Identification of Differentially Regulated Splice Variants and Novel Exons in Glial Brain Tumors Using Exon Expression Arrays

French, Pim J.; Peeters, Justine K.; Horsman, Sebastiaan; Duijm, Elza; Siccama, Ivar; Bent, Martin J. van den; Luider, Theo M.; Kros, Johan M.; Spek, Peter van der; Smitt, Peter A. Sillevis

doi:10.1158/0008-5472.can-06-2869

Cited by 82 publications

(92 citation statements)

References 40 publications

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“…The total number of detected splice variants between normal and cancer samples was comparable to other reports (33,45). The number was relatively low, which could be due to our conservative approach where only splice variants that occurred in most of the samples were identified; the stringent filtering applied to the data also lowered the number of splice variants detected.…”

Section: Discussionsupporting

confidence: 81%

Alternative Splicing in Colon, Bladder, and Prostate Cancer Identified by Exon Array Analysis

Thorsen¹,

Sørensen²,

Brems-Eskildsen³

et al. 2008

Molecular & Cellular Proteomics

219

222

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Alternative splicing enhances proteome diversity and modulates cancer-associated proteins. To identify tissueand tumor-specific alternative splicing, we used the GeneChip Human Exon 1.0 ST Array to measure wholegenome exon expression in 102 normal and cancer tissue samples of different stages from colon, urinary bladder, and prostate. We identified 2069 candidate alternative splicing events between normal tissue samples from colon, bladder, and prostate and selected 15 splicing events for RT-PCR validation, 10 of which were successfully validated by RT-PCR and sequencing. Furthermore 23, 19, and 18 candidate tumor-specific splicing alterations in colon, bladder, and prostate, respectively, were selected for RT-PCR validation on an independent set of 81 normal and tumor tissue samples. In total, seven genes with tumor-specific splice variants were identified (ACTN1, CALD1, COL6A3, LRRFIP2, PIK4CB, TPM1, and VCL). The validated tumor-specific splicing alterations were highly consistent, enabling clear separation of normal and cancer samples and in some cases even of different tumor stages. A subset of the tumor-specific splicing alterations (ACTN1, CALD1, and VCL) was found in all three organs and may represent general cancer-related splicing events. In silico protein predictions suggest that the identified cancerspecific splice variants encode proteins with potentially altered functions, indicating that they may be involved in pathogenesis and hence represent novel therapeutic targets. In conclusion, we identified and validated alternative splicing between normal tissue samples from colon, bladder, and prostate in addition to cancerspecific splicing events in colon, bladder, and prostate Alternative splicing is a key component in expanding a relatively limited number of genes into very complex proteomes. It has been estimated that about three-quarters of all human genes undergo alternative splicing (1-3), which may affect function, localization, binding properties, and stability of the encoded proteins (4). The recent results from the ENCODE (Encyclopedia of DNA Elements) consortium (5) extend and confirm the ubiquity of alternative splicing (6). Several splice variants with antagonistic functions have been described, e.g. BCL-X has an antiapoptotic long isoform and a proapoptotic short isoform (7,8). Alternative splicing can also lead to degradation of the transcript, thereby abrogating protein expression; examples include certain Serine/Arginine-rich (SR) protein splicing factors for which the inclusion of a particular exon causes mRNA degradation by nonsense-mediated decay (9, 10).Single nucleotide polymorphisms and somatic splice site mutations leading to aberrant splicing patterns have been described for a number of tumor suppressor genes, including APC, TP53, and BRCA1 (11). Deregulation of trans-acting proteins, such as splicing factors and heterogeneous nuclear ribonucleoproteins, may cause a more general change in RNA splicing in cancer cells. The SFRS1 gene, encoding the splicing factor 2/alternate spli...

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Section: Discussionsupporting

confidence: 81%

Alternative Splicing in Colon, Bladder, and Prostate Cancer Identified by Exon Array Analysis

Thorsen¹,

Sørensen²,

Brems-Eskildsen³

et al. 2008

Molecular & Cellular Proteomics

219

222

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“…We have recently demonstrated that exon-level profiling can detect exon-skipping mutants and splice variants (French et al, 2007;Schutte et al, 2008). In this study, we used these expression profiles to screen for fusion transcripts.…”

Section: Identifying a Novel Translocation Involving Est Ai364529 Andmentioning

confidence: 99%

“…Tissues adjacent to the paraffin-embedded hematoxylin and eosin-stained frozen sections were used for nucleic-acid isolation as described (French et al, 2007). For this study, we also isolated protein from the Trizol fraction according to the manufacturer's protocol (Invitrogen, Breda, The Netherlands).…”

Section: Rna Dna and Protein Isolationmentioning

confidence: 99%

“…As such translocations often show aberrant expression of the fusion partners, screening for aberrant expression can identify novel fusion genes (Tomlins et al, 2005). Exonlevel expression profiling is a method that can identify differentially expressed splice variants (Clark et al, 2007;French et al, 2007). We have recently shown that genomic mutations in cancer that lead to skipping of one or more exons are also detected by such arrays (Schutte et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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The CASPR2 cell adhesion molecule functions as a tumor suppressor gene in glioma

et al. 2010

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Genomic translocations have been implicated in cancer.In this study, we performed a screen for genetic translocations in gliomas based on exon-level expression profiles. We identified a translocation in the contactinassociated protein-like 2 (CASPR2) gene, encoding a cell adhesion molecule. CASPR2 mRNA was fused to an expressed sequence tag that likely is part of the nuclear receptor coactivator 1 gene. Despite high mRNA expression levels, no CASPR2 fusion protein was detected. In a set of 25 glioblastomas and 22 oligodendrogliomas, mutation analysis identified two additional samples with genetic alterations in the CASPR2 gene and all three identified genetic alterations are likely to reduce CASPR2 protein expression levels. Methylation of the CASPR2 gene was also observed in gliomas and glioma cell lines. CASPR2-overexpressing cells showed decreased proliferation rates, likely because of an increase in apoptosis. Moreover, high CASPR2 mRNA expression level is positively correlated with survival and is an independent prognostic factor. These results indicate that CASPR2 acts as a tumor suppressor gene in glioma.

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“…This study provides further evidence that microarrays, and in particular exon arrays where probes are designed to detect individual exons, are excellent tools to discover novel alternative transcripts (Clark et al 2007, French et al 2007, Skotheim & Nees 2007, Rahman et al 2010, Menghi et al 2011. The exon-junction probe in our custom array was designed to target GenBank sequences deposited by Affymetrix as a result of RT-PCR validation of testisenriched exon array data (Clark et al 2007).…”

Section: Discussionmentioning

confidence: 87%

Cloning of a novel insulin-regulated ghrelin transcript in prostate cancer

Seim¹,

Lubik²,

Lehman³

et al. 2012

Journal of Molecular Endocrinology

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Ghrelin is a multifunctional hormone, with roles in stimulating appetite and regulating energy balance, insulin secretion and glucose homoeostasis. The ghrelin gene locus (GHRL) is highly complex and gives rise to a range of novel transcripts derived from alternative first exons and internally spliced exons. The wild-type transcript encodes a 117 amino acid preprohormone that is processed to yield the 28 amino acid peptide ghrelin. Here, we identified insulin-responsive transcription corresponding to cryptic exons in intron 2 of the human ghrelin gene. A transcript, termed in2c-ghrelin (intron 2-cryptic), was cloned from the testis and the LNCaP prostate cancer cell line. This transcript may encode an 83 amino acid preproghrelin isoform that codes for ghrelin, but not obestatin. It is expressed in a limited number of normal tissues and in tumours of the prostate, testis, breast and ovary. Finally, we confirmed that in2c-ghrelin transcript expression, as well as the recently described in1-ghrelin transcript, is significantly upregulated by insulin in cultured prostate cancer cells. Metabolic syndrome and hyperinsulinaemia have been associated with prostate cancer risk and progression. This may be particularly significant after androgen deprivation therapy for prostate cancer, which induces hyperinsulinaemia, and this could contribute to castrate-resistant prostate cancer growth. We have previously demonstrated that ghrelin stimulates prostate cancer cell line proliferation in vitro. This study is the first description of insulin regulation of a ghrelin transcript in cancer and should provide further impetus for studies into the expression, regulation and function of ghrelin gene products.

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Identification of Differentially Regulated Splice Variants and Novel Exons in Glial Brain Tumors Using Exon Expression Arrays

Cited by 82 publications

References 40 publications

Alternative Splicing in Colon, Bladder, and Prostate Cancer Identified by Exon Array Analysis

Alternative Splicing in Colon, Bladder, and Prostate Cancer Identified by Exon Array Analysis

The CASPR2 cell adhesion molecule functions as a tumor suppressor gene in glioma

Cloning of a novel insulin-regulated ghrelin transcript in prostate cancer

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