Alternative splicing and cell survival: from tissue homeostasis to disease

Abstract

Most human genes encode multiple mRNA variants and protein products through alternative splicing of exons and introns during pre-mRNA processing. In this way, alternative splicing amplifies enormously the coding potential of the human genome and represents a powerful evolutionary resource. Nonetheless, the plasticity of its regulation is prone to errors and defective splicing underlies a large number of inherited and sporadic diseases, including cancer. One key cellular process affected by alternative splicing is the programmed cell death or apoptosis. Many apoptotic genes encode for splice variants having opposite roles in cell survival. This regulation modulates cell and tissue homeostasis and is implicated in both developmental and pathological processes. Furthermore, recent evidence has also unveiled splicing-mediated regulation of genes involved in autophagy, another essential process for tissue homeostasis. In this review, we highlight some of the best-known examples of alternative splicing events involved in cell survival. Emphasis is given to the role of this regulation in human cancer and in the response to chemotherapy, providing examples of how alternative splicing of apoptotic genes can be exploited therapeutically.

Figure 1

The apoptotic pathways. Schematic representation of the the extrinsic (death receptor) pathway (left) and of the intrinsic (mitochondrial) pathway of apoptosis (right). Binding of ligands of the TNF family leads to oligomerization of death receptors and subsequent recruitment and activation of initiator caspases (caspase-8 and -10) via adaptor proteins. The initiator caspases target effector caspases for proteolytic cleavage and activation. In response to an apoptotic stimulus pro-apoptotic BCL-2 proteins lead to permeabilization of the outer mitochondrial membrane and release of cytochrome c, which then binds APAF-1. Next, a conformational change leads to recruitment of an initiator caspase (caspase-9) and formation of the apoptosome. Caspase-9 in turn activates effector caspases (for example, caspase-3). In both pathways the activated caspases cleave selected nuclear and cytoplasmic target proteins for the accomplishment of the apoptotic program

Figure 2

Regulation of alternative splicing in apoptotic genes. (a) FAS protein scheme showing the three main domains (upper): signal peptide (SP), transmembrane domain (TM) and death domain (DD). DD is a protein module composed of a bundle of six α-helices. The lower panel shows the annotated FAS transcripts. Exons encoding each domain are indicated. NM000043 is the longest mRNA variant. The NM152871 variant 2 (FASΔEx6) lacks an in-frame exon encoding the transmembrane region. The NM152872 variant 3 (FASΔEx8) lacks a coding segment, which leads to a translation frameshift and a distinct, shorter C-terminus compared with isoform 1. The NR028034 (5), NR028035 (6), NR028033 (4) and NR028036 (7) variants lack, respectively, three (5), two (6 and 4) or one (7) alternative coding exons compared with variant 1 and are candidates for NMD. (b) In the upper panel, the three main domains of the CASP9 protein are depicted: CARD domain and the two catalytic domains (p18 and p10). The lower panel shows the annotated CASP9 transcripts. Exons encoding each protein domain are indicated. The NM001229 variant (α) encodes the longest isoform. The NM032996 variant differs in the 5'-UTR, lacks a portion of the 5'-coding region and yields a shorter N-terminus. The NM001278054 variant (β) lacks four alternative in-frame exons in the coding region and encodes the caspase-9 S isoform. (c) BCL-X comprises four BCL-2 homology (BH) domains. The anti-apoptotic BCL-X_L isoform (NM138578 variant) contains all BH domains and a transmembrane (TM) domain that anchors the protein to cellular membranes, including the mitochondrial outer membrane. The pro-apoptotic BCL-X_S isoform (NM001191 variant) uses an alternate in-frame 5'-splice site in exon 2, yielding a smaller protein that lacks BH2. (d) Alternative splicing of FAS is controlled by TIA-1, EWS, PTB and HUR, which directly bind the pre-mRNA and either promote or inhibit exon 6 inclusion. The lncRNA FAS-AS1 affects FAS alternative splicing by sequestering RBM5 and preventing its inhibitory effect of FAS exon 6 inclusion. (e) Regulation of BCL2L1 alternative splicing. Enhancer and silencer cis-elements are indicated in either in green or in red. Representative splicing factors (SAP155, hnRNP K, hnRNP F/H, PTBP1 and SRSF9) and lncRNAs (INXS) affecting the recognition of the proximal (upper factors) or distal (lower factors) 5' splice site are depicted

Figure 3

Cancer therapies based on antisense oligonucleotide-mediated splicing modulation. (a) Treatment of cancer cells with an ASO masking the proximal 5' splice site effectively switches BCL-X splicing and induces apoptosis. (b) MDM4 protein is highly expressed in embryonic tissues and in cancers as a result of enhanced exon 6 inclusion, driven mainly by SRSF3. ASO-promoting skipping of exon 6 is a suitable approach to inhibit p53 degradation by MDM4 in cancer cells. (c) Example of the hypothetical application of ASO therapy to FAS. An ASO masking the PTBP1-binding site in FAS exon 6 could be rescue FAS exon 6 inclusion, thus enhancing FAS-mediated cell death of cancer cells

Figure 4

Alternative splicing and chemoresistance. Schematic representation of representative splicing events involved in genotoxic stress response. Gemcitabine resistance in pancreatic cancer is favored by short-term upregulation of SRSF1, MNK2b splicing (A) and eIF4E phosphorylation, or by long-term upregulation of PTBP1 and splicing of the pro-oncogenic PKM2 isoform (B). In AML, hnRNPs H1/H2 cause NMD-linked splicing of TP (C), leading to resistance to capecitibine. In prostate cancer, mitoxantrone causes a relocalization of SAM68 in active transcription sites (D), and leads to the inclusions of CD44 variable exons which confers pro-oncogenic features. In prostate cancer, malignant transformation correlates also with the upregulation of SAM68 and SRSF1, which promote the pro-oncogenic Cyclin D1b variant (E). In breast cancer, alternative splicing of HER2 confers different sensitivity to trastuzumab (F). Genotoxic stress causes the inclusion of exon 6 in DHX9 transcript in Ewing sarcoma cells (G), leading to NMD of DHX9 mRNA and sensitizing cells to apoptosis. In AML, expression of the shorter isoform of CFLAR gene, c-FLIPs, correlates with sensitivity to Vorinostat (H)