Alternative splicing in prostate cancer progression and therapeutic resistance

Abstract

Prostate cancer (CaP) remains the second leading cause of cancer deaths in western men. CaP mortality results from diverse molecular mechanisms that mediate resistance to the standard of care treatments for metastatic disease. Recently, alternative splicing has been recognized as a hallmark of CaP aggressiveness. Alternative splicing events cause treatment resistance and aggressive CaP behavior and are determinants of the emergence of the two major types of late-stage treatment-resistant CaP, namely castration-resistant CaP (CRPC) and neuroendocrine CaP (NEPC). Here, we review recent multi-omics data that are uncovering the complicated landscape of alternative splicing events during CaP progression and the impact that different gene transcript isoforms can have on CaP cell biology and behavior. We discuss renewed insights in the molecular machinery by which alternative splicing occurs and contributes to the failure of systemic CaP therapies. The potential for alternative splicing events to serve as diagnostic markers and/or therapeutic targets is explored. We conclude by considering current challenges and promises associated with splicing-modulating therapies, and their potential for clinical translation into CaP patient care.

Fig. 1. Genomics, transcriptomics and epigenomics alterations during the progression to advanced treatment-resistant CaP.

Localized treatment-naïve CaP progresses to CRPC and, in some cases, to NEPC. Each CaP stage and transition is characterized by recurring genomic, transcriptomics and epigenomics changes, for which representative examples are listed. A growing body of evidence supports that shifts occur also in the alternative splicing landscape as CaP progresses and develops resistance to ADT. Such recurring alternative splicing events are listed. ADT, androgen derivation therapy, CaP, prostate cancer; Surgery and radiation, standard of care treatments for localized untreated CaP. The figure was generated using Biorender.

Fig. 2. Schematic overview of the mechanisms of action of the major spliceosome and the minor spliceosome.

During major spliceosome formation and action, U1 snRNP first recognizes the 5′end splice site (5′SS) of the target pre-mRNA and binds to it along with other non-snRNP splice factors to form the early spliceosome E complex. The non-snRNP SF1 (not shown) recognizes the branch point sequence (BPS) pre-bulging the BPS adenosine for base-pairing with the U2 snRNA, followed by U2AF2 interaction with SF1 (not shown) and recruitment of the U2 snRNP to the spliceosome to form an intermediate A complex. The U4/U5/U6 tri-snRNP is recruited to the spliceosome to form another intermediate complex B. The B complex undergoes extensive conformational rearrangements due to U1 and U4 dissociation and by the action of the RNA helicase DHX16 (not shown) resulting in the formation of the catalytically active Bact complex. The Bact complex catalyzes the first step of splicing, generating the cleaved 5′ exon and intron-3′ exon lariat intermediates and forming complex C. After additional RNP rearrangements, the C complex catalyzes the second step of splicing, resulting in the ligation of the 5′ and 3′ exons and release of the intron in the form of a lariat. The minor spliceosome assembly is similar to that of major spliceosome except that it requires U11, U12 and U4atac/U6atac snRNPs as the functional analogs of the U1, U2 and U4/U6 snRNPs along with the U5 snRNP in the major spliceosome. The figure was generated using Biorender.

Fig. 3. Overview of major molecular determinants that control the spectrum of alternative transcripts that are generated from a pre-mRNA (site).

Alternative splicing, and the use of specific splice or regulatory sites, is controlled by both trans-acting factors (top part) and cis-acting factors (bottom part). For example, overexpression of SRGs can stimulate splicing at a receptive sites. Mutations in SRGs on the other hand can result in disruption of the spliceosome and decreased generation of transcripts at receptive sites. Strengths of the cis-acting elements and of their interaction with trans-acting factors can favor the use of specific sites, motifs and thus specific transcript isoforms. Site-specific mutations in RNA sequences can lead to loss of RNA-protein interactions and/or skipping the use of specific site (combinations), resulting in decreased levels of the associated transcripts. The figure was generated using Biorender.

Fig. 4. Therapeutic strategies that have been tested in CaP to interfere with alternative splicing.

Therapeutic strategies tested in CaP so far have targeted both trans-acting factors (top part) and cis-acting factors (bottom part). The former has involved the preclinical use of inhibitors of spliceosome components such as SF3B and several small molecule drugs/compounds that were designed to inhibit other SRGs. These inhibitors either interfere with splicing factor(SF)-RNA interactions, lead to proteasomal degradation of SRGs, inhibit the activity of kinases that control SF RNA binding and action, or consisted of non-splicing-specific compounds such as Hsp90 inhibitors that were found to impact also CaP-relevant alternative splicing events. Targeting the cis-elements has involved the use of splicing switch oligonucleotides (SSOs) that were designed to target specific pre-mRNA sequences and thereby control the transcript isoforms generated from those sites. The figure was generated using Biorender.