Sensitivity to ferroptosis was associated with m6A modification level in HNSCC.
RSL3 is an activator of ferroptosis, which inhibits the activity of GPX4 by covalent bonding with GPX4 and leads to lipid peroxides accumulation22. Firstly, we performed different HNSCC cell lines exposed to RSL3 with various concentrations for 24 hours. AlamarBlueassay and LDH release assay showed that sensitivity to RSL3 treatment differed considerably between the various HNSCC cell lines. RSL3 reduced the cellviability of HPSCC (Detroit 562 and FaDu cells)(Fig.1a, b)and colonyformation(Fig.S1a)with the sensitivity greatly decreased inOSCC (SCC9 and SCC25) cells.Cell death induced in HPSCC cells by RSL3 treatment was reversed by co-treatment with a ferroptosisinhibitor liproxstatin-1(Fig.S1b, c).
Meanwhile, RSL3 induced cellular lipid peroxidation (lipid ROS) levels to varying degrees among HNSCC cell lines.Changes in cellular lipid ROS measured by BODIPY C11 after RSL3
treatment showed a similar trend in these cell lines(Fig.1c).RSL3 reduced the expression of GPX4 protein in a dose-dependent manner in Detroit 562 HPSCC cell, but not in SCC25 cells(Fig.1d).
To explore the correlation between m6A modificationswith ferroptosis in HNSCC,m6A dot blot experiments and m6A RNA Methylation Quantification were performed. The results showed that the m6A modification level was significantly decreased in HPSCC cells which showed higher sensitivity to ferroptosis) (Fig.1e, f).Furthermore, qRT-PCR analysis was performed to compare theregulated- m6A gene expression profiles in HPSCC cells, SCC9 and SCC25. In line with m6A modification level lower in HPSCC , we observed that there was a most significant higher ALKBH5 expression in HPSCC (Fig.1g). It implies that dysregulation of ALKBH5 may be involved in ferroptosis of HPSCC.
Inhibition of ALKBH5 expression decrease sensitivity toferroptosis in HPSCC.
HPSCC cells were treated with the lentivirus encoding a short hairpin RNA specific for ALKBH5.The transfection efficiency was validated by qRT-PCR and western blotting (Fig. S2a, b). A noticeable increase in m6A levels was observed in polyadenylated RNAs (poly(A) RNAs) of the ALKBH5-knockdown cells, compared to the wild-type (WT)HPSCC cells, asmeasured by m6A dot blot (Fig. 2a) and EpiQuik™ m6A quantificationassay (Fig. S2c).
To elucidate whether ALKBH5 plays a role in HPSCC ferroptosis, we performed western blotting to compare the novel ferroptosis-related gene expression of ALKBH5-knockdown Detroit 562 cells and control cells. Knocking down ALKBH5 remarkedly increased the expression of GPX4, HO-1and NRF2 protein in shALKBH5 cells, but not in control cells, along with the decreased expression of TFRCand Keap1 protein(Fig. 2b). Immunofluorescence staining showed higher positivity of GPX4 in shALKBH5 cells with RSL3 treatment, compared with control cells (Fig. 2c). Inhibition of the ALKBH5 gene significantly reversed RSL3-induced cell death and cellular lipid ROS levels (Fig. 2d-f). Along with that, using electron microscopy we observed much more shrunken mitochondria in shCON cells than shALKBH5 FaDu cells with RSL3 treatment (Fig. 2g). These data suggested that ALKBH5 positively regulates RSL3-induced ferroptosis.
The overexpression of ALKBH5 sensitized HPSCC cells to RSL3 treatment in vitro and vivo
We next examined the effect of ALKBH5 overexpression on cell growth and viability of HPSCC cells. The transfection efficiency was validated by qRT-PCR and western blotting(Fig. S2d). Inhibition of NRF2 and GPX4 could be induced by the ALKBH5 transfection. When the ALKBH5 gene was overexpressed by plasmid transfection, NRF2 and GPX4 expression decreased in HPSCC with or without RSL3 treatment (Fig.3a). Meanwhile, ALKBH5 overexpression inhibitsthe growth and cell viability of HPSCC cells (Fig.3b-d). The cell death was enhanced by a combination of RSL3 with ALKBH5 plasmid transfection (Fig.3b-d). To clarify the effects of ALKBH5 on HPSCCin vivo, ALKBH5-overexpressing and negative control FaDu cells were implanted into BALB/c mice via subcutaneous injection.RSL3 treatment reduced HPSCC tumor growth and tumor weight in xenograft mouse models (Fig. 3e, f).In addition, overexpression of ALKBH5 significantly enhanced the RSL3-inducded tumor suppression. The data strongly suggests that ALKBH5 may be a tumor-suppress gene and mediate ferroptosis in HPSCC.
MeRIP-seq combined with RNA-seq identified potential targets of ALKBH5.
To identify potential mRNA targets of ALKBH5 whose m6A levels were decreased by ALKBH5 in HPSCC cells, ALKBH5-knockdown and control FaDu cells were selected for transcriptome-wide m6A-sequencing (m6A-seq, MeRIP-seq) and RNA-sequencing (RNA-seq) assays. Principal component analysis (PCA) showed that two repeats (shCON: shCON_1 and shCON_2; shALKBH5: shK5_1 and shK5_2) of each sample clustered together, suggesting good repeatability among the two replicates of each group (Fig. S3a). Consisted with the previous research, the most common m6A motif“GGAC” is significantly enriched in the m6A peaks (Fig. 4a).RNA-seq uncovered 548 down-regulated and 564 up-regulated transcripts (p < 0.05) upon ALKBH5 overexpression(Fig. S3b). Peaks were located in protein-coding transcripts and enriched in the 5’UTR and 3′UTR, especially near stop codons, which was coincidence with the m6A distribution(Fig. 4b, c).
We next compared the genes with altered-m6A modifications between the control and ALKBH5-knockdown cells. The analysis of m6A-seq revealed a global hypermethylation of m6A in the transcription level after knockdown of ALKBH5 in FaDu cells (Fig. 4d). Considering the role of ALKBH5 in the m6A methyltransferase complex, mRNA transcripts carrying hypermethylated
m6A peaks in FaDu ALKBH5 knockdown cells were likely potential targets. Through analysis of the RNA-seq data, we identified 967hyper-methylated m6A genes whose mRNA transcripts were down-regulated (p < 0.05, Hyper-down) and 49 hyper-methylated m6A genes whose mRNA transcripts were up-regulated (p < 0.05, Hyper-up) in ALKBH5-knockdown cells, compared with shCON cells (Fig. 4e). GO enrichment and KEGG pathway analysis revealed that these hyper-methylated m6Agene transcripts (p < 0.05) were predominantly enriched in “protein and lipid binding”, “lipid metabolic process”, “membrane” , “endocytosis“ and “ferroptosis” (Fig. S3c). Moreover, GSEA showed that these genes are involved in response_to_oxidized_phospholipids, KEGG_oxidative_phosphorylation, missiaglia _regulated _by_methylation_up (Fig. S3d).It is consisting with the acknowledge that lipid peroxidation of long chain fatty acid and oxidative phosphorylation were correlated with cell membrane oxidative damage in ferroptosis process23-26.182 genes involved in ferroptosis and lipid oxidation andoxidoreductase activity were selected for further validation. Six genes STEAP3, AIFM2, NFE2L2/NRF2, FH(Fumarate hydratase), ZNRF2, PEBP1 whose alterations suppress ferroptosis were finally selected as candidate targets of ALKBH5(Fig. 4f).
Further qRT-PCR analyses showed the mRNA levels in NFE2L2/NRF2 genes were dramatically increased in ALKBH5-knockdown FaDu cells (Fig. 4g).The Integrative Genomics View (IGV) data peak revealed m6A peak enrichment in the 5′-UTR of NFE2L2/NRF2 mRNA that was diminished upon ALKBH5 knockdown (Fig. 4h, i).Consistent with our results, previously reported m6A, RIP and CLIP data from the m6A2Target Database (http://m6a2target.canceromics.org/#/search/TFR) identified NFE2L2/NRF2 mRNA as a potential target of ALKBH5 (GSE87515).
NFE2L2/NRF2 plays the crucial role in the regulation of cellular antioxidant molecules. It controls cellular antioxidant systems in cancer cells, playing a key role in protecting against intracellular and environmental stress27, 28. NFE2L2/NRF2 is constantly degraded by Keap1 and is activated by the inhibition of Keap129, 30. Ferroptosis could be a result of aberrant NRF2 signaling31. Inhibition of NFE2L2/NRF2 and the AREs HO-1, FTH1, and NQO1 in HNCcells has been shown to significantly enhance the antitumor activity of ferroptosis inducers32, 33. Furthermore, accumulation of the NFE2L2/NRF2-ARE proteins during ferroptosis reduced the sensitivity of head and neck cells to the GPX4 inhibitors RSL3 and ML-16234. Taken together, NFE2L2/NRF2 may be the direct downstream target of ALKBH5.
ALKBH5 abolishes NFE2L2/NRF2mRNA levels and stability depending on its m6A methyltransferase activity.
MeRIP-qPCR assays with specific primers aiming at potential m6A sites was then applied to confirm the ALKBH5-mediated m6A demethylation of NFE2L2/NRF2mRNAin 3’ UTR. When compared to the IgG group (pulldown control), an enrichment of NFE2L2/NRF2 mRNA in 3’ UTR was obtained by the reaction to m6A-specific antibody (Fig. 5a).Western blotting and qRT-PCR was then applied to confirm NFE2L2/NRF2expression was upregulated in ALKBH5-knockdown FaDu cells(Fig. S4a).
To elucidate whether the ALKBH5 demethyltransferase function decreased NFE2L2/NRF2 expression, ALKBH5 mutant (ALKBH5-mut, NM_017758, H204A) recombination plasmids was constructed. Then we measured m6A levels of NFE2L2/NRF2 after transfection of control, ALKBH5-WT and ALKBH5-mut plasmids, The MeRIP-qPCR data showed that overexpression of ALKBH5-WT, but not ALKBH5-mut, dramatically led to a decreased m6A levelsof NFE2L2/NRF2 in Detroit562 cells (Fig. 5b).In addition, overexpression of ALKBH5, but not ALKBH5-mut dramatically decreased NFE2L2/NRF2expression (Fig. S4b).
Thereafter, to address the effect of m6A modification on NFE2L2/NRF2 gene,we constructed both wild-type and mutant NFE2L2/NRF2 luciferase reporter plasmids, whichN6-methylated adenosine (A)was replaced with C (cytosine) ofNFE2L2/NRF2 mRNA that have intact m6A sites (Fig. 4i, Fig. 5c, Fig. S4c).As expected, compared with shCON, ALKBH5-knockdown substantially increased luciferase activity of the individual reporter (Firefly/Renilla) constructs containing wild-type NFE2L2/NRF2 3’UTR. However, ALKBH5-knockdown had no such significant effect on the NFE2L2/NRF2 -Mut luciferase reporter plasmid in Detroit 562 and FaDu cells (Fig. 5d). Furthermore, overexpression of ALKBH5, but not ALKBH5 mutant, significantly reduced luciferase activity of individual reporter (Firefly/Renilla) constructs containing wild-type NFE2L2/NRF23’ UTR (Fig. 5e). Overexpression of ALKBH5 and ALKBH5-mut had no such significant effect on NFE2L2/NRF23’ UTR luciferase reporterplasmids (Fig. 5e).
The m6A-modification is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate; insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3), to promote the stability and storage of their target mRNAs in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output35. To analyze the effect of m6A modification on the stability of ALKBH5 target transcripts, we conducted RNA stability assays. The RNA stability curves showed that knockdown of ALKBH5 prolong the half-life of NFE2L2/NRF2 mRNA in HPSCC cells (Fig. 5f). As YTHDF2, YTHDF3 and YTHDC2 are responsible for mRNA decay, they were hardly the potential effectors participating in the current process.Nevertheless, qRT-PCR data showed that knockdown of IGF2BP2, but not IGF2BP1/3 significantly reduced the mRNA level of NFE2L2/NRF2 in HPSCC cells(Fig. S4d,e). Furthermore, the half-life of NFE2L2/NRF2 mRNA was significantly decreased when IGF2BP1 was impaired (Fig. S4f). Notably, NFE2L2/NRF2 expression was positively correlated with the IGF2BP2 level in the TCGA HNSCC dataset (Fig. S4g)36. RIP- assay showed that IGF2BP2 directly bound to the 3’ UTR of NFE2L2/NRF2 mRNA (Fig. 5g). RIP-PCR indicated that knockdown of ALKBH5 significantly enhanced the binding efficiency of IGF2BP2 to the 3’ UTR of NFE2L2/NRF2 mRNA(Fig. 5h).In addition, overexpression of ALKBH5-WTbut not ALKBH5-Mut significantly reduced the binding efficiency of IGF2BP2 to the 3’ UTR of NFE2L2/NRF2 mRNA (Fig. 5i).
NFE2L2/NRF2 is functionally essential target gene of ALKBH5 in HPSCC.
We next performed rescue-experiments to illustrate whether NFE2L2/NRF2 participated in the biological function of ALKBH5 in HPSCC. Detroit 562 control and ALKBH5-knockdown cells were transfected with control and siNFE2L2/NRF2 (siNRF2), while FaDu control and ALKBH5-knockdown cells were transfected with control and NFE2L2/NRF2-WT plasmids. The transfection efficiency of were confirmedby qRT-PCR and Western blot analysis (Fig. S5a, b). NFE2L2/NRF2-overexpression downregulated protein levels of GPX4 and FTH1 of ALKBH5-knockdowncells (Fig. S5c). Cell viability assay, LDH release assay and Colonyformation assays indicated that silencing of NFE2L2/NRF2 impairedcell growth and viability of ALKBH5-knockdown cells (Fig. 6a-c). Silencing of NFE2L2/NRF2 also significantly increased cellular lipid ROS levelsof ALKBH5-knockdown cells (Fig. 6d). NFE2L2/NRF2 overexpression also led to significantly restored cell growth and viability of ALKBH5-overexpression cells (Fig. S5d, e). These results strongly indicated that NFE2L2/NRF2 is a critical target gene of ALKBH5 in HPSCC cells. Inhibition of GPX4 renders HNC cells susceptible to ferroptosis, while activation of the Nrf2–ARE system caused the resistance to GPX4 inhibition22,34. Both the light chain and heavy chain of ferritin (FTL/FTH1), stores iron in ferritin, as well as ferroportin (SLC40A1), which is responsible for iron efflux out of the cell, are controlled by NRF237, 38. Furthermore, western blot analysis showed that NFE2L2/NRF2 silencing restored the protein levels of GPX4 and FTH1 of ALKBH5-knockdown cells (Fig. 6e). Analysis of TCGA HNSCC datasets also revealed high GPX4 levels positively correlated with NFE2L2/NRF2 expression (Fig. S5f)36.
So far, we have proved that ALKBH5 may function as a tumor suppresser through ferroptosis by abolishing NFE2L2/NRF2 mRNA stability and expression in HPSCC cells. Several small molecular NRF2 inhibitors have been developed as anti-tumor drug candidates39, 40.We treated the mice baring shALKBH5 and control xenograft tumor with one NRF2 inhibitor-ML38541, 42. ML385 is efficient to inhibit tumor growth and tumor weight in nude mice bearing shALKBH5 cells (Fig. 6f-h). We next performed IHC analyses of HPSCC and paracancerous tissues from patients, to further explore the correlation between expression of ALKBH5 and NFE2L2/NRF2 in HPSCC tissues. As expected, ALKBH5 negatively interrelated with NFE2L2/NRF2 expression (Fig. 6i, j). Taken together, the data show that NFE2L2/NRF2 mediates the ferroptosis regulation of ALKBH5 in HPSCC cells.