==== Front Int J Mol Sci Int J Mol Sci ijms International Journal of Molecular Sciences 1422-0067 MDPI 33261131 10.3390/ijms21239032 ijms-21-09032 Review Alternative Splicing: Expanding the Landscape of Cancer Biomarkers and Therapeutics Bessa Cláudia 12 https://orcid.org/0000-0002-9379-9696Matos Paulo 12 https://orcid.org/0000-0002-1425-9211Jordan Peter 12*† https://orcid.org/0000-0003-1934-4279Gonçalves Vânia 12*† 1 Department of Human Genetics, National Health Institute Dr. Ricardo Jorge, 1649-016 Lisbon, Portugal; claudia.bessa@insa.min-saude.pt (C.B.); paulo.matos@insa.min-saude.pt (P.M.) 2 BioISI—Biosystems & Integrative Sciences Institute, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal * Correspondence: peter.jordan@insa.min-saude.pt (P.J.); vania.goncalves@insa.min-saude.pt (V.G.); Tel.: +351-217-519-380 (P.J.)† These authors contributed equally to this work. 27 11 2020 12 2020 21 23 903231 10 2020 25 11 2020 © 2020 by the authors.2020Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Alternative splicing (AS) is a critical post-transcriptional regulatory mechanism used by more than 95% of transcribed human genes and responsible for structural transcript variation and proteome diversity. In the past decade, genome-wide transcriptome sequencing has revealed that AS is tightly regulated in a tissue- and developmental stage-specific manner, and also frequently dysregulated in multiple human cancer types. It is currently recognized that splicing defects, including genetic alterations in the spliced gene, altered expression of both core components or regulators of the precursor messenger RNA (pre-mRNA) splicing machinery, or both, are major drivers of tumorigenesis. Hence, in this review we provide an overview of our current understanding of splicing alterations in cancer, and emphasize the need to further explore the cancer-specific splicing programs in order to obtain new insights in oncology. Furthermore, we also discuss the recent advances in the identification of dysregulated splicing signatures on a genome-wide scale and their potential use as biomarkers. Finally, we highlight the therapeutic opportunities arising from dysregulated splicing and summarize the current approaches to therapeutically target AS in cancer. pre-messenger RNAalternative splicingmutationcancer progressionsplicing factorsignal transductionbiomarkertherapeutic targettumor biology ==== Body 1. Introduction In higher eukaryotes, the primary gene transcripts, also called precursor messenger RNAs (pre-mRNAs), undergo a finely tuned post-transcriptional regulatory process that removes the non-coding regions (introns) and splices together the coding sequences (exons), thus generating the mature mRNAs. This mechanism is designated as pre-mRNA splicing and is a critical step in gene expression. In addition, it is well known that the splicing patterns of a gene vary widely as result of the process of alternative splicing (AS) that differentially retains or excludes certain exons from the pre-mRNA transcript. Consequently, various combinations of exons from a single gene can produce a diversity of mRNA variants, which is determinant to structural transcript variation and proteome diversity [1] and can generate different protein isoforms with related, distinct, or even opposing functions [2,3]. Remarkably, AS is a widespread event affecting more than 95% of transcribed human genes, as suggested by data provided by whole transcriptome sequencing projects [2,4]. This complex and tightly regulated mechanism is shared across different tissues and developmental stages, and frequently dysregulated in various human diseases, including cancer [5]. This dysregulation was verified in various types of cancer through detection of aberrant splicing patterns in tumor tissues when compared to their normal counterparts by high-throughput sequencing techniques [6,7,8,9]. Additionally, accumulating evidence clearly supports that the aberrant splicing profiles found in cancer are contributing to neoplastic transformation, cancer progression, and therapy resistance [10,11]. Therefore, it is of utmost relevance to identify pathological splicing isoforms for the development of new effective biomarkers, as well as to clarify the mechanisms behind aberrant AS, thereby elucidating its impact on cancer and providing novel therapeutic strategies. Hence, this review summarizes our current understanding of splicing alterations in cancer and emphasizes the need for a deeper understanding of cancer-specific splicing programs in order to provide new insights in oncology. Particularly, we highlight the relevance of identifying cancer-specific AS events for the development of novel biomarkers and discuss part of the current therapeutic landscape regarding splicing-based therapies for cancer treatment. 2. Alternative RNA Splicing: An Overview Pre-mRNA splicing consists of a multistep process orchestrated by the spliceosome, a huge RNA/protein complex comprising five small nuclear ribonucleoproteins (snRNPs; U1, U2, U4, U5, and U6) and numerous associated proteins [12,13]. Briefly, the reaction initiates with the assembly of an initial spliceosome complex through recognition of critical consensus splice sites at the pre-mRNA transcript, as schematically represented in Figure 1A. It comprises a stepwise process that begins with the recruitment of U1 snRNP to the 5′ splice site. Then, the splicing factor 1 (SF1), U2 snRNP auxiliary factor 2 (U2AF2), and U2 snRNP auxiliary factor (U2AF) 2, and U2AF 1recognize the branch point site (BPS), the polypyrimidine (poly-Y) tract, and the AG dinucleotide of the 3′ splice site region, respectively. The occupancy of these three consensus sequences induces the association of U2 snRNP with the BPS, which is further stabilized by the U2 snRNP component SF3B1. Consequently, intronic recognition prompts the engagement of U4/U6/U5 tri-snRNP with the complex, and subsequent formation of a catalytically inactive complex. This leads to several conformational and compositional rearrangements of spliceosomal components, including the dissociation of U1 and U4 snRNPs, which in turn promotes the formation of the activated spliceosome that catalyzes the splicing reaction [14]. Transcripts from nearly all protein-coding genes undergo one or more types of AS, giving rise to different mRNAs that differ in transcript degradation or are translated into alternative protein isoforms in a cell type-, organ-, or tissue-specific manner [2,4,15]. In higher eukaryotes, among the currently known AS events represented in Figure 1B, the most common is exon skipping [16], accounting for approximately 40% of all AS events, in which a cassette exon is removed from the pre-mRNA together with its flaking introns. Besides this, switching between alternative 5′ and 3′ splice site positions, mutually exclusive splicing of adjacent exons and differential retention of introns are also important variations of AS (Figure 1B). Other types of AS events include the use of alternative transcription start sites and alternative polyadenylation. In AS, the regulated process consists of the recognition of an exon by the spliceosome. For this, splice site utilization is further regulated by cis-acting splicing-regulatory elements, which either promote or inhibit the use of adjacent splice sites by recruiting trans-acting splicing factors [17]. Thus, they are classified into exonic or intronic splicing enhancers (ESE/ISE) or silencers (ESS/ISS), depending on their positions and functions (Figure 1C). In general, enhancers are recognized by trans-acting factors belonging to the serine/arginine-rich (SR) protein family to facilitate splice site recognition and exon inclusion [18]. On the other hand, silencers usually interact with other types of trans-acting factors such as heterogeneous ribonucleoproteins (hnRNPs) to inhibit splice site recognition and promote exon skipping [2]. However, several AS events exist in which SR or hnRNP proteins act as inhibitors or enhancers of splicing, respectively. 2.1. Dysregulation of Alternative Splicing in Cancer Cancer mainly evolves through successive genetic alterations and genomic dysregulation, but is also affected by the tumor microenvironment. These render oncogenes constitutively active and inactivate tumor-suppressor genes. As a result, cancer cells acquire specific abilities during tumor development, including self-sufficiency in growth signals, insensitivity to growth inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis [19]. These processes can also be dysregulated by AS, which in turn can generate variant proteins with altered physiological function [3]. Particularly, a recent systematic study performed by Kahles et al. reported that AS events are more frequent in cancer tissues compared to normal ones, and many of them are cancer-type specific [20]. Among the factors that can trigger aberrant AS, somatic mutations that disrupt splicing regulatory motifs, as well as mutations or expression changes in components of the core splicing machinery or splicing auxiliary factors, are frequently described [6,7,21,22,23,24]. Aberrant splicing in cancer has been widely linked to mutations creating cis-regulatory motifs that generate novel splice sites, as demonstrated by the discovery of almost 2000 splice site-creating mutations through a robust whole-exome analysis encompassing more than 8000 tumor samples across 33 cancer types [25]. One of the AS events frequently associated with these somatic mutations is intron retention, and mainly affects tumor suppressor genes such as TP53, ARID1A, and PTEN [7]. Importantly, most of the intron retention events are able to induce frameshifts in pre-mRNA sequence, resulting in the generation of premature termination codons (PTCs), which in turn leads to the degradation of the transcript through nonsense-mediated mRNA decay (NMD) or to the production of truncated proteins (e.g., dominant negative isoforms or neo-antigens). Interestingly, somatic exonic mutations have also been reported in oncogenes, particularly in ESE and ESS sequences [6], and associated with the generation of pro-tumorigenic variants. Recurrent somatic mutations affecting the components of the early spliceosome complex formation have frequently been described in cancer, particularly in hematological malignancies, including myelodysplastic syndromes (MDS), other myeloid neoplasms, and chronic lymphocytic leukemia (CLL) [26,27,28]. Among the genes most affected by these mutations that almost always occur in a mutually exclusive manner are SF3B1 (splicing factor 3b subunit 1), SRSF2 (serine/arginine-rich splicing factor 2), U2AF1 (U2 small nuclear RNA auxiliary factor 1), and ZRSR2 (zinc finger RNA binding motif and serine/arginine rich 2) [26]. SF3B1, a subunit of the U2 snRNP that recognizes the BPS, is the most commonly mutated splicing regulator in numerous cancers, with a prevalence ranging from 5% in breast cancer to 81% in an MDS subtype [29]. Cancer-associated SF3B1 mutations are located within HEAT (Huntingtin, Elongation factor 3, protein phosphatase 2A, Targets of rapamycin 1) domains, which are involved in protein–protein interactions and clustered in hotspots, namely K700, E622, R625, H662, and K666. Specifically, they are mainly related with the binding of SF3B1 to cryptic 3′ splice sites, located in regions with shorter and weaker poli-Y tracts, and consequently linked to aberrant BPS usage [22,30,31]. This abnormal assembly of spliceosome originates many mRNAs with a PTC, which are subsequently degraded by NMD. Although the mechanism that induces the change of 3′ splice site usage by SF3B1 is not fully elucidated, it is hypothesized that these mutations alter the interaction of SF3B1 with other spliceosomal components required for BPS recognition. SRSF2 is a member of the SR protein family that binds to specific ESE sequences, namely CCNG or GGNG, through its RNA recognition motif (RRM) domain, and recruits U1 snRNP and U2AF to the 5′ and 3′ flanking splice sites, respectively [32]. This splicing regulator has also been found recurrently mutated, particularly in patients with MDS and chronic myelomonocytic leukemia (CMML) [26]. SRSF2 mutations predominantly occur at the P95 residue, which is located near the RRM domain [26]. According to several reports, these mutations change the RNA-binding affinity of SRSF2, favoring the recognition of C-rich CCNG over G-rich GGNG motifs in ESE consensus sites, which in turn leads to misregulation of exon inclusion [33,34]. The gene encoding UA2F1 is also mutated in myeloid malignancies, as well as in lung adenocarcinomas [26,35,36]. U2AF1 hotspot mutations occur almost exclusively at S34 and Q157 residues within the two conserved zinc-finger domains, thus affecting the recognition of the 3′ splice site AG motif [37,38]. In contrast to mutually exclusive hotspot mutations described for SF3B1, SRSF2, and U2AF1, ZRSR2 mutations are distributed throughout the gene and most are consistent with a loss-of-function phenotype [23]. In 2015, in addition to the major (or U2) spliceosome, ZRSR2 was also characterized as an essential component of the minor (or U12) spliceosome that catalyzes the processing of a distinct class of introns (U12-type introns). Particularly, it is involved in 3′ splice site recognition in U12 snRNA-dependent splicing, so that mutations in this gene are associated with an increase in the retention of U12-type introns [23]. Apart from genomic mutations, the pre-mRNA splicing of many genes related to cancer pathogenesis can also be disturbed by changes of the copy number or expression levels of splicing factors [39]. Actually, abnormal expression of several splicing factors have frequently been reported in solid tumors and closely associated with cancer development and progression, even in the absence of mutations [40,41,42,43]. One of the best characterized is the serine-arginine splicing factor 1 (SRSF1; formerly known as ASF or SF2), an SR protein involved in both constitutive and AS, as well as in other cellular processes. It is upregulated in several human tumors, including colon, breast, thyroid, small intestine, kidney, and lung, and its experimentally induced overexpression leads to the transformation of human and mouse mammary epithelial cells, suggesting that it acts as a proto-oncogene [44,45,46]. Until now, SRSF1 upregulation has been shown to affect many AS events in cancer-associated genes. In particular, SRSF1 overexpression induces an increase in the levels of oncogenic protein isoforms of RON [47], MNK2, and S6K1 [44] and of the anti-apoptotic isoforms Bcl-xL and MCL-1L [48], and a loss of the tumor suppressor isoform of BIN1 [44]. Curiously, the overexpression of hnRNP A1 and hnRNP A2/B1, two factors previously suggested to antagonize SR proteins, was also reported in lung, breast, and brain tumors [49,50,51,52]. Interestingly, in glioblastoma (GBM) cells, hnRNP A2/B1 showed splicing effects similar to the proto-oncogenic SR protein SRSF1 [52]. More recently, hnRNP A2 (as well as B1 and K) has been associated with enhanced expression of anti-apoptotic variants of BIN1 and CASP9, and decreased expression of the pro-apoptotic variant Bcl-xS [48], promoting the same phenotypic response as SRSF1 overexpression. The major drivers of aberrant splicing profiles appear to be changes in the expression levels of splicing factors; however, the mechanisms behind the altered expression of the splicing factors in tumors are not yet fully understood. Although sporadic somatic mutations in genes encoding splicing factors have already been recurrently detected in solid tumors [43], it is widely recognized that oncogenic signaling has a central role [53]. Actually, abnormal activation of signaling pathways has been extensively reported in cancer. For instance, in colon cancer, oncogenic Kirsten rat sarcoma viral (KRAS) activates the RAS–MAPK pathway, leading to an increase in the expression levels of the AS factor polypyrimidine tract-binding protein 1 (PTBP1), activated via transcription factor ELK1. In turn, increased PTBP1 levels induce a shift in the AS of tumor-associated transcripts, namely, the small GTPase Ras-related C3 botulinum toxin substrate 1 (RAC1), adaptor protein NUMB, and PKM [54]. In addition to transcriptional stimulation of PTBP1 downstream of RAS, ERK was reported to phosphorylate the splicing factor SAM68, thereby inducing the binding of phospho-SAM68 to the 3′UTR of the SRSF1 transcript [55]. This binding promotes the retention of an intron required for production of full-length SRSF1 and prevents the downregulation of SRSF1 transcripts through the NMD pathway. Consequently, the increased SRSF1 levels, comparable in effect to the above described SRSF1 gene amplifications [44], induce a switch in AS of the RON gene transcripts, favoring the production of the oncogenic isoform RONΔex11. Phosphorylated SAM68 further stimulates inclusion of the variable exon 5 sequence into the CD44 mRNA, generating a pro-invasive cell adhesion protein variant [56]. Another MAPK pathway responds when cells experience physiologic stress. Osmotic stress triggers the MKK(3/6)-signaling cascade, leading to p38-activation, which upon nuclear translocation induces hnRNP A1 phosphorylation, followed by its export into the cytoplasm [57,58]. The corresponding decrease in nuclear splicing factor abundance is sufficient to change AS patterns. The PI3K/AKT signaling is another key pathway involved in cell survival and escape from apoptosis in numerous solid tumors. In non-small cell lung cancers (NSCLC), it was demonstrated that the activation of the PI3K/AKT pathway by oncogenic factors mediates the exclusion of the exon 3,4,5,6 cassette of CASP9 transcripts’ via the phosphorylation state of SRSF1, thus generating the anti-apoptotic Casp-9b isoform [59]. At the same time, AKT-mediated phosphorylation of hnRNPL induces its binding to a splice silencer element in Casp-9 pre-mRNA, further enhancing the exclusion of the exon cassette [60,61]. AKT activation also leads to phosphorylation and nuclear translocation of SR proteins, causing alternative exon inclusion in the fibronectin pre-mRNA [62]. Interestingly, in colorectal cells, inhibition of PI3K/AKT signaling led to increased expression of endogenous SRSF1, leading to the inclusion of an alternative exon, termed 3b, in the mRNA of the small GTPase RAC1, which generates the pro-tumorigenic splice variant RAC1B [63]. Later, it was described that SRPK1 and GSK3β act upstream of SRSF1, and are required to sustain RAC1B splicing in colorectal cancer (CRC) cells [64]. Particularly, it was shown that GSK3β indirectly regulates the levels of SRSF1 and RAC1B via SRPK1, since its depletion leads to a reduction of SRPK1 activity towards SRSF1, and a concomitant decrease in nuclear SRSF1 levels, resulting in less RAC1B generated. Another central hub of oncogenic signaling is the Wnt pathway, which is activated in many colorectal tumors. Remarkably, this pathway also modulates RAC1B splicing in CRC cells: It was described that the SRSF3 gene encoding splicing factor SRSF3/SRp20 is a transcriptional target for activated β-catenin/TCF4 complexes, leading to increased SRSF3 protein levels [65]. In a subsequent work, it was demonstrated that increased SRSF3 transcription following activation of the β-catenin/TCF4 pathway suppresses RAC1B splicing through SRSF3-mediated exclusion of exon 3b from the RAC1 mRNA [63]. Together, these examples show how signaling mechanisms affect alternative pre-mRNA splicing and change tumor-related gene expression. 2.2. Examples of Cancer-Associated Alternatively Spliced Variants Several splice variants have been associated with different hallmarks of cancer, including initiation, progression, and metastasis. In Table 1, we highlight some of the most relevant AS events in cancer-associated genes involved in different steps of oncogenic transformation, as well as the types of cancer they are most often associated with. Other examples were listed in a recent review [66]. 3. RNA Splice Variants as Potential Biomarkers in Cancer Early detection and diagnosis of cancer as well as the identification of the most effective personalized therapy for each patient remain the main challenges in oncology. Over the past few years, cancer biomarkers have emerged as valuable screening, diagnostic, and prognostic tools, enabling us to classify the extent of disease, define the prognosis, select the most appropriate treatment regimens, or follow up on the clinical response after treatment or surgical intervention [113]. Despite the advances, the development of more efficient biomarkers is still needed. Indeed, the amount of candidate cancer biomarkers that have been approved for clinical practice is too low, indicating that the majority of them are poor predictors of disease and treatment outcome, and are thus not reliable clinical tools [114]. In order to fill this gap, the biomarker potential of AS in cancer is currently being explored. Notably, the technological developments in sequencing and bioinformatics have provided extensive information to identify AS targets on a genome-wide scale, and in turn pathways and cellular programs that are differentially regulated in cancer cells [115,116,117,118,119,120,121]. However, from this large-scale approach, hundreds of splicing alterations are obtained that result either from mutations or abnormal expression of splicing factors, but do not readily allow for the identification of the critical cancer-driving splicing events. On the other hand, although individual pathogenic splicing events have already been described, systematic studies of the functional impact of widespread splicing alterations in cancer have yet to be performed. Actually, it is crucial to determine the outcome induced by the observed splicing changes in a tumor-specific manner with corresponding resolution at the proteome level. Therefore, in order to overcome this issue of data science and explore the splicing opportunities in precision medicine, it is extremely important to use robust analysis methods able to predict and validate reliable cancer-associated splicing changes. To date, several cancer-specific alternative transcripts with potential prognostic and predictive value in clinical settings were identified. For instance, the presence of the alternatively spliced androgen receptor variant 7 (AR-V7) in castration-resistant prostate cancer patients has been linked to a decrease in the effectiveness of hormone-directed therapy [122]. In pancreatic ductal adenocarcinoma patients receiving radical surgery and adjuvant chemotherapy, it was suggested that the tumors with higher basal expression of PKM2 exhibit more aggressive behavior and worse response to chemotherapy [123]. Additionally, EGFR variants have been widely reported in various tumor types and related to tumor progression [103,104,105,124,125]. In some cases, however, the evidence is less clear. For example, the prognostic value of the CD44 variant 6 (CD44v6) in CRC was debated for years due to contradictory results [126,127,128]. Nevertheless, further studies reinforced the relevance of CD44v6 as an independent negative prognostic factor and a promising therapeutic target in CRC [94,95,129]. Another example of a splicing biomarker with predictive potential in CRC is the upregulation of RAC1B. The overexpression of this RAC1 splice variant is frequent in CRCs carrying BRAFV600E mutation, which in advanced-stage tumors is a recognized poor prognostic biomarker [130]. Moreover, it was also reported that RAC1B expression impacts the clinical outcome of metastatic CRC patients treated with first-line 5-fluorouracil/leucovorin plus oxaliplatin or capecitabine plus oxaliplatin (FOLFOX/XELOX) chemotherapy. Indeed, the results obtained indicate that RAC1B overexpression represents an independent predictive marker of poor outcome in KRAS/BRAF wild-type metastatic CRC patients treated with this adjuvant therapy [131]. In 2013, the overexpression of RAC1B in papillary thyroid carcinomas (PTCs) was documented for the first time, and a possible interplay between BRAFV600E mutation and RAC1B postulated that may contribute to an unfavorable prognosis [132], which was also proposed for follicular thyroid carcinomas [101]. Later, the pro-tumorigenic advantage of RAC1B overexpression in thyroid carcinomas was linked to the induction of apoptosis resistance through NFκB activation [102]. Curiously, in CRC, RAC1B expression was also described as conferring chemoresistance to oxaliplatin through activation of NFκB signaling [133]. Despite being preliminary, these results indicate that RAC1B may be a clinically useful prognostic molecular biomarker for disease progression as well as a marker of resistance to therapy. Genome-Wide Identification of Cancer-Associated Splicing Signatures Recent advances in high-throughput screening (HTS) technologies covering whole-genome and -exome sequencing, such as RNA sequencing (RNA-seq), have greatly contributed to improving the diagnosis and treatment of human diseases. Particularly, RNA-seq currently represents one of the most powerful tools to investigate AS at the genome-wide level [134,135,136,137,138,139]. Compared with gene expression microarrays, also designed to sample AS events on a genome scale, RNA-seq exhibits various potential advantages, including the ability of estimating the abundance of known and novel alternative transcripts, and providing better resolution, deeper coverage, and higher accuracy [139]. However, this technology still presents some drawbacks, namely the high cost of sequencing at deep coverage and the need to continually optimize the bioinformatics protocols for processing and analyzing RNA-seq data. On the other hand, most standard RNA-seq-based analyses have mainly focused on changes in gene expression level, thus lacking information about slight differences in alternative isoform usage and exon inclusion or exclusion. The importance of investigating AS profiles in RNA-seq data was recently highlighted by a study in a preclinical model of progressive diabetic nephropathy [135]. Using the isoform- and exon-level analysis of RNA-seq data, the authors identified AS patterns in genes implicated in disease pathogenesis, such as SHC1, SERPINC1, EPB4.1L5, and IL-33, which would have been overlooked by standard gene-level analysis. Similarly, the profiling of AS signatures can be expected to provide insight into the disease process or identify potential prognostic or therapeutic biomarkers for cancer. Indeed, with the advent of HTS technologies, several studies have focused on detecting cancer-specific AS events by comparing cancer tissues with normal controls. In 2013, Eswaran et al. described for the first time splicing signatures specific to one of the three breast cancer sub-types. They further revealed that exon skipping and intron retention were the predominantly occurring splicing changes and also uncovered previously unknown isoforms of CDK4, LARP1, ADD3, and PHLPP2 [140]. This example highlights how the accumulation of RNA-seq data derived from clinical samples holds great potential to yield not only cancer-specific isoforms but also biomarkers of patient prognosis or response to therapy. In fact, certain other AS events have recently been reported to show prognostic value for ovarian, lung, pancreatic, prostate, and colorectal cancer patients [141,142,143,144,145]. For instance, in lung cancer, a genome-wide profiling identified various AS events significantly associated with patient survival [142], including EGFR, CD44, AR, RRAS2, MAPKAP1, and FGFR2. In CRC, two differently expressed AS events, namely, CSTF3-RI (intron retention) and CXCL12-AT (alternate terminator), were validated as independent prognostic indicators for both overall survival (OS) and disease-free survival [145]. Recently, the combination of high expression levels of COL6A3 E5-E6 junction and HKDC1 E1-E2 junction was for the first time associated with a better CRC patient OS [146]. Interestingly, it was previously reported that high gene expression of COL6A3 in stroma is linked to poor OS in CRC [147], while high expression of the HKDC1 gene is related to poor OS in hepatocarcinoma [148], indicating that the biomarker value of some AS events is tumor-type or -stage specific. Additionally, some reports have focused on the identification of predictive biomarkers for drug response. For instance, Safikhani et al., based on a combined approach between pharmacological data and genome-wide transcriptomics, validated AS isoforms of IGF2BP2, NECTIN4, ITGB6, and KLHDC9 as predictive biomarkers for drug response to AZD6244, lapatinib, erlotinib, and paclitaxel, respectively [149]. As a whole, despite the potential biomarkers identified to date, they still require validation in independent patient cohorts and translation into clinical practice. 4. Therapeutic Strategies Targeting Alternative Splicing in Cancer The identification of cancer-specific AS variations has guided the development of a multitude of promising therapeutic strategies. Actually, due to the different origins of AS dysregulation, previously discussed in Section 2.1, aberrant splicing programs in cancer can be targeted in diverse ways, as exemplified in Figure 2, including strategies such as blocking of protein kinases that post-translationally regulate splicing factors, disruption of signaling pathways regulating AS programs, use of oligonucleotides that modulate splicing factor recruitment to the pre-mRNA, targeting of protein isoforms derived from aberrant AS events, or targeting of the components of RNA spliceosome machinery. The latter has been discussed in detail elsewhere [29,150,151], and thus will not be addressed here. 4.1. Inhibiting Post-Translational Modifications of Splicing Factors or RNA Binding Proteins Although several types of post-translational modifications of splicing factors were described, their phosphorylation has a key role, and the development of small-molecule inhibitors targeting protein kinases has emerged as a promising therapeutic strategy to reverse aberrant RNA splicing [152,153]. The two main targets of these molecules are the SR-rich protein-specific kinases (SRPKs) and the dual-specificity Cdc2-like kinases (CLKs), which primarily regulate pre-mRNA splicing by phosphorylating SR proteins, controlling both their nucleo-cytoplasmic shuttling and their interactions with the spliceosome [154]. Increased levels of these splicing regulatory protein kinases have been found in several types of cancers, which highlights the therapeutic potential of their pharmacological targeting. Particularly, upregulated expression of SRPK1 is frequently associated with an oncogenic activity in a variety of cancer types [155]. Accordingly, pharmacological inhibition of SRPK1 with the first-generation drug SRPIN340 induced splice switching of pro-angiogenic VEGFA165 to anti-angiogenic VEGFA165b (Figure 2A) in prostate cancer and leukemic cells [156,157]. Another study also showed that SRPIN340 significantly reduces tumor growth in metastatic melanoma in vivo via reduced expression of pro-angiogenic VEGF isoforms [158]. More recently, a covalent inhibitor of SPRK1 and SPRK2—SRPKIN-1—was developed, which efficiently reduced SR protein phosphorylation, promoted splice switching of VEGFA165 to VEGFA165b, and blocked neovascularization. This was achieved by local application in mice [159], but a corresponding benefit in tumor therapy remains to be demonstrated. The first CLK inhibitor to be discovered was the benzothiazole compound TG-003, which demonstrated selective potency toward CLK1, CLK2, and CLK4 and regulated splicing by reducing the phosphorylation of several SR proteins, including SRSF1 [160]. Two other compounds, leucettine L41 and T-025, were also identified as CLK inhibitors that modulate AS by the same mechanism of action [161,162]. Lastly, in a large-scale screening, a set of related compounds was identified, namely Cpd-1, Cpd-2, and Cpd-3, capable of targeting CLK1 and CLK2 and, to a lesser extent, SPRK1 and SRPK2 [163]. Despite the apparent success of these compounds in vitro, further studies are needed in order to improve their efficacy and narrow the window of off-target effects on splicing before moving to the clinical trial setting. Another strategy to target splicing is exemplified by the use of sulfonamides, including E7820, indisulam, tasisulam, and chloroquinoxaline sulphonamide. These agents are known to show antitumor activity, and some of them have already been tested in clinical trials [164,165,166]. Later, it was confirmed that several sulphonamides interfere with splicing by promoting ubiquitin-mediated degradation of U2AF-related splicing factor RBM39 (also called CAPERα) via CRL4 E3 ubiquitin ligase complex [167,168]. For example, it was found that after indisulam treatment of cultured cancer cells, RBM39 degradation led to altered pre-mRNA splicing, including intron retention and exon skipping, in hundreds of genes [167]. However, it was also shown that RBM39 degradation is limited to certain cancer cells. Actually, mutations in RBM39, specifically in RRM2 domain, prevent its proteasomal degradation, thus conferring sulphonamide resistance. Sensitivity to these compounds also correlates with the expression levels of DCAF15 in hematopoietic and lymphoid lineages because the CUL4-DCAF15 complex regulates the ubiquitination and degradation of RBM39 [167]. 4.2. Modulation of Signaling Pathways Regulating Alternative Splicing Events The involvement of signaling pathways in the regulation of AS is well recognized, as referred to above. The mechanisms through which signal transduction pathways interfere directly or indirectly with splicing, typically involve regulation of either the cellular localization or the activation status of splicing-regulatory proteins. Therefore, modulation of signaling pathways represents a promising approach to target dysregulated AS. Importantly, despite the existence of a wide range of compounds able to target these pathways (reviewed in [169]), some of which have already been tested in clinical trials, they were not specifically developed to modulate AS. However, they proved to be valuable tools to further elucidate the mechanisms involved in the regulation of AS by oncogenic signaling pathways. For instance, the AKT inhibitor MK2206 was used to validate the results obtained in a study that aimed to unravel the differential regulation of the phosphoproteome by AKT isoforms [170]. Briefly, it was demonstrated that the specific RNA processing protein IWS1 is phosphorylated by AKT1 and AKT3 in lung cancer. IWS1 phosphorylation allows the recruitment of SETD2 to the RNA polymerase II complex. SETD2 trimethylates histone H3 during transcription, creating a docking site for PTBP1 splicing factor. In turn, PTBP1 promotes the skipping of exon IIIb in the fibroblast growth factor receptor 2 (FGFR-2) gene, shifting the balance of FGFR-2 splicing from the IIIb to the IIIc isoform, which promotes cell proliferation, migration, and invasiveness in response to FGF-2. Moreover, in a work carried out in Ewing sarcoma cells, it was found that hnRNP M was strongly upregulated both at the mRNA and protein level upon inhibition of the PI3K/AKT/mTOR pathway with BEZ235, and located in the soluble nucleoplasmic fraction, where it modulated U1 snRNP recruitment to a 5′ splice site and triggered a splicing program contributing to drug resistance (Figure 2B) [171]. Overall, these types of inhibitory drugs have the ability to change splicing outcomes; however, it is crucial to invest in the development of compounds targeting specific abnormal AS events in order to limit the occurrence of undesired side effects. 4.3. Antisense Oligonucleotides RNA-targeted therapies emerged in 1978 when Zamecnik and Stephenson described for the first time a chemically modified oligonucleotide that inhibited gene expression and viral replication of Rous sarcoma virus [172,173]. From there, antisense oligonucleotides (ASOs) have been extensively explored in the process of drug development and proven to be a useful alternative approach for the target-specific treatment of splicing-related human diseases, including cancer. Briefly, ASOs are synthetic molecules consisting of short single-stranded nucleic-acid sequences, generally 15–25 nucleotides in length, that specifically bind through Watson–Crick base-pairing to complementary pre-mRNA sequences [174]. RNA-targeted therapies are already used in the clinic and numerous clinical trials with therapeutic ASOs are currently underway [175,176,177]. The antisense therapies can be subdivided into two groups according to their downstream mechanisms of action and functional outcomes. The majority of ASOs are designed to promote the cleavage of targeted mRNA by endogenous cellular nucleases, such as RNase H, which recognizes double-stranded RNA:DNA hybrids and subsequently degrades the disease-causing gene product. A different strategy aims to interfere with the access of the splicing machinery to the regulatory sequences in the pre-mRNA instead of causing the transcript degradation. So-called splice-switching antisense oligonucleotides (SSOs) are designed to compete with and sterically block the binding of certain splicing factors to their specific sites in the pre-mRNA, which in turn changes exon recognition by the spliceosome [178]. As such, this strategy intends to specifically shift the splicing pattern of a targeted pre-mRNA transcript, favoring the production of one of the splicing variants with potential therapeutic benefits. To date, two SSOs were approved by the US Food and Drug Administration (FDA), Eteplirsen and Nusinersen, for the clinical treatment of the genetic diseases Duchenne muscular dystrophy and spinal muscular atrophy, respectively [179,180]. Eteplirsen hybridizes at exon 51 of the DMD pre-mRNA (which encodes the dystrophin protein), sterically blocking the recognition of this exon by the spliceosome and thereby promoting the skipping of exon 51 to correct the disease-causing frameshift mutation and generating a shorter but functional variant of the protein (Figure 2C) [181]. In a distinct way, Nusinersen binds to an intronic region upstream of exon 7 in the SMN2 pre-mRNA that encodes the survival motor neuron protein [180]. The binding blocks recruitment of an inhibitory splicing factor that would normally impede the recognition of exon 7 by the spliceosome, thus enhancing the inclusion of the formerly missing exon 7 of SMN2, and the subsequent production of a fully functional protein that is absent in patients with spinal muscular atrophy. Although the application of SSOs in anticancer therapy is still under evaluation, the modulation of RNA splicing of cancer-related genes has been successfully achieved in various pre-clinical cancer models. One of the most used strategies targets the BCL2L1 gene that is alternatively spliced, originating either anti-apoptotic Bcl-xL or pro-apoptotic Bcl-xS proteins. Thus, in order to abolish the high expression levels of Bcl-xL reported in many cancers, Bcl-x SSOs were designed to induce a splicing switch, favoring the production of the pro-apoptotic isoform Bcl-xS. It was shown in vitro that treatment with these SSOs shifted splicing from Bcl-xL to Bcl-xS in various cancer cell lines [182]. Moreover, it was found that Bcl-xS proteins induced by the SSOs sensitized the cancer cells to treatment with chemotherapeutic agents or ultraviolet (UV) radiation [70]. Additionally, the antitumor activity of SSOs was also demonstrated in vivo in a mouse model of melanoma lung metastases where the systemic administration of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and led to a significant reduction in tumor burden in treated mice [183]. Another important antitumor target is the hnRNP-regulated splicing of the PKM gene [184], which is a critical player in the regulation of glucose metabolism by producing either the PKM1 isoform (that stimulates oxidative phosphorylation) or the PKM2 isoform (that promotes aerobic glycolysis, a metabolic shift also recognized as the Warburg effect). PKM2 is frequently upregulated in cancer cell lines and various tumor types, including CRC, and SSOs used to switch the expression back to PKM1 induced apoptosis [185]. Further examples of SSO-mediated splicing modulation of other genes, including BCL2L11, BRCA1, ERBB2, MDM4, MKNK2, and STAT3, were recently reviewed in [150]. A related SSO approach is the design of decoy oligonucleotides composed of the RNA motif recognized by a given splicing factor, which can downmodulate its splicing activity. This could be a promising therapeutic approach whenever a splicing factor is either overexpressed or hyperactived in cancer cells [186]. 4.4. Targeting the Alternative Protein Isoform The presence of specific AS variant proteins in tumor cells suggest them as potential therapeutic targets. Some variants may result in the translation of immunogenic neoantigens, either as a result of frameshifts or re-expression of developmental variants. As such, some strategies have been developed to target cancer-specific isoforms by immunotherapies. One of the most explored therapeutic targets are EGFR variants de4 and vIII. Although in GBM and other cancers EGFRvIII results from a genomic deletion of exons 2–7 [187], an AS variant with skipping exon 4 leads to a comparable phenotype in other tumors: a lack of amino acids in the extracellular ligand-binding domain, resulting in a constitutively active variant able to stimulate downstream signaling in a ligand-independent manner. Several studies have supported the oncogenic role of these EGFR variants and their association with a poor prognosis [103,104,105,124,125,188,189,190,191,192]. Being tumor-specific cell surface molecules, these receptor variants were successfully targeted by therapeutic antibodies [104,189]. Notably, in 2015, the vaccine rindopepimut (also known as CDX-110), consisting of an EGFRvIII-specific peptide conjugated to keyhole limpet haemocyanin, was approved by FDA for the treatment of GBM. Actually, the results obtained in phase I and II clinical trials showed that the treatment with rindopepimut increases both OS and progression-free survival of GBM patients expressing EGFRvIII [189]. Additionally, the role of cell adhesion molecule CD44 and its isoforms containing the exon v6 have been broadly implicated in the metastatic tumor process, and as such they have also been explored as targets for anticancer therapy [193]. One of the most recognized anti-CD44v6 therapy consists of using bivatuzumab, a humanized IgG1 monoclonal antibody labelled with rhenium-186 (Figure 2D). Particularly in phase I clinical trials for patients with the head and neck squamous cell carcinoma (HNSCC), a tissue that expresses high amounts of CD44v6 antigen, bivatuzumab showed promising antitumor effects with consistent stable disease at higher radioactive dose levels and with low toxicity [194,195]. Based on these results, a novel strategy comprising the coupling of bivatuzumab with a non-radioactive cytotoxic drug, mertansine, was developed [196]. Interestingly, in phase I clinical trials, the intravenous injection of bivatuzumab mertansine in adult patients with recurrent or metastatic HNSCC induced a partial response in three of the 30 patients tested, which presented a stabilization of the disease and regression of tumors [197]. Despite the promising results, the toxic side effects observed in the skin led to the discontinuation of the clinical trials with bivatuzumab mertansine. Another example is the tight junction molecule claudin-18 isoform 2 (CLDN18.2). In 2008, CLDN18.2 was identified as a highly selective cell lineage marker, whose expression in normal tissues is restricted to differentiated epithelial cells of the gastric mucosa, being absent from the gastric stem cell zone [198]. Additionally, it was also reported that CLDN18.2 is expressed in a significant proportion of primary gastric cancers and their metastases. Since CLDN18.2 exposes extracellular loops available for antibody binding, a targeted therapy based on the monoclonal antibody IMAB362 (claudiximab) was developed [199]. According to the promising results obtained in previous clinical trials, a phase III global study of IMAB362 plus FOLFOX versus FOLFOX plus placebo as first-line treatment was initiated in 2018 in gastric cancer patients (NCT03504397). Besides these immunotherapeutic approaches, protein–protein interaction inhibitors could become a promising precision-medicine approach for targeting AS-derived protein isoforms. Many AS variants generate proteins following exon inclusion or intron retention and can contain extra protein domains that participate in protein–protein interactions involved in their downstream function. Small-molecule drugs that compete with these interactions are being developed [200,201]. For example, the BCL-2-selective inhibitor ABT-199 competes for anti-apoptotic interaction with BAK/BAD proteins [202], and inhibitors of the MDM2–p53 complex can restore p53 function in cancerous cells, leading to their growth arrest and apoptosis [203]. 5. Concluding Remarks and Future Perspectives From a large body of experimental data, it has become increasingly clear that AS is tightly associated with human health and disease [3]. However, despite AS being the major driver of biological diversity and playing a role in every hallmark of cancer, it was neglected for a long time in the profiling of tumor characteristics and overlooked as a source of new biomarkers and therapeutic targets for drug development. Nevertheless, with the emergence of advanced sequencing technologies that provide a landscape of AS at a genome-wide level, additional insights into the splicing programs were achieved [6,7,8,9]. Remarkably, these studies have contributed in a decisive way to the identification of aberrant AS events in cancer development and progression, a prerequisite for the identification of potential biomarkers and development of new therapeutic strategies towards cancer precision medicine. Despite the described progress, the tumor-specific splicing alterations are far from being characterized and further efforts are needed to provide a comprehensive view of splicing regulation and of its dysregulation in cancer. Another important aspect that has emerged from advanced sequencing technology is the need to move our understanding from individual AS variants to the overall pattern of splicing changes in tumors. Any change in activity or localization of a splicing factor will potentially trigger a plethora of AS decisions in many different genes. Thus, AS signature profiles or patterns may represent more meaningful biomarkers. Regarding AS-targeting drug development, existing small-molecule compounds do mostly interfere with early spliceosome assembly or post-translational modification of SR proteins, but lack efficient antitumor activity. As such, the recent efforts focused on the targeting of pathological RNA isoforms or tumor-specific protein variants represent the most promising attempts to develop more effective drug candidates. Unfortunately, these targeted anticancer therapies based on AS are still far from reaching the clinic. To address this issue, it is a priority to reveal in more detail how altered AS actually drives tumorigenesis, and how it is connected to altered genotypes or signaling pathways that characterize tumor phenotypes. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Author Contributions Conceptualization, P.J. and V.G.; writing—original draft preparation, C.B.; writing—review and editing, P.M., V.G., and P.J. All authors have read and agreed to the published version of the manuscript. Funding Work in the authors’ laboratory was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal (through grant UID/MULTI/04046/2019 to Research Unit BioISI since January 2019 and grant PTDC/BIA-MOL/28386/2017 to V.G. since October 2018) and by the Portuguese association Maratona da Saúde—Cancro 2014 to P.J. since January 2016. Conflicts of Interest The authors declare no conflict of interest. The funders had no role in the collection of data, the writing of the manuscript, or in the decision to publish the manuscript. Figure 1 Regulation of pre-mRNA splicing. (A) Stepwise assembly of spliceosome on the pre-mRNA and catalysis of the splicing reaction to generate mature spliced mRNA. (B) Schematic representation of the most common alternative splicing AS events. The grey, yellow, red, and blue boxes represent different exons. The solid black and dotted grey lines indicate distinct splicing events. (C) Complex interplay between cis- and trans-acting factors in the regulation of AS. RNA-binding motif (RBM) proteins, serine/arginine-rich (SR) proteins, and heterogeneous (hn) ribonucleoproteins (hnRNPs) bind to exonic or intronic regulatory elements to promote or prevent the recognition of either 3′ or 5′ splice sites (ss) by the small nuclear (sn) RNPs (snRNPs) and splicing factors. The solid and dotted black arrows represent binding stimulation and inhibition, respectively; (ss—splice sites; BPS—branch point site; poly-Y—polypyrimidine tract; pre-mRNA—precursor messenger RNA; snRNPs—small nuclear ribonucleoprotein particle; SF1—splicing factor 1; U2AF—U2 snRNP auxiliary factor). Figure 2 Examples of therapeutic strategies targeting alternative splicing (AS). (A) Targeting of protein kinases by small molecules to inhibit the post-translational phosphorylation of splicing factors. (B) Inhibition of signaling pathways by small molecules. (C) Splice-switching antisense oligonucleotides. (D) Targeting of cancer-specific isoforms by therapeutic monoclonal antibodies; (pre-mRNA—precursor messenger RNA; ss—splice site; RTK—receptor tyrosine kinase; hnRNP—heterogeneous nuclear ribonucleoprotein; DMD—dystrophin gene). ijms-21-09032-t001_Table 1Table 1 Tumor-associated AS variants and the respective cancer-promoting process. Gene Splicing Event Biological Function Cancer Types References BCL2L1 5′ alternative splice site usage in exon 2 Bcl-xL inhibits apoptosis Lymphoma, glioma, breast, prostate, and liver cancer [67,68,69,70,71] MKNK2 Skipping of exon 14a and inclusion of exon 14b MNK2b acts p38-MAPK-independent and promotes cell growth Breast, colon, and lung cancer [44,72,73] PKM Skipping of exon 9 and inclusion of exon 10 PKM2 stimulates aerobic glycolysis Ovarian, gastric, liver, and colon cancer [74,75,76,77] MST1R (RON) Skipping of exon 11 RONΔex11 induces cell motility and invasion Colon, ovarian, brain, lung, and gastric cancer [78,79,80,81,82] RPS6KB1 Inclusion of three cassette exons 6a, 6b, and 6c with a PTC in exon 6c RPS6KB1-2 promotes cell proliferation and tumor growth Breast and lung cancer [83,84] CCND1 5′ alternative splice site usage in exon 4 introduces a PTC Cyclin D1b induces invasion and metastasis Breast, lung, and prostate cancer [85,86,87] VEGFA Alternative 3′ splice site in exon 8 VEGFA165 has pro-angiogenic activity Colon, prostate, renal, and skin cancer [88,89,90,91] CEACAM1 Inclusion of exon 7 CEACAM1-L accelerates metastasis progression Colon cancer and metastatic melanoma [92,93] CD44 Inclusion of variable exon 6 CD44-v6 induces migration and expression of mesenchymal markers Colon cancer [94,95,96] RAC1 Inclusion of exon 3b RAC1B increases cell survival and transformation Colon, pancreas, thyroid, breast, and lung cancer [63,97,98,99,100,101,102] EGFR Skipping of exon 4 de4-EGFR promotes malignant transformation as constitutively active receptor variant Glioma, prostate, and ovarian cancer [103,104,105] KLF6 5′ alternative splice site usage in exon 2 KLF6-SV1 lacks nuclear localization and contributes to mesenchymal phenotype Breast, lung, pancreatic, prostate, and liver cancer [106,107,108,109,110] CTTN Inclusion of exon 11 Cortactin isoform-a increases cell migration Colorectal cancer [111] FAK Deletion of exon 26 The −26-exon FAK isoform is caspase-resistant and inhibits apoptosis Breast cancer [112] The listed genes are B-cell CLL/lymphoma 2-like 1 (BCL2L1), MAPK interacting serine/threonine kinase 2 (MKNK2), pyruvate kinase M (PKM), macrophage stimulating 1 receptor (MST1R), ribosomal protein S6 kinase B1 (RPS6KB1), cyclin D1 (CCND1), vascular endothelial growth factor A (VEGFA), CEA cell adhesion molecule 1 (CEACAM1), clusters of differentiation 44 (CD44), ras-related C3 botulinum toxin substrate 1 (RAC1), epidermal growth factor receptor (EGFR), Krüppel-like factor 6 (KLF6), cortactin (CTTN), and focal adhesion kinase (FAK); (PTC—premature termination codon). ==== Refs References 1. Blencowe B.J. The Relationship between Alternative Splicing and Proteomic Complexity Trends Biochem. Sci. 2017 42 407 408 10.1016/j.tibs.2017.04.001 28483376 2. Wang E.T. Sandberg R. Luo S. Khrebtukova I. Zhang L. Mayr C. Kingsmore S.F. Schroth G.P. Burge C.B. Alternative isoform regulation in human tissue transcriptomes Nature 2008 456 470 476 10.1038/nature07509 18978772 3. Kim H.K. Pham M.H.C. Ko K.S. Rhee B.D. Han J. Alternative splicing isoforms in health and disease Pflug. Arch. 2018 470 995 1016 10.1007/s00424-018-2136-x 29536164 4. Pan Q. Shai O. Lee L.J. Frey B.J. Blencowe B.J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing Nat. Genet. 2008 40 1413 1415 10.1038/ng.259 18978789 5. Venables J.P. Aberrant and alternative splicing in cancer Cancer Res. 2004 64 7647 7654 10.1158/0008-5472.CAN-04-1910 15520162 6. Supek F. Minana B. Valcarcel J. Gabaldon T. Lehner B. Synonymous mutations frequently act as driver mutations in human cancers Cell 2014 156 1324 1335 10.1016/j.cell.2014.01.051 24630730 7. Jung H. Lee D. Lee J. Park D. Kim Y.J. Park W.Y. Hong D. Park P.J. Lee E. Intron retention is a widespread mechanism of tumor-suppressor inactivation Nat. Genet. 2015 47 1242 1248 10.1038/ng.3414 26437032 8. Chen L. Tovar-Corona J.M. Urrutia A.O. Increased levels of noisy splicing in cancers, but not for oncogene-derived transcripts Hum. Mol. Genet. 2011 20 4422 4429 10.1093/hmg/ddr370 21862452 9. Dvinge H. Bradley R.K. Widespread intron retention diversifies most cancer transcriptomes Genome Med. 2015 7 45 10.1186/s13073-015-0168-9 26113877 10. Dvinge H. Kim E. Abdel-Wahab O. Bradley R.K. RNA splicing factors as oncoproteins and tumour suppressors Nat. Rev. Cancer 2016 16 413 430 10.1038/nrc.2016.51 27282250 11. Paronetto M.P. Passacantilli I. Sette C. Alternative splicing and cell survival: From tissue homeostasis to disease Cell Death Differ. 2016 23 1919 1929 10.1038/cdd.2016.91 27689872 12. Rappsilber J. Ryder U. Lamond A.I. Mann M. Large-scale proteomic analysis of the human spliceosome Genome Res. 2002 12 1231 1245 10.1101/gr.473902 12176931 13. Jurica M.S. Moore M.J. Pre-mRNA splicing: Awash in a sea of proteins Mol. Cell 2003 12 5 14 10.1016/S1097-2765(03)00270-3 12887888 14. Coltri P.P. Dos Santos M.G.P. da Silva G.H.G. Splicing and cancer: Challenges and opportunities Wiley Interdiscip. Rev. RNA 2019 10 e1527 10.1002/wrna.1527 30773852 15. Liu Y. Gonzalez-Porta M. Santos S. Brazma A. Marioni J.C. Aebersold R. Venkitaraman A.R. Wickramasinghe V.O. Impact of Alternative Splicing on the Human Proteome Cell Rep. 2017 20 1229 1241 10.1016/j.celrep.2017.07.025 28768205 16. Keren H. Lev-Maor G. Ast G. Alternative splicing and evolution: Diversification, exon definition and function Nat. Rev. Genet. 2010 11 345 355 10.1038/nrg2776 20376054 17. Wang Z. Burge C.B. Splicing regulation: From a parts list of regulatory elements to an integrated splicing code RNA 2008 14 802 813 10.1261/rna.876308 18369186 18. Lee Y. Rio D.C. Mechanisms and Regulation of Alternative Pre-mRNA Splicing Annu Rev. Biochem. 2015 84 291 323 10.1146/annurev-biochem-060614-034316 25784052 19. Hanahan D. Weinberg R.A. Hallmarks of cancer: The next generation Cell 2011 144 646 674 10.1016/j.cell.2011.02.013 21376230 20. Kahles A. Lehmann K.V. Toussaint N.C. Huser M. Stark S.G. Sachsenberg T. Stegle O. Kohlbacher O. Sander C. Cancer Genome Atlas Research Network Comprehensive Analysis of Alternative Splicing Across Tumors from 8,705 Patients Cancer Cell 2018 34 211 224 10.1016/j.ccell.2018.07.001 30078747 21. Bechara E.G. Sebestyen E. Bernardis I. Eyras E. Valcarcel J. RBM5, 6, and 10 differentially regulate NUMB alternative splicing to control cancer cell proliferation Mol. Cell 2013 52 720 733 10.1016/j.molcel.2013.11.010 24332178 22. Darman R.B. Seiler M. Agrawal A.A. Lim K.H. Peng S. Aird D. Bailey S.L. Bhavsar E.B. Chan B. Colla S. Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3′ Splice Site Selection through Use of a Different Branch Point Cell Rep. 2015 13 1033 1045 10.1016/j.celrep.2015.09.053 26565915 23. Madan V. Kanojia D. Li J. Okamoto R. Sato-Otsubo A. Kohlmann A. Sanada M. Grossmann V. Sundaresan J. Shiraishi Y. Aberrant splicing of U12-type introns is the hallmark of ZRSR2 mutant myelodysplastic syndrome Nat. Commun. 2015 6 6042 10.1038/ncomms7042 25586593 24. Zong F.Y. Fu X. Wei W.J. Luo Y.G. Heiner M. Cao L.J. Fang Z. Fang R. Lu D. Ji H. The RNA-binding protein QKI suppresses cancer-associated aberrant splicing PLoS Genet. 2014 10 e1004289 10.1371/journal.pgen.1004289 24722255 25. Jayasinghe R.G. Cao S. Gao Q. Wendl M.C. Vo N.S. Reynolds S.M. Zhao Y. Climente-Gonzalez H. Chai S. Wang F. Systematic Analysis of Splice-Site-Creating Mutations in Cancer Cell Rep. 2018 23 270 281 10.1016/j.celrep.2018.03.052 29617666 26. Yoshida K. Sanada M. Shiraishi Y. Nowak D. Nagata Y. Yamamoto R. Sato Y. Sato-Otsubo A. Kon A. Nagasaki M. Frequent pathway mutations of splicing machinery in myelodysplasia Nature 2011 478 64 69 10.1038/nature10496 21909114 27. Bejar R. Stevenson K.E. Caughey B.A. Abdel-Wahab O. Steensma D.P. Galili N. Raza A. Kantarjian H. Levine R.L. Neuberg D. Validation of a prognostic model and the impact of mutations in patients with lower-risk myelodysplastic syndromes J. Clin. Oncol. 2012 30 3376 3382 10.1200/JCO.2011.40.7379 22869879 28. Papaemmanuil E. Gerstung M. Malcovati L. Tauro S. Gundem G. Van Loo P. Yoon C.J. Ellis P. Wedge D.C. Pellagatti A. Chronic Myeloid Disorders Working Group of the International Cancer Genome, C., Clinical and biological implications of driver mutations in myelodysplastic syndromes Blood 2013 122 3616 3627 10.1182/blood-2013-08-518886 24030381 29. Bonnal S.C. Lopez-Oreja I. Valcarcel J. Roles and mechanisms of alternative splicing in cancer—implications for care Nat. Rev. Clin. Oncol. 2020 17 457 474 10.1038/s41571-020-0350-x 32303702 30. DeBoever C. Ghia E.M. Shepard P.J. Rassenti L. Barrett C.L. Jepsen K. Jamieson C.H. Carson D. Kipps T.J. Frazer K.A. Transcriptome sequencing reveals potential mechanism of cryptic 3′ splice site selection in SF3B1-mutated cancers PLoS Comput. Biol. 2015 11 e1004105 10.1371/journal.pcbi.1004105 25768983 31. Alsafadi S. Houy A. Battistella A. Popova T. Wassef M. Henry E. Tirode F. Constantinou A. Piperno-Neumann S. Roman-Roman S. Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage Nat. Commun. 2016 7 10615 10.1038/ncomms10615 26842708 32. Daubner G.M. Clery A. Jayne S. Stevenin J. Allain F.H. A syn-anti conformational difference allows SRSF2 to recognize guanines and cytosines equally well EMBO J. 2012 31 162 174 10.1038/emboj.2011.367 22002536 33. Kim E. Ilagan J.O. Liang Y. Daubner G.M. Lee S.C. Ramakrishnan A. Li Y. Chung Y.R. Micol J.B. Murphy M.E. SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition Cancer Cell 2015 27 617 630 10.1016/j.ccell.2015.04.006 25965569 34. Zhang J. Lieu Y.K. Ali A.M. Penson A. Reggio K.S. Rabadan R. Raza A. Mukherjee S. Manley J.L. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities Proc. Natl. Acad. Sci. USA 2015 112 4726 4734 10.1073/pnas.1514105112 26261309 35. Kandoth C. McLellan M.D. Vandin F. Ye K. Niu B. Lu C. Xie M. Zhang Q. McMichael J.F. Wyczalkowski M.A. Mutational landscape and significance across 12 major cancer types Nature 2013 502 333 339 10.1038/nature12634 24132290 36. Seiler M. Peng S. Agrawal A.A. Palacino J. Teng T. Zhu P. Smith P.G. Cancer Genome Atlas Research N. Buonamici S. Yu L. Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types Cell Rep. 2018 23 282 296 10.1016/j.celrep.2018.01.088 29617667 37. Ilagan J.O. Ramakrishnan A. Hayes B. Murphy M.E. Zebari A.S. Bradley P. Bradley R.K. U2AF1 mutations alter splice site recognition in hematological malignancies Genome Res. 2015 25 14 26 10.1101/gr.181016.114 25267526 38. Fei D.L. Motowski H. Chatrikhi R. Prasad S. Yu J. Gao S. Kielkopf C.L. Bradley R.K. Varmus H. Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival PLoS Genet. 2016 12 e1006384 10.1371/journal.pgen.1006384 27776121 39. Zhang J. Manley J.L. Misregulation of pre-mRNA alternative splicing in cancer Cancer Discov. 2013 3 1228 1237 10.1158/2159-8290.CD-13-0253 24145039 40. Ghigna C. Valacca C. Biamonti G. Alternative splicing and tumor progression Curr. Genom. 2008 9 556 570 10.2174/138920208786847971 41. Ghigna C. Moroni M. Porta C. Riva S. Biamonti G. Altered expression of heterogenous nuclear ribonucleoproteins and SR factors in human colon adenocarcinomas Cancer Res. 1998 58 5818 5824 9865741 42. Grosso A.R. Martins S. Carmo-Fonseca M. The emerging role of splicing factors in cancer EMBO Rep. 2008 9 1087 1093 10.1038/embor.2008.189 18846105 43. Sveen A. Kilpinen S. Ruusulehto A. Lothe R.A. Skotheim R.I. Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes Oncogene 2016 35 2413 2427 10.1038/onc.2015.318 26300000 44. Karni R. de Stanchina E. Lowe S.W. Sinha R. Mu D. Krainer A.R. The gene encoding the splicing factor SF2/ASF is a proto-oncogene Nat. Struct. Mol. Biol. 2007 14 185 193 10.1038/nsmb1209 17310252 45. Anczukow O. Rosenberg A.Z. Akerman M. Das S. Zhan L. Karni R. Muthuswamy S.K. Krainer A.R. The splicing factor SRSF1 regulates apoptosis and proliferation to promote mammary epithelial cell transformation Nat. Struct. Mol. Biol. 2012 19 220 228 10.1038/nsmb.2207 22245967 46. Anczukow O. Akerman M. Clery A. Wu J. Shen C. Shirole N.H. Raimer A. Sun S. Jensen M.A. Hua Y. SRSF1-Regulated Alternative Splicing in Breast Cancer Mol. Cell 2015 60 105 117 10.1016/j.molcel.2015.09.005 26431027 47. Ghigna C. Giordano S. Shen H. Benvenuto F. Castiglioni F. Comoglio P.M. Green M.R. Riva S. Biamonti G. Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene Mol. Cell 2005 20 881 890 10.1016/j.molcel.2005.10.026 16364913 48. Kedzierska H. Piekielko-Witkowska A. Splicing factors of SR and hnRNP families as regulators of apoptosis in cancer Cancer Lett. 2017 396 53 65 10.1016/j.canlet.2017.03.013 28315432 49. Fielding P. Turnbull L. Prime W. Walshaw M. Field J.K. Heterogeneous nuclear ribonucleoprotein A2/B1 up-regulation in bronchial lavage specimens: A clinical marker of early lung cancer detection Clin. Cancer Res. 1999 5 4048 4052 10632338 50. Zhou J. Nong L. Wloch M. Cantor A. Mulshine J.L. Tockman M.S. Expression of early lung cancer detection marker: hnRNP-A2/B1 and its relation to microsatellite alteration in non-small cell lung cancer Lung Cancer 2001 34 341 350 10.1016/S0169-5002(01)00254-9 11714531 51. Zhou J. Allred D.C. Avis I. Martinez A. Vos M.D. Smith L. Treston A.M. Mulshine J.L. Differential expression of the early lung cancer detection marker, heterogeneous nuclear ribonucleoprotein-A2/B1 (hnRNP-A2/B1) in normal breast and neoplastic breast cancer Breast Cancer Res. Treat. 2001 66 217 224 10.1023/A:1010631915831 11510693 52. Golan-Gerstl R. Cohen M. Shilo A. Suh S.S. Bakacs A. Coppola L. Karni R. Splicing factor hnRNP A2/B1 regulates tumor suppressor gene splicing and is an oncogenic driver in glioblastoma Cancer Res. 2011 71 4464 4472 10.1158/0008-5472.CAN-10-4410 21586613 53. Goncalves V. Pereira J.F.S. Jordan P. Signaling Pathways Driving Aberrant Splicing in Cancer Cells Genes 2017 9 9 10.3390/genes9010009 29286307 54. Hollander D. Donyo M. Atias N. Mekahel K. Melamed Z. Yannai S. Lev-Maor G. Shilo A. Schwartz S. Barshack I. A network-based analysis of colon cancer splicing changes reveals a tumorigenesis-favoring regulatory pathway emanating from ELK1 Genome Res. 2016 26 541 553 10.1101/gr.193169.115 26860615 55. Valacca C. Bonomi S. Buratti E. Pedrotti S. Baralle F.E. Sette C. Ghigna C. Biamonti G. Sam68 regulates EMT through alternative splicing-activated nonsense-mediated mRNA decay of the SF2/ASF proto-oncogene J. Cell Biol. 2010 191 87 99 10.1083/jcb.201001073 20876280 56. Matter N. Herrlich P. Konig H. Signal-dependent regulation of splicing via phosphorylation of Sam68 Nature 2002 420 691 695 10.1038/nature01153 12478298 57. van der Houven van Oordt W. Diaz-Meco M.T. Lozano J. Krainer A.R. Moscat J. Caceres J.F. The MKK(3/6)-p38-signaling cascade alters the subcellular distribution of hnRNP A1 and modulates alternative splicing regulation J. Cell Biol. 2000 149 307 316 10.1083/jcb.149.2.307 10769024 58. Allemand E. Guil S. Myers M. Moscat J. Caceres J.F. Krainer A.R. Regulation of heterogenous nuclear ribonucleoprotein A1 transport by phosphorylation in cells stressed by osmotic shock Proc. Natl. Acad. Sci. USA 2005 102 3605 3610 10.1073/pnas.0409889102 15738418 59. Shultz J.C. Goehe R.W. Wijesinghe D.S. Murudkar C. Hawkins A.J. Shay J.W. Minna J.D. Chalfant C.E. Alternative splicing of caspase 9 is modulated by the phosphoinositide 3-kinase/Akt pathway via phosphorylation of SRp30a Cancer Res. 2010 70 9185 9196 10.1158/0008-5472.CAN-10-1545 21045158 60. Goehe R.W. Shultz J.C. Murudkar C. Usanovic S. Lamour N.F. Massey D.H. Zhang L. Camidge D.R. Shay J.W. Minna J.D. hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing J. Clin. Investig. 2010 120 3923 3939 10.1172/JCI43552 20972334 61. Vu N.T. Park M.A. Shultz J.C. Goehe R.W. Hoeferlin L.A. Shultz M.D. Smith S.A. Lynch K.W. Chalfant C.E. hnRNP U enhances caspase-9 splicing and is modulated by AKT-dependent phosphorylation of hnRNP L J. Biol. Chem. 2013 288 8575 8584 10.1074/jbc.M112.443333 23396972 62. Blaustein M. Pelisch F. Tanos T. Munoz M.J. Wengier D. Quadrana L. Sanford J.R. Muschietti J.P. Kornblihtt A.R. Caceres J.F. Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT Nat. Struct. Mol. Biol. 2005 12 1037 1044 10.1038/nsmb1020 16299516 63. Goncalves V. Matos P. Jordan P. Antagonistic SR proteins regulate alternative splicing of tumor-related Rac1b downstream of the PI3-kinase and Wnt pathways Hum. Mol. Genet. 2009 18 3696 3707 10.1093/hmg/ddp317 19602482 64. Goncalves V. Henriques A.F. Pereira J.F. Neves Costa A. Moyer M.P. Moita L.F. Gama-Carvalho M. Matos P. Jordan P. Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b in colorectal cells RNA 2014 20 474 482 10.1261/rna.041376.113 24550521 65. Goncalves V. Matos P. Jordan P. The beta-catenin/TCF4 pathway modifies alternative splicing through modulation of SRp20 expression RNA 2008 14 2538 2549 10.1261/rna.1253408 18952824 66. El Marabti E. Younis I. The Cancer Spliceome: Reprograming of Alternative Splicing in Cancer Front. Mol. Biosci. 2018 5 80 10.3389/fmolb.2018.00080 30246013 67. Xerri L. Hassoun J. Devilard E. Birnbaum D. Birg F. BCL-X and the apoptotic machinery of lymphoma cells Leuk. Lymphoma 1998 28 451 458 10.3109/10428199809058352 9613974 68. Li Z. Li Q. Han L. Tian N. Liang Q. Li Y. Zhao X. Du C. Tian Y. Pro-apoptotic effects of splice-switching oligonucleotides targeting Bcl-x pre-mRNA in human glioma cell lines Oncol. Rep. 2016 35 1013 1019 10.3892/or.2015.4465 26718027 69. Olopade O.I. Adeyanju M.O. Safa A.R. Hagos F. Mick R. Thompson C.B. Recant W.M. Overexpression of BCL-x protein in primary breast cancer is associated with high tumor grade and nodal metastases Cancer J. Sci. Am. 1997 3 230 237 9263629 70. Mercatante D.R. Mohler J.L. Kole R. Cellular response to an antisense-mediated shift of Bcl-x pre-mRNA splicing and antineoplastic agents J. Biol. Chem. 2002 277 49374 49382 10.1074/jbc.M209236200 12381725 71. Takehara T. Liu X. Fujimoto J. Friedman S.L. Takahashi H. Expression and role of Bcl-xL in human hepatocellular carcinomas Hepatology 2001 34 55 61 10.1053/jhep.2001.25387 11431734 72. Cohen-Eliav M. Golan-Gerstl R. Siegfried Z. Andersen C.L. Thorsen K. Orntoft T.F. Mu D. Karni R. The splicing factor SRSF6 is amplified and is an oncoprotein in lung and colon cancers J. Pathol. 2013 229 630 639 10.1002/path.4129 23132731 73. Maimon A. Mogilevsky M. Shilo A. Golan-Gerstl R. Obiedat A. Ben-Hur V. Lebenthal-Loinger I. Stein I. Reich R. Beenstock J. Mnk2 alternative splicing modulates the p38-MAPK pathway and impacts Ras-induced transformation Cell Rep. 2014 7 501 513 10.1016/j.celrep.2014.03.041 24726367 74. Chao T.K. Huang T.S. Liao Y.P. Huang R.L. Su P.H. Shen H.Y. Lai H.C. Wang Y.C. Pyruvate kinase M2 is a poor prognostic marker of and a therapeutic target in ovarian cancer PLoS ONE 2017 12 e0182166 10.1371/journal.pone.0182166 28753677 75. Shiroki T. Yokoyama M. Tanuma N. Maejima R. Tamai K. Yamaguchi K. Oikawa T. Noguchi T. Miura K. Fujiya T. Enhanced expression of the M2 isoform of pyruvate kinase is involved in gastric cancer development by regulating cancer-specific metabolism Cancer Sci. 2017 108 931 940 10.1111/cas.13211 28235245 76. Liu W.R. Tian M.X. Yang L.X. Lin Y.L. Jin L. Ding Z.B. Shen Y.H. Peng Y.F. Gao D.M. Zhou J. PKM2 promotes metastasis by recruiting myeloid-derived suppressor cells and indicates poor prognosis for hepatocellular carcinoma Oncotarget 2015 6 846 861 10.18632/oncotarget.2749 25514599 77. Takahashi H. Nishimura J. Kagawa Y. Kano Y. Takahashi Y. Wu X. Hiraki M. Hamabe A. Konno M. Haraguchi N. Significance of Polypyrimidine Tract-Binding Protein 1 Expression in Colorectal Cancer Mol. Cancer Ther. 2015 14 1705 1716 10.1158/1535-7163.MCT-14-0142 25904505 78. Zhou Y.Q. He C. Chen Y.Q. Wang D. Wang M.H. Altered expression of the RON receptor tyrosine kinase in primary human colorectal adenocarcinomas: Generation of different splicing RON variants and their oncogenic potential Oncogene 2003 22 186 197 10.1038/sj.onc.1206075 12527888 79. Mayer S. Hirschfeld M. Jaeger M. Pies S. Iborra S. Erbes T. Stickeler E. RON alternative splicing regulation in primary ovarian cancer Oncol. Rep. 2015 34 423 430 10.3892/or.2015.3995 25997828 80. Eckerich C. Schulte A. Martens T. Zapf S. Westphal M. Lamszus K. RON receptor tyrosine kinase in human gliomas: Expression, function, and identification of a novel soluble splice variant J. Neurochem. 2009 109 969 980 10.1111/j.1471-4159.2009.06027.x 19519771 81. Krishnaswamy S. Mohammed A.K. Tripathi G. Alokail M.S. Al-Daghri N.M. Splice variants of the extracellular region of RON receptor tyrosine kinase in lung cancer cell lines identified by PCR and sequencing BMC Cancer 2017 17 738 10.1186/s12885-017-3747-x 29121914 82. Collesi C. Santoro M.M. Gaudino G. Comoglio P.M. A splicing variant of the RON transcript induces constitutive tyrosine kinase activity and an invasive phenotype Mol. Cell. Biol. 1996 16 5518 5526 10.1128/MCB.16.10.5518 8816464 83. Ben-Hur V. Denichenko P. Siegfried Z. Maimon A. Krainer A. Davidson B. Karni R. S6K1 alternative splicing modulates its oncogenic activity and regulates mTORC1 Cell Rep. 2013 3 103 115 10.1016/j.celrep.2012.11.020 23273915 84. Mei H. Wang Y. Fan J. Lin Z. Alternative splicing of S6K1 promotes non-small cell lung cancer survival Tumour Biol. 2016 37 13369 13376 10.1007/s13277-016-5253-1 27460085 85. Burd C.J. Petre C.E. Morey L.M. Wang Y. Revelo M.P. Haiman C.A. Lu S. Fenoglio-Preiser C.M. Li J. Knudsen E.S. Cyclin D1b variant influences prostate cancer growth through aberrant androgen receptor regulation Proc. Natl. Acad. Sci. USA 2006 103 2190 2195 10.1073/pnas.0506281103 16461912 86. Li R. An S.J. Chen Z.H. Zhang G.C. Zhu J.Q. Nie Q. Xie Z. Guo A.L. Mok T.S. Wu Y.L. Expression of cyclin D1 splice variants is differentially associated with outcome in non-small cell lung cancer patients Hum. Pathol. 2008 39 1792 1801 10.1016/j.humpath.2008.05.008 18715616 87. Wang Y. Dean J.L. Millar E.K. Tran T.H. McNeil C.M. Burd C.J. Henshall S.M. Utama F.E. Witkiewicz A. Rui H. Cyclin D1b is aberrantly regulated in response to therapeutic challenge and promotes resistance to estrogen antagonists Cancer Res. 2008 68 5628 5638 10.1158/0008-5472.CAN-07-3170 18632615 88. Varey A.H. Rennel E.S. Qiu Y. Bevan H.S. Perrin R.M. Raffy S. Dixon A.R. Paraskeva C. Zaccheo O. Hassan A.B. VEGF 165 b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: Balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy Br. J. Cancer 2008 98 1366 1379 10.1038/sj.bjc.6604308 18349829 89. Rennel E. Waine E. Guan H. Schuler Y. Leenders W. Woolard J. Sugiono M. Gillatt D. Kleinerman E. Bates D. The endogenous anti-angiogenic VEGF isoform, VEGF165b inhibits human tumour growth in mice Br. J. Cancer 2008 98 1250 1257 10.1038/sj.bjc.6604309 18349828 90. Bates D.O. Cui T.G. Doughty J.M. Winkler M. Sugiono M. Shields J.D. Peat D. Gillatt D. Harper S.J. VEGF165b, an inhibitory splice variant of vascular endothelial growth factor, is down-regulated in renal cell carcinoma Cancer Res. 2002 62 4123 4131 12124351 91. Pritchard-Jones R.O. Dunn D.B. Qiu Y. Varey A.H. Orlando A. Rigby H. Harper S.J. Bates D.O. Expression of VEGF(xxx)b, the inhibitory isoforms of VEGF, in malignant melanoma Br. J. Cancer 2007 97 223 230 10.1038/sj.bjc.6603839 17595666 92. Ieda J. Yokoyama S. Tamura K. Takifuji K. Hotta T. Matsuda K. Oku Y. Nasu T. Kiriyama S. Yamamoto N. Re-expression of CEACAM1 long cytoplasmic domain isoform is associated with invasion and migration of colorectal cancer Int. J. Cancer 2011 129 1351 1361 10.1002/ijc.26072 21413011 93. Ortenberg R. Galore-Haskel G. Greenberg I. Zamlin B. Sapoznik S. Greenberg E. Barshack I. Avivi C. Feiler Y. Zan-Bar I. CEACAM1 promotes melanoma cell growth through Sox-2 Neoplasia 2014 16 451 460 10.1016/j.neo.2014.05.003 24931667 94. Todaro M. Gaggianesi M. Catalano V. Benfante A. Iovino F. Biffoni M. Apuzzo T. Sperduti I. Volpe S. Cocorullo G. CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis Cell Stem Cell 2014 14 342 356 10.1016/j.stem.2014.01.009 24607406 95. Saito S. Okabe H. Watanabe M. Ishimoto T. Iwatsuki M. Baba Y. Tanaka Y. Kurashige J. Miyamoto Y. Baba H. CD44v6 expression is related to mesenchymal phenotype and poor prognosis in patients with colorectal cancer Oncol. Rep. 2013 29 1570 1578 10.3892/or.2013.2273 23404221 96. Yan Y. Zuo X. Wei D. Concise Review: Emerging Role of CD44 in Cancer Stem Cells: A Promising Biomarker and Therapeutic Target Stem Cells Transl. Med. 2015 4 1033 1043 10.5966/sctm.2015-0048 26136504 97. Singh A. Karnoub A.E. Palmby T.R. Lengyel E. Sondek J. Der C.J. Rac1b, a tumor associated, constitutively active Rac1 splice variant, promotes cellular transformation Oncogene 2004 23 9369 9380 10.1038/sj.onc.1208182 15516977 98. Pelisch F. Khauv D. Risso G. Stallings-Mann M. Blaustein M. Quadrana L. Radisky D.C. Srebrow A. Involvement of hnRNP A1 in the matrix metalloprotease-3-dependent regulation of Rac1 pre-mRNA splicing J. Cell Biochem. 2012 113 2319 2329 10.1002/jcb.24103 22345078 99. Melzer C. Hass R. Lehnert H. Ungefroren H. RAC1B: A Rho GTPase with Versatile Functions in Malignant Transformation and Tumor Progression Cells 2019 8 21 10.3390/cells8010021 100. Matos P. Jordan P. Increased Rac1b expression sustains colorectal tumor cell survival Mol. Cancer Res. 2008 6 1178 1184 10.1158/1541-7786.MCR-08-0008 18644982 101. Faria M. Capinha L. Simoes-Pereira J. Bugalho M.J. Silva A.L. Extending the Impact of RAC1b Overexpression to Follicular Thyroid Carcinomas Int. J. Endocrinol. 2016 2016 1972367 10.1155/2016/1972367 27127508 102. Faria M. Matos P. Pereira T. Cabrera R. Cardoso B.A. Bugalho M.J. Silva A.L. RAC1b overexpression stimulates proliferation and NF-kB-mediated anti-apoptotic signaling in thyroid cancer cells PLoS ONE 2017 12 e0172689 10.1371/journal.pone.0172689 28234980 103. Wang H. Zhou M. Shi B. Zhang Q. Jiang H. Sun Y. Liu J. Zhou K. Yao M. Gu J. Identification of an exon 4-deletion variant of epidermal growth factor receptor with increased metastasis-promoting capacity Neoplasia 2011 13 461 471 10.1593/neo.101744 21532887 104. Wang H. Shi B. Zhang Q. Jiang H. Hu S. Kong J. Yao M. Yang S. Li Z. Growth and metastasis suppression of glioma xenografts expressing exon 4-deletion variant of epidermal growth factor receptor by monoclonal antibody CH12-mediated receptor degradation FASEB J. 2012 26 73 80 10.1096/fj.11-191064 21917986 105. Zhang P. Zhang P. Zhou M. Jiang H. Zhang H. Shi B. Pan X. Gao H. Sun H. Li Z. Exon 4 deletion variant of epidermal growth factor receptor enhances invasiveness and cisplatin resistance in epithelial ovarian cancer Carcinogenesis 2013 34 2639 2646 10.1093/carcin/bgt216 23764753 106. Hatami R. Sieuwerts A.M. Izadmehr S. Yao Z. Qiao R.F. Papa L. Look M.P. Smid M. Ohlssen J. Levine A.C. KLF6-SV1 drives breast cancer metastasis and is associated with poor survival Sci. Transl. Med. 2013 5 169 10.1126/scitranslmed.3004688 107. DiFeo A. Feld L. Rodriguez E. Wang C. Beer D.G. Martignetti J.A. Narla G. A functional role for KLF6-SV1 in lung adenocarcinoma prognosis and chemotherapy response Cancer Res. 2008 68 965 970 10.1158/0008-5472.CAN-07-2604 18250346 108. Hartel M. Narla G. Wente M.N. Giese N.A. Martignoni M.E. Martignetti J.A. Friess H. Friedman S.L. Increased alternative splicing of the KLF6 tumour suppressor gene correlates with prognosis and tumour grade in patients with pancreatic cancer Eur. J. Cancer 2008 44 1895 1903 10.1016/j.ejca.2008.06.030 18691883 109. Narla G. DiFeo A. Yao S. Banno A. Hod E. Reeves H.L. Qiao R.F. Camacho-Vanegas O. Levine A. Kirschenbaum A. Targeted inhibition of the KLF6 splice variant, KLF6 SV1, suppresses prostate cancer cell growth and spread Cancer Res. 2005 65 5761 5768 10.1158/0008-5472.CAN-05-0217 15994951 110. Yea S. Narla G. Zhao X. Garg R. Tal-Kremer S. Hod E. Villanueva A. Loke J. Tarocchi M. Akita K. Ras promotes growth by alternative splicing-mediated inactivation of the KLF6 tumor suppressor in hepatocellular carcinoma Gastroenterology 2008 134 1521 1531 10.1053/j.gastro.2008.02.015 18471523 111. Wang Z.N. Liu D. Yin B. Ju W.Y. Qiu H.Z. Xiao Y. Chen Y.J. Peng X.Z. Lu C.M. High expression of PTBP1 promote invasion of colorectal cancer by alternative splicing of cortactin Oncotarget 2017 8 36185 36202 10.18632/oncotarget.15873 28404950 112. Yao L. Li K. Peng W. Lin Q. Li S. Hu X. Zheng X. Shao Z. An aberrant spliced transcript of focal adhesion kinase is exclusively expressed in human breast cancer J. Transl. Med. 2014 12 136 10.1186/1479-5876-12-136 24885534 113. Ludwig J.A. Weinstein J.N. Biomarkers in cancer staging, prognosis and treatment selection Nat. Rev. Cancer 2005 5 845 856 10.1038/nrc1739 16239904 114. Drucker E. Krapfenbauer K. Pitfalls and limitations in translation from biomarker discovery to clinical utility in predictive and personalised medicine EPMA J. 2013 4 7 10.1186/1878-5085-4-7 23442211 115. David C.J. Manley J.L. Alternative pre-mRNA splicing regulation in cancer: Pathways and programs unhinged Genes Dev. 2010 24 2343 2364 10.1101/gad.1973010 21041405 116. Venables J.P. Klinck R. Koh C. Gervais-Bird J. Bramard A. Inkel L. Durand M. Couture S. Froehlich U. Lapointe E. Cancer-associated regulation of alternative splicing Nat. Struct. Mol. Biol. 2009 16 670 676 10.1038/nsmb.1608 19448617 117. Gardina P.J. Clark T.A. Shimada B. Staples M.K. Yang Q. Veitch J. Schweitzer A. Awad T. Sugnet C. Dee S. Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array BMC Genomics 2006 7 325 10.1186/1471-2164-7-325 17192196 118. Shapiro I.M. Cheng A.W. Flytzanis N.C. Balsamo M. Condeelis J.S. Oktay M.H. Burge C.B. Gertler F.B. An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype PLoS Genet. 2011 7 e1002218 10.1371/journal.pgen.1002218 21876675 119. Misquitta-Ali C.M. Cheng E. O’Hanlon D. Liu N. McGlade C.J. Tsao M.S. Blencowe B.J. Global profiling and molecular characterization of alternative splicing events misregulated in lung cancer Mol. Cell. Biol. 2011 31 138 150 10.1128/MCB.00709-10 21041478 120. Germann S. Gratadou L. Dutertre M. Auboeuf D. Splicing programs and cancer J. Nucleic Acids 2012 2012 269570 10.1155/2012/269570 22132318 121. Lapuk A. Marr H. Jakkula L. Pedro H. Bhattacharya S. Purdom E. Hu Z. Simpson K. Pachter L. Durinck S. Exon-level microarray analyses identify alternative splicing programs in breast cancer Mol. Cancer Res. 2010 8 961 974 10.1158/1541-7786.MCR-09-0528 20605923 122. Qu Y. Dai B. Ye D. Kong Y. Chang K. Jia Z. Yang X. Zhang H. Zhu Y. Shi G. Constitutively active AR-V7 plays an essential role in the development and progression of castration-resistant prostate cancer Sci. Rep. 2015 5 7654 10.1038/srep07654 25563505 123. Calabretta S. Bielli P. Passacantilli I. Pilozzi E. Fendrich V. Capurso G. Fave G.D. Sette C. Modulation of PKM alternative splicing by PTBP1 promotes gemcitabine resistance in pancreatic cancer cells Oncogene 2016 35 2031 2039 10.1038/onc.2015.270 26234680 124. Nagane M. Coufal F. Lin H. Bogler O. Cavenee W.K. Huang H.J. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis Cancer Res. 1996 56 5079 5086 8895767 125. Zhu H. Acquaviva J. Ramachandran P. Boskovitz A. Woolfenden S. Pfannl R. Bronson R.T. Chen J.W. Weissleder R. Housman D.E. Oncogenic EGFR signaling cooperates with loss of tumor suppressor gene functions in gliomagenesis Proc. Natl. Acad. Sci. USA 2009 106 2712 2716 10.1073/pnas.0813314106 19196966 126. Gotley D.C. Fawcett J. Walsh M.D. Reeder J.A. Simmons D.L. Antalis T.M. Alternatively spliced variants of the cell adhesion molecule CD44 and tumour progression in colorectal cancer Br. J. Cancer 1996 74 342 351 10.1038/bjc.1996.364 8695347 127. Coppola D. Hyacinthe M. Fu L. Cantor A.B. Karl R. Marcet J. Cooper D.L. Nicosia S.V. Cooper H.S. CD44V6 expression in human colorectal carcinoma Hum. Pathol. 1998 29 627 635 10.1016/S0046-8177(98)80014-2 9635685 128. Mikami T. Mitomi H. Hara A. Yanagisawa N. Yoshida T. Tsuruta O. Okayasu I. Decreased expression of CD44, alpha-catenin, and deleted colon carcinoma and altered expression of beta-catenin in ulcerative colitis-associated dysplasia and carcinoma, as compared with sporadic colon neoplasms Cancer 2000 89 733 740 10.1002/1097-0142(20000815)89:4<733::AID-CNCR3>3.0.CO;2-# 10951334 129. Fan C.W. Wen L. Qiang Z.D. Chen T. Zhou Z.G. Mo X.M. Hu J.K. Prognostic significance of relevant markers of cancer stem cells in colorectal cancer—A meta analysis Hepatogastroenterology 2012 59 1421 1427 22683959 130. Matos P. Oliveira C. Velho S. Goncalves V. da Costa L.T. Moyer M.P. Seruca R. Jordan P. B-Raf(V600E) cooperates with alternative spliced Rac1b to sustain colorectal cancer cell survival Gastroenterology 2008 135 899 906 10.1053/j.gastro.2008.05.052 18602919 131. Alonso-Espinaco V. Cuatrecasas M. Alonso V. Escudero P. Marmol M. Horndler C. Ortego J. Gallego R. Codony-Servat J. Garcia-Albeniz X. RAC1b overexpression correlates with poor prognosis in KRAS/BRAF WT metastatic colorectal cancer patients treated with first-line FOLFOX/XELOX chemotherapy Eur. J. Cancer 2014 50 1973 1981 10.1016/j.ejca.2014.04.019 24833563 132. Silva A.L. Carmo F. Bugalho M.J. RAC1b overexpression in papillary thyroid carcinoma: A role to unravel Eur. J. Endocrinol. 2013 168 795 804 10.1530/EJE-12-0960 23482591 133. Goka E.T. Chaturvedi P. Lopez D.T.M. Garza A. Lippman M.E. RAC1b Overexpression Confers Resistance to Chemotherapy Treatment in Colorectal Cancer Mol. Cancer Ther. 2019 18 957 968 10.1158/1535-7163.MCT-18-0955 30926638 134. Zhao S. Fung-Leung W.P. Bittner A. Ngo K. Liu X. Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells PLoS ONE 2014 9 e78644 10.1371/journal.pone.0078644 24454679 135. Zhang C. Dower K. Zhang B. Martinez R.V. Lin L.L. Zhao S. Computational identification and validation of alternative splicing in ZSF1 rat RNA-seq data, a preclinical model for type 2 diabetic nephropathy Sci. Rep. 2018 8 7624 10.1038/s41598-018-26035-x 29769602 136. Zhang C. Zhang B. Lin L.L. Zhao S. Evaluation and comparison of computational tools for RNA-seq isoform quantification BMC Genom. 2017 18 583 10.1186/s12864-017-4002-1 28784092 137. Wang Z. Gerstein M. Snyder M. RNA-Seq: A revolutionary tool for transcriptomics Nat. Rev. Genet. 2009 10 57 63 10.1038/nrg2484 19015660 138. Mortazavi A. Williams B.A. McCue K. Schaeffer L. Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq Nat. Methods 2008 5 621 628 10.1038/nmeth.1226 18516045 139. Byron S.A. Van Keuren-Jensen K.R. Engelthaler D.M. Carpten J.D. Craig D.W. Translating RNA sequencing into clinical diagnostics: Opportunities and challenges Nat. Rev. Genet. 2016 17 257 271 10.1038/nrg.2016.10 26996076 140. Eswaran J. Horvath A. Godbole S. Reddy S.D. Mudvari P. Ohshiro K. Cyanam D. Nair S. Fuqua S.A. Polyak K. RNA sequencing of cancer reveals novel splicing alterations Sci. Rep. 2013 3 1689 10.1038/srep01689 23604310 141. Zhu J. Chen Z. Yong L. Systematic profiling of alternative splicing signature reveals prognostic predictor for ovarian cancer Gynecol. Oncol. 2018 148 368 374 10.1016/j.ygyno.2017.11.028 29191436 142. Li Y. Sun N. Lu Z. Sun S. Huang J. Chen Z. He J. Prognostic alternative mRNA splicing signature in non-small cell lung cancer Cancer Lett. 2017 393 40 51 10.1016/j.canlet.2017.02.016 28223168 143. Yang C. Wu Q. Huang K. Wang X. Yu T. Liao X. Huang J. Zhu G. Gong Y. Han C. Genome-Wide Profiling Reveals the Landscape of Prognostic Alternative Splicing Signatures in Pancreatic Ductal Adenocarcinoma Front. Oncol. 2019 9 511 10.3389/fonc.2019.00511 31275849 144. Zhang D. Hu Q. Liu X. Ji Y. Chao H.P. Liu Y. Tracz A. Kirk J. Buonamici S. Zhu P. Intron retention is a hallmark and spliceosome represents a therapeutic vulnerability in aggressive prostate cancer Nat. Commun. 2020 11 2089 10.1038/s41467-020-15815-7 32350277 145. Xiong Y. Deng Y. Wang K. Zhou H. Zheng X. Si L. Fu Z. Profiles of alternative splicing in colorectal cancer and their clinical significance: A study based on large-scale sequencing data EBioMedicine 2018 36 183 195 10.1016/j.ebiom.2018.09.021 30243491 146. Lian H. Wang A. Shen Y. Wang Q. Zhou Z. Zhang R. Li K. Liu C. Jia H. Identification of novel alternative splicing isoform biomarkers and their association with overall survival in colorectal cancer BMC Gastroenterol. 2020 20 171 10.1186/s12876-020-01288-x 32503434 147. Qiao J. Fang C.Y. Chen S.X. Wang X.Q. Cui S.J. Liu X.H. Jiang Y.H. Wang J. Zhang Y. Yang P.Y. Stroma derived COL6A3 is a potential prognosis marker of colorectal carcinoma revealed by quantitative proteomics Oncotarget 2015 6 29929 29946 10.18632/oncotarget.4966 26338966 148. Li G.H. Huang J.F. Inferring therapeutic targets from heterogeneous data: HKDC1 is a novel potential therapeutic target for cancer Bioinformatics 2014 30 748 752 10.1093/bioinformatics/btt606 24162464 149. Safikhani Z. Smirnov P. Thu K.L. Silvester J. El-Hachem N. Quevedo R. Lupien M. Mak T.W. Cescon D. Haibe-Kains B. Gene isoforms as expression-based biomarkers predictive of drug response in vitro Nat. Commun. 2017 8 1126 10.1038/s41467-017-01153-8 29066719 150. Desterro J. Bak-Gordon P. Carmo-Fonseca M. Targeting mRNA processing as an anticancer strategy Nat. Rev. Drug Discov. 2020 19 112 129 10.1038/s41573-019-0042-3 31554928 151. Rahman M.A. Nasrin F. Bhattacharjee S. Nandi S. Hallmarks of Splicing Defects in Cancer: Clinical Applications in the Era of Personalized Medicine Cancers 2020 12 1381 10.3390/cancers12061381 32481522 152. Lee S.C. Abdel-Wahab O. Therapeutic targeting of splicing in cancer Nat. Med. 2016 22 976 986 10.1038/nm.4165 27603132 153. Antonopoulou E. Ladomery M. Targeting Splicing in Prostate Cancer Int. J. Mol. Sci. 2018 19 1287 10.3390/ijms19051287 29693622 154. Aubol B.E. Wu G. Keshwani M.M. Movassat M. Fattet L. Hertel K.J. Fu X.D. Adams J.A. Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing Mol. Cell 2016 63 218 228 10.1016/j.molcel.2016.05.034 27397683 155. Toker A. Chin Y.R. Akt-ing up on SRPK1: Oncogene or tumor suppressor? Mol. Cell 2014 54 329 330 10.1016/j.molcel.2014.04.020 24813709 156. Amin E.M. Oltean S. Hua J. Gammons M.V. Hamdollah-Zadeh M. Welsh G.I. Cheung M.K. Ni L. Kase S. Rennel E.S. WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing Cancer Cell 2011 20 768 780 10.1016/j.ccr.2011.10.016 22172722 157. Siqueira R.P. Barbosa Ede A. Poleto M.D. Righetto G.L. Seraphim T.V. Salgado R.L. Ferreira J.G. Barros M.V. de Oliveira L.L. Laranjeira A.B. Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N -(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340) PLoS ONE 2015 10 e0134882 10.1371/journal.pone.0134882 26244849 158. Gammons M.V. Lucas R. Dean R. Coupland S.E. Oltean S. Bates D.O. Targeting SRPK1 to control VEGF-mediated tumour angiogenesis in metastatic melanoma Br. J. Cancer 2014 111 477 485 10.1038/bjc.2014.342 25010863 159. Hatcher J.M. Wu G. Zeng C. Zhu J. Meng F. Patel S. Wang W. Ficarro S.B. Leggett A.L. Powell C.E. SRPKIN-1: A Covalent SRPK1/2 Inhibitor that Potently Converts VEGF from Pro-angiogenic to Anti-angiogenic Isoform Cell Chem. Biol. 2018 25 460 470 10.1016/j.chembiol.2018.01.013 29478907 160. Muraki M. Ohkawara B. Hosoya T. Onogi H. Koizumi J. Koizumi T. Sumi K. Yomoda J. Murray M.V. Kimura H. Manipulation of alternative splicing by a newly developed inhibitor of Clks J. Biol. Chem. 2004 279 24246 24254 10.1074/jbc.M314298200 15010457 161. Debdab M. Carreaux F. Renault S. Soundararajan M. Fedorov O. Filippakopoulos P. Lozach O. Babault L. Tahtouh T. Baratte B. Leucettines, a class of potent inhibitors of cdc2-like kinases and dual specificity, tyrosine phosphorylation regulated kinases derived from the marine sponge leucettamine B: Modulation of alternative pre-RNA splicing J. Med. Chem. 2011 54 4172 4186 10.1021/jm200274d 21615147 162. Iwai K. Yaguchi M. Nishimura K. Yamamoto Y. Tamura T. Nakata D. Dairiki R. Kawakita Y. Mizojiri R. Ito Y. Anti-tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC-dependent vulnerability EMBO Mol. Med. 2018 10 e8289 10.15252/emmm.201708289 29769258 163. Araki S. Dairiki R. Nakayama Y. Murai A. Miyashita R. Iwatani M. Nomura T. Nakanishi O. Inhibitors of CLK protein kinases suppress cell growth and induce apoptosis by modulating pre-mRNA splicing PLoS ONE 2015 10 e0116929 10.1371/journal.pone.0116929 25581376 164. Assi R. Kantarjian H.M. Kadia T.M. Pemmaraju N. Jabbour E. Jain N. Daver N. Estrov Z. Uehara T. Owa T. Final results of a phase 2, open-label study of indisulam, idarubicin, and cytarabine in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome Cancer 2018 124 2758 2765 10.1002/cncr.31398 29660836 165. Talbot D.C. von Pawel J. Cattell E. Yule S.M. Johnston C. Zandvliet A.S. Huitema A.D. Norbury C.J. Ellis P. Bosquee L. A randomized phase II pharmacokinetic and pharmacodynamic study of indisulam as second-line therapy in patients with advanced non-small cell lung cancer Clin. Cancer Res. 2007 13 1816 1822 10.1158/1078-0432.CCR-06-0249 17363538 166. Supuran C.T. Indisulam: An anticancer sulfonamide in clinical development Expert Opin. Investig. Drugs 2003 12 283 287 10.1517/13543784.12.2.283 167. Han T. Goralski M. Gaskill N. Capota E. Kim J. Ting T.C. Xie Y. Williams N.S. Nijhawan D. Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15 Science 2017 356 eaal3755 10.1126/science.aal3755 28302793 168. Uehara T. Minoshima Y. Sagane K. Sugi N.H. Mitsuhashi K.O. Yamamoto N. Kamiyama H. Takahashi K. Kotake Y. Uesugi M. Selective degradation of splicing factor CAPERalpha by anticancer sulfonamides Nat. Chem. Biol. 2017 13 675 680 10.1038/nchembio.2363 28437394 169. Black A.J. Gamarra J.R. Giudice J. More than a messenger: Alternative splicing as a therapeutic target Biochim. Biophys. Acta Gene Regul. Mech. 2019 1862 194395 10.1016/j.bbagrm.2019.06.006 31271898 170. Sanidas I. Polytarchou C. Hatziapostolou M. Ezell S.A. Kottakis F. Hu L. Guo A. Xie J. Comb M.J. Iliopoulos D. Phosphoproteomics screen reveals akt isoform-specific signals linking RNA processing to lung cancer Mol. Cell 2014 53 577 590 10.1016/j.molcel.2013.12.018 24462114 171. Passacantilli I. Frisone P. De Paola E. Fidaleo M. Paronetto M.P. hnRNPM guides an alternative splicing program in response to inhibition of the PI3K/AKT/mTOR pathway in Ewing sarcoma cells Nucleic Acids Res. 2017 45 12270 12284 10.1093/nar/gkx831 29036465 172. Stephenson M.L. Zamecnik P.C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide Proc. Natl. Acad. Sci. USA 1978 75 285 288 10.1073/pnas.75.1.285 75546 173. Zamecnik P.C. Stephenson M.L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide Proc. Natl. Acad. Sci. USA 1978 75 280 284 10.1073/pnas.75.1.280 75545 174. Dean N.M. Bennett C.F. Antisense oligonucleotide-based therapeutics for cancer Oncogene 2003 22 9087 9096 10.1038/sj.onc.1207231 14663487 175. Levin A.A. Treating Disease at the RNA Level with Oligonucleotides N. Engl. J. Med. 2019 380 57 70 10.1056/NEJMra1705346 30601736 176. Bennett C.F. Therapeutic Antisense Oligonucleotides Are Coming of Age Annu Rev. Med. 2019 70 307 321 10.1146/annurev-med-041217-010829 30691367 177. Liu J. Guo B. RNA-based therapeutics for colorectal cancer: Updates and future directions Pharmacol. Res. 2020 152 104550 10.1016/j.phrs.2019.104550 31866285 178. Havens M.A. Hastings M.L. Splice-switching antisense oligonucleotides as therapeutic drugs Nucleic Acids Res. 2016 44 6549 6563 10.1093/nar/gkw533 27288447 179. Aartsma-Rus A. Krieg A.M. FDA Approves Eteplirsen for Duchenne Muscular Dystrophy: The Next Chapter in the Eteplirsen Saga Nucleic Acid Ther. 2017 27 1 3 10.1089/nat.2016.0657 27929755 180. Michelson D. Ciafaloni E. Ashwal S. Lewis E. Narayanaswami P. Oskoui M. Armstrong M.J. Evidence in focus: Nusinersen use in spinal muscular atrophy: Report of the Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology Neurology 2018 91 923 933 10.1212/WNL.0000000000006502 30315070 181. Charleston J.S. Schnell F.J. Dworzak J. Donoghue C. Lewis S. Chen L. Young G.D. Milici A.J. Voss J. DeAlwis U. Eteplirsen treatment for Duchenne muscular dystrophy: Exon skipping and dystrophin production Neurology 2018 90 e2146 e2154 10.1212/WNL.0000000000005680 29752304 182. Mercatante D.R. Bortner C.D. Cidlowski J.A. Kole R. Modification of alternative splicing of Bcl-x pre-mRNA in prostate and breast cancer cells. analysis of apoptosis and cell death J. Biol. Chem. 2001 276 16411 16417 10.1074/jbc.M009256200 11278482 183. Bauman J.A. Kole R. Modulation of RNA splicing as a potential treatment for cancer Bioeng. Bugs 2011 2 125 128 10.4161/bbug.2.3.15165 21637003 184. Chen M. Zhang J. Manley J.L. Turning on a fuel switch of cancer: hnRNP proteins regulate alternative splicing of pyruvate kinase mRNA Cancer Res. 2010 70 8977 8980 10.1158/0008-5472.CAN-10-2513 20978194 185. Wang Z. Jeon H.Y. Rigo F. Bennett C.F. Krainer A.R. Manipulation of PK-M mutually exclusive alternative splicing by antisense oligonucleotides Open Biol. 2012 2 120133 10.1098/rsob.120133 23155487 186. Denichenko P. Mogilevsky M. Clery A. Welte T. Biran J. Shimshon O. Barnabas G.D. Danan-Gotthold M. Kumar S. Yavin E. Specific inhibition of splicing factor activity by decoy RNA oligonucleotides Nat. Commun. 2019 10 1590 10.1038/s41467-019-09523-0 30962446 187. Moscatello D.K. Holgado-Madruga M. Godwin A.K. Ramirez G. Gunn G. Zoltick P.W. Biegel J.A. Hayes R.L. Wong A.J. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors Cancer Res. 1995 55 5536 5539 7585629 188. Aldape K.D. Ballman K. Furth A. Buckner J.C. Giannini C. Burger P.C. Scheithauer B.W. Jenkins R.B. James C.D. Immunohistochemical detection of EGFRvIII in high malignancy grade astrocytomas and evaluation of prognostic significance J. Neuropathol. Exp. Neurol. 2004 63 700 707 10.1093/jnen/63.7.700 15290895 189. Swartz A.M. Li Q.J. Sampson J.H. Rindopepimut: A promising immunotherapeutic for the treatment of glioblastoma multiforme Immunotherapy 2014 6 679 690 10.2217/imt.14.21 25186601 190. Johns T.G. Perera R.M. Vernes S.C. Vitali A.A. Cao D.X. Cavenee W.K. Scott A.M. Furnari F.B. The efficacy of epidermal growth factor receptor-specific antibodies against glioma xenografts is influenced by receptor levels, activation status, and heterodimerization Clin. Cancer Res. 2007 13 1911 1925 10.1158/1078-0432.CCR-06-1453 17363548 191. Fukai J. Nishio K. Itakura T. Koizumi F. Antitumor activity of cetuximab against malignant glioma cells overexpressing EGFR deletion mutant variant III Cancer Sci. 2008 99 2062 2069 10.1111/j.1349-7006.2008.00945.x 19016767 192. Heimberger A.B. Learn C.A. Archer G.E. McLendon R.E. Chewning T.A. Tuck F.L. Pracyk J.B. Friedman A.H. Friedman H.S. Bigner D.D. Brain tumors in mice are susceptible to blockade of epidermal growth factor receptor (EGFR) with the oral, specific, EGFR-tyrosine kinase inhibitor ZD1839 (iressa) Clin. Cancer Res. 2002 8 3496 3502 12429640 193. Orian-Rousseau V. CD44, a therapeutic target for metastasising tumours Eur. J. Cancer 2010 46 1271 1277 10.1016/j.ejca.2010.02.024 20303742 194. Borjesson P.K. Postema E.J. Roos J.C. Colnot D.R. Marres H.A. van Schie M.H. Stehle G. de Bree R. Snow G.B. Oyen W.J. Phase I therapy study with (186)Re-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with head and neck squamous cell carcinoma Clin. Cancer Res. 2003 9 3961S 3972S 14506195 195. Colnot D.R. Roos J.C. de Bree R. Wilhelm A.J. Kummer J.A. Hanft G. Heider K.H. Stehle G. Snow G.B. van Dongen G.A. Safety, biodistribution, pharmacokinetics, and immunogenicity of 99mTc-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with squamous cell carcinoma of the head and neck Cancer Immunol. Immunother. 2003 52 576 582 10.1007/s00262-003-0396-5 14627130 196. Tijink B.M. Buter J. de Bree R. Giaccone G. Lang M.S. Staab A. Leemans C.R. van Dongen G.A. A phase I dose escalation study with anti-CD44v6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck or esophagus Clin. Cancer Res. 2006 12 6064 6072 10.1158/1078-0432.CCR-06-0910 17062682 197. Riechelmann H. Sauter A. Golze W. Hanft G. Schroen C. Hoermann K. Erhardt T. Gronau S. Phase I trial with the CD44v6-targeting immunoconjugate bivatuzumab mertansine in head and neck squamous cell carcinoma Oral Oncol. 2008 44 823 829 10.1016/j.oraloncology.2007.10.009 18203652 198. Sahin U. Koslowski M. Dhaene K. Usener D. Brandenburg G. Seitz G. Huber C. Tureci O. Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development Clin. Cancer Res. 2008 14 7624 7634 10.1158/1078-0432.CCR-08-1547 19047087 199. Singh P. Toom S. Huang Y. Anti-claudin 18.2 antibody as new targeted therapy for advanced gastric cancer J. Hematol. Oncol. 2017 10 105 10.1186/s13045-017-0473-4 28494772 200. Arkin M.R. Tang Y. Wells J.A. Small-molecule inhibitors of protein-protein interactions: Progressing toward the reality Chem. Biol. 2014 21 1102 1114 10.1016/j.chembiol.2014.09.001 25237857 201. Jin L. Wang W. Fang G. Targeting protein-protein interaction by small molecules Annu Rev. Pharmacol. Toxicol. 2014 54 435 456 10.1146/annurev-pharmtox-011613-140028 24160698 202. Souers A.J. Leverson J.D. Boghaert E.R. Ackler S.L. Catron N.D. Chen J. Dayton B.D. Ding H. Enschede S.H. Fairbrother W.J. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets Nat. Med. 2013 19 202 208 10.1038/nm.3048 23291630 203. Zhao Y. Aguilar A. Bernard D. Wang S. Small-molecule inhibitors of the MDM2-p53 protein-protein interaction (MDM2 Inhibitors) in clinical trials for cancer treatment J. Med. Chem. 2015 58 1038 1052 10.1021/jm501092z 25396320