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. 2024 Oct:56:101034.
doi: 10.1016/j.neo.2024.101034. Epub 2024 Aug 10.

LncRNA HOTAIRM1 promotes radioresistance in nasopharyngeal carcinoma by modulating FTO acetylation-dependent alternative splicing of CD44

Jinglin Mi  1 , Yiru Wang  1 , Siyi He  1 , Xinling Qin  1 , Zhixun Li  1 , Tingting Zhang  1 , Weimei Huang  2 , Rensheng Wang  3
Affiliations

LncRNA HOTAIRM1 promotes radioresistance in nasopharyngeal carcinoma by modulating FTO acetylation-dependent alternative splicing of CD44

Jinglin Mi et al. Neoplasia. 2024 Oct.

Abstract

Background: Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC); however, almost 20% of patients experience treatment failure due to radioresistance. Therefore, understanding the mechanisms of radioresistance is imperative. HOTAIRM1 is deregulated in various human cancers, yet its role in NPC radioresistance are largely unclear.

Methods: This study investigated the association between HOTAIRM1 and radioresistance using CCK8, flow cytometry, and comet assays. Additionally, xenograft mice and patient-derived xenografts (PDX) models were employed to elucidate the biological functions of HOTAIRM1, and transcriptomic RNA sequencing was utilized to identify its target genes.

Results: Our study revealed an upregulation of HOTAIRM1 levels in radioresistant NPC cell lines and tissues. Furthermore, a positive correlation was noted between high HOTAIRM1 expression and increased NPC cell proliferation, reduced apoptosis, G2/M cell cycle arrest, and diminished cellular DNA damage following radiotherapy. HOTAIRM1 modulates the acetylation and stability of the FTO protein, and inhibiting FTO elevates the m6A methylation level of CD44 precursor transcripts in NPC cells. Additionally, silencing the m6A reading protein YTHDC1 was found to increase the expression of CD44V. HOTAIRM1 enhances NPC cell resistance to ferroptosis and irradiation through the HOTAIRM1-FTO-YTHDC1-CD44 axis. Mechanistically, HOTAIRM1 interacts with the FTO protein and induces m6A demethylation of the CD44 transcript. The absence of m6A modification in the CD44 transcript prevents its recognition by YTHDC1, resulting in the transition from CD44S to CD44V. An abundance of CD44V suppresses ferroptosis induced by irradiation and contributes to NPC radioresistance.

Conclusions: In conclusion, the results in this study support the idea that HOTAIRM1 stimulates CD44 alternative splicing via FTO-mediated demethylation, thereby attenuating ferroptosis induced by irradiation and promoting NPC radioresistance.

Keywords: Alternative splicing; Ferroptosis and radioresistance; HOTAIRM1; NPC.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author is not involved in the editorial review or the decision to publish this article.

Figures

Fig 1
Fig. 1
Elevated expression of HOTAIRM1 in radioresistant NPC. (A) RT‒qPCR analysis of HOTAIRM1 expression in radioresistant and radiosensitive NPC tissues. (B) Detection of HOTAIRM1 expression in radioresistant and radiosensitive NPC tissues using ISH. (C) Development of radioresistant NPC cell lines (C666-1R and HONE1R) employing a single-hit multitarget model. (D) Comparative analysis of HOTAIRM1 expression in radioresistant and radiosensitive NPC cell lines (C666-1R and HONE1R). * P < 0.05, ** P < 0.01. NPC: nasopharyngeal carcinoma. ISH: in situ hybridization.
Fig 2
Fig. 2
Impact of HOTAIRM1 knockdown on NPC radioresistance. (A, B) Verification of HOTAIRM1 silencing efficiency using RT‒qPCR. (C, D) Assessment of proliferation in C666-1R and HONE1R cells with HOTAIRM1 knockdown post 4 Gy irradiation using a CCK8 assay. (E, F) Analysis of apoptosis in C666-1R and HONE1R cells post 4 Gy irradiation with HOTAIRM1 knockdown using flow cytometry. (G, H) Examination of DNA damage in C666-1R and HONE1R cells post 4 Gy irradiation with HOTAIRM1 knockdown using comet assays. (I, J) Macroscopic images and measurements of xenograft tumor volumes in nude mice in shNC and shHOTAIRM1 groups following 20 Gy irradiation. (K) Evaluation of average xenograft tumor weights in each group. (L) RT‒qPCR analysis of HOTAIRM1 expression levels in xenograft tumors. (M) Tumor tissue analysis via HE staining and immunohistochemistry. * P < 0.05, ** P < 0.01, *** P < 0.001. NPC: nasopharyngeal carcinoma.
Fig 3
Fig. 3
HOTAIRM1 overexpression enhances NPC cell radioresistance. (A, B) Overexpression efficiency was confirmed using RT‒qPCR. (C, D) Proliferation of C666-1 and HONE1 cells, stably transfected with HOTAIRM1 and exposed to 4 Gy irradiation, was assessed using a CCK8 assay. (E, F) Apoptosis in C666-1 and HONE1 cells, stably transfected with HOTAIRM1 and exposed to 4 Gy irradiation, was evaluated via flow cytometry. (G) DNA damage in these cells post-irradiation was investigated using comet assays. (I, J) Macroscopic images and volumes of xenograft tumors in nude mice from the oeNC and oeHOTAIRM1 groups, exposed to 20 Gy radiotherapy, are presented. (K) Changes in the average weight of xenograft tumors in each group are shown. (L) RT‒qPCR assay was used to measure HOTAIRM1 expression in xenograft tumors. (M) Tumor tissue was analyzed by HE staining and immunohistochemical staining. * P < 0.05, ** P < 0.01, *** P < 0.001. NPC: nasopharyngeal carcinoma.
Fig 4
Fig. 4
HOTAIRM1 inhibition augments radiosensitivity in NPC PDX model. (A) Description of PDX model construction. (B) HE staining of PDX founder (P0) tumors and original patient-derived specimens. (C) Representative images of subcutaneous tumors from mice. (D) Growth curves of tumors from mice treated with ASO-Ctrl or ASO-HOTAIRM1 under 20 Gy radiotherapy. (E) Comparison of tumor weights between the two groups is provided. ** P < 0.01. NPC: nasopharyngeal carcinoma. PDX, patient-derived xenografts.
Fig 5
Fig. 5
HOTAIRM1 affects the acetylation and stability of the FTO protein. (A) RNA pull-down confirmed the physical interaction between HOTAIRM1 and FTO. (B) FTO protein half-life was examined by western blotting. (C, D) RIP assay and DNA gel electrophoresis demonstrated HOTAIRM1′s affinity for FTO. (E) FISH assay showed colocalization of HOTAIRM1 and FTO. (F) Interaction between truncated HOTAIRM1 and FTO was evaluated using Western blotting. (G) Western blot analysis of shHOTAIRM1 and shNC NPC cells co-transfected with wild type (WT) or mutant FTO. (H) Western blotting detected acetylation and SUMOylation of FTO protein in shHOTAIRM1 and shNC NPC cells. TAK-981, sumoylation inhibitor. A-485, acetylation inhibitor. FISH, fluorescence in situ hybridization.
Fig 6
Fig. 6
HOTAIRM1 and FTO modulate the alternative splicing of the CD44 transcript. (A) Transcriptome sequencing revealed that HOTAIRM1 silencing impacts alternative splicing events in C666-1R cells. (B) m6A peak density and frequency on CD44 transcripts base on GSE103496 datasets. (C) TCGA database analysis indicated that higher PSI rates of CD44 precursor transcripts correlate with worse HNSCC prognosis. (D) MeRIP-qPCR assay evaluated the m6A modification level in CD44T (total) mRNA. (E) RIP assay evaluated the CD44T mRNA enrichment of FTO protien. (F-H) RT‒qPCR and Western blotting assessed HOTAIRM1 and FTO's impact on CD44V (variant), CD44S (standard), and CD44T mRNA and protein expression. PSI: percent spliced-in. * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 7
Fig. 7
YTHDC1 recognizes the m6A sites of CD44 transcripts and regulates CD44 alternative splicing. (A, B) YTHDC1 and CD44 interaction was assessed using RIP assays and DNA gel electrophoresis. (C, D) CD44T enrichment in YTHDC1 protein was examined using RIP assays. (E-G) The effects of HOTAIRM1, FTO and YTHDC1 on CD44T, CD44S, and CD44V mRNA and protein expression were determined using RT‒qPCR and western blot. * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 8
Fig. 8
Expression analysis of FTO, YTHDC1, and CD44V in radiosensitive and radioresistant NPC tissues. Immunohistochemistry was conducted to analyze FTO, YTHDC1 and CD44V protein levels in radiosensitive and radioresistant NPC tissues. NPC: nasopharyngeal carcinoma.
Fig 9
Fig. 9
HOTAIRM1-FTO-YTHDC1-CD44V axis affects radioresistance of C666-1R cells. Ferroptosis markers, including lipid peroxidation (A), MDA (B), Fe2+ (C), GSH levels (D) and mitochondria damage (E) were assessed. Cell proliferation was evaluated by CCK8 assay (F). The apoptotic rate was measured by flow cytometry (G). DNA damage was determined using comet assays (H). Black arrows showed the shrunken mitochondria. * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig 10
Fig. 10
The HOTAIRM1-FTO-YTHDC1-CD44V axis affects radioresistance of C666-1R cells in vivo. (A) Macroscopic images of xenograft tumors in four experimental groups. (B) Average tumor volumes in the groups are presented. (C) Electron micrographs depicted mitochondrial injury in each group. (D) HE staining and IHC for Ki67 and Phospho-Histone H2A.X in tumor tissues. IHC, immunohistochemical. *** P < 0.001.
Fig 11
Fig. 11
Proposed working model of HOTAIRM1. High HOTAIRM1 expression in NPC cells promotes FTO acetylation through direct binding, competitively inhibiting ubiquitination-like modification at the same site, thus enhancing FTO stability. Increased nuclear FTO mediates m6A demethylation of CD44 transcripts. Without m6A methylation, CD44 transcripts are not recognized or spliced by YTHDC1, leading to a higher proportion of CD44V transcripts. The upregulated CD44V inhibits radiation-induced cell ferroptosis via the stable xCT system (XC cysteine glutamate reverse transport system), culminating in NPC radioresistance.
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