Two-Dimensional Transcriptome Profiling: Identification of Messenger RNA Isoform Signatures in Prostate Cancer from Archived Paraffin-Embedded Cancer Specimens

Li, Hairi; Wang‐Rodriguez, Jessica; Nair, T. Murlidharan; Yeakley, Joanne M.; Kwon, Young‐Soo; Bibikova, Marina; Zheng, Christina; Zhou, Lixin; Zhang, Kui; Downs, Tracy M.; Fu, Xiang‐Dong; Fan, Jian‐Bing

doi:10.1158/0008-5472.can-05-4264

Cited by 73 publications

(68 citation statements)

References 51 publications

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“…Further characterization of the identified splice variants, along with other markers, could also improve the staging of cancer samples as several of the identified splice variants change their expression pattern with progression of the disease. Indeed splice variants have been used in prostate tumor classification (17). One may also hypothesize that the cancer-specific alternatively spliced isoforms could be used as cancerspecific drug targets.…”

Section: Discussionmentioning

confidence: 99%

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

Thorsen¹,

Sørensen²,

Brems-Eskildsen³

et al. 2008

Molecular & Cellular Proteomics

218

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: Discussionmentioning

confidence: 99%

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

Thorsen¹,

Sørensen²,

Brems-Eskildsen³

et al. 2008

Molecular & Cellular Proteomics

218

222

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“…Culturing LNCaP cells and DHT treatment were as described in ref. 28. Primers used for qPCR are listed in Table S4.…”

Section: Methodsmentioning

confidence: 99%

Determination of tag density required for digital transcriptome analysis: Application to an androgen-sensitive prostate cancer model

Lovci

Kwon

et al. 2008

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

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High-throughput sequencing has rapidly gained popularity for transcriptome analysis in mammalian cells because of its ability to generate digital and quantitative information on annotated genes and to detect transcripts and mRNA isoforms. Here, we described a double-random priming method for deep sequencing to profile double poly(A)-selected RNA from LNCaP cells before and after androgen stimulation. From Ϸ20 million sequence tags, we uncovered 71% of annotated genes and identified hormone-regulated gene expression events that are highly correlated with quantitative real time PCR measurement. A fraction of the sequence tags were mapped to constitutive and alternative splicing events to detect known and new mRNA isoforms expressed in the cell. Finally, curve fitting was used to estimate the number of tags necessary to reach a ''saturating'' discovery rate among individual applications. This study provides a general guide for analysis of gene expression and alternative splicing by deep sequencing.alternative splicing ͉ androgen-regulated gene expression in prostate cancer cells ͉ curve regression ͉ high-throughput sequencing M icroarray-based approaches, especially unbiased tiling arrays, suggest that up to 80% of the genome may be transcribed to produce a huge number of uncharacterized transcripts relative to current gene annotation (1-4). In contrast, recent transcriptome analysis by deep sequencing indicates that the vast majority of expressed transcripts in mammalian tissues and cell lines are confined to annotated genes and exons (5, 6). Although microarraybased approaches suffer from a great degree of uncertainty in relating detected hybridization signals to defined transcripts, sequencing-based approaches tend to be overwhelmed by abundant transcripts in the cell. Construction of ''normalized'' libraries for deep sequencing might facilitate the discovery of low abundance transcripts, many of which may act as noncoding, regulatory RNA in mammalian cells.An advantage of transcriptome analysis by deep sequencing is the ability to detect structural variation of individual transcripts. It is well known that different transcripts from the same genes may be generated by differential promoter usage, heterogeneous transcriptional start sites and alternative 3Ј end formation (2). A recent Pol II ChIP-chip study indicates that protein-coding genes may have an average of 3 to 5 promoters in both the mouse and human genomes (7). Further adding to the diversity in the transcriptome is alternative RNA processing of most protein-coding genes as a consequence of alternative 5Ј and 3Ј splice site choices, exon inclusion/ skipping, intron retention, and combinatorial use of alternative exons (8). It is estimated that up to 74% of human genes undergo alternative splicing, which is believed to contribute to the complexity of the proteome in mammalian cells (9).It is striking to note that individual laboratories now have the capacity to generate sequenced tags that are on the same order of sequenced mRNA/ESTs in publicly availab...

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“…One source of such biomarkers, locus hCG_1815491 on chromosome 16, is of particular interest, as it has negligible expression in normal colorectal tissue but substantial expression in both colorectal adenomas and adenocarcinomas, suggesting that this gene is active early in neoplastic progression (LaPointe et al, submitted manuscript). In recognition of these observations, the HUGO Gene Nomenclature Committee accepted our proposal to name the locus colorectal neoplasia differentially expressed (CRNDE).…”

Section: Introductionmentioning

confidence: 99%

“…15 In some cancers, the upregulation of specific splice variants amounts to a "molecular signature" for neoplasia. 16,17 Here, we investigate the relationship of each CRNDE transcript to colorectal neoplasia. In addition, we define the boundaries and features of the CRNDE locus through exon microarray analyses, 5′-RACE, and transcript-specific RT-PCR assays.…”

Section: Introductionmentioning

confidence: 99%

Colorectal Neoplasia Differentially Expressed (CRNDE), a Novel Gene with Elevated Expression in Colorectal Adenomas and Adenocarcinomas

Pedersen²,

et al. 2011

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An uncharacterized gene locus (Chr16:hCG_1815491), now named colorectal neoplasia differentially expressed (gene symbol CRNDE), is activated early in colorectal neoplasia. The locus is unrelated to any known protein-coding gene. Microarray analysis of 454 tissue specimens (discovery) and 68 previously untested specimens (validation) showed elevated expression of CRNDE in >90% of colorectal adenomas and adenocarcinomas. These findings were confirmed and extended by exon microarray studies and RT-PCR assays. CRNDE transcription start sites were identified in CaCo2 and HCT116 cells by 5′-RACE. The major transcript isoforms in colorectal cancer (CRC) cell lines and colorectal tissue are CRNDE-a, -b, -d, -e, -f, -h, and -j. Except for CRNDE-d, the known CRNDE splice variants are upregulated in neoplastic colorectal tissue; expression levels for CRNDE-h alone demonstrate a sensitivity of 95% and specificity of 96% for adenoma versus normal tissue. A quantitative RT-PCR assay measuring CRNDE-h RNA levels in plasma was (with a threshold of 2 -ΔCt = 2.8) positive for 13 of 15 CRC patients (87%) but only 1 of 15 healthy individuals (7%). We conclude that individual CRNDE transcripts show promise as tissue and plasma biomarkers, potentially exhibiting high sensitivity and specificity for colorectal adenomas and cancers.

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Two-Dimensional Transcriptome Profiling: Identification of Messenger RNA Isoform Signatures in Prostate Cancer from Archived Paraffin-Embedded Cancer Specimens

Cited by 73 publications

References 51 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

Determination of tag density required for digital transcriptome analysis: Application to an androgen-sensitive prostate cancer model

Colorectal Neoplasia Differentially Expressed (CRNDE), a Novel Gene with Elevated Expression in Colorectal Adenomas and Adenocarcinomas

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