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Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5.

ABSTRACT: Similar to many genes involved in programmed cell death (PCD), the caspase 2 (casp-2) gene generates both proapoptotic and antiapoptotic isoforms by alternative splicing. Using a yeast RNA-protein interaction assay, we identified RBM5 (also known as LUCA-15) as a protein that binds to casp-2 pre-mRNA. In both transfected cells and in vitro splicing assay, RBM5 enhances the formation of proapoptotic Casp-2L. RBM5 binds to a U/C-rich sequence immediately upstream of the previously identified In100 splicing repressor element. Our mutagenesis experiments demonstrate that RBM5 binding to this intronic sequence regulates the ratio of proapoptotic/antiapoptotic casp-2 splicing isoforms, suggesting that casp-2 splicing regulation by RBM5 may contribute to its tumor suppressor activity. Our work has uncovered a player in casp-2 alternative splicing regulation and revealed a link between the alternative splicing regulator and the candidate tumor suppressor gene. Together with previous studies, our work suggests that splicing control of cell death genes may be an important aspect in tumorigenesis. Enhancing the expression or activities of splicing regulators that promote the production of proapoptotic splicing isoforms might provide a therapeutic approach to cancer.

SUBMITTER: Fushimi K

PROVIDER: S-EPMC2572934 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5.

Fushimi Kazuo K Ray Payal P Kar Amar A Wang Lei L Sutherland Leslie C LC Wu Jane Y JY

Proceedings of the National Academy of Sciences of the United States of America 20081007 41

Similar to many genes involved in programmed cell death (PCD), the caspase 2 (casp-2) gene generates both proapoptotic and antiapoptotic isoforms by alternative splicing. Using a yeast RNA-protein interaction assay, we identified RBM5 (also known as LUCA-15) as a protein that binds to casp-2 pre-mRNA. In both transfected cells and in vitro splicing assay, RBM5 enhances the formation of proapoptotic Casp-2L. RBM5 binds to a U/C-rich sequence immediately upstream of the previously identified In100 ...[more]

PMID: 18840686

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Project description:BackgroundApoptosis-stimulating Protein of TP53-2 (ASPP2) is a tumor suppressor enhancing TP53-mediated apoptosis via binding to the TP53 core domain. TP53 mutations found in cancers disrupt ASPP2 binding, arguing for an important role of ASPP2 in TP53-mediated tumor suppression. We now identify an oncogenic splicing variant, ASPP2κ, with high prevalence in acute leukemia.MethodsAn mRNA screen to detect ASPP2 splicing variants was performed and ASPP2κ was validated using isoform-specific PCR approaches. Translation into a genuine protein isoform was evaluated after establishing epitope-specific antibodies. For functional studies cell models with forced expression of ASPP2κ or isoform-specific ASPP2κ-interference were created to evaluate proliferative, apoptotic and oncogenic characteristics of ASPP2κ.FindingsExon skipping generates a premature stop codon, leading to a truncated C-terminus, omitting the TP53-binding sites. ASPP2κ translates into a dominant-negative protein variant impairing TP53-dependent induction of apoptosis. ASPP2κ is expressed in CD34+ leukemic progenitor cells and functional studies argue for a role in early oncogenesis, resulting in perturbed proliferation and impaired induction of apoptosis, mitotic failure and chromosomal instability (CIN) - similar to TP53 mutations. Importantly, as expression of ASPP2κ is stress-inducible it defines a novel class of dynamic oncogenes not represented by genomic mutations.InterpretationOur data demonstrates that ASPP2κ plays a distinctive role as an antiapoptotic regulator of the TP53 checkpoint, rendering cells to a more aggressive phenotype as evidenced by proliferation and apoptosis rates - and ASPP2κ expression results in acquisition of genomic mutations, a first initiating step in leukemogenesis. We provide proof-of-concept to establish ASPP2κ as a clinically relevant biomarker and a target for molecule-defined therapy. FUND: Unrestricted grant support from the Wilhelm Sander Foundation for Cancer Research, the IZKF Program of the Medical Faculty Tübingen, the Brigitte Schlieben-Lange Program and the Margarete von Wrangell Program of the State Ministry Baden-Wuerttemberg for Science, Research and Arts and the Athene Program of the excellence initiative of the Eberhard-Karls University, Tübingen.

Project description:PurposeCell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer.Experimental designThe CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR.ResultsWe report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression.ConclusionsCADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers.

Dataset Information

Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5.

Publications

Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5.

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