Aberrant RNA Splicing in Cancer and Drug Resistance

Abstract

More than 95% of the 20,000 to 25,000 transcribed human genes undergo alternative RNA splicing, which increases the diversity of the proteome. Isoforms derived from the same gene can have distinct and, in some cases, opposing functions. Accumulating evidence suggests that aberrant RNA splicing is a common and driving event in cancer development and progression. Moreover, aberrant splicing events conferring drug/therapy resistance in cancer is far more common than previously envisioned. In this review, aberrant splicing events in cancer-associated genes, namely BCL2L1, FAS, HRAS, CD44, Cyclin D1, CASP2, TMPRSS2-ERG, FGFR2, VEGF, AR and KLF6, will be discussed. Also highlighted are the functional consequences of aberrant splice variants (BCR-Abl35INS, BIM-γ, IK6, p61 BRAF V600E, CD19-∆2, AR-V7 and PIK3CD-S) in promoting resistance to cancer targeted therapy or immunotherapy. To overcome drug resistance, we discuss opportunities for developing novel strategies to specifically target the aberrant splice variants or splicing machinery that generates the splice variants. Therapeutic approaches include the development of splice variant-specific siRNAs, splice switching antisense oligonucleotides, and small molecule inhibitors targeting splicing factors, splicing factor kinases or the aberrant oncogenic protein isoforms.

Keywords: alternative splicing; drug resistance; splice switching oligonucleotide; spliceosome; splicing factor; splicing factor inhibitor.

Figure 1

Different modes of alternative RNA splicing. Different modes of alternative splicing (AS), including constitutive splicing, intron retention, exon skipping, alternative splice site selection (5′ and 3′), mutually exclusive splicing, alternative promoter selection and alternative polyadenylation sites [19,20]. The pre-mRNAs are shown on the left panel, and the mature mRNA variants following AS are shown on the right panel.

Figure 2

Alternative splicing events in cancers. Examples of aberrant splice variants and oncogenic consequences for Bcl-xL, FAS-S, p21H-RAS, Cyclin D1b, CASP-2S, TMPRSS2-ERG+72bp, FGFR2-IIIc, VEGF-165, AR-V7 and KLF6-SV1 [20,43,47]. Abbreviations: TMPRSS2–ERG+72bp, TMPRSS2–ERG fusion transcript with inclusion of a 72-bp exon [48]; BCa, breast cancer; PCa, prostate cancer; CE3, cryptic exon 3. Overexpression of splice variant TMPRSS2–ERG+72bp has been correlated to aggressive prostate cancer with poor clinical outcome [49]. The pre-mRNAs are shown on the left panel, and the mature mRNA variants (only the exons surrounding the differential splicing event are shown) following AS are shown on the right panel.

Figure 3

Aberrant mRNA splice variants in cancer drug resistance. AS events involving oncogenes, BCR-ABL, BIM, IKZF1, BRCA1, TP53, BRAF, CD19, AR, ER and PIK3CD, leading to resistance to targeted therapies, chemotherapy, hormone therapy or immunotherapy [121,122,123,124,125,126,127]. ATG1: Start codon for full-length TP53 and ERα. ATG2: Start codon for ∆40p53 and ERα36 splice variants. TGA: Stop codon for ERα36 splice variant. See text for additional references.

Figure 4

Different re-sensitization approaches to reverse RNA splicing events causing drug resistance. (A) Top panel: SiRNA-mediated knockdown of oncogenic splice variants. SiRNAs targeting exon 1of AR-V7 variant. Bottom panel: SiRNA targeting the junction of exons 19 and 21 to specifically inhibit expression of PIK3CD-S. (B) Splice switching oligonucleotides (SSO) as a strategy to interfere with aberrant splicing. Example illustrated is an SSO (or splice switching anti-sense oligonucleotide, ASO) bound to the intron-exon junction to prevent splice generation of the MKNK2-2b variant, thereby re-sensitizing cancer cells to drug treatment. (C) Pharmacologic inhibition of splicing factors. Spliceostatin A (SF3B1 inhibitor) and quercetin (HNRNPA1 inhibitor) suppress formation of p61 BRAF V600E and AR-V7 splice variants, respectively. These approaches will induce re-sensitization of cancer cells to drug treatments. See text for corresponding references.

Figure 5

Potential therapeutic approaches to correct splicing errors. Small molecule inhibitors (Cpd-1, Cpd-2, Cpd-3, SRPIN340, SPHINX) can be used to block the activity of splicing factor kinases (CLKs and SRPKs) thereby reversing aberrant mRNA splicing. Small molecule inhibitors of splicing factors (Spliceostatin A, Meayamycin B, H3B-8800, Quercetin) can reverse aberrant splicing by blocking spliceosome assembly or directly targeting splicing factors. Splice switching oligonucleotides (SSOs) can prevent SRSF and HNRNP proteins from interacting with exon splicing enhancers (ESEs) and exonic splicing silencers (ESSs) located on the pre-mRNA, respectively. SSOs targeting exon-intron junctions can disrupt alternative splicing. Future small molecules can be developed to specifically target aberrant protein isoforms. All of these strategies may be used to correct splicing errors and overcome drug resistance. See text for corresponding references.