Aberrant Bcl-x splicing in cancer: from molecular mechanism to therapeutic modulation

Abstract

Bcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy.

Keywords: Alternative splicing; Bcl-x; Cell apoptosis; Small molecular modulators; Splice-switching oligonucleotides; Splicing correction.

Fig. 1

Alternative splicing and the effect of aberrant alternative splicing on cancer progression. The spliceosome, consists of five small nuclear ribonucleoproteins particles (U1, U2, U4, U5 and U6) and hundreds of additional proteins, recognizes the consensus sequence of each intron and assembles reversibly on splice sites to catalytic pre-mRNA splicing. SR  proteins  and hnRNPs bound to exonic or intronic regulatory elements to promote or prevent the use of splice sites thus affecting alternative splicing decisions. The figure displays some examples of cancer-specific splicing events that contribute to distinct hallmarks of cancer. Arrows up and down indicate the corresponding isoforms contributing or suppressing the hallmark respectively

Fig. 2

Bcl-X pre-mRNA splicing and structures of splicing isoforms. a. Alternative splicing mode and splicing regulation. Splicing occurred closer to the 5'PSS of exon 2 produces the long isoform Bcl-xL. Alternative splicing occurred near the 5' DSS of exon 2 produces the short isoform Bcl-xS. In addition, distinct cis-elements and splice factors bind to cis-elements to influence the alternative 5' splice site selection of Bcl-x pre-mRNA. b. General characteristics of isoforms spliced from Bcl-x pre-mRNA. c. The protein structures of Bcl-xL. The secondary structure of Bcl-xL and the position in the space of BH domains (up). Tertiary structure of Bcl-xL and the BH domain and hydrophobic groove are showed (down)

Fig. 3

Cell apoptosis regulated by Bcl-x isoforms. a. Three modes that had been proposed to explain how Bcl-xL regulate MOMP. Mode 0: Bcl-xL prevented the binding of apoptotic effectors Bax to mitochondrial outer membrane through retrotranslocating Bax from the mitochondria into cytosol constantly. Mode 1: Bcl-xL sequestered BH3-only activators (tBid) to prevent them from binding to and activating Bax. Mode 2: Bcl-xL directly bound to activated Bax to prevent its oligomerization and MOMP. b. Cell apoptosis pathways regulated by Bcl-xL and Bcl-xS

Fig. 4

Cell autophagy mediated by Bcl-xL. Bcl-xL inhibited initial steps of autophagy by interacting with the core regulators of autophagy Beclin-1, which disrupted the hVps34–Beclin-1 complex and limited its ability to stimulate autophagosome formation. Bcl-xL also could inhibit PINK1/Parkin-dependent mitophagy through directly interacting with PINK1 and Parkin to inhibit the translocation of Parkin from cytoplasm into mitochondria

Fig. 5

Chemical modifications of splice switching oligonucleotides. a. Chemical modifications on phosphate backbone and ribose ring of SSOs. Unmodified RNA is shown for reference. PS, one of the phosphate backbone oxygen atom is replaced by a sulphur atom; 2′-MOE and 2′-OMe, PS-SSOs are often combined with ribose modifications including 2′-O-(2-methoxyethyl) or 2′O-methyl; PMO, charge-neutral nucleic acid, in which the six-membered morpholine ring replaces the five-membered ribose heterocycle; PPMO, positively charged peptides in PPMO dramatically improve intracellular uptake of PMO. VPMO, covalently linking MO to an octaguanidine dendrimer to improve delivery efficacy. LNA, the second and fourth of ribose form a rigid structure by shrinkage. PNA, a pseudo peptide polymer backbone substitutes for the phosphate backbone of RNA. b. Properties comparison of the common chemistries of antisense oligonucleotides

Fig. 6

Strategies modulating Bcl-x splicing in cancer. a. An SSO that binds to the proximal 5' splice site (5'PSS) prevents binding of spliceosome, leading to a splicing shift to the short isoform Bcl-xS. b. a. The small molecular modulators that bind to spliceosomal components affect splice-site accessibility, leading to an inhibition of Bcl-xL splicing. c. At the protein level, BH3-mimetics could occupy the hydrophobic pockets of Bcl-Xl, thus blocking their anti-apoptotic activity and resulting in the ignition of apoptosis