CNE2 cells of shFTO, shALKBH5, shFTO+shALKBH5. B The number of m⁶A peaks found in m⁶A-seq in CNE2 cells of shCtrl, shFTO, shALKBH5 and shFTO+shALKBH5. C The number of m⁶A-modified genes identified in m⁶A-seq. D The distribution map of m⁶A peaks shows the proportion of m⁶A peaks specific to the designated area in shCtrl, shFTO, shALKBH5, shFTO+shALKBH5 cells. E Downstream analysis diagram of FTO and ALKBH5 joint regulation. F Positioning of the downstream of the joint regulation of FTO and ALKBH5 in the 1/4 quadrant diagram.

Fig. 4. The synergistic down-regulation of FTO and ALKBH5 affects the expression and clinical significance of ARHGAP35.

A Western blot of ARHGAP35 protein in CNE2 and C666-1 knockdown by FTO and ALKBH5 respectively or jointly. B RT-qPCR was used to analyze the expression of ARHGAP35 mRNA in CNE2 and C666-1 before and after FTO and ALKBH5 were respectively or jointly knocked down. The sample was normalized to GAPDH mRNA. one-way ANOVA, ****p < 0.0001. C IHC staining of representative ARHGAP35 of normal tissue, scale: 100 μm. D IHC staining of representative ARHGAP35 of NPC tissue microarray, scale: 100 μm. E The expression of ARHGAP35 in different clinical stages was statistically compared. Single factor analysis of variance. F, G Spearman correlation analysis of FTO score and ARHGAP35 score, ALKBH5 score and ARHGAP35 score. H Spearman correlation analysis of FTO score, ALKBH5 score and ARHGAP35 score in xyz-three-dimensional coordinate system. I The Kaplan-Meier survival time of NPC patients with ARHGAP35 was analyzed. J The Kaplan-Meier survival with differential gene expression of the three biomarkers was assessed. The scale bar in the images represents 100μm. Mean ± SD, ****p < 0.0001, one-way ANOVA.

Fig. 5. It promotes the stability of ARHGAP35 mRNA and translation by jointly downregulating FTO and ALKBH5 related m⁶A modification.

A The m⁶A peak showed the relative abundance of the m⁶A sites on ARHGAP35 mRNA in shFTO+shALKBH5 CNE2 cells. B Detection of m⁶A enrichment in endogenous ARHGAP35 mRNA in NPC cells. Using MeRIP method, quantitative RT-PCR was used to detect the ARHGAP35 mRNA level in the same number of NPC cells with and without FTO, ALKBH5 combined knockdown. *p < 0.05, student’s test. C, D The stability of ARHGAP35 mRNA in CNE2 and C666-1 cells was determined by RT-qPCR after treatment with transcription inhibitor: actinomycin D (ActD). GADPH is used as internal parameter. The data were expressed as the average ± SD value of the three biological repeats. *p < 0.05, **p < 0.01, and ***p < 0.001, student’s test. E–J NPC cells were pre-transfected with vector control or FTO construct for 24 h and then further treated with CHX (10 μg/ml) or MG-132 (5 μM) for 6 h, the expression of ARHGAP35 was detected by western blot analysis (up) and quantitatively analyzed (down). K, L Western Blot assay was used to detect the protein expression of ARHGAP35 in CNE2 and C666-1 cells treated with cycloheximide (CHX) for the indicated time periods (left) and quantitatively analyzed (right).

Fig. 6. Overexpression of ARHGAP35 inhibits the growth and metastasis of NPC in vivo.

A, D The corresponding images of xenogeneic tumor grafts. B, E After being injected with NPC cells, the tumor volume of the two groups was compared. (***p < 0.001, ****p < 0.0001, one-way ANOVA). C, F The volume of xenograft removed. (***p < 0.001, ****p < 0.0001, one-way ANOVA). G, I Representative images of Ki67 and Cleaned Caspase3 IHC in xenograft tissues of NPC in two groups. H, J IHC staining score of Ki67 and Cleaned Caspase3 expression. (****p < 0.0001, one-way ANOVA). K–N The lung metastasis in BALB/c mice have been visualized and quantitative analyzed. (**p < 0.01, one-way ANOVA). O, P HE staining image of metastatic lung tissue. Scale: 100 μm.

Fig. 7. In vivo experiment verifies that FTO/ALKBH5-ARHGAP35 signal axis promotes the malignant behavior of NPC.

A, C Images of subcutaneous xenograft tumor in BALB/c mice. B, D The volume of xenografts removed by surgery. (*p < 0.0001, one-way ANOVA). E–H representative image and staining score of Ki67 and Cleaned Caspase3 after IHC staining of xenograft. (****p < 0.0001, one-way ANOVA). I–L Visualization and analysis of mouse lung metastasis model. (*p < 0.05, **p < 0.01, one-way ANOVA) (M, N) HE staining image of metastatic tumor of lung tissue. Scale: 100 μm.

Fig. 8

Schematic diagram of the effect of FTO and ALKBH5 on promoting the malignant phenotype of NPC by negatively regulating the m⁶A modification level.