An integrative analysis of colon cancer identifies an essential function for PRPF6 in tumor growth

Adler, Adam S.; McCleland, Mark L.; Yee, Sharon; Yaylaoglu, Murat; Hussain, Sofia; Cosino, Ely; Quiñones, Gabriel; Modrušan, Zora; Seshagiri, Somasekar; Torres, Eric; Chopra, Vivek S.; Haley, Benjamin; Zhang, Zemin; Blackwood, Elizabeth M.; Singh, Mallika; Junttila, Melissa R.; Stéphan, Jean-Philippe; Liu, Jinfeng; Pau, Grégoire; Fearon, Eric R.; Jiang, Zhaoshi; Firestein, Ron

doi:10.1101/gad.237206.113

Cited by 88 publications

(73 citation statements)

References 69 publications

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“…Consistent with this, reduction of either hnRNP K or PRPF6, direct EWS-FLI1 partners, leads to alternative splicing. PRPF6 has been independently implicated as a driver of colorectal cancer (46). However, reduction of SF3A2, which is an RNA-dependent, indirect EWS-FLI1 partner, did not alter splicing for those genes analyzed.…”

Section: Discussionmentioning

confidence: 91%

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Selvanathan

Graham

Erkizan

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

118

135

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The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNAseq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.T he alternative splicing of mRNA expands the diversity of the human proteome through evolution and ontogeny (1, 2). Spliceosomal network interactions, including proteins that recognize splice enhancer and silencer regions, are critical for the regulation of alternative splicing leading to protein isoforms with disparate functionality (3). Alternative splicing provides both a method by which to categorize subsets of cancers and an avenue for more effective targeted treatments (4). However, the spliceosomal protein interaction networks that are specific to cancer have not been systematically defined; alternative splicing can also change protein-protein interactions within networks (5). A systems biology approach can be used to study the relationship between splicing and oncogenesis by using tumor models with chromosomal translocations whose expressed fusion proteins have putative roles in splicing (6, 7).Fusion proteins produced by chromosomal translocations in sarcomas often contain the amino-terminal portion of EWS and are classified as transcription factors due to the presence of a canonical carboxyl-terminal DNA-binding domain (6). EWS-FLI1 is a well-established ES oncoprotein and is re...

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Section: Discussionmentioning

confidence: 91%

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Selvanathan

Graham

Erkizan

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

118

135

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“…Changes in RNA splicing are recognized as major contributors to cancer, particularly hematologic malignancy (37). Recently, other genetics screens have identified RNA-splicing machinery as preferentially required in cancer cells, with PRPF6 important in colon cancer (38) and PHF5a in brain cancer (39). These data suggest that aberrant RNA splicing in osteosarcoma are important in maintaining the tumor state and warrant further investigation.…”

Section: Discussionmentioning

confidence: 97%

Systematic Screening Identifies Dual PI3K and mTOR Inhibition as a Conserved Therapeutic Vulnerability in Osteosarcoma

Gupte

Baker

Wan

et al. 2015

Clinical Cancer Research

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Purpose: Osteosarcoma is the most common cancer of bone occurring mostly in teenagers. Despite rapid advances in our knowledge of the genetics and cell biology of osteosarcoma, significant improvements in patient survival have not been observed. The identification of effective therapeutics has been largely empirically based. The identification of new therapies and therapeutic targets are urgently needed to enable improved outcomes for osteosarcoma patients.Experimental Design: We have used genetically engineered murine models of human osteosarcoma in a systematic, genomewide screen to identify new candidate therapeutic targets. We performed a genome-wide siRNA screen, with or without doxorubicin. In parallel, a screen of therapeutically relevant small molecules was conducted on primary murine-and primary human osteosarcoma-derived cell cultures. All results were validated across independent cell cultures and across human and mouse osteosarcoma. Results:The results from the genetic and chemical screens significantly overlapped, with a profound enrichment of pathways regulated by PI3K and mTOR pathways. Drugs that concurrently target both PI3K and mTOR were effective at inducing apoptosis in primary osteosarcoma cell cultures in vitro in both human and mouse osteosarcoma, whereas specific PI3K or mTOR inhibitors were not effective. The results were confirmed with siRNA and small molecule approaches. Rationale combinations of specific PI3K and mTOR inhibitors could recapitulate the effect on osteosarcoma cell cultures.Conclusions: The approaches described here have identified dual inhibition of the PI3K-mTOR pathway as a sensitive, druggable target in osteosarcoma, and provide rationale for translational studies with these agents.

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“…These specifically contribute to colon tumorigenesis through proteomic changes that cause Notch pathway activation and promotion of aerobic glycolysis, genomic instability, and epithelial-mesenchymal transition (Radisky et al 2005;Christofk et al 2008;Misquitta-Ali et al 2010;Bechara et al 2013;Myant et al 2013). In addition, PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, was shown to positively affect colon cancer proliferation (Adler et al 2014).…”

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confidence: 99%

A network-based analysis of colon cancer splicing changes reveals a tumorigenesis-favoring regulatory pathway emanating from ELK1

et al. 2016

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Splicing aberrations are prominent drivers of cancer, yet the regulatory pathways controlling them are mostly unknown. Here we develop a method that integrates physical interaction, gene expression, and alternative splicing data to construct the largest map of transcriptomic and proteomic interactions leading to cancerous splicing aberrations defined to date, and identify driver pathways therein. We apply our method to colon adenocarcinoma and non-small-cell lung carcinoma. By focusing on colon cancer, we reveal a novel tumor-favoring regulatory pathway involving the induction of the transcription factor MYC by the transcription factor ELK1, as well as the subsequent induction of the alternative splicing factor PTBP1 by both. We show that PTBP1 promotes specific RAC1, NUMB, and PKM splicing isoforms that are major triggers of colon tumorigenesis. By testing the pathway's activity in patient tumor samples, we find ELK1, MYC, and PTBP1 to be overexpressed in conjunction with oncogenic KRAS mutations, and show that these mutations increase ELK1 levels via the RAS-MAPK pathway. We thus illuminate, for the first time, a full regulatory pathway connecting prevalent cancerous mutations to functional tumorinducing splicing aberrations. Our results demonstrate our method is applicable to different cancers to reveal regulatory pathways promoting splicing aberrations.

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An integrative analysis of colon cancer identifies an essential function for PRPF6 in tumor growth

Cited by 88 publications

References 69 publications

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Systematic Screening Identifies Dual PI3K and mTOR Inhibition as a Conserved Therapeutic Vulnerability in Osteosarcoma

A network-based analysis of colon cancer splicing changes reveals a tumorigenesis-favoring regulatory pathway emanating from ELK1

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