Alternative splicing in lung cancer

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BBA - Gene Regulatory Mechanisms journal homepage: www.elsevier.com/locate/bbagrm

Alternative splicing in lung cancer☆ ⁎

Alice O. Coomera, , Fiona Blackb, Alastair Greystokec, Jennifer Munkleya, David J. Elliotta,

⁎

a

Institute of Genetic Medicine, Newcastle University, Central Parkway, Newcastle upon Tyne NE1 3BZ, United Kingdom of Great Britain and Northern Ireland Cellular Pathology Department, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, United Kingdom of Great Britain and Northern Ireland Northern Institute for Cancer Research, Paul O'Gorman Building, Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom of Great Britain and Northern Ireland b c

A B S T R A C T

Lung cancer has the highest mortality rate of all cancers worldwide. Lung cancer is a very heterogeneous disease that is often diagnosed at later stages which have a poor prognosis. Aberrant alternative splicing patterns found in lung cancer contribute to important cell functions. These include changes in splicing for the BCL2L1, MDM2, MDM4, NUMB and MET genes during lung tumourigenesis, to affect pathways involved in apoptosis, cell proliferation and cellular cohesion. Global analyses of RNASeq datasets suggest there may be many more potentially influential aberrant splicing events that need to be investigated in lung cancer. Changes in expression of the splicing factors that regulate alternative splicing events have also been identified in lung cancer. Of these, changes in expression of QKI, RBM4, RBM5, RBM6, RBM10 and SRSF1 proteins regulate many of the most frequently referenced aberrant splicing events in lung cancer. The expanding list of genes known to be aberrantly spliced in lung cancer along with the altered expression of splicing factors that regulate them are providing new clues as to how lung cancer develops, and how these events can be exploited for better treatment. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.

1. Introduction Lung cancer is the second most common malignant cancer, and in 2016 accounted for 12.6% of all new cancer registrations in England [1]. Lung is also the leading cause of cancer-related death worldwide, with a 10-year survival rate of only 5% in the UK [2,3]. The poor prognosis for lung cancer is partly attributable to the majority of cancer cases being diagnosed at a late disease stage, often after cancer metastasis to sites such as the liver, bone, nervous or respiratory systems [4,5]. Lung cancer is primarily caused by environmental factors such as tobacco smoke and atmospheric pollutants including coal dust and asbestos, as well as indoor pollution from solid fuel smoke [6]. It is estimated that 79% of lung cancer cases are preventable by not smoking tobacco [7]. Whilst it is thought that familial cases of lung cancer are more connected to similar lifestyle and environment than any heritable factors, mutations in EGFR and other genes could confer a slightly heightened inherited risk of developing lung cancer, as reviewed by Clamon et al. [8]. Lung cancers are broadly classified as small-cell lung cancer (SCLC), and non-small cell lung cancer (NSCLC). Of these, NSCLC forms the larger group (88.5%) [9]. NSCLC can be further subdivided into histological sub-types, although it can sometimes be difficult to differentiate exactly between sub-types. The most common of these NSCLC subtypes are lung adenocarcinoma (LUAD) (36%), lung squamous cell

carcinoma (LUSC) (22%) and large cell carcinoma (< 11%) [9]. Fig. 1A shows how the normal lung tissue is organised, with the airway structures such as the bronchiole and thin walled alveoli surrounded by blood vessels. This organisation becomes disrupted in lung tumours. The tumour cells and stroma replace the normal structures of the lung, and elicit a variable host inflammatory response (Fig. 1B & C). Lung tumours are staged according to the extent of disease using the TNM (tumour, node, metastasis) system [10]. Stage I tumours have relatively small tumours (T) ≤ 4 cm but no lymph node metastasis (N) or distant metastasis (M). Stage II tumours consist of larger tumours or smaller tumours with localised lymph node metastases; Stage III tumours are either very large ≥7 cm, are invading local structures, have multiple lymph node metastases, or a combination of these features but not have any distant metastases; Stage IV tumours can have any T and N numbers but have distant metastases [10]. Standard treatment for NSCLC depends on the stage at diagnosis and can include surgery or radiotherapy in medically fit patients with stage I-II cancers. Stage III cancers may still be cured using multi-modality treatment including combinations of surgery or radiotherapy and chemotherapy with or without immunotherapy. The treatment paradigm for metastatic NSCLC is rapidly changing, and now involves combinations of platinum-based chemotherapy and immunotherapy in fit patients where a targetable oncogene driver cannot be identified; in these patients, primary treatment uses tyrosine kinase inhibitors [11].

☆ ⁎

This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm. Corresponding authors. E-mail addresses: [email protected] (A.O. Coomer), [email protected] (D.J. Elliott).

https://doi.org/10.1016/j.bbagrm.2019.05.006 Received 4 April 2019; Accepted 20 May 2019 1874-9399/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Alice O. Coomer, et al., BBA - Gene Regulatory Mechanisms, https://doi.org/10.1016/j.bbagrm.2019.05.006

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Fig. 1. Lung tissue visualised from primary NSCLC subtypes. A – Lung tissue from normal city dweller. B –Lung Squamous Cell Carcinoma. C –Lung Adenocarcinoma. All panels are H&E stained histological sections.

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Fig. 2. Splice isoforms of Bcl-X and their encoded protein domains. An alternative 5′ splice site in the second exon of Bcl-X pre-mRNA leads to the formation of a long (Bcl-XL) and short (Bcl-XS) version of the gene, both of which can be transcribed into functional proteins with opposing functions. The Bcl-XS isoform retains its transmembrane (TM) localisation domain required for its function, along with two of the Bcl-2 homology (BH) domains, BH3 and BH4.

of alternative splicing in lung cancer a decade ago, here we highlight key subsequent advances in the field that have identified new mechanisms driving development of aberrant alternative splicing. We will also discuss new information about splicing regulators that change expression in lung cancer, summarise recently discovered system-wide analyses of splicing patterns in lung cancer and how the above can be used to influence therapeutic management of the disease.

In this article we will specifically review the role of splicing aberrations in lung cancer. This is an important topic for both understanding the molecular causes and progression of lung cancer, as well as informing treatment. The majority of human genes are split up into exons and introns, and are transcribed as long precursor mRNAs (premRNAs). A macromolecular structure called the spliceosome assembles on the pre-mRNA to join exons together at splice sites to create mRNAs, with the usually much longer introns being discarded [12]. Spliceosome assembly on pre-mRNAs is often influenced by the binding of splicing regulator proteins with exons and nearby flanking introns. A proportion of exons – called alternative exons – are variably included into mRNAs. In different patterns of alternative splicing, entire exons can be either spliced into mRNAs or excluded (called exon skipping), different 5′ and 3′ splice sites can be used to change exon sizes, and introns can be retained within mRNAs. Whilst the maintenance of normal alternative splicing results in the generation of a diverse and multi-functional proteome to ensure healthy cellular functions, aberrant alternative splicing can contribute to of both the original and emerging hallmarks of cancer, as reviewed recently by Oltean and Bates [13,14]. Aberrant splicing is relevant to multiple different cancers and can result from up/downregulation of associated splicing factors, alterations in upstream signalling pathways, or mutations in splice site sequences. Aberrant splicing in prostate cancer contributes to angiogenesis and the avoidance of apoptosis [15]. In gastric cancer, splicing changes lead to the activation of invasion and metastasis [16]. A vast range of splicing targets can be mis-spliced to contribute to all of the cancer hallmarks, to such an extent that it has been suggested that ‘Epigenetic and RNA deregulation’ form a new, separate hallmark [17]. Alternative splicing changes can promote cell proliferation, angiogenesis, drug resistance and inhibit apoptosis, all contributing to the cancer phenotype. Alternative splicing in lung cancer was previously reviewed by Pio and Montuenga, who highlighted some of the known splice variants of genes associated with lung cancer, in particular BCL-X, as well as relevant regulatory splicing factors [18]. Following this previous review

2. Target RNAs mis-spliced in lung cancer This first section will review splicing changes of key target RNAs that have been identified in lung cancer, and how they might contribute to the disease. 2.1. Alternative splicing switches in the BCL-X gene inhibit apoptosis of lung cancer cells Apoptosis is the process of programmed cell death, and is essential for organisms to respond to damage [19]. In cases where the DNA has been badly damaged, it is important for the organism to remove the cell before mutations can trigger a malignant cancer. Many types of chemotherapy and radiotherapy rely on DNA damage to cause tumour cell death by inducing apoptosis. This means that defects in the apoptosis pathway can cause resistance to treatment. Resisting cell death is therefore considered one of the hallmarks of cancer relevant in all subtypes of the disease [14]. Apoptosis can be induced by either extrinsic or intrinsic cellular signals via distinct pathways. The extrinsic pathway relies on signals received from outside the cell, often if the cell is diseased or no longer needed. The extrinsic apoptosis pathway is centred around death receptors such as FAS and TRAILR that instigate a chain of events to initiate cell death through the activation of caspases and of the BID protein [20]. In contrast, the intrinsic pathway is often a response to stresses in the cell, such as DNA damage or hypoxia. The intrinsic pathway is the cell's own internal defence mechanism and depends on 3

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within cells. Some p53 splice isoforms, including one called p47, have been shown to affect the ubiquitination, degradation and cellular localisation of wild-type p53 protein [40]. Alternative splicing of transcripts from a gene called MDM2 can also be very important for regulating p53 protein levels in cancer cells. MDM2 is a proto-oncogene which is overexpressed in lung cancer. MDM2 protein is involved in an auto-regulatory feedback loop with p53 [41]. MDM2 protein binds to p53 and directs its proteasomal degradation. MDM2 expression leads to the continuous proteasomal degradation of p53 in unstressed cells, whilst the MDM2 gene itself is induced by p53 [42]. MDM2 expression can be induced by a variety of genotoxic agents. Interestingly, it has been suggested that carcinogens found in cigarette smoke, such as benzo[a]pyrene and benzo[a]pyrene diolepoxide, lead to increased levels of aberrant MDM2 splicing and worse prognosis in NSCLC [43]. Over forty splice variants of MDM2 have been described, and six of these have been found in human cancers [44]. The protein products of splice variants MDM2-A, -B, -C, and -D cannot bind and inactivate p53, whilst MDM2-E and -FB26 produce proteins that can bind p53 but not lead to its degradation. MDM2-A, -B and -C are expressed in NSCLC, accounting in part for the counter-intuitive finding that MDM2 (which is a tumour suppressor) is overexpressed in lung tumours [45]. MDM2A may also be a dominant negative protein, since it can bind full-length MDM2, preventing it from inhibiting p53 activity. MDM2-B in particular is thought to promote p53- independent cell growth and lead to the inhibition of apoptosis [43]. Disruption of the spliceosome through the targeting of U2 and U5 snRNP complex components and of the U4/ U6·U5 tri-snRNP splicing complex, has been shown to reduce protein levels of p53 repressors MDM4 and MDM2, stabilising p53 as a result [46]. MDM4 splice variants have also been identified in NSCLC, although much less is known about these splicing events than those of MDM2. The HDMX211 isoform of MDM4 lacks eight exons and has a cryptic 3′ splice site within the eleventh exon, meaning that the encoded protein loses nearly the entire N-terminal p53-binding domain, but retains its ring finger domain responsible for binding MDM2 [47]. As a result, HDMX211 protein is able to bind and stabilise MDM2, also contributing to the overexpression of MDM2 protein in NSCLC. The HDMX211 isoform inhibits MDM2-induced degradation of p53, thereby stabilising inactive p53 protein levels in lung tumours which express high levels of MDM2 [45]. The interactions of these splicing changes in MDM2 and MDM4, and their respective involvement in p53 regulation, establish them as key aberrant events in lung cancer. The p53 protein is not the only important protein to be regulated by MDM2. MDM2 also regulates degradation of the NUMB endocytic adaptor protein. NUMB is a tumour suppressor gene that inhibits Notch signalling pathways. Importantly for cancer cells, NUMB protein also stabilises p53 through its interactions with MDM2, and the complex formed by NUMB and MDM2 inhibits the MDM2-dependent degradation of p53. The protein interaction between NUMB and MDM2 depends on peptide sequences encoded by (and so the splicing inclusion of) NUMB exon 3 (Fig. 3). NUMBΔ3 splice isoforms have been found in breast cancer. These splice isoforms encode proteins that lack the ability to bind MDM2 and therefore promote degradation of p53, thereby contributing to oncogenesis [48]. Thus far the NUMB splicing events known to be present in lung cancer have not been connected to the NUMB-MDM2 interaction. However, aberrant NUMB splicing is involved in multiple other crucial pathways such as the QKI/NUMB/ Notch signalling interaction as discussed below [49].

the activation and expression of BH3-only pro-/anti-apoptotic proteins [21]. Both the extrinsic and intrinsic cell death pathways are multi-step processes, and thus changes in pathway components at any stage could have a severe effect on cell death. A family of Bcl-2 proteins regulates apoptosis by forming heteroand homo-dimers. Bcl-2 protein itself controls commitment to apoptosis, particularly in the intrinsic pathway, by controlling permeability of the mitochondrial membrane to regulate cytochrome c release [19]. Anti-apoptotic members of the Bcl-2 family also inhibit cell death through binding to BH3-only proteins and to activated BAX and BAK in the intrinsic pathway [21]. Bcl-X, encoded by the BCL2L1 gene, is an apoptotic regulator which controls cell death by regulating the voltagedependent anion receptor in the mitochondrial membrane that controls the mitochondrial membrane potential. BCL2L1 pre-mRNA is spliced into two mRNA isoforms which encode proteins with quite different physiological properties. These are an anti-apoptotic isoform called BclXL, and a pro-apoptotic isoform called Bcl-XS (Fig. 2). Shifts in BCL2L1 splicing patterns between its pro- and anti- apoptotic isoforms play a crucial role in the progression of SCLC [22]. BCL2L1 splicing switches affect coding information for key protein domains. The BH1 and BH2 domains within Bcl-XL protein are required for heterodimerisation with BAX to inhibit apoptosis [23]. Within Bcl-XS the BH3 domain is necessary for Bcl-XS apoptotic activity [24]. Present in both Bcl-X isoforms, the TM domain is responsible for localisation of the protein to the mitochondria. Altered patterns of Bcl-X splicing isoforms are not restricted to lung cancer, but also important diseases such as liver fibrosis [25], multiple myeloma [26], and prostate cancer [27]. This makes the understanding of splicing mechanisms that switch between pro- and anti-apoptotic versions of Bcl-X extremely important. Splicing control of Bcl-X opens up the possibility of therapeutic strategies for manipulating apoptotic pathways in lung cancer. The use of antisense oligonucleotides to push the Bcl-X splicing switch towards the pro-apoptotic Bcl-XS isoform has been shown to sensitise lung adenocarcinoma A549 cells to apoptosis following radiotherapy in vitro [28]. In vivo modelling of antisense oligonucleotides targeting Bcl-X splicing is yet to be demonstrated in lung tumour models, but successfully induces apoptosis and subsequent cell death in a dose-dependent manner in melanoma, breast and colorectal cancer in vivo [29,30]. Cytokine treatment of NSCLC cells using MDA-7/IL-24 is another method that has been used to shift towards production of the BclXS isoform in vitro to cause cancer cell death [31]. 2.2. Alternative splicing of MDM2 controls activity of tumour suppressor p53 Relatively little is still known about the global impact of splicing aberrations in lung cancer. However, there has been particular focus on investigating variant splicing patterns of key oncogenes and tumour suppressors already implicated in lung cancer. These include the TP53 tumour suppressor gene which is mutated in 50% of NSCLC and 80% of SCLC cases [32]. TP53 encodes p53 protein, the normal function of which is to control the cell cycle in response to DNA damage. Following input from cell stress sensors, p53 protein halts cell cycle progression in response to excessive DNA damage. Irreversible DNA damage triggers p53-induced cell death via apoptosis [33]. However, loss of p53 is one way in which tumour cells avoid undergoing apoptosis. Mutations in TP53 are a particular hallmark of Li-Fraumeni syndrome, a heritable autosomal dominant syndrome predisposing families to certain rare cancers [34,35]. Although lung cancer is not one of the most common cancers to be associated with Li-Fraumeni syndrome, a small number of cases have been documented, including EGFR-mutant NSCLC and lung adenocarcinoma [36–38]. Although they might not provide a familial predisposition to lung cancer, acquired TP53 mutations have been shown to have a significantly negative effect on the prognosis in lung adenocarcinoma cases [39]. Alternative splicing is relevant to the regulation of TP53 expression

2.3. Mutations in splice site sequences in lung cancer change splicing patterns of the proto-oncogene MET The MET proto-oncogene encodes a receptor tyrosine kinase that is activated by HGF ligand binding, to control RAS/ERK/MAPK and other signalling pathways [50]. Once activated, MET initiates a series of inter-related signal transduction cascades that contribute to cell 4

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Fig. 3. Differentially spliced events in lung cancer. Volcano plot shows differentially spliced events in LUAD and LUSC datasets from TCGA, generated using the psichomics alternative splicing quantification tool [80]. A – LUAD splice events (27,314 events plotted) with labels identifying those commonly referred to in the literature as detailed in Table 1; B – LUSC splice events (27,740 events plotted) with labels identifying those commonly referred to in the literature as detailed in Table 1. Data highlighted by x-value as -0.05 ≤ x ≤ 0.05 where x displays ΔMedian percentage splicing index and y shows -log10(t-test p-value (BH adjusted)) highlighted with a lower limit of -log10(0.05).

MET inhibitors against METΔ14-mutated NSCLC tumours [53–57]. Whereas a number of chemotherapeutic tyrosine kinase inhibitors are potentially made less effective by the aberrant splicing of their targets (Table 2), it is especially interesting that MET inhibitors remain effective against the METΔ14 isoform.

proliferation, reduced apoptosis, decreased cellular cohesion and induction of colony dispersal, each of which are key processes in wound healing and embryogenesis [51]. Mutations in the MET gene have been found in several tumour types, but in lung cancer frequently lead to exon skipping in MET mRNAs [52]. A range of mutations have been found within the 5′ and 3′ splice sites of MET within lung tumour samples. These mutations all result in the skipping of MET exon 14. Whilst multiple different mutations have been identified that affect splicing of MET exon 14, the flanking exons remain in phase, so retaining the reading frame for a MET protein which lacks a phosphorylation site needed for binding a key protein called CBL. CBL is a ubiquitin ligase needed for ubiquitin-mediated degradation of MET, so the loss of CBL binding improves the stability of MET protein [52]. This METΔ14 splice variant has been shown to sustain ligand-dependant signalling longer than the wild-type MET, suggesting that this is an oncogenic gene product [53]. The METΔ14 mutation particularly has been detected within a unique subgroup of NSCLC patients that include older female non-smokers that are diagnosed at an earlier pathology stage [54,55]. The identification of this group is important for directing therapy, since several studies have demonstrated the effectiveness of

2.4. Mutations in lung cancer can create de novo splice sites The majority of cancer genomics research has focussed on mutations which disrupt splice sites. However, mutations which create new splice sites have also been detected in lung cancer patients. Use of the MiSplice tool developed by Jayasinghe and Cao has allowed the identification of > 1500 previously mis-annotated mutations which create new splice sites, several of which occur in lung cancer [81]. For example, a silent PARP1 mutation found in LUSC creates a de novo 5′ splice site. PARP1 encodes a protein involved in the base excision repair and non-homologous end joining pathways that is essential to DNA repair. Administering PARP inhibitors is a common therapeutic strategy to increase the response to chemotherapeutic agents. Whilst PARP inhibitors are not currently listed as standard chemotherapy for lung 5

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genes. Ranking of the most important functional switches in cancer drivers based on patient count identified RAC1 and TP53 as common changes in both LUAD and LUSC. Additionally, NTRK2, PLXNA1 and ADCY1 show some of the greatest functional changes in LUSC, whilst ERBB2 has a significant functional change in LUAD [109]. This functional analysis identified novel aberrant splice events for further investigation.

cancers (Table 2), there is currently growing interest in the use of PARP inhibitors, including in NSCLC [82]. The action of those currently in trial such as Olaparib (https://clinicaltrials.gov/ct2/show/ NCT01788332) could be impeded by a splice creating mutation in PARP1 that affected the coding information for the drug-binding pocket. Bioinformatic analysis of mutation-driven aberrant splicing events which either create or disrupt splice sites, indicate that both LUAD and LUSC have higher numbers of aberrant splicing events than would be expected based on the mean total variant count. Smokers and reformed smokers also have higher aberrant splicing events than non-smokers in both LUAD and LUSC [83].

3. Mis-regulation of splicing factors in lung cancer What causes aberrant splicing in lung cancer? The production of splice isoform patterns is regulated by the cellular concentrations and activity of splicing regulator proteins. These splicing regulator proteins frequently bind to pre-mRNAs within intronic and exonic splicing enhancer and repressor sequences, and this binding activates or represses splice site selection [110]. Changes in the concentrations and activities of particular splicing regulator proteins can drive the splicing patterns of important genes in lung cancer. Changes in splicing regulator levels can be via gene copy number variation, changes in gene expression levels and mutations affecting protein function. Genome wide analysis using datasets such as TCGA has allowed for the identification of millions of mutations and genetic alterations in over 25 different cancer types over the last decade [111]. System-wide approaches have identified amplification, copy number variants and mutations in genes encoding splicing regulators within lung cancer, as well as differences in these between LUAD and LUSC. In this second section, we will review some of the key splicing factors to be mis-regulated in lung cancer and how they contribute to some of the aberrant splicing patterns of target RNAs.

2.5. Mis-spliced therapeutic targets can affect efficacy of chemotherapeutic agents in lung cancer Since lung cancer is often diagnosed in an advanced stage, surgical resection of the tumour is not an option. Instead, many late stage tumours are treated with chemotherapy, primarily with tyrosine kinase inhibitors (TKIs) (Table 2). Interestingly, nearly all targets for TKIs used to treat lung cancer inhibit tyrosine kinases that are also alternatively spliced in some lung tumours (namely EGFR, ALK, RET, MET, VEGFR and FGFR) [84,85]. This raises the possibility that alternative splicing patterns might modify patient drug responses. Alternative splicing isoforms encoding tyrosine kinases that are found in cancer cells, including MET, are already being targeted with specific therapies [53–57]. In addition to this, aberrant splice variants which affect the epitope of monoclonal antibody targets such as PD-L1 could be a potential mechanism of immunotherapy resistance. Targeting production of aberrant splice variants could reduce resistance and increase the range of treatments suitable for individual patients. Alternative splicing signatures important for improved LUSC and LUAD survival have been identified, via genome-wide analysis of splicing network changes reported in The Cancer Genome Atlas (TCGA) datasets [84]. Whether these splicing signatures could be exploited for personalised therapies based on a patient's splicing signature is yet to be established.

3.1. QKI is the most commonly downregulated splicing factor in lung cancer QKI is the most commonly downregulated splicing factor in lung cancer, and QKI protein expression positively correlates with overall patient survival [49]. The QKI gene encodes an RNA-binding protein (RBP) and member of the Signal Transduction and Activators of RNA (STAR) protein family. QKI is essential for embryonic and postnatal development and has key roles in myelinisation and oligodendrocyte differentiation [112]. A recent genome-wide analysis showed QKI regulates more than half of the top 20 validated splicing events in lung cancer [113]. QKI has three main splice variants, QKI-5, QKI-6 and QKI7, which differ in the length of their encoded C-terminal amino acid sequences. Isoforms QKI-5 and QKI-6 are both expressed in lung cancer, however QKI-5 is the predominant isoform in tumour tissue, whilst QKI6 is dominant in matched normal tissue in NSCLC patients [113]. The balance of these isoforms is suggested to be more influential in LUAD than LUSC, based on recent system-wide analyses [114]. NUMB is one of the most important alternative splicing targets controlled by QKI in lung cancer. NUMB encodes a protein involved in the MDM2/p53 interaction. Several NUMB splice isoforms are expressed in multiple cancers including breast [61] and cervical [115]. A QKI/NUMB/Notch interaction has been suggested whereby the QKI-5 protein competes with splicing factor SF1 for sequence-specific binding of NUMB, resulting in the skipping of NUMB exon 11 and inhibition of Notch signalling [49,113]. Since QKI expression is significantly reduced in lung cancer, splicing inclusion of NUMB exon 11 co-ordinately increases, leading to the activation of the Notch signalling pathway and hence increased cell proliferation. Kaplan-Meier plots of data from the TCGA dataset show that NUMB exon 11 splicing inclusion levels above 65% significantly correlate with a negative overall survival in LUAD (Fig. 4). Hence QKI may operate as a tumour suppressor in lung cancer by repressing NUMB exon 11 splicing. QKI also regulates splicing of FN1, which encodes a glycoprotein responsible for cell adhesion and migration called fibronectin [113]. FN1 is known to have three different splice isoforms. The ED-B FN1 isoform has been shown to be upregulated in both NSCLC and SCLC and

2.6. How frequent are splicing changes in lung cancer? Whilst the aberrant splicing events discovered in MDM2, MDM4, Bcl-X and MET discussed above display some of the variety of target RNAs mis-spliced in lung cancer, both in terms of the type of aberrant splicing shown and the role of the target RNA involved. However, these are just some of the many aberrant events which have been identified in lung cancer. A larger but by no means exhaustive list is discussed in Table 1. The wide range of splicing events and their connections with many hallmarks of cancer are consistent with high complexity of aberrant alternative splicing in lung cancer. Additionally, common splicing factors are responsible for a number of different aberrant events in lung cancer, so there is the possibility that some aberrant splicing events may be causally related. How frequent might aberrant splicing patterns be in lung cancer? Is it possible that the events shown in Table 1 might still be the tip of the iceberg? Data from TCGA suggest many genes may be aberrantly spliced in lung cancer. Fig. 3 shows volcano plots displaying differentially spliced events in the LUAD and LUSC datasets respectively. Of these splice events globally identified as either up- or down-regulated in lung cancers, the most characterised splice events from Table 1 form just a small group. This is consistent with the theory that there are many additional splice events associated with lung cancer that still need to be investigated. The limited or often entire lack of literature investigating many of the splice events shown Fig. 3 in relation to lung cancer identifies this an area worthy of investigation. A recent study investigating the functional impact of aberrant alternative splicing in multiple different cancers identified a subgroup of aberrant events which affect protein domain families in cancer driver 6

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Table 1 Aberrant splicing events in lung cancer. Examples of the variety of aberrant splicing events found in lung cancer are shown, including exon skipping and alternative splice sites, and a combined event where intron retention causes the introduction of a premature polyadenylation site. Solid lines indicate normal splicing pattern; dashed lines indicate the aberrant event described. Skipped exon events are shown in gold; Alternative splice site events are shown in blue; cassette exon events are shown in red. AS = Alternative splicing; SE = Skipped exon; Cr3SS = Cryptic 3′ splice site; A5SS = Alternative 5′ splice site; IR = Intron retention; A3SS = Alternative 3′ splice site; CE = cassette exon. Gene

AS event

Sub-type

Role in cancer

Splice factor

Ref.

MDM2c

SE ex 4–9

NSCLC

Oncogene, apoptosis

Unclear

[45,58,59]

SE ex 4–11

SE ex 5–9

MDM4c

SE ex 3–10 (Cr3SS ex 11)

NSCLC

Apoptosis

Unclear

[45,47,58]

Bcl-Xc

A5SS ex 2

SCLC/NSCLC

Apoptosis

RBM4, SRSF1

[22,60]

METc

SE ex 14

NSCLC

Oncogene

NUMBd

SE ex 3

NSCLC

Tumour suppressor

RBM5, RBM6, RBM10, QKI-5

[49,61]

[52,53]

SE ex 11

VEGFR1c VEGF-Ac

IR int 13, premature polyadenylationa A3SS ex 8

NSCLC NSCLC

Angiogenesis Angiogenesis

VEGF165 SRPK1, SRSF1

[62] [63–65]

MST1Rc

SE ex 11

SCLC/NSCLC

Invasion, inflammation

SRSF1

[66–70]

SE ex 11–13

SE ex 18–19

MKNK2c

A3SS ex 14

NSCLC

Oncogenic transformation

SRSF1

[71]

CFLARc DNAJA3c

IR int 5, premature polyadenylationa SE ex 5

NSCLC NSCLC

Apoptosis, chemo-resistance Apoptosis

RBM5, RBM6, RBM10 hnRNPA1/A2b

[72,73] [74–76]

SE ex 11

PRRC2Cd

CE ex 33/34

NSCLC

Apoptosis, cell proliferation

SRSF1

[77]

PTPMT1d

SE ex 3

NSCLC

Sensitisation to radiotherapy

SRSF1

[78]

(continued on next page)

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Table 1 (continued) Gene c

FAS

a b c d

AS event

Sub-type

Role in cancer

Splice factor

Ref.

SE ex 6

NSCLC

Apoptosis

RBM5, RBM6, RBM10, SRSF7

[72,79]

Splice event causes retention of part of the indicated intron, which itself introduces a premature polyadenylation site. hnRNPA1/A2 are suggested as splice factors for exon 11 skipping of DNAJA3, splice factors for the exon 5 skipping event are unclear. Splice events were identified by a candidate gene method. Splice events were identified through genome-wide analyses.

RBM5 is a component of the spliceosome A complex. RBM5 protein controls apoptosis and cell cycle arrest through regulating TP53 (encoding the tumour suppressor p53) splicing patterns [125]. RBM5 expression is downregulated in NSCLC, as in prostate cancer and some breast tumours. RBM5 downregulation is in fact considered a signature of metastasis in solid tumours [61,126]. Interestingly, an in vivo model of RBM5 loss of function showed a dramatic increase in the aggressiveness of lung tumours without RBM5 expression, further enhanced when combining this model with cigarette smoke, thus supporting the role of RBM5 as a tumour suppressor [127]. RBM5 regulates the splicing of c-FLIP, which also regulates apoptosis. Isoforms of c-FLIP switch between two functionally distinct splice isoforms under the control of RBM5 to activate or inhibit the Fas pathway. In NSCLC, decreased expression of RBM5, RBM6 and RBM10 lead to increased levels of the anti-apoptotic c- FLIP(s) isoform, contributing to tumourigenesis [72]. Despite being identified two decades ago, there is still very little known about RBM6 function. RBM6 was initially isolated alongside RBM5 and thought to have comparable functions, but the majority of the literature describing RBM6 function still combines its role with other RBM proteins [128]. RBM6 is often deleted or mutated in lung cancer. RBM6 along with RBM5 works antagonistically to RBM10 to regulate the splicing inclusion of NUMB exon 11 in NSCLC, resulting in enhanced Notch signalling and subsequent increased cell proliferation [61]. Further investigation is required to establish the individual contribution to disease progression of RBM6 downregulation in NSCLC. The RNA splicing regulator RBM4 controls switches between Bcl-X splice isoforms. RBM4 shifts towards the production of the Bcl-XS isoform by competing with the splicing regulator SRSF1. This means that expression levels of both RBM4 and SRSF1 are thus critical for disease progression. RBM4 expression was reported to be decreased in NSCLC by Wang et al., which would result in an increase in Bcl-XL levels and corresponding reduction in cancer cell apoptosis. This implicates RBM4 as a tumour suppressor that becomes downregulated in lung cancer [60]. However, more recent work has contested this protective effect, and suggested instead that RBM4 is actually overexpressed in lung

possibly contributes to increased angiogenesis in the tumour [116,117]. Whilst there is little known about the splicing of FN1 in lung cancer, it is identified as one of the most statistically significant differential splicing events in LUAD in Fig. 3. Combined with the system-wide analyses identifying FN1 aberrant splicing as an event regulated by QKI, this shows how large scale approaches broaden the field of alternative splicing in lung cancer and suggest how therapeutic targeting of splicing factors could be potentially multi-targeted in their efficacy. 3.2. Mis-regulation of RBM proteins leads to aberrant alternative splicing in lung cancer RBM4, RBM5, RBM6 and RBM10 are each downregulated in lung cancer and have been suggested to act as tumour suppressors [60,61]. RBM is an acronym of RNA Binding Motif, and RBM proteins each contain an RNA Recognition Motif (RRM). RRM domains enable RBM proteins to bind their target RNAs. RBM5, RBM6 and RBM10 are ancient paralogs (http://www.ensembl.org/Homo_sapiens/Gene/ Compara_Paralog?db=core;g=ENSG00000003756;r=3:5008890850119021) that encode proteins with similar domain structures. An OCRE domain in RBM5, RBM6 and RBM10 proteins mediates interactions with the U4/U5·U6 tri-snRNP (Fig. 5) [60,118–120]. RBM10 is downregulated and one of the most frequently mutated splicing factors in lung cancer [17,121,122]. Despite the majority of known mutations in spliceosome components occurring in haematological malignancies, RBM10 is one of a small number of genes also mutated in solid tumours (along with U2AF1, also mutated in NSCLC) [123]. Interestingly, the majority of RBM10 mutations in LUAD encode protein truncating variants, which in turn can lead to loss of function, or produce proteins with toxic functions [122]. One of the key splicing targets for RBM10 protein is NUMB exon 11. Mutations in RBM10 found in NSCLC disrupt its regulation of NUMB alternative splicing, causing increased inclusion of exon 11 and thus increased cell proliferation, mirroring the effects of reduced QKI expression on NUMB splicing as discussed above [113,124].

Fig. 4. NUMB skipped exon survival curve in LUAD. Kaplan-Meier plot showing survival in LUAD based on the TCGA dataset for the NUMB exon skipping event chr14:73749067-73744001, negative strand, hg19. This data was plotted using the Psichomics alternative splicing quantification tool with PSI cutoff: 0.65, log-rank p-value = 2.46e-05 [80].

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Fig. 5. Modular organisation of RBM protein domains implicated in lung cancer. Schematic representation of RBM protein domains. Protein domains are placed according to organisation within the protein, locations and domain sizes are not to scale. RRM = RNA Recognition Motif; ZnF = Zinc Finger; A = Alanine-rich region; RS = Arginine/Serine-rich region; OCRE = OCtamer REpeat domain; G-patch = Glycine patch; RG = Arginine-Glycine-Glycine box.

apoptosis [77]. SRSF1 overexpression has also been shown to contribute to radio-resistance in lung cancer cells through its control of PTPMT1 splicing. PTPMT1, which encodes a PTEN-like phosphatase, switches towards its full-length splice isoform, PTPMT1-A, following SRSF1 overexpression. This in turn reduces p-AMPK levels, inhibits DNA damage and renders cells radio-resistant [78]. Reducing SRSF1 expression levels in lung cancer could thus be effective in sensitising cells to radiotherapy. Tra2β has both N- and C-terminal RS domains, separated by a central RRM (Fig. 6). Despite being downregulated in kidney and thyroid cancers, TRA2B is amplified in LUSC [121]. Overexpression of TRA2B promotes cell proliferation in NSCLC cells in vitro and is associated with poorer patient prognosis in NSCLC [136]. Tra2β upregulation in NSCLC may induce multiple aberrant splicing events. In breast cancer, Tra2β controls splicing of exon 3 of CHK1, allowing CHK1 protein to successfully monitor DNA damage and control cell cycle progression, and reducing replication stress in the rapidly dividing cancer cells [137]. The significant amplification of TRA2B in LUSC, along with its importance to many splicing events in breast cancer suggest a potential role for the gene as a master regulator of aberrant splicing in lung cancer.

cancer leading to increased cell proliferation, i.e. having a more oncogenic role in tumourigenesis [129]. It is still unclear how RBM4 expression changes in NSCLC [60,130]. This creates a conundrum. If RBM4 is downregulated in NSCLC, this would lead to the increase of Bcl-XL levels and reduced apoptosis, aiding tumourigenesis. However, if RBM4 is in fact upregulated, Bcl-XS would be the dominant isoform, resulting in increased apoptosis and reduced cell proliferation. 3.3. Changes in expression of Serine/Arginine-rich splicing regulators activate splicing of exons in lung cancer Serine/Arginine-rich (SR) proteins usually bind to exons to activate splicing inclusion. SR proteins contain an arginine/serine (RS) domain, which facilitates the protein-protein interactions with other RS domaincontaining sequences, and at least one RRM that binds to RNA (Fig. 6) [131]. Mis-regulated expression of two SR proteins has been reported in lung cancer – SRSF1 and Tra2β. SRSF1 is a member of the classic group of SR proteins, as defined by their in vitro splicing activity. S100 extracts are derived from lysed HeLa cells and contain most core spliceosome components except SR proteins. Any classic SR protein can be added to S100 extracts, and this is needed to enable in vitro splicing activity [132]. TRA2B encodes an SR-like protein called Tra2β. SR-like proteins share many features with the classic SR protein family, including containing RS domains and RRMs, but cannot restore in vitro splicing activity to S100 extracts [133]. The SRSF1 gene encoding the classic SR protein SRSF1 becomes amplified in NSCLC (and many other cancers), leading to a more aggressive phenotype in LUAD and contributing to chemo-resistance [134]. SRSF1 overexpression has been shown to deregulate the splicing of multiple targets in NSCLC, including proline rich coiled-coil 2C (PRRC2C), a gene involved in proliferation and cell cycle regulation [135]. PRRC2C contains a cassette exon which is increasingly spliced in response to SRSF1 overexpression, favouring the PRRC2C-L (long) isoform that in turn increases proliferative capacity and resistance to

4. Conclusion The last decade of lung cancer and splicing research has been incredibly informative in terms of identifying previously unreported target RNAs mis-spliced in lung cancer, but also with regard to furthering the understanding of how known aberrant splicing events are regulated. The Bcl-X splicing switch was previously characterised in several cancers including lung, however the controversy over the regulation of this splicing event by RBM4 adds to the complexity of this mechanism. Bcl-X splicing gives an example of an area where targeting of splicing using antisense oligonucleotides is already being tested as a potential therapeutic strategy. Since there are a number of Fig. 6. Modular organisation of SRSF1 and Tra2β proteins implicated in lung cancer. Schematic representation of the protein domains of SRSF1 and Tra2β. Protein domains are placed according to organisation within the protein, locations and domain sizes are not to scale. RRM = RNA Recognition Motif; RS = Arginine/Serine-rich region; G = Glycine-rich hinge region.

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Table 2 Chemotherapeutic agents licensed in the UK for treating lung cancer. Abbreviations used are AS = Alternative Splicing; TKI = Tyrosine Kinase Inhibitor; EGFR = Epidermal Growth Factor Receptor; ALK = Anaplastic Lymphoma Kinase; VEGFR = Vascular Endothelial Growth Factor Receptor; PD-L1 = Programmed Death Ligand 1; FGFR = Fibroblast Growth Factor Receptor 1; PDGFR = Platelet-derived Growth Factor Receptor 1; PD-1 = Programmed cell death protein 1; LA/ Met = Locally advanced or metastatic. Drug

Chemotherapy class

Target

AS of target

Range of use

Method of action a

Afatinib Alectinib

TKI TKI

EGFR and HER2 ALK

EGFR ALK

LA/Met NSCLC Advanced NSCLCa

Ceritinib

TKI

ALK

ALK

Advanced NSCLC

Crizotinib Erlotinib Gefitinib

TKI TKI TKI

C-Met and ALK, ROS1 EGFR1 EGFR1

MET, ALK EGFR EGFR

Advanced NSCLC LA/Met NSCLC LA/Met NSCLC

Nintedanib

TKI

VEGFR, FGFR, PDGFR

VEGFR, FGFR

Osimertinib Atezolizumab Nivolumab

TKI Monoclonal antibody Monoclonal antibody

EGFR PD-L1 PD-1

EGFR – –

Pembrolizumab Gemcitabine Pemetrexed Docetaxel Vinorelbine Carboplatin

Monoclonal antibody Anti-metabolite Anti-metabolite Taxane Vinca alkaloid Platinating agent

– – – – – –

Cisplatin

Platinating agent

Topotecan

Topoisomerase I inhibitor

PD-1 Pyrimidine analogue Folic acid analogue binds Tubulin Binds tubulin Heavy metal alkylation of DNA Heavy metal alkylation of DNA Topoisomerase I

LA/Met or locally recurrent NSCLC LA/Met NSCLCa LA/Met NSCLCa LA/Met squamous/nonsquamous NSCLC LA/Met NSCLC Advanced NSCLC LA/Met NSCLC LA/Met NSCLC Advanced NSCLC Limited-stage SCLCa

–

Limited-stage SCLC, extensivestage SCLC Relapsed SCLCa

Bevacizumab Necitumumab Paclitaxel Ramucirumab

Monoclonal antibody Monoclonal antibody Taxane Monoclonal antibody

VEGFR EGFR Binds tubulin VEGFR2

VEGFR EGFR – VEGFR

No No No No

a

–

longer longer longer longer

recommended recommended recommended recommended

Inhibits EGFR/HER2 [88] Blocks STAT3 and PI3K/AKT/mTOR pathway [89] Inhibits mutated ALK, prevents cell proliferation [90] Inhibits action of secondary messengers [91] Inhibits auto-phosphorylation of EGFR [92] Inhibits EGFR signal transduction pathways [93] Inhibits angiogenesis [94] Inhibits EGFR [95] Activation of the immune response [96] Activate T cell proliferation and cytokine production [97] Reduces immune evasion of the cancer [98] Prevents DNA synthesis [99] Prevents DNA synthesis [100] Prevents microtubule disassembly [101] Prevents tubulin polymerisation [102] Crosslinking of DNA/RNA/Protein [103] Crosslinking of DNA/RNA/Protein [103] Prevents unwinding of supercoiled DNA for replication [104] Inhibits tumour angiogenesis [105] Inhibits EGFR [106] Prevents microtubule disassembly [107] Prevents angiogenesis [108]

Only for a limited number of cases (see NICE guidelines) Based on the Lung Cancer NICE pathway and ESMO guidelines [10,86,87].

over the last decade. With the popularity of system-wide analyses pulling out increasing numbers of potential new events and prognostic splicing signatures being characterised in lung cancer, the next decade of research for the field has the potential to further define lung cancer sub-types and provide a more personalised approach to therapy.

chemotherapeutic agents whose efficacy may be affected by aberrant splicing in lung cancer (Table 2), the establishment of techniques with which to target splicing events is a crucial area for further investigation. Where aberrant splicing has occurred following mutations such as those seen in the MET gene, correction of these mutations by gene editing techniques such as CRISPR could prove effective, however the wide variety of mutations present and the complexity of accurate CRISPR targeting might make this an undesirable technique. To date it seems that many of the known aberrant splicing events in lung cancer are regulated by a relatively small pool of splicing factors (Table 1). Added to the alternative splicing signatures of prognostic value already being identified in LUSC and LUAD, there is potential that therapeutic targeting of the splicing factors may have caused effects on multiple splicing events. Inhibition of overexpressed factors, or increase gene expression of reduced splicing factors could help restore normal alternative splicing in lung tumours. In addition to exploiting the currently known aberrant splicing events in lung cancer, the increase in system-wide analysis of large datasets has revealed that there are many significant differential splicing events which are not currently being investigated. Whilst some of these events may not impact on tumourigenesis, further investigation into these gene events is required. NUMB splicing in particular could prove an important target. Prognostic alternative splicing signatures are still being uncovered in several cancers including in NSCLC [84]. The identification of a diagnostic splicing signature could allow aberrant splice patterns to be exploited as biomarkers to guide personalised treatment. This has been proposed for stomach adenocarcinoma, but no such signature has been identified as of yet [138]. The identification of a diagnostic aberrant splicing signature in lung cancer could offer further personalisation to current therapeutic options. The field of alternative splicing in lung cancer has come a long way

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