Alternative splicing in human cancer cells is modulated by the amiloride derivative 3,5-diamino-6-chloro-N-(N-(2,6-dichlorobenzoyl)carbamimidoyl)pyrazine-2-carboxide.

Chien‐Chin Lee,Kai‐Cheng Hsu,Jinn‐Moon Yang,Chih‐Shiang Chang,Shyr‐Yi Lin,Yang‐Chang Wu,Yu‐Chia Chen,Yun‐Ti Chen,Wen‐Hsin Chang,Ya‐Sian Chang,Jan‐Gowth Chang,Ting‐Yuan Liu

doi:10.1002/1878-0261.12524

Abstract

Alternative splicing (AS) is a process that enables the generation of multiple protein isoforms with different biological properties from a single mRNA. Cancer cells often use the maneuverability conferred by AS to produce proteins that contribute to growth and survival. In our previous studies, we identified that amiloride modulates AS in cancer cells. However, the effective concentration of amiloride required to modulate AS is too high for use in cancer treatment. In this study, we used computational algorithms to screen potential amiloride derivatives for their ability to regulate AS in cancer cells. We found that 3,5‐diamino‐6‐chloro‐N‐(N‐(2,6‐dichlorobenzoyl)carbamimidoyl)pyrazine‐2‐carboxamide (BS008) can regulate AS of apoptotic gene transcripts, including HIPK3, SMAC, and BCL‐X, at a lower concentration than amiloride. This splicing regulation involved various splicing factors, and it was accompanied by a change in the phosphorylation state of serine/arginine‐rich proteins (SR proteins). RNA sequencing was performed to reveal that AS of many other apoptotic gene transcripts, such as AATF, ATM, AIFM1, NFKB1, and API5, was also modulated by BS008. In vivo experiments further indicated that treatment of tumor‐bearing mice with BS008 resulted in a marked decrease in tumor size. BS008 also had inhibitory effects in vitro, either alone or in a synergistic combination with the cytotoxic chemotherapeutic agents sorafenib and nilotinib. BS008 enabled sorafenib dose reduction without compromising antitumor activity. These findings suggest that BS008 may possess therapeutic potential for cancer treatment.

Highlights

In eukaryotes, alternative splicing (AS) is a crucial mechanism for generating transcriptome diversity and can result in single-gene coding for multiple proteins
The binding sites were defined by residues situated ≤ 8 A from the Abbreviations AS, alternative splicing; DSBs, DNA double-strand breaks; HCC, hepatocellular carcinoma; HIPK3, homeodomain-interacting protein kinase 3; hnRNPs, heterogeneous RNPs; OA, okadaic acid; PP1, protein phosphatase-1; PTMs, post-translational modifications; RNAbinding proteins (RBPs), RNA-binding proteins; SiMMaps, site-moiety maps; SMAC/DIABLO, second mitochondria-derived activator of caspases; SR proteins, serine/arginine-rich proteins; SREs, splicing regulatory sequence elements; SRPKs, SR protein-specific kinases
Since we have previously reported that amiloride regulates the AS of BCL-X and HIPK3 premRNA in various cancer cell lines including human HCC cell line Huh-7 (Chang et al, 2011a,2011b), it would be of interest to investigate if BS008 had the same effects on AS in Huh-7

Summary

Introduction

Alternative splicing (AS) is a crucial mechanism for generating transcriptome diversity and can result in single-gene coding for multiple proteins. Numerous RBPs, which function to regulate AS, have been studied, and the well-known regulators of splice site selection are serine/arginine-rich (SR) proteins and heterogeneous RNPs (hnRNPs). Serine/arginine-rich proteins (SR proteins) bind to exonic splicing enhancers through their RNA-binding domain and promote exon inclusion by recruiting spliceosome components. Cancer cells often take advantage of these specific splicing events to produce oncogenes or aberrant tumor suppressors that promote growth and survival (Sveen et al, 2016; Venables, 2004). This implies that shifting splicing toward the appropriate isoform may offer therapeutic potential in cancer treatment

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