Alternative splicing and tumor progression

Abstract

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

Keywords: Alternative splicing; EMT; biomarkers.; cancer; splicing correction; splicing factors.

Fig. (1)

Cis-acting sequences required for the splicing reaction and different types of alternative splicing events. (A) Splicing consensus sequences of a typical eukaryotic gene (exon/intron splice site signals, branch site and polypyrimidine tract). (B) Alternatively spliced mRNAs result from exon skipping, intron retention, usage of alternative 3’- (acceptor) or 5’- (donor) sites and from selection of mutually exclusive exons. At the protein level, alternative splicing drastically affects the amino acid sequence by deletion or insertion of domains, frame-shifts or stop codons. Alternative splicing in non-coding regions of the mature mRNA might impact on translation and mRNA stability.

Fig. (2)

Cis- and trans-acting regulatory elements that control alternative splicing and models for the function of splicing enhancers and silencers. (A) Alternatively spliced exons are usually characterized by weak splice sites. Recognition of these sites depends on splicing regulatory elements: exonic splicing enhancers (ESE) and silencers (ESS) and intronic splicing enhancers (ISE) and silencers (ISS). (B) ESE elements are bound by splicing factors of the SR family. Via interactions with proteins of the splicing apparatus, the RS domain of SR factors promotes the assembly of the splicesome on the adjacent intron (left). In addition, SR factors can counteract the inhibitory activity of hnRNP proteins bound to ESS elements (right). (C) The mechanism of action of the silencer elements depend on the their position along the pre-mRNA. In some cases, inhibitory factors (for example hnRNP proteins) bind to ISSs sequences flanking an alternative exon and cause looping out and skipping of this exon (left). Alternatively, inhibitory factors bound to ESSs may polymerize along the exon and displace the ESE-bound SR proteins (right).

Fig. (3)

Schematic representation of the Ron receptor and of the downstream signalling pathways. (A) Ron is a single-pass, disulfide-linked α/β heterodimer. The α chain is an extracellular glycoprotein while the β chain is a transmembrane subunit that comprises an extra-cellular sequence, a short trans-membrane segment, a large cytoplasmic portion with intrinsic tyrosine kinase domain (TK) and a C-terminal tail. Residues and domains important for the Ron activity are indicated. In particular, the intracellular domain includes the tyrosine kinase catalytic site (white oval) flanked by distinctive juxtamembrane and carboxy-terminal sequences. Phosphorylation of two tyrosines within the kinase domain positively regulates the enzyme activity, whereas a serine residue in the juxtamembrane domain has a negative regulatory role. Two tyrosine residues in the carboxy-terminal region, when phosphorylated, form a specific docking site for multiple signal transducers and adaptors. Activation of Ron by MSP can initiate signaling through many pathways implicated in tumor progression and metastasis, such adhesion, invasion, mobility, proliferation and inhibition of apoptosis. (B) The constitutively active ΔRon isoform is generated through skipping of exon 11. This event is controlled by two adjacent splicing elements, a silencer and an enhancer, located in the central part and at the 3’ end of exon 12, respectively. These two regulatory elements may form a ‘‘control cassette’’ that tunes the strength of the acceptor site of intron 11 and thus the ratio between Ron and ΔRon transcripts. Splicing factor SF2/ASF directly binds to the enhancer and governs its activity. High levels of SF2/ASF increase the strength of the acceptor site of the intron 11, promote the production of ΔRon isoform and trigger morphological and molecular changes typical of the epithelial to mesenchymal transition (EMT). The primary function of the silencer could be to antagonize the enhancer and prevent exon skipping.

Fig. (4)

Potential use of therapeutic approaches that target alternative splicing. Various strategies are currently being used to exploit alternative splicing for treatment of cancer. (A) In many cases, cancer-restricted splice variants contain unique epitopes that could be used as the targets for specific antibodies conjugated to tumor-cell toxins. (B) Many chemical compounds have been found to affect splicing of numerous genes. Although the mechanisms by which splicing patterns are altered are still poorly understood, several compounds are able to block the activity of SR-protein kinases (SRpKs) and, consequently, reduce the phosphorylation state of SR splicing factors. (C) Synthetically modified oligonucleotides, such as phosphorothioate, morpholino phosphorodiamide and 2´-O-methylethyl, are able to block the interaction of the spliceosome machinery with a specific site. Moreover, they are more stable and active than regular nucleotide backbones and display low toxicity in vivo. (D) Cancer-specific mRNA transcripts that contain unique sequences can be targeted using RNAi mediated degradation.