ALKBH5-HOXA10 loop-mediated JAK2 m6A demethylation and cisplatin resistance in epithelial ovarian cancer

Abstract

Background: Chemotherapy resistance remains a barrier to improving the prognosis of epithelial ovarian cancer (EOC). ALKBH5 has recently been shown to be one of the RNA N6-methyladenosine (m6A) demethyltransferases associated with various cancers, but its role in cancer therapeutic resistance remains unclear. This study aimed to investigate the role of AlkB homolog 5 (ALKBH5) in cisplatin-resistant EOC.

Methods: Functional assays were performed both in vitro and in vivo. RNA sequencing (RNA-seq), m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq), chromatin immunoprecipitation, RNA immunoprecipitation, and luciferase reporter and actinomycin-D assays were performed to investigate RNA/RNA interaction and m6A modification of the ALKBH5-HOXA10 loop.

Results: ALKBH5 was upregulated in cisplatin-resistant EOC and promoted cancer cell cisplatin resistance both in vivo and in vitro. Notably, HOXA10 formed a loop with ALKBH5 and was found to be the upstream transcription factor of ALKBH5. HOXA10 overexpression also facilitated EOC cell chemoresistance both in vivo and in vitro. Collective results of MeRIP-seq and RNA-seq showed that JAK2 is the m6A-modified gene targeted by ALKBH5. The JAK2/STAT3 signaling pathway was activated by overexpression of the ALKBH5-HOXA10 loop, resulting in EOC chemoresistance. Cell sensitivity to cisplatin was rescued by ALKBH5 and HOXA10 knockdown or inhibition of the JAK2/STAT3 signaling pathway in EOC cells overexpressing ALKBH5-HOXA10.

Conclusions: The ALKBH5-HOXA10 loop jointly activates the JAK2/STAT3 signaling pathway by mediating JAK2 m6A demethylation, promoting EOC resistance to cisplatin. Thus, inhibition of the expression of the ALKBH5-HOXA10 loop may be a potential strategy to overcome cisplatin resistance in EOC.

Keywords: ALKBH5; Cisplatin resistance; Epithelial ovarian cancer; N6-methyladenosine.

Fig. 1

ALKBH5 overexpression promotes EOC cell proliferation and resistance to cisplatin in vitro and in vivo. (a) ALKBH5 mRNA expression is up-regulated in cisplatin-resistant EOC cells (upper); ALKBH5 mRNA expression is up-regulated in platinum-resistan EOC samples (down). (b) Results of the western-blot assays shows ALKBH5 was up-regulated both in cisplatin-resistant EOC cells (upper) and platinum-resistant EOC samples (down). (c) Expression of ALKBH5 protein in platinum-resistant (left) and platinum-sensitive (right) EOC tissues by IHC assay. (d) The transfection efficiency of oe-ALKBH5 lentivirus in A2780 and HO8910. (e and f) CCK8 and EdU proliferation assays confirm that ALKBH5 overexpression promotes cell proliferation in EOC cells. (g) The chemosensitivity assay shows that the IC50 of cisplatin is higher in EOC cells with ALKBH5 overexpression. (h) The IF assay shows that γH2AX expression is reduced in EOC cells with ALKBH5 overexpression after cisplatin treatment (5 μM, 6 h). (i) The cell cycle assays show that ALKBH5 overexpression can attenuate blocking in the G2/M phase induced by cisplatin (5 μM, 48 h). (j) The apoptosis assay shows that ALKBH5 overexpression can decrease the percentage of cell apoptosis induced by cisplatin (5 μM, 48 h). (k) The animal study shows that ALKBH5 overexpression promotes tumor growth and chemoresistance to cisplatin in vivo

Fig. 2

ALKBH5-HOXA10 loop maintains ALKBH5 and HOXA10 overexpression in EOC. (a) The results of the RIP-qPCR assay show that HOXA10 mRNA is enriched in Flag-specific antibody in EOC cells on transfection with oe-ALKBH5 using Flag-tag. (b) The Act-D assay shows that ALKBH5 overexpression can maintain HOXA10 mRNA stability in EOC cells. (c and d) ALKBH5 regulates HOXA10 expression in EOC. (e) The ChIP-qPCR assay suggests that the ALKBH5 promoter fragment is enriched in HOXA10 (top), and the AGE assay validates the sonicated products and immunoprecipitated DNA of the ChIP assay (middle). EOC cells are transfected with HOXA10-Flag-tag (bottom). (f) Based on the TF binding motif of HOXA10 predicted by the JASPAR database (top), the luciferase reporter assay is conducted, and the results show that HOXA10 could interact with the region containing TAAA of ALKBH5 promoter (bottom). (g) The transfection efficiency of oe-HOXA10 lentivirus in A2780 and HO8910. (h) ALKBH5 is up-regulated in EOC cells with HOXA10 overexpression. (i) The transfection efficiency of specific siRNAs targeting HOXA10. (j) ALKBH5 expression is down-regulated by knocking down HOXA10 in EOC cells

Fig. 3

HOXA10 overexpression promotes EOC cell proliferation and cisplatin resistance in vitro and in vivo. (a) HOXA10 mRNA is upregulated in cisplatin-resistant EOC cells and platinum-resistant EOC samples. (b) Results of the western-blot assays shows HOXA10 was up-regulated both in cisplatin-resistant EOC cells (upper) and platinum-resistant EOC samples (down). (c) Expression of HOXA10 protein in platinum-resistant (left) and platinum-sensitive (right) EOC tissues by IHC assay. (d and e) CCK8 and EdU proliferation assays confirm that HOXA10 overexpression promotes cell proliferation in EOC cells. (f) The chemosensitivity assay demonstrates higher IC50 of cisplatin in EOC cells with oe-HOXA10 transfection. (g) The IF assay shows that γH2AX foci is decreased in EOC cells with HOXA10 overexpression after cisplatin treatment (5 μM, 6 h). (h) The cell cycle assays show that HOXA10 overexpression can alleviate the blocking in the G2/M phase induced by cisplatin (5 μM 48 h). (i) The apoptosis assays show that HOXA10 overexpression can lower the percentage of cell apoptosis induced by cisplatin (5 μM, 48 h). (j) The animal study shows that HOXA10 overexpression promotes cell proliferation and resistance to cisplatin in vivo

Fig. 4

ALKBH5 erases m6A modifications of JAK2 mRNA and maintains JAK2 mRNA expression by lowering YTHDF2-mediated mRNA degradation. (a) The two differentially enriched m6A-modification motif in the immunopurified RNA in A2780. (b) Distribution of regulated m6A peaks in mRNA is detected by MeRIP-seq after ALKBH5 overexpression. (c) Schematic of downstream analysis for MeRIP-Seq and RNA-seq. (d) The quadrantal diagram graph displays the transcripts with different m6A peaks and regulated gene expression based on MeRIP-seq and RNA-seq. (e) The Venn diagram shows the genes detected by MeRIP-seq and RNA-seq; the 19 candidate target genes of ALKBH5 are shown on the right. (f) m6A abundances in JAK2 mRNA transcripts in cells with ALKBH5 overexpression (MeRIP and input) and in negative control (MeRIP and input). m6A regulation is calculated as the ratio of m6A abundances of MeRIP to input (log2FC = − 2, p = 0.03). (g) MeRIP-qPCR confirms that ALKBH5 down-regulates the m6A peak in 3’UTR of JAK2 mRNA. (h) RIP-qPCR confirmed JAK2 mRNA binding to ALKBH5. (i) The Act-D assay shows an increased lifespan of JAK2 mRNA after ALKBH5 overexpression. (j) Relative luciferase activity of the wild-type or mutant JAK2 3′UTR luciferase reporter in EOC cells with ALKBH5 overexpression and the negative control. (k) ALKBH5 positively regulates JAK2 mRNA expression in EOC cells. (l and m) YTHDF2 remarkably regulates JAK2 expression in A2780 and HO8910. (n) RIP-qPCR confirms YTHDF2 binding to JAK2 mRNA. (o) Increased lifespan of JAK2 mRNA after YTHDF2 silencing

Fig. 5

ALKBH5-HOXA10 loop steadily promotes cell proliferation and resistance to cisplatin. (a and b) CCK8 and EdU proliferation assays show that using specific siRNAs to respectively knock down HOXA10 or ALKBH5 expression in EOC cells with ALKBH5 or HOXA10 expression could significantly suppress cancer cell proliferation. (c and d) HOXA10 or ALKBH5 knockdown rescues resistance to cisplatin and DDR in EOC cells with ALKBH5- HOXA10 loop overexpression

Fig. 6

ALKBH5-HOXA10 loop overexpression activates the JAK2/STAT3 signaling pathway. (a) Upregulation of the ALKBH5-HOXA10 loop promotes JAK2 expression and the phosphorylation level of STAT3. (b) Downregulation of the ALKBH5-HOXA10 loop inhibits JAK2 expression and the phosphorylation level of STAT3. (c) Activation of the JAK2/STAT3 pathway can be rescued by knocking down ALKBH5 or HOXA10 respectively in cells with HOXA10 and ALKBH5 overexpression. (d and e) The IHC assays show ALKBH/HOXA10/JAK2 regulation in the xenograft model

Fig. 7

Graphic abstract. Overview of the present study