EMT and stemness: Flexible processes tuned by alternative splicing in development and cancer progression

Epithelial-to-mesenchymal transition (EMT) is associated with metastasis formation as well as with generation and maintenance of cancer stem cells. In this way, EMT contributes to tumor invasion, heterogeneity and chemoresistance. Morphological and functional changes involved in these processes require robust reprogramming of gene expression, which is only partially accomplished at the transcriptional level. Alternative splicing is another essential layer of gene expression regulation that expands the cell proteome. This step in post-transcriptional regulation of gene expression tightly controls cell identity between epithelial and mesenchymal states and during stem cell differentiation. Importantly, dysregulation of splicing factor function and cancer-specific splicing isoform expression frequently occurs in human tumors, suggesting the importance of alternative splicing regulation for cancer biology. In this review, we briefly discuss the role of EMT programs in development, stem cell differentiation and cancer progression. Next, we focus on selected examples of key factors involved in EMT and stem cell differentiation that are regulated post-transcriptionally through alternative splicing mechanisms. Lastly, we describe relevant oncogenic splice-variants that directly orchestrate cancer stem cell biology and tumor EMT, which may be envisioned as novel targets for therapeutic intervention

Nova2 alternative splicing regulation of the netrin receptor Unc5b during angiogenesis

L'angiogenesi, la crescita di nuovi vasi sanguinei da una vascolatura pre-esistente, è un processo fondamentale per la progressione tumorale e la formazione di metastasi. Nonostante il processo di angiogenesi rappresenti un bersaglio terapeutico ideale per le terapie anti-tumorali, le strategie adottate finora hanno mostrato risultati modesti. Una migliore comprensione dei meccanismi molecolari alla base della crescita dei vasi tumorali è quindi fondamentale per lo sviluppo di nuove e più specifiche terapie anti-angiogeniche. Lo splicing alternativo (Alternative Splicing, AS) è una modificazione post-trascrizionale in grado di generare diversi mRNAs che portano alla produzione di isoforme proteiche con differenti proprietà funzionali. Numerose evidenze indicano un ruolo causativo di AS nel cancro. Infatti, all'incirca 15'000 eventi di AS sono specifici delle cellule tumorali ma non delle cellule normali. Queste evidenze suggeriscono che AS possa essere utilizzato come un nuovo strumento diagnostico, prognostico e terapeutico per la cura del cancro. Il ruolo di AS nell'angiogenesi è stato a lungo ignorato. Per la prima volta il nostro gruppo ha dimostrato che la formazione del lume vascolare durante l'angiogenesi è controllata a livello post-trascrizionale dal fattore di splicing Nova2, finora considerato specifico del sistema nervoso. Attraverso RNA-seq e CLIP-seq, il nostro gruppo ha recentemente identificato nuove isoforme di AS regolate da Nova2 di geni coinvolti nello sviluppo del sistema vascolare. Tra queste abbiamo trovato una nuova variante del gene Unc5b, il recettore della Netrina-1 che svolge importanti funzioni sia nel sistema vascolare che in quello nervoso. Durante il mio dottorato, ho dimostrato che Nova2 promuove la formazione di una nuova isoforma di Unc5b deleta di 11 amminoacidi nella porzione extra-cellulare del recettore (Unc5b-Δ8). Ho dimostrato che Unc5b-Δ8, a differenza del recettore wild-type, non risponde all’inibizione dell’apoptosi indotta dalla Netrina-1. Inoltre, ho dimostrato che Unc5b-Δ8 è fondamentale per la formazione di specifici vasi in vivo. Infine, ho trovato che l’espressione di Unc5b-Δ8 aumenta in campioni di tumore ovarico. In conclusione, i miei dati indicano che la regolazione di Unc5b da parte di Nova2 ha un ruolo chiave durante l'angiogenesi, identificando quindi un nuovo “pathway molecolare” che potrebbe rivelarsi importante per la progressione tumorale.Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential to sustain tumor growth and metastasis formation. Although targeting angiogenesis is a promising anti-cancer therapy, all attempted strategies so far have shown modest therapeutic effects. This indicates that tumor angiogenesis is a more complex phenomenon than previously anticipated. Hence, a better understanding of the molecular mechanisms supporting growth of tumor vessels will be crucial to identify novel and more specific anti-angiogenic therapies. Alternative splicing (AS) is the post-transcriptional molecular process that generates, from a single gene, multiple mRNAs leading to the production of protein isoforms with different structural and functional properties. Notably, increasing evidence supports a causative role of AS regulation in cancer. In particular, as many as 15'000 cancer-specific AS events have been identified suggesting that AS could serve as a powerful source of new diagnostic, prognostic and therapeutic tools for human cancer. Despite its well-established function(s) in tumor progression, the role of AS during angiogenesis has so far been largely ignored. Indeed, the molecular pathways involved in angiogenesis have been traditionally suggested to act primarily through regulation of transcription. For the first time, our group demonstrated an important role for the regulation of AS during angiogenesis. We found that the organization of the vascular lumen during angiogenesis is controlled at post-transcriptional level by the AS factor Nova2, previously considered neural cell-specific. Consequently, Nova2 ablation in zebrafish embryos causes vascular lumen formation defects, reminiscent of aberrant morphology of tumor vasculature. By RNA-seq and CLIP-seq, we recently identified novel Nova2-mediated AS variants of several receptors implicated in vascular patterning. Among these is Unc5b, encoding for a Netrin-1 receptor involved in both axon guidance and vessel morphogenesis. During my Ph.D., I found that Nova2 directly promotes the production of a novel Unc5b isoform deleted of 11 amino acids in the extracellular portion of the receptor (Unc5b-Δ8). I found that Unc5b-Δ8 is increased in ovarian cancer and I showed that the Nova2-dependent production of Unc5b-Δ8 plays a fundamental role in the formation of specific vessels during vascular development in vivo. Mechanistically, I found that Unc5b-Δ8 is not able to respond to the canonical Netrin-1 pro-survival signal. Collectively, my data suggest that Nova2 regulation of Unc5b AS plays a key role in angiogenesis thus identifying a novel pathway that could be relevant for cancer progression

SAM68: Signal Transduction and RNA Metabolism in Human Cancer

Alterations in expression and/or activity of splicing factors as well as mutations in cis-acting splicing regulatory sequences contribute to cancer phenotypes. Genome-wide studies have revealed more than 15,000 tumor-associated splice variants derived from genes involved in almost every aspect of cancer cell biology, including proliferation, differentiation, cell cycle control, metabolism, apoptosis, motility, invasion, and angiogenesis. In the past decades, several RNA binding proteins (RBPs) have been implicated in tumorigenesis. SAM68 (SRC associated in mitosis of 68 kDa) belongs to the STAR (signal transduction and activation of RNA metabolism) family of RBPs. SAM68 is involved in several steps of mRNA metabolism, from transcription to alternative splicing and then to nuclear export. Moreover, SAM68 participates in signaling pathways associated with cell response to stimuli, cell cycle transitions, and viral infections. Recent evidence has linked this RBP to the onset and progression of different tumors, highlighting misregulation of SAM68-regulated splicing events as a key step in neoplastic transformation and tumor progression. Here we review recent studies on the role of SAM68 in splicing regulation and we discuss its contribution to aberrant pre-mRNA processing in cancer

Gene expression profiles controlled by the alternative splicing factor nova2 in endothelial cells

Alternative splicing (AS) plays an important role in expanding the complexity of the human genome through the production of specialized proteins regulating organ development and physiological functions, as well as contributing to several pathological conditions. How AS programs impact on the signaling pathways controlling endothelial cell (EC) functions and vascular development is largely unknown. Here we identified, through RNA-seq, changes in mRNA steady-state levels in ECs caused by the neuro-oncological ventral antigen 2 (Nova2), a key AS regulator of the vascular morphogenesis. Bioinformatics analyses identified significant enrichment for genes regulated by peroxisome proliferator-activated receptor-gamma (Ppar-γ) and E2F1 transcription factors. We also showed that Nova2 in ECs controlled the AS profiles of Ppar-γ and E2F dimerization partner 2 (Tfdp2), thus generating different protein isoforms with distinct function (Ppar-γ) or subcellular localization (Tfdp2). Collectively, our results supported a mechanism whereby Nova2 integrated splicing decisions in order to regulate Ppar-γ and E2F1 activities. Our data added a layer to the sequential series of events controlled by Nova2 in ECs to orchestrate vascular biology

Widespread Alternative Splicing Changes in Metastatic Breast Cancer Cells.

Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types

Widespread Alternative Splicing Changes in Metastatic Breast Cancer Cells.

Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types

Widespread Alternative Splicing Changes in Metastatic Breast Cancer Cells

Aberrant alternative splicing (AS) is a hallmark of cancer and a potential target for novel anti-cancer therapeutics. Breast cancer-associated AS events are known to be linked to disease progression, metastasis, and survival of breast cancer patients. To identify altered AS programs occurring in metastatic breast cancer, we perform a global analysis of AS events by using RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq). We demonstrate that, relative to low-metastatic, high-metastatic breast cancer cells show different AS choices in genes related to cancer progression. Supporting a global reshape of cancer-related splicing profiles in metastatic breast cancer we found an enrichment of RNA-binding motifs recognized by several splicing regulators, which have aberrant expression levels or activity during breast cancer progression, including SRSF1. Among SRSF1-regulated targets we found DCUN1D5, a gene for which skipping of exon 4 in its pre-mRNA introduces a premature termination codon (PTC), thus generating an unstable transcript degraded by nonsense-mediated mRNA decay (NMD). Significantly, distinct breast cancer subtypes show different DCUN1D5 isoform ratios with metastatic breast cancer expressing the highest level of the NMD-insensitive DCUN1D5 mRNA, thus showing high DCUN1D5 expression levels, which are ultimately associated with poor overall and relapse-free survival in breast cancer patients. Collectively, our results reveal global AS features of metastatic breast tumors, which open new possibilities for the treatment of these aggressive tumor types

Global Alternative Splicing Defects in Human Breast Cancer Cells.

Breast cancer is the most frequently occurred cancer type and the second cause of death in women worldwide. Alternative splicing (AS) is the process that generates more than one mRNA isoform from a single gene, and it plays a major role in expanding the human protein diversity. Aberrant AS contributes to breast cancer metastasis and resistance to chemotherapeutic interventions. Therefore, identifying cancer-specific isoforms is the prerequisite for therapeutic interventions intended to correct aberrantly expressed AS events. Here, we performed RNA-mediated oligonucleotide annealing, selection, and ligation coupled with next-generation sequencing (RASL-seq) in breast cancer cells, to identify global breast cancer-specific AS defects. By RT-PCR validation, we demonstrate the high accuracy of RASL-seq results. In addition, we analyzed identified AS events using the Cancer Genome Atlas (TCGA) database in a large number of non-pathological and breast tumor specimens and validated them in normal and breast cancer samples. Interestingly, aberrantly regulated AS cassette exons in cancer tissues do not encode for known functional domains but instead encode for amino acids constituting regions of intrinsically disordered protein portions characterized by high flexibility and prone to be subjected to post-translational modifications. Collectively, our results reveal novel AS errors occurring in human breast cancer, potentially affecting breast cancer-related biological processes