The human genome can be described as a complex, biological manual that encodes for the genetic instructions required to build and maintain an individual. Furthermore, as Charles Darwin famously discovered that human beings are no stranger to evolution, it should come as to no surprise to us of the complicated and dynamic nature of the genetic code . It is the main reason that our strong efforts placed into developing a greater understanding of the inherent causes and mechanisms driving the plethora of human disorders have been frequently undermined.
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One of the culprits that has an unsuspecting role impacting human pathologies such as cancers, given its defining characteristic in establishing the distinct and functional role of many cells in the human body is alternative splicing (AS). AS of mRNA is a crucial molecular mechanism that contributes greatly to the transcriptome and consequently proteome diversity. It is a controlled process that allows for different protein isoforms to be expressed via a single pre-mRNA transcript. Its discovery has therefore been labelled as one of the few exclusions to the original ideology of ‘One gene, one polypeptide’. During this splicing process, certain exons within a gene could potentially be included or excluded which as a result will alter the nucleotide sequences of the generated final mature mRNA. Hence, the amino acids encoded for by the different nucleotide sequences will lead to the production of structurally and functionally dissimilar proteins.
So what exactly is the relevance of AS to tumorigenesis and the formation of cancer cells ? Abnormal phenotypes exhibited by the transformed cells are expressed through aberrant mRNA transcripts due to alternative splicing changes, mutation in splicing factors or cis-acting splicing elements. As a matter of fact, Braun S et al. has reported that alternative splicing (AS) of exon 11 in proto-oncogene RON directed by various point mutations can cause the formation of isoform RON∆165 which possess oncogenic potential and the capability of inducing invasive tumorigenesis.1
There is a great variety of contributing factors leading up to AS changes and its further participation in cancer initiation or progression. For instance, by using a high throughput mutagenesis screen of minigenes, Braun S et al. has revealed that more than 80% of the positions of the cis regulating landscape has an effect on the AS of RON.1 Moreover, through mathematical modelling of splicing kinetics, more than 1000 mutations which were related to the skipping of exon 11 have been identified to cause the synthesis of isoform RON∆165.1 To recapitulate, the ramifications following AS changes along with further triggers can be seen in Figure 1 below.
Figure 1. Factors contributing to alternative splicing (AS) changes such as DNA damage, alterations in chromatin state and level of transcription, mutations in splicing factors, changes in expression levels of splicing factors and modified sequences of splicing regulatory elements. These modifications can cause the development of cancer hallmarks such as inducing angiogenesis, resisting cell death, sustaining proliferative signalling, enabling replicative immortality and activating invasion and metastasis. AS changes are also potential biomarkers for different tumour subtypes and clinical stages which can dictate the possibilities of therapy resistance. Lastly, different splicing patterns can be informative in the selection of targeted therapies enabling an increase in specificity and effectiveness. 2
As mentioned above, mutations that inflict structural and sequential changes including altered expression of splicing factors can trigger malignant cell transformations. For example, an upregulated expression of SFRS1 is found to be present in breast and colon cancers leading to the generation of an alternative form of tyrosine kinase protein known as Delta Ron which confers an increase in cell motility and matrix invasion, leading to tumour metastasis. 3Conversely, a decrease in the expression of U2AF35 has been linked to pancreatic cancer due to the production of a misspliced form of cholecystokinin-B/gastrin (CCK-B) receptor.4 Braun S et al. have also observed via iCLIP and synergy analysis that RBPs (RNA Binding Proteins) such as heterogeneous nuclear ribonucleoprotein H (HNRNPH) which has multiple independent binding sites that exhibit strong cooperativity has a regulatory effect on AS of RON.1 The authors then further postulated its functional role as a splicing switch for RON.1
More importantly, since AS switches are tissue specific, studies have shown that its detection can function as a prospective biomarker to detect different types of cancer. The evidence was provided by a study conducted using a high-throughput reverse transcription-PCR-based system that confirmed the 41 different splicing events present in breast tumours in relative to normal breast tissues. 5 Furthermore, the presence of different AS switches can also predict drug and therapy resistance in certain types of cancers. Clinical trials that used monoclonal antibodies targeting RON to prevent ligand binding have proved to be ineffective as the nature of its pathophysiological isoform, RON∆165 no longer relies upon ligand activation.1 Besides that, CART-19 immunotherapy failed to yield response in some leukemic patients due to the skipping of exon 2 that prevented tumour antigen recognition by CART-19. Breast cancer patients can also develop resistance towards chemotherapy cisplatin combined with PARP inhibitors due to mutations on exon 11 of BRCA1.6
Finally, AS has garnered the attention of being a target for developing specific therapies and for discovering novel drugs. Recent therapeutic approach involves the usage of antisense oligonucleotides (AONs) that can activate or inhibit specific splicing events, enabling the possibility of reverting the cell to its original phenotype. This has been proven to work in other splicing related disorders like Duchenne muscular dystrophy (DMD). In addition to that, depending on tumour type and the stages of mutation in the splicing factors, small molecule compounds can also be used to regulate the activity of splicing factors. 2
Consequently, it has become increasingly clear to us that it would be beneficial to deepen our physiological understanding of the molecular mechanisms of alternative splicing given its relevance towards many cancer pathologies. Only then, we can strive to improve and enhance present therapeutics and perhaps to even increase therapeutic specificity by personalizing treatments for individual cancer patients.
1 Simon Braun1, Mihaela Enculescu1, Samarth T. Setty2, Mariela Cortés-López1, Bernardo P. de Almeida3,4, F.X.Reymond Sutandy1, Laura Schulz1, Anke Busch1, Markus Seiler2, Stefanie Ebersberger1, Nuno L. Barbosa-Morais 3, Stefan Legewie1, Julian König1 & Kathi Zarnack. Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis. Nature Communications volume 9, Article number: 3315 (2018)
2 Singh B, Eyras E. The role of alternative splicing in cancer. Transcription. 2016;8(2):91-98.
3 Ghigna C, Giordano S, Shen H, Benvenuto F, Castiglioni F, Comoglio PM, Green MR, Riva S, Biamonti G (2005) Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene. Mol Cell 20: 881–890
4 Ding WQ, Kuntz SM, Miller LJ (2002) A misspliced form of the cholecystokinin-B/gastrin receptor in pancreatic carcinoma: role of reduced cellular U2AF35 and a suboptimal 3′-splicing site leading to retention of the fourth intron. Cancer Res 62: 947–952
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5 Venables JP, Klinck R, Bramard A, Inkel L, Dufresne-Martin G, Koh C, Gervais-Bird J, Lapointe E, Froehlich U, Durand M, Gendron D, Brosseau JP, Thibault P, Lucier JF, Tremblay K, Prinos P, Wellinger RJ, Chabot B, Rancourt C, Elela SA. Identification of alternative splicing markers for breast cancer. Cancer Res. 2008 Nov 15; 68(22):9525-31.
6 ZahavaSiegfried, RotemKarni. The role of alternative splicing in cancer drug resistance. Current Opinion in Genetics and Development. 2018; 48:16-21. Available from: https://doi.org/10.1016/j.gde.2017.10.001