EMT and stemness: flexible processes tuned by alternative splicing in development and cancer progression

Abstract

Epithelial-to-mesenchymal transition (EMT) is associated with metastasis formation as well as with generation and maintenance of cancer stem cells. In this way, EMT contributes to tumor invasion, heterogeneity and chemoresistance. Morphological and functional changes involved in these processes require robust reprogramming of gene expression, which is only partially accomplished at the transcriptional level. Alternative splicing is another essential layer of gene expression regulation that expands the cell proteome. This step in post-transcriptional regulation of gene expression tightly controls cell identity between epithelial and mesenchymal states and during stem cell differentiation. Importantly, dysregulation of splicing factor function and cancer-specific splicing isoform expression frequently occurs in human tumors, suggesting the importance of alternative splicing regulation for cancer biology.In this review, we briefly discuss the role of EMT programs in development, stem cell differentiation and cancer progression. Next, we focus on selected examples of key factors involved in EMT and stem cell differentiation that are regulated post-transcriptionally through alternative splicing mechanisms. Lastly, we describe relevant oncogenic splice-variants that directly orchestrate cancer stem cell biology and tumor EMT, which may be envisioned as novel targets for therapeutic intervention.

Keywords: Alternative splicing; Cancer stem cells; EMT; RNA binding proteins; Stem cell differentiation; Tumor progression.

Fig. 1

Significant alternative splicing changes occurring during EMT. a Key transcription factors upregulated during EMT; gradient color represents their expression increase from epithelial to mesenchymal phenotype. b Schematic representation of EMT progression. From left to right: (i) polarized epithelial cell with strong cell-cell junctions. Par complex and actin filaments localize to the junctions; (ii) epithelial cell with residual junctions starts to re-organize its cytoskeleton and change its morphology. E-cadherin disappears from cell membrane (small yellow square). The Par complex is disassembled and PAR6/aPKC move to the apical cell surface; (iii) the epithelial cell loses its epithelial features and begins to acquire an elongated and spindle-like morphology, while PAR6/aPKC, with other polarity complexes (not shown), allow the establishment of a front-rear polarity. Metalloproteases are secreted in order to degrade the ECM; (iv) a motile mesenchymal cell is able to invade the surrounding tissues. c Expression gradients of key splicing factors regulated during EMT. d Center. AS of genes involved in different EMT programs, including migration and invasion (FGFR2, RON and CD44), polarity and cytoskeleton organization (NUMB, RAC and p120) and transcription regulation (TCFL2). Alternative exons are represented in red, mutually exclusive exon in blue. Left. Scheme of epithelial-specific AS variants. Alternative exons and the encoded amino acids are indicated in red. Right. Mesenchymal-specific isoforms are also shown. Differences in functional properties of epithelial versus mesenchymal isoforms are highlighted: FGFR2 exons IIIb and IIIc confer different ligand binding specificity; ΔRON and Rac1b are constitutively active cytoplasmic isoforms; inclusion of exon 6 in NUMB allows it to interact with Par complex and E-cadherin; p120 isoforms 1-2 localize to AJ, whereas p120 isofoms 3-4 localize with the activate RAC and repress RHOA signaling thus promoting re-organization of the actin cytoskeleton; skipping of exon 4 in TCFL2 generates the more active transcriptional factor TCFL2-Δ4

Fig. 2

Alternative Splicing regulation. a Scheme of the different AS modalities: (i) cassette exons; (ii) mutually exclusive exons; (iii) intron retention; (iv) alternative 5′ splice sites; (v) alternative 3′ splice sites; (vi) inclusion of a poison exon containing a premature stop-codon (yellow) leading to mRNA degradation through NMD. Precursor transcripts and final spliced products are shown. b AS regulation by combined action of trans- and cis-acting elements. Intronic and exonic splicing enhancers (ISE and ESE) promote the inclusion (+) of the AS exon (red) by providing the binding sites for activators (orange circles), whereas intronic and exonic splicing silencers (ISS and ESS) are bound by repressors (yellow circles) and promote exon skipping (-). Generally, ESE-bound SR factors stimulate the assembly of the splicesome on the variant exon or counteract the inhibitory activity of hnRNPs bound to ESS elements. On the contrary, hnRNPs interfere with the assembly of spliceosome to the variant exon leading to exon skipping. In addition, hnRNPs by binding ISSs located in the introns flanking a variant exon cause its looping out and skipping, whereas when bound to ESSs they may polymerize along the exon and displace the ESE-bound SR proteins (not shown). c Some members of the SR and hnRNP families mentioned in the text are shown with their characteristic domains. SR proteins have a modular structure with one or two RNA recognition motifs (RRM) in the N-terminus able to interact with the pre-mRNA, whereas at C-terminus all members of this family present a domain of variable length rich in serine-arginine dipeptides (RS domain) involved in protein-protein interactions with spliceosomal components. HnRNPs possess one or more RNA-binding domains associated with different “auxiliary” domains that are diverse in sequence and involved in sub-cellular localization or protein-protein interactions. Tissue-specific AS regulators (RBFOX, MBNL, ESRP and NOVA families) are indicated with their own RNA-binding domains

Fig. 3

Significant alternative splicing changes occurring during stem cell differentiation. Center. Splicing factors and AS of genes involved in somatic cell reprogramming; gradient color represents splicing factor expression increase/decrease from ESCs or iPSCs to differentiated cells. Left. Scheme of ESCs or iPSCs-specific AS variants. Alternative exons and the encoded amino acids are indicated in red. Right. Differentiated cells-specific isoforms are also shown. Differences in functional properties of pluripotent versus differentiated isoforms are highlighted: FOXP1 mutually exclusive exons confer different DNA binding properties; MBD2 AS variants c and a are both enriched at the promoters of Oct4 and Nanog, but only MBD2a is able to recruit chromatin remodeling complexes to repress pluripotency factors transcription; PRDM14-ES, ZNF207 A/C and GRHL1-FL enhance somatic cells reprogramming, whereas their AS isoforms, lacking the alternative exon, counteract reprogramming