Di Matteo et al. Journal of Experimental & Clinical Cancer Research (2020) 39:275 https://doi.org/10.1186/s13046-020-01753-1 REVIEW Open Access Alternative splicing in endothelial cells: novel therapeutic opportunities in cancer angiogenesis Anna Di Matteo1†, Elisa Belloni1†, Davide Pradella1†, Ambra Cappelletto2†, Nina Volf2†, Serena Zacchigna2,3*† and Claudia Ghigna1*† Abstract Alternative splicing (AS) is a pervasive molecular process generating multiple protein isoforms, from a single gene. It plays fundamental roles during development, differentiation and maintenance of tissue homeostasis, while aberrant AS is considered a hallmark of multiple diseases, including cancer. Cancer-restricted AS isoforms represent either predictive biomarkers for diagnosis/prognosis or targets for anti-cancer therapies. Here, we discuss the contribution of AS regulation in cancer angiogenesis, a complex process supporting disease development and progression. We consider AS programs acting in a specific and non-redundant manner to influence morphological and functional changes involved in cancer angiogenesis. In particular, we describe relevant AS variants or splicing regulators controlling either secreted or membrane-bound angiogenic factors, which may represent attractive targets for therapeutic interventions in human cancer. Keywords: Alternative splicing; RNA binding proteins, Endothelial cells, Angiogenesis, Vascular biology, Anti- angiogenic therapy Background sustained viability of dog thyroid glands ex vivo. To prove Introduction: from the theory of angiogenesis to an tissue viability, he implanted mouse tumor cells into dog orchestra of alternatively spliced angiogenic genes glands and observed that the neoplastic mass stopped In the 1970s Judah Folkman revolutionized the field of growing after having reached a modest size, but grew rap- angiogenesis with his radical idea that tumor growth could idly again if transplanted back into a living mouse. He also be halted by depriving it of blood supply. It all started as a noticed that retro-transplanted tumors were decorated by by-product of an investigation originally designed to test a network of tiny blood vessels, which were not present in the efficacy of haemoglobin-plasma solution as a blood tumors grown inside the thyroid glands [1]. Later, experi- substitute for prolonged extracorporeal perfusion. Folk- ments in the hamster cheek pouch showed that capillary man was testing whether haemoglobin-plasma solution sprouts grew even if tumor cells were separated from the host stroma by a porous filter, suggesting the existence of an active humoral factor capable of driving tumor neovas- * Correspondence: [email protected]; [email protected] †Anna Di Matteo, Elisa Belloni, Davide Pradella, Ambra Cappelletto and Nina cularization (also named angiogenesis) [2, 3]. This factor Volf contributed equally to this work. † was isolated by Folkman and initially named tumor- Claudia Ghigna and Serena Zacchigna are Co-last authors angiogenesis factor, TAF. It could be purified from human 2Cardiovascular Biology Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), 34149 Trieste, Italy and animal tumors, as well as from the placenta, and 1Istituto di Genetica Molecolare, “Luigi Luca Cavalli-Sforza”, Consiglio showed remarkable mitogenic activity toward endothelial Nazionale delle Ricerche, via Abbiategrasso 207, 27100 Pavia, Italy cells (ECs) in multiple assays [2–4]. This was the first Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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AS new blood vessel formation and paved the way to the idea correlates with organism complexity, affecting 95% of of blocking angiogenesis to halt tumor growth. In its ori- human protein-coding genes [10, 11] and only 25–60% ginal assumption, the concept of anti-angiogenesis would of Drosophila melanogaster and Caenorhabditis elegans prevent new vessel sprouts from penetrating into an early genes, respectively [12–14]. tumor and keep it in an avascular and dormant state, in Alternatively spliced mRNAs frequentlydisplayatissue- which it cannot exceed 2–3mmsize[5]. While this con- specific expression [11] and encode for specialized proteins cept was initially widely criticized, its potential efficacy in involved in development, differentiation and maintenance treating cancer started to emerge a few years later, when of tissue homeostasis [15]. AS often affects domains in- Folkman teamed up with his students and monitored the volved in protein-protein interaction, suggesting its crucial grow of cancer cells when implanted into either the avas- role in controlling connected signaling cascades [15]. cular anterior chamber of the eye or the iris, which con- Splicing signals (for example 3′ splice sites) are often tains abundant blood vessels. Avascular implants in the short and degenerated. The intrinsic weakness of these anterior chamber barely grew and soon became dormant. motifs determines their low affinity for spliceosome In contrast, the same tumors grew 4000-fold faster in the components. This, in combination with auxiliary se- vascularized iris. This clearly demonstrated that tumor quences that are located either within exons or in the growth depends on blood supply and tumor dormancy is adjacent introns, creates the opportunity to realize AS caused by lack of vascularization and not by cell cycle ar- schemes. Auxiliary splicing signals are recognized by rest or immune control, as previously believed [6]. Discov- RNA binding proteins (RBPs), which either stimulate ery of TAF triggered the search for numerous angiogenic (enhancers) or inhibit (silencers) spliceosome assembly molecules, including vascular endothelial growth factor on the pre-mRNA [16] (Fig. 1d). The majority of the (VEGF), fibroblast growth factor (FGF), angiogenin, and splicing enhancers are purine-rich motifs and are bound many others [7]. by Serine-Arginine-rich (SR) proteins [17]. On the con- Over the years, a more complex situation has emerged, trary, splicing silencers are diverse in sequence and they and the original names (i.e. VEGF or FGF) are currently are mainly bound by heterogeneous nuclear ribonucleo- used to indicate families of proteins, each one existing in proteins (hnRNPs) [18]. Similar to transcription regula- multiple splicing isoforms. It has also become clear that tory sequences, splicing enhancers and silencers are members of the same family, but also alternative splicing often clustered on the pre-mRNA. Consequently, several (AS) variants of the same protein, can elicit either pro- SR proteins and hnRNPs act in either synergistic or an- or anti-angiogenic activities. Their relative abundance in tagonistic manner. For example, SR proteins can block cancer significantly contributes to the effective formation the binding of hnRNPs to a nearby silencer sequence of new blood vessels and thus AS represents an attract- and thus inhibit their negative effect on splicing (Fig. 1d). ive target for the development of innovative therapies. Therefore, the relative levels of SR proteins and hnRNPs determine the outcome of the AS reaction. While SR Alternative splicing proteins are ubiquitously expressed, a few splicing regu- In eukaryotic cells, intron removal from primary tran- latory factors (SRFs) display a more restricted pattern of scripts (pre-mRNAs) by splicing is an obligatory step be- expression, thus contributing to tissue-specific gene ex- fore mature mRNAs are transported into the cytoplasm pression programs [15]. Finally, reading of the “splicing for their translation (Fig. 1a). Splicing is realized in the code” depends on multiple elements that can mask spli- nucleus by a complex and dynamic molecular machin- cing signals, including secondary structures in the pre- ery, the spliceosome, which recognizes short consensus mRNA [19], chromatin organization, epigenetic modifi- motifs close to the exon-intron and intron-exon junc- cations [20], and RNA pol II elongation rate [21]. tions: the 5′ and the 3′ splices sites, the branch point, AS dysregulation has emerged as an important genetic and the polypyrimidine tract [8] (Fig. 1a). These se- modifier in tumorigenesis [22]. Mutations in splicing se- quences are bound by spliceosome components (such as quences and/or