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. 2024 Jun 19:14:1402126.
doi: 10.3389/fonc.2024.1402126. eCollection 2024.

The two-faced role of RNA methyltransferase METTL3 on cellular response to cisplatin in head and neck squamous cell carcinoma in vitro model

Kamila Ostrowska  1   2 , Agnieszka A Rawłuszko-Wieczorek  3 , Julia Ostapowicz  1   2   4 , Wiktoria M Suchorska  2   4 , Wojciech Golusiński  1
Affiliations

The two-faced role of RNA methyltransferase METTL3 on cellular response to cisplatin in head and neck squamous cell carcinoma in vitro model

Kamila Ostrowska et al. Front Oncol. .

Abstract

Background: RNA methyltransferase-like 3 (METTL3) is responsible for methyl group transfer in the progression of N 6-methyladenosine (m6A) modification. This epigenetic feature contributes to the structural and functional regulation of RNA and consequently may promote tumorigenesis, tumor progression, and cellular response to anticancer treatment (chemo-, radio-, and immunotherapy). In head and neck squamous cell carcinoma (HNSCC), the commonly used chemotherapy is cisplatin. Unfortunately, cisplatin resistance is still a major cause of tumor relapse and patients' death. Thus, this study aimed to investigate the role of METTL3 on cellular response to cisplatin in HNSCC in vitro models.

Materials and methods: HNSCC cell lines (H103, FaDu, and Detroit-562) with stable METTL3 knockdown (sgMETTL3) established with CRISPR-Cas9 system were treated with 0.5 tolerable plasma level (TPL) and 1 TPL of cisplatin. Further, cell cycle distribution, apoptosis, CD44/CD133 surface marker expression, and cell's ability to colony formation were analyzed in comparison to controls (cells transduced with control sgRNA).

Results: The analyses of cell cycle distribution and apoptosis indicated a significantly higher percentage of cells with METTL3 knockdown 1) arrested in the G2/S phase and 2) characterized as a late apoptotic or death in comparison to control. The colony formation assay showed intensified inhibition of a single cell's ability to grow into a colony in FaDu and Detroit-562 METTL3-deficient cells, while a higher colony number was observed in H103 METTL3 knockdown cells after cisplatin treatment. Also, METTL3 deficiency significantly increased cancer stem cell markers' surface expression in all studied cell lines.

Conclusion: Our findings highlight the significant influence of METTL3 on the cellular response to cisplatin, suggesting its potential as a promising therapeutic target for addressing cisplatin resistance in certain cases of HNSCC.

Keywords: RNA methylation; RNA methyltransferase-like 3; chemotherapy; cisplatin; head and neck squamous cell carcinoma.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Validation of METTL3 knockdown by CRISPR-Cas9 in H103, FaDu, and Detroit-562 cell lines. Clonal selection of each cell line was performed to select the cells with statistically significant decreased levels of METTL3 at protein (A) and mRNA (B) levels in comparison to control sgRNA. Per cell line, two sgMETTL3 cell clones were selected (marked with red rectangle on protein blots). *p ≤ 0.05. **p < 0.01. ***p < 0.001.
Figure 2
Figure 2
Flow cytometry detected the distribution of cell cycle phase (A) and apoptosis (B) after METTL3 knockdown in three independent cell lines—H103, FaDu, and Detroit-562—with different METTL3 knockdown efficiency and by three independent repeats. Each cell line was analyzed as two independent cell clones and control (transduced with control sgRNA). The cells were treated with doses of 0 TPL, 0.5 TPL, and 1 TPL of cisplatin for 72 h and analyzed. Data are represented as mean ± SD (n = 3) of the percentage number of cells; two-way ANOVA for multiple comparisons (here, we showed p-values for sgControl 0 TPL vs. all sgMETTL3 constructs 0 TPL; sgControl 0.5 TPL vs. all sgMETTL3 constructs 0.5 TPL; sgControl 1 TPL vs. all sgMETTL3 constructs 1 TPL). *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.
Figure 3
Figure 3
Colony formation assay analysis of three independent cell lines—H103, FaDu, and Detroit-562—with different METTL3 knockdown efficiency and by six independent repeats. Each cell line was analyzed as two independent cell clones and control (transduced with control sgRNA). The cells were treated with doses of 0 TPL, 0.5 TPL, and 1 TPL of cisplatin for 14 days and analyzed. Data are represented as mean ± SD (n = 6) number of colonies; ordinary one-way ANOVA comparison. *p < 0.05. **p < 0.01. ****p < 0.0001.
Figure 4
Figure 4
Flow cytometry analysis of CD44 (A) and CD133 (B) surface expression in three independent cell lines—H103, FaDu, and Detroit-562—with different METTL3 knockdown efficiency and by three independent repeats. Each cell line was analyzed as two independent cell clones and control (transduced with control sgRNA). The cells were treated with doses of 0 TPL, 0.5 TPL, and 1 TPL of cisplatin for 72 h and analyzed. Data are represented as mean ± SD (n = 3) median fluorescence intensity (MFI); ordinary one-way ANOVA comparison. *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001.
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