Dataset Information

Depletion of the m⁶A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma.

ABSTRACT:

Background

N⁶-methyladenosine (m⁶A) is one of the most common RNA modifications in eukaryotes and has effects on RNA structure and stability. Recent studies have shown that m⁶A methylation is involved in human carcinogenesis. In the present study, we investigated the effects of m⁶A demethylases FTO and ALKBH5 on renal cell carcinoma (RCC) cell lines.

Methods

The epithelial-mesenchymal in vitro knockdowns of FTO and ALKBH5 induced by antisense oligonucleotides (LNA-GapmeR system) were established in RCC cell lines. Their effects on migration and proliferation were investigated subsequently. The influence of FTO and ALKBH5 knockdown on key epithelial-mesenchymal transition (EMT) genes was analyzed.

Results

Inactivation of FTO and ALKBH5 resulted in decreased proliferation and motility in all cell lines examined (ACHN, Caki-1, 769-P). Vimentin (VIM) was downregulated after the knockdown of FTO and ALKBH5, indicating an EMT switch.

Conclusions

Knockdown of the m⁶A erasers FTO and ALKBH5 inhibits the malignant potential in the cell cultures studied by means of an EMT switch.

SUBMITTER: Hu W

PROVIDER: S-EPMC10086911 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Publications

Depletion of the m<sup>6</sup>A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma.

Hu Wei W Klümper Niklas N Schmidt Doris D Ritter Manuel M Ellinger Jörg J Hauser Stefan S

American journal of translational research 20230315 3

<h4>Background</h4>N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) is one of the most common RNA modifications in eukaryotes and has effects on RNA structure and stability. Recent studies have shown that m<sup>6</sup>A methylation is involved in human carcinogenesis. In the present study, we investigated the effects of m<sup>6</sup>A demethylases FTO and ALKBH5 on renal cell carcinoma (RCC) cell lines.<h4>Methods</h4>The epithelial-mesenchymal in vitro knockdowns of FTO and ALKBH5 induced by anti ...[more]

PMID: 37056835

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Project description:Translation is one of the main gene expression steps targeted by cellular stress, commonly referred to as translational stress, which includes treatment with anticancer drugs. While translational stress blocks the translation initiation of bulk mRNAs, it nonetheless activates the translation of specific mRNAs known as short upstream open reading frames (uORFs)-mRNAs. Among these, the ATF4 mRNA encodes a transcription factor that reprograms gene expression in cells responding to various stresses. Although the stress-induced translation of the ATF4 mRNA relies on the presence of uORFs (upstream to the main ATF4 ORF), the mechanisms mediating this effect, particularly during chemoresistance, remain elusive. Here, we report that ALKBH5 (AlkB Homolog 5) and FTO (FTO: Fat mass and obesity-associated protein), the two RNA demethylating enzymes, promote the translation of ATF4 mRNA in a transformed liver cell line (Hep3B) treated with the chemotherapeutic drug sorafenib. Using the in vitro luciferase reporter translational assay, we found that depletion of both enzymes reduced the translation of the reporter ATF4 mRNA upon drug treatment. Consistently, depletion of either protein abrogates the loading of the ATF3 mRNA into translating ribosomes as assessed by polyribosome assays coupled to RT-qPCR. Collectively, these results indicate that the ALKBH5 and FTO-mediated translation of the ATF4 mRNA is regulated at its initiation step. Using in vitro methylation assays, we found that ALKBH5 is required for the inhibition of the methylation of a reporter ATF4 mRNA at a conserved adenosine (A235) site located at its uORF2, suggesting that ALKBH5-mediated translation of ATF4 mRNA involves demethylation of its A235. Preventing methylation of A235 by introducing an A/G mutation into an ATF4 mRNA reporter renders its translation insensitive to ALKBH5 depletion, supporting the role of ALKBH5 demethylation activity in translation. Finally, targeting either ALKBH5 or FTO sensitizes Hep3B to sorafenib-induced cell death, contributing to their resistance. In summary, our data show that ALKBH5 and FTO are novel factors that promote resistance to sorafenib treatment, in part by mediating the translation of ATF4 mRNA.

Dataset Information

Depletion of the m⁶A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma.

Background

Methods

Results

Conclusions

Publications

Depletion of the m<sup>6</sup>A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Depletion of the m6A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma.

Background

Methods

Results

Conclusions

Publications

Depletion of the m<sup>6</sup>A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Depletion of the m⁶A demethylases FTO and ALKBH5 impairs growth and metastatic capacity through EMT phenotype change in clear cell renal cell carcinoma.