Jerantinine A induces tumor-specific cell death through modulation of splicing factor 3b subunit 1 (SF3B1)

www.nature.com/scientificreports OPEN received: 08 September 2016 accepted: 13 January 2017 Published: 15 February 2017 Jerantinine A induces tumorspecific cell death through modulation of splicing factor 3b subunit 1 (SF3B1) Felicia Fei-Lei Chung1, Perry Faith Tze Ming Tan2, Vijay Joseph Raja3, Boon-Shing Tan4, Kuan-Hon Lim5, Toh-Seok Kam6, Ling-Wei Hii1,7, Si Hoey Tan1,7, Sze-Jia See1, Yuen-Fen Tan1,7, Li-Zhe Wong1,7, Wai Keat Yam8, Chun Wai Mai8, Tracey D. Bradshaw9 & Chee-Onn Leong1,8 Precursor mRNA (pre-mRNA) splicing is catalyzed by a large ribonucleoprotein complex known as the spliceosome. Numerous studies have indicated that aberrant splicing patterns or mutations in spliceosome components, including the splicing factor 3b subunit 1 (SF3B1), are associated with hallmark cancer phenotypes. This has led to the identification and development of small molecules with spliceosome-modulating activity as potential anticancer agents. Jerantinine A (JA) is a novel indole alkaloid which displays potent anti-proliferative activities against human cancer cell lines by inhibiting tubulin polymerization and inducing G2/M cell cycle arrest. Using a combined pooled-genome wide shRNA library screen and global proteomic profiling, we showed that JA targets the spliceosome by up-regulating SF3B1 and SF3B3 protein in breast cancer cells. Notably, JA induced significant tumor-specific cell death and a significant increase in unspliced pre-mRNAs. In contrast, depletion of endogenous SF3B1 abrogated the apoptotic effects, but not the G2/M cell cycle arrest induced by JA. Further analyses showed that JA stabilizes endogenous SF3B1 protein in breast cancer cells and induced dissociation of the protein from the nucleosome complex. Together, these results demonstrate that JA exerts its antitumor activity by targeting SF3B1 and SF3B3 in addition to its reported targeting of tubulin polymerization. Precursor mRNA (pre-mRNA) splicing is a fundamental process in eukaryotic cells, which is catalyzed by the spliceosome, a macromolecular ribonucleoprotein (RNP) complex composed of five small nuclear ribonucleoproteins (U1, U2, U4, U5 and U6 snRNPs) and more than 200 polypeptides1–3. The splicing factor 3b subunit 1 (SF3B1) protein is a core component of the U2 snRNP at the catalytic center of the spliceosome, which recognizes and defines the 3′splice site at the intron-exon junctions4. Through pre-mRNA splicing, a single pre-mRNA transcript may give rise to multiple different combinations of introns and exons, resulting in increased transcript diversity and the synthesis of alternative proteins5. While changes in alternative splicing patterns play an integral role in normal development and cell differentiation, numerous cancer-specific aberrant splicing patterns have been documented6,7. However, it is currently unclear whether the observed splicing abnormalities are a by-product of cellular transformation or an intrinsic characteristic of transformed cells. Recently, growing evidence has demonstrated that aberrant splicing contributes to essential phenotypes associated with transformed cells. For instance, alternative protein products of epidermal growth factor 1 Center for Cancer and Stem Cell Research, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia. 2School of Medicine, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia. 3 Department of Biochemistry, Weill Cornell Medical College, New York, NY 10021, USA. 4Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. 5School of Pharmacy, University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor, Malaysia. 6Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia. 7School of Postgraduate Studies, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia. 8School of Pharmacy, International Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia. 9School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD, UK. Correspondence and requests for materials should be addressed to C.-O.L. (email: ) Scientific Reports | 7:42504 | DOI: 10.1038/srep42504 1 www.nature.com/scientificreports/ receptor (EGFR)8, p539, vascular endothelial growth factor (VEGF)10, and E-cadherin11 reportedly promoted cancer-associated pathways, including the evasion of apoptosis, increased cell proliferation, angiogenesis, and invasion. Mutations in SF3B1 have also been reported in myelodysplastic syndromes (MDS) as well as numerous cancers, including acute myeloid leukemia, primary myelofibrosis, chronic myelomonocytic leukemia (CML)12, chronic lymphocytic leukemia (CLL)13,14, multiple myeloma, uveal melanoma15–18 and breast cancers19–21. While it is currently unclear as to how SF3B1 mutations might alter its function, previous studies have shown that the dysregulation of spliceosomal components can alter splicing patterns, causing intron retention or exon skipping, and affect protein isoform balances leading to abnormal cell proliferation or differentiation2,22. As such, the spliceosome has emerged as an attractive target for anticancer treatment. Several spliceosome modulators have already been identified, including natural products derived from bacterial fermentation (e.g. pladienolides, GEX1, FR901463, etc.) and their synthetic analogues (spliceostatin A, meayamycin and E7107) as well as natural plant products (e.g. isoginkgetin)23. Indole alkaloids represent a large and highly structurally diverse group of secondary metabolites with remarkable bioactivities against the different targets in cancer. The importance of this group of compounds is best represented by the Vinca alkaloid vinblastine, which is currently among the foremost drugs used in cancer chemotherapy24. Previously, we have described the potent and selective antitumor activity of seven new Aspidosperma indole alkaloids, jerantinines A-G, isolated from the leaf extracts of the Malayan plant Tabernaemontana corymbosa (Fig. 1A)25. Jerantinines A-E were found to display pronounced in vitro anti-proliferative activities against human cancer cell lines in the nanomolar range26–28. Furthermore, we have recently demonstrated that jerantinine A and B and the acetate derivative inhibited tubulin polymerization, polo-like kinase 1 (PLK1) activity and induced G2/M cell cycle arrest in a panel of human cancer cell lines consisting of vincristine-resistant nasopharyngeal carcinoma cells25, as well as breast, colorectal, lung and pancreatic carcinoma cells27,28. Similarly, jerantinine E was also shown to disrupt microtubules, and displayed significant antitumor activity against human cervical carcinoma cells29. Importantly, no cross-resistance to jerantinines was observed in vincristine-resistant HCT-116 cells, suggesting that jerantinines overcome p-glycoprotein-mediated multidrug resistance and might affect other cancer-relevant targets besides tubuli (...truncated)