RNA N6‐methyladenosine connects YTHDF3 and LOXL3 to promote melanoma metastasis

Abstract

N⁶‐methyladenosine (m6A) modifications of RNAs are associated with diverse biological processes in normal and cancer cells. In melanoma cancers, mutations in genes that function in the writing or erasure of RNA m6A have been shown to affect cancer progression and metastasis. More recently, Shi et al. find that a protein which binds to the m6A modification, called YTHDF3, is widely overexpressed in melanoma cell lines and patient samples. Through multi‐omics and targeted RNA analyses, they uncover a novel YTHDF3‐LOXL3 axis which contribute to in vitro melanoma invasion and in vivo metastasis. Here, we discuss these findings and highlight several possible new and exciting research directions stemming from this research.

Chemical modifications of RNA play important roles in tumorigenesis and cancer progression. Of these, N⁶-methyladenosine (m6A) is the most abundant mRNA modification in humans and is known to be tightly associated with fundamental cellular processes such as RNA splicing, localization, stability, and translation. In humans, m6A can be added onto mRNAs by the ‘writer’ proteins METTL3/14 and WTAP. Conversely, m6A can be removed by the ‘eraser’ proteins ALKBH5 and FTO. In the context of melanomas, which is the most malignant skin cancer, it has been demonstrated that the misregulation of m6A dynamics affects cancer progression. Induced knockdown of the m6A writer protein, METTL3, reduces colony formation and invasiveness in A375 and WM793 human melanoma cell lines. Conversely, overexpression of METTL3 increases the expression of metastasis-associated protein matrix metalloproteinase 2. In ocular melanoma cells, knockdown of the m6A writer METTL3 increases colony formation rates and migratory potential. Conversely, knockdown of the m6A eraser ALKBH5 decreases both cancer-associated phenotypes. Another melanoma study implicated the m6A eraser FTO as a potential pro-tumorigenesis factor in Mel624, CHL-1, and B16F10 melanoma cell lines. Thus, while the link between m6A and melanoma cancer progression is clear, the underlying molecular mechanisms of how m6A modifications of RNAs regulate melanomas remain unclear.

To uncover a possible mechanism, Shi et al. found that a protein ‘reader’ of the m6A modification, YTHDF3, is widely overexpressed across multiple melanoma patient tissues and cell lines, compared to normal epidermal melanocytes. Mis-regulation of YTHDF3 is likely important for melanoma progression because knockdown led to reduced migration and invasion in vitro and decreased pulmonary metastases in nude mouse models. As many mRNAs contain m6A, they went further by systematically performing multi-omics analyses to discover specific m6A-modified mRNAs that YTHDF3 acts through to potentially promote melanoma. Amongst the identified YTHDF3 RNA targets was LOXL3. Through mining cancer databases such as the Cancer Genome Atlas, the authors found evidence that LOXL3 is upregulated across multiple melanoma cell lines. They further showed that induced knockdown of LOXL3 promoted melanoma cell death, a phenotype which could be prevented by re-expression of LOXL3. Together, this suggested that YTHDF3 may affect melanoma progression through LOXL3 regulation. Strikingly, knockdown of YTHDF3 decreased LOXL3 at the protein level but not at the RNA level. To address the disconnect, the authors showed that YTHDF3 binds to m6A modifications of the LOXL3 transcript to likely promote translation of the mRNA without affecting LOXL3 mRNA steady-state levels. They suggest that the impact of YTHDF3 on LOXL3 mRNA is at the level of translation and not RNA decay. They proposed that elevated YTHDF3 in melanoma could increase LOXL3 protein levels which would promote melanoma metastasis. Prior work had suggested that LOXL3 might promote melanoma survival by maintaining genomic stability. Altogether, this work pinpoints specific translation of the LOXL3 mRNA as a possible mechanism for m6A/YTHDF3-mediated melanoma progression.

This study revealed a novel YTHDF3-LOXL3 axis that regulates melanoma progression in human melanoma cell lines and mouse models. Outstanding unknowns for this axis include the biological relevance of the multiple m6A sites across the LOXL3 transcript. Global mapping of m6A sites revealed numerous sites across the LOXL3 RNA. Removal of all m6A sites specifically on the LOXL3 RNA, by targeting catalytically-dead Cas13b fused with the m6A eraser protein FTO, disrupted YTHDF3-LOXL3 binding. However, it remains to be tested whether all the m6A sites contribute to the YTHDF3-LOXL3-melanoma association. Moreover, as m6A has been implicated in regulating multiple aspects of RNA processing and metabolism, it would be interesting to understand how the multiple sites regulate LOXL3 transcripts. Furthermore, it is unclear whether the YTHDF3-LOXL3 interaction is direct or indirect, and whether it involves additional factors. Lastly, this study supports the prospect that the YTHDF3-LOXL3 axis may have clinical significance. Future clinical studies will be needed to address this possibility.

As the abundant m6A modification is involved in normal and diseased cellular processes, treating melanoma by a global disruption of m6A, through inhibiting ‘writer’ and ‘eraser’ factors, might not be an ideal therapeutic intervention. Precision medicine to specifically target those m6A-modified mRNAs that cause melanoma phenotypes may produce better patient outcomes. The study by Shi et al. highlights that targeting LOXL3 translation could be a useful therapeutic approach in treating melanoma. Additionally, the authors identified other YTHDF3-targeted transcripts such as CHD7 and PDE3A. Presently, it is unclear how these other transcripts might contribute to YTHDF3-mediated melanoma progression. Further investigations will be required to fully identify all actionable modes of m6A-driven melanoma.

ACKNOWLEDGEMENT

Tommy V. Vo is supported by start-up funds from Michigan State University.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

References

Yang Y, Hsu PJ, Chen Y‐S, Yang Y‐G. Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism. Cell Res. 2018;28:616‐624.

Desrosiers R, Friderici K, Rottman F. Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc Nat Acad Sci. 1974;71:3971‐3975.

Dahal U, Le K, Gupta M. RNA m6A methyltransferase METTL3 regulates invasiveness of melanoma cells by matrix metallopeptidase 2. Melan Res. 2019;29:382‐389.

Jia R, Chai P, Wang S, et al. m6A modification suppresses ocular melanoma through modulating HINT2 mRNA translation. Mol Cancer. 2019;18:1‐21.

Yang S, Wei J, Cui Y‐H, et al. m6A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti‐PD‐1 blockade. Nat Commun. 2019;10:1‐14.

Shi Hz, Xiong J‐S, Gan L, et al. N6‐methyladenosine reader YTHDF3 regulates melanoma metastasis via its ‘executor'LOXL3. Clin Transl Med. 2022;12: [eLocator: e1075].

Shi H, Wang X, Lu Z, et al. YTHDF3 facilitates translation and decay of N⁶‐methyladenosine‐modified RNA. Cell Res. 2017;27:315‐328.

Santamaría PG, Floristán A, Fontanals‐Cirera B, et al. Lysyl oxidase‐like 3 is required for melanoma cell survival by maintaining genomic stability. Cell Death Diff. 2018;25:935‐950.