Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N⁶,2’-O-dimethyladenosine (m⁶A_m) demethylase activity. While m⁶A_m is strategically located next to the m⁷G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m⁶A_m level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m⁶A_m methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m⁶A_m modification in stem-like properties acquisition. FTO-mediated regulation of m⁶A_m marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m⁶A_m modification and its potential adverse consequences for colorectal cancer management.

Alternative splicing (AS) enables differential inclusion of exons from a given transcript, thereby contributing to the transcriptome and proteome diversity. Aberrant AS patterns play major roles in the development of different pathologies, including breast cancer.N⁶-methyladenosine (m⁶A), the most abundant internal modification of eukaryotic mRNA, influences tumor progression and metastasis of breast cancer, and it has been recently linked to AS regulation. Here, we identify a specific AS signature associated with breast tumorigenesis in vitro. We characterize for the first time the role of METTL3 in modulating breast cancer-associated AS programs, expanding the role of the m⁶A-methyltransferase in tumorigenesis. Specifically, we find that both m⁶A deposition in splice site boundaries and in splicing and transcription factor transcripts, such asMYC, direct AS switches of specific breast cancer-associated transcripts. Finally, we show that five of the AS events validated in vitro are associated with a poor overall survival rate for patients with breast cancer, suggesting the use of these AS events as a novel potential prognostic biomarker.

We are delighted to share with you our fifth Journal Club and highlight some of the most interesting papers published recently.

RNA modification pathways are often mis-regulated in various cancers, with N ⁶ -methyladenosine (m ⁶ A) having a pivotal role in cancer progression and metastasis. Methyltransferase-like 3 (METTL3), a core component of the m ⁶ A methyltransferase complex, functions not only as an m ⁶ A writer but also promotes tumorigenesis through m ⁶ A-independent mechanisms. Here, we show that METTL3 is mislocalized to the cytoplasm in breast cancer tumors from patients, contributing to the oncogenic phenotype. Cytoplasmic METTL3 interacts with EXOC7, a key regulator of exocytosis, promoting its stabilization. Additionally, METTL3 regulates m ⁶ A-dependent alternative splicing of EXOC7 . Silencing METTL3 impairs vesicle trafficking and the breast cancer secretome – effects that do not rely on its enzymatic activity but instead involve METTL3-mediated stabilization of EXOC7 and potentially other exocyst components. Furthermore, METTL3 knockdown impairs invadopodia formation, collagen matrix invasion, and focal adhesion morphology in vitro, while inhibition of METTL3 catalytic activity does not. Our findings uncover non-catalytic roles of METTL3 in regulating exocytosis and the cancer secretome.

Alternative splicing (AS) enables differential inclusion of exons from a given transcript, thereby contributing to the transcriptome and proteome diversity. Aberrant AS patterns play major roles in the development of different pathologies, including breast cancer. N ⁶ -methyladenosine (m ⁶ A), the most abundant internal modification of eukaryotic mRNA, influences tumor progression and metastasis of breast cancer, and it has been recently linked to AS regulation. Here, we identify a specific AS signature associated with breast tumorigenesis in vitro . We characterize for the first time the role of METTL3 in modulating breast cancer-associated AS programs, expanding the role of the m ⁶ A-methyltransferase in tumorigenesis. Specifically, we find that both m ⁶ A deposition in splice site boundaries and in splicing and transcription factor transcripts, such as PHF5A and MYC , direct AS switches of specific breast cancer-associated transcripts. Finally, we show that five of the AS events validated in vitro are associated with a poor overall survival rate for patients with breast cancer, suggesting the use of these AS events as a novel potential prognostic biomarker.

Peroxiredoxin 1 (PRDX1) is a highly conserved, thiol-dependent peroxidase that rapidly scavenges reactive oxygen species to modulate redox signaling. PRDX1-null mice exhibited genomic instability, shortened life span, and accelerated tumorigenesis, including development of lymphomas, sarcomas, and carcinomas. Despite extensive characterization of these phenotypes, the molecular mechanism by which PRDX1 loss causes genomic instability remains poorly understood. Here, we show that PRDX1 deficiency alters nucleolar morphology, impairs RNA polymerase I (POL-I)–dependent transcription of pre–ribosomal RNAs, and triggers nucleolar genomic instability. This oxidative stress–induced nucleolar dysfunction promotes the stability of secondary DNA structures, such as RNA-DNA hybrids and G-quadruplex DNA, contributing to nucleolar genomic instability. We demonstrate that PRDX1 loss reduces nascent ribosomal RNA (rRNA) levels and impairs rRNA processing, further affecting ribosome biogenesis. Mechanistically, we established that PRDX1 loss triggers activation of the nucleolar DNA damage response characterized by activation of the DNA repair kinase ATM and elevated TCOF1 within the nucleolus. In addition, we observed recruitment of the MRE11-RAD50-NBS1 (MRN) complex subunit NBS1 to ribosomal DNA (rDNA) loci and this was further increased under oxidative stress. NBS1 accumulation correlates with the repression of rDNA transcription by POL-I, potentially delaying rRNA synthesis, and safeguarding the nucleolar genome from further oxidative damage. Collectively, these findings uncover a previously unrecognized, but critical role, for PRDX1 in maintaining nucleolar integrity and ribosomal biogenesis through redox-dependent regulation of rDNA transcription and processing machinery.