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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 as 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.

Gene expression is spatially and temporally regulated at multiple levels. N⁶-methyladenosine (m⁶A) is the most prevalent internal modification in messenger RNA (mRNA) and long noncoding RNA (lncRNAs). m⁶A plays important roles in multiple cellular processes including stem cell pluripotency, adipogenesis, spermatogenesis, neurogenesis, circadian rhythm and development by modulating RNA splicing, export, stability, degradation and translation. Although aberrant m⁶A methylation has been reported in various types of cancer, the underlying molecular functions of METTL3, the solely catalytic subunit of the m⁶A-methylase complex, has yet to be defined.

m⁶A has been recently identified in nascent pre-mRNA, and more specifically intronic m⁶A has been linked to exon skipping events. The occurrence of impaired alternative splicing (AS) is frequently found during the development of cancer. We performed transcriptome wide analysis in breast cancer cell lines and explored AS events. Our results define an AS signature for breast tumorigenesis. We found that METTL3 modulates AS directly through m⁶A deposition at the intron-exon junctions or indirectly by the m⁶A deposition in transcripts encoding for splicing factors and transcription factors. In particular, we show that MYC mRNA harbours the m⁶A mark, suggesting that METTL3 regulates AS indirectly via the regulation of MYC expression. Indeed, the targets of MYC overlapped with METTL3-associated AS events. Importantly, five of the AS events identified and validated in vitro, are linked to a worse prognosis in breast cancer patients. Additionally, we show that METTL3 enhances the breast cancer phenotype through a dual mechanism depending on its sub-cellular localization. We find that the canonical nuclear function of METTL3 decorates transcripts that are involved in cell proliferation and migration. We observe that METTL3 is highly expressed in the cytoplasmic compartment of breast cancer cells from patients. Remarkably, we uncover that the cytoplasmic METTL3 interacts with subunits of the exocyst, whose subunit EXOC7 has been linked to cell adhesion, migration and invasion. Notably, we show that breast cancer cell lines depleted of METTL3 display less gelatinase activity and invadopodia formation, supporting the role of METTL3 in cell invasion via exocytosis.

m⁶A is a reversible modification, which can be demethylated by the erasers FTO and ALKBH5. Depletion of FTO has been shown to increase the level of m⁶A in mRNA, however recent studies have reported that FTO could demethylate N⁶,2´-O-dimethyladenosine (m⁶A_m), adjacent to the 7-methylguanosine cap on mRNA. In the cellular model of colorectal cancer CRC1, depletion of FTO leads to a cancer stem cell phenotype and confers chemotherapy resistance. By performing m⁶A-RNA immunoprecipitation followed by sequencing (MeRIP), we show that knockdown of FTO in CRC1 cells does not affect the global level of m⁶A in mRNA but of m⁶A_m level.

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 (m6Am) demethylase activity. While m6Am 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 m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.

Comprehensive analysis of the mammalian genome uncovered the discovery of pervasive transcription of large RNA transcripts that do not code for proteins, namely, long noncoding RNAs (lncRNAs). LncRNAs play important roles in the regulation of gene expression from integration of chromatin remodeling complexes to transcriptional and posttranscriptional regulation of protein-coding genes. Application of next-generation sequencing technologies to cancer transcriptomes has revealed that aberrant expression of lncRNAs is associated with tumor progression and metastasis. Although thousands of lncRNAs have been shown to be dysregulated in different cancer types, just few of them have been fully characterized. In this book chapter, we aim to highlight recent findings of the mechanistic function of lncRNAs in breast cancer and summarize key examples of lncRNAs that are misregulated during breast tumorigenesis. We have categorized breast cancer–associated lncRNA according to their contribution to tumor suppression or tumor progression based on recent studies. Because some of them are expressed in a specific molecular breast cancer subtype, we have outlined lncRNAs that can potentially serve as diagnostic and prognostic markers, in which expression is linked to chemotherapy resistance. Finally, we have discussed current limitations and perspectives on potential lncRNA targets for use in therapies against breast cancer.

High-throughput analyses have revealed that the vast majority of the transcriptome does not code for proteins. These non-translated transcripts, when larger than 200 nucleotides, are termed long non-coding RNAs (lncRNAs), and play fundamental roles in diverse cellular processes. LncRNAs are subject to dynamic chemical modification, adding another layer of complexity to our understanding of the potential roles that lncRNAs play in health and disease. Many lncRNAs regulate transcriptional programs by influencing the epigenetic state through direct interactions with chromatin-modifying proteins. Among these proteins, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) have been shown to be recruited by lncRNAs to silence target genes. Aberrant expression, deficiency or mutation of both lncRNA and Polycomb have been associated with numerous human diseases, including cancer. In this review, we have highlighted recent findings regarding the concerted mechanism of action of Polycomb group proteins (PcG), acting together with some classically defined lncRNAs including X-inactive specific transcript (XIST), antisense non-coding RNA in the INK4 locus (ANRIL), metastasis associated lung adenocarcinoma transcript 1 (MALAT1), and HOX transcript antisense RNA (HOTAIR).

RNA modification pathways are often mis-regulated in various cancers, with N6-methyladenosine (m6A) having a pivotal role in cancer progression and metastasis. Methyltransferase-like 3 (METTL3), a core component of the m6A methyltransferase complex, functions not only as an m6A writer but also promotes tumorigenesis through m6A-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 m6A-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.