Alternative Splicing and Tumor Progression

Alternative splicing is a key molecular mechanism for increasing the functional diversity of the eukaryotic proteomes. A large body of experimental data implicates aberrant splicing in various human diseases, including cancer. Both mutations in cis-acting splicing elements and alterations in the expression and/or activity of splicing regulatory factors drastically affect the splicing profile of many cancer-associated genes. In addition, the splicing profile of several cancer-associated genes is altered in particular types of cancer arguing for a direct role of specific splicing isoforms in tumor progression. Deciphering the mechanisms underlying aberrant splicing in cancer may prove crucial to understand how splicing machinery is controlled and integrated with other cellular processes, in particular transcription and signaling pathways. Moreover, the characterization of splicing deregulation in cancer will lead to a better comprehension of malignant transformation. Cancer-associated alternative splicing variants may be new tools for the diagnosis and classification of cancers and could be the targets for innovative therapeutical interventions based on highly selective splicing correction approaches

Oncogenic Alternative Splicing Switches: Role in Cancer Progression and Prospects for Therapy

Alterations in the abundance or activities of alternative splicing regulators generate alternatively spliced variants that contribute to multiple aspects of tumor establishment, progression and resistance to therapeutic treatments. Notably, many cancer-associated genes are regulated through alternative splicing suggesting a significant role of this post-transcriptional regulatory mechanism in the production of oncogenes and tumor suppressors. Thus, the study of alternative splicing in cancer might provide a better understanding of the malignant transformation and identify novel pathways that are uniquely relevant to tumorigenesis. Understanding the molecular underpinnings of cancer-associated alternative splicing isoforms will not only help to explain many fundamental hallmarks of cancer, but will also offer unprecedented opportunities to improve the efficacy of anti-cancer treatments

Regulation of the Ras-MAPK and PI3K-mTOR Signalling Pathways by Alternative Splicing in Cancer

Alternative splicing is a fundamental step in regulation of gene expression of many tumor suppressors and oncogenes in cancer. Signalling through the Ras-MAPK and PI3K-mTOR pathways is misregulated and hyperactivated in most types of cancer. However, the regulation of the Ras-MAPK and PI3K-mTOR signalling pathways by alternative splicing is less well established. Recent studies have shown the contribution of alternative splicing regulation of these signalling pathways which can lead to cellular transformation, cancer development, and tumor maintenance. This review will discuss findings in the literature which describe new modes of regulation of components of the Ras-MAPK and PI3K-mTOR signalling pathways by alternative splicing. We will also describe the mechanisms by which signals from extracellular stimuli can be communicated to the splicing machinery and to specific RNA-binding proteins that ultimately control exon definition events

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

Sam68 regulates EMT through alternative splicing–activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene

Expression levels of SF2/ASF are controlled by Sam68 mediated activation of splicing-induced mRNA decay

Identification of bi-allelic LFNG variants in three patients and further clinical and molecular refinement of spondylocostal dysostosis 3

: Spondylocostal dysostosis (SCD), a condition characterized by multiple segmentation defects of the vertebrae and rib malformations, is caused by bi-allelic variants in one of the genes involved in the Notch signaling pathway that tunes the "segmentation clock" of somitogenesis: DLL3, HES7, LFNG, MESP2, RIPPLY2, and TBX6. To date, seven individuals with LFNG variants have been reported in the literature. In this study we describe two newborns and one fetus with SCD, who were found by trio-based exome sequencing (trio-ES) to carry homozygous (c.822-5C>T) or compound heterozygous (c.[863dup];[1063G>A]) and (c.[521G>T];[890T>G]) variants in LFNG. Notably, the c.822-5C>T change, affecting the polypyrimidine tract of intron 5, is the first non-coding variant reported in LFNG. This study further refines the clinical and molecular features of spondylocostal dysostosis 3 and adds to the numerous investigations supporting the usefulness of trio-ES approach in prenatal and neonatal settings

Transcription of Satellite III non-coding RNAs is a general stress response in human cells

In heat-shocked human cells, heat shock factor 1 activates transcription of tandem arrays of repetitive Satellite III (SatIII) DNA in pericentromeric heterochromatin. Satellite III RNAs remain associated with sites of transcription in nuclear stress bodies (nSBs). Here we use real-time RT-PCR to study the expression of these genomic regions. Transcription is highly asymmetrical and most of the transcripts contain the G-rich strand of the repeat. A low level of G-rich RNAs is detectable in unstressed cells and a 104-fold induction occurs after heat shock. G-rich RNAs are induced by a wide range of stress treatments including heavy metals, UV-C, oxidative and hyper-osmotic stress. Differences exist among stressing agents both for the kinetics and the extent of induction (>100- to 80.000-fold). In all cases, G-rich transcripts are associated with nSBs. On the contrary, C-rich transcripts are almost undetectable in unstressed cells and modestly increase after stress. Production of SatIII RNAs after hyper-osmotic stress depends on the Tonicity Element Binding Protein indicating that activation of the arrays is triggered by different transcription factors. This is the first example of a non-coding RNA whose transcription is controlled by different transcription factors under different growth conditions

Alternative Splicing: Recent Insights into Mechanisms and Functional Roles

Alternative splicing generates multiple protein isoforms from one primary transcript and represents one of the major drivers of proteomic diversity in human cells [...

Alternative Splicing: Role in Cancer Development and Progression

Alternative splicing of precursor messenger RNAs (premRNAs) is a fundamental step in the regulation of gene expression. This processing step of the nascent messenger amplifies the coding potential of eukaryotic genomes by allowing the production of multiple protein isoforms with distinct structural and functional properties. The advent of high-throughput sequencing techniques has recently revealed that alternative splicing of exons and introns represents a major source of proteomic diversity in complex organisms characterized by a limited number of protein-coding genes. Nevertheless, the evolutionary advantage provided by alternative splicing can also turn into a source of deleterious problems for the organism. Indeed, the extreme flexibility of its regulation, which relies on the combinatorial action of multiple non stringent factors, is subject to errors and the aberrant splicing of key genes can result in the onset of many human genetic and sporadic diseases. In this regard, mounting evidence illustrates how changes in alternative splicing patterns of specific genes is an important tool used by cancer cells to produce protein isoforms involved in all areas of cancer cell biology, including numerous aspects of tumor establishment, progression, and resistance to therapeutic treatments. Importantly, cancer-specific splice variants have the potential to become suitable therapeutic targets for human cancer, as novel tools to correct splicing defects are being developed and, in some cases, have entered clinical trials for other human diseases, such as spinal muscular atrophy. Nevertheless, these findings are likely to represent just the tip of the iceberg and important questions regarding the role of alternative splicing in cancer still remain to be addressed. The main focus of this special issue is to emphasize key mechanisms involved in oncogenic splicing changes, their connection with other steps of gene expression, and the therapeutic potential of cancer-associated alternative splicing isoforms. More specifically, M. Ladomery discusses alternative splicing in the context of the so-called hallmarks of cancer, originally proposed by Hanahan and Weinberg in 2000. The list of hallmarks was originally six; recently it was augmented to ten. M. Ladomery proposes that a comprehensive dysregulation of alternative splicing could, in itself, be considered yet another hallmark of cancer. The idea is that the aberrant expression and activity of key oncogenic splicing factors and/or their regulatory kinases could lead to a systematic change in gene expression by favouring the concurrent production of several oncogenic splice variants of genes involved in critical biological aspects of tumour cells. S. C. Lenzken et al. review our current knowledge of the role of alternative splicing in the multiple and various aspects of the DNA damage response (DDR) and the control of genome stability. This review illustrates several mechanisms through which pre-mRNA splicing and genomic stability can influence each other and contribute to tumorigenesis. M. Romano and colleagues draw attention to the function that pseudoexons and pseudointrons can play directly in cancer pathology. These sequences can be found in genes that have well-established roles in cancer, including BRCA1, 2 International Journal of Cell Biology BRCA2, NF-1, and ATM. They describe the mechanisms through which pseudoexons and pseudointrons can be activated or repressed. In addition, they discuss their potential use as tumour biomarkers to provide a more detailed staging and grading of cancer. C. Naro and C. Sette discuss the key role that reversible phosphorylation plays in the regulation of alternative splicing. Both splice factors and core components of the spliceosome are affected by phosphorylation. The review focuses on the role of protein kinases and phosphatases whose activity has specifically been linked to aberrant alternative splicing associated with neoplastic transformation. Moreover, it illustrates the fact that signal transduction routes that are frequently altered in cancer cells, such as the RAS/ERK and the PI3K/AKT pathways, can modulate alternative splicing events through the direct or indirect phosphorylation of splicing regulatory proteins. Thus, protein kinases and phosphatases involved in this step of gene expression regulation may provide exciting opportunities for novel drug design. A. Best et al. describe the emerging role of Tra2, an SR-related protein, in human cancer. The gene encoding this splicing factor is amplified in various types of cancer and the increased expression of Tra2 is associated with cancer cell survival. Interestingly, the Tra2 gene is a transcriptional target of the proto-oncogene ETS-1, whereas known Tra2 splicing targets play key roles in cancer cells, where they affect metastasis, proliferation, and cell survival. These observations point to regulation of Tra2 expression in cancer cells as an important step in tumorigenesis. The review by Z. Siegfried et al. gives a series of specific examples that cover misregulated alternative splicing events affecting both the Ras-MAPK and PI3K-mTOR signalling pathways during carcinogenesis.These pathways show extensive crosstalk and are commonly altered in many cancers by genetic and epigenetic aberrations. This article also addresses how these signalling pathways play key roles in the transmission of extracellular signals to the splicing machinery and to specific RNA-binding proteins that ultimately regulate exon definition events. C. Jackson et al. give an overview on a topic of significant clinical interest: the roles (often opposed) of the HER2 splice isoforms in breast cancer progression and drug resistance. M. P. Paronetto describes the function of the Ewing Sarcoma protein (EWS) in cancer biology. EWS is best known for its involvement in translocations associated with sarcomas. Recent evidence has implicated EWS in the regulation of DNA damage response (DDR) in cancer. EWS is a multifunctional protein thought to help coordinate multiple steps in the synthesis and processing of pre-mRNAs. This review illustrates in detail the biochemical features and the physiological roles of this RNA binding protein and provides some hints on its possible contribution to human cancer. Other two reviews give a series of specific examples of cancer-associated splicing variants. C. Sette discusses the growing evidence that dysregulated alternative splicing is a major factor in the remarkable biological heterogeneity of prostate cancer. Key genes, including the androgen receptor itself, are alternatively spliced, thereby expressing isoforms with opposing functions. The review also illustrates how the regulation of alternative splicing is likely to present novel opportunities in the diagnosis, prognosis, and treatment of prostate cancer. S. Bonomi et al. deal with novel information on how alternative splice variants of many cancer-related genes can directly contribute to the oncogenic phenotype, focusing on a number of processes involved in cancer progression, such as response to hypoxia, migration, invasion, and metastasis. Furthermore, they discuss some significant examples of alternative splicing isoforms selectively expressed by tumors and not by normal tissues, which may not only represent diagnostic and prognostic tumor biomarkers, but also provide potential targets for the development of new therapeutic strategies. In their article, L. Spraggon and L. Cartegni focus on the role of U1 snRNP, an essential component of the splicing machinery, in the regulation of alternative polyadenylation and they strongly emphasize its implications in cancer pathogenesis. Moreover, this review underlines the interesting possibility of manipulating this U1 snRNP function for anticancer therapeutic purposes. Lastly, S. Barberan-Soler and J. M. Ragle give an overview of the advantages of using the nematode Caenorhabditis elegans to study splicing regulation in vivo. Importantly, a large percentage of genes undergo alternative splicing also in this simple and genetically useful organism. A big proportion of these events are functional, conserved, and under strict regulation across development, suggesting that their investigation is likely to provide general mechanisms of regulation that can be applied also to human genes. Notably, the review illustrates several examples of alternatively spliced genes that have human homologues implicated in cancer biology. We hope that this special issue will attract the attention of researchers on new progresses in the fields of alternative splicing and cancer. In particular, the articles presented herein might highlight how this posttranscriptional mechanism of gene expression plays important roles in the generation of oncogenes and tumor suppressors, describe its interplay with signaling pathways, and suggest how our knowledge of these processes is leading to a better comprehension of malignant transformation, thus helping develop novel therapeutic strategies for the treatment of cancers