Poly (A)+ Transcriptome Assessment of ERBB2-Induced Alterations in Breast Cell Lines

Abstract

We report the first quantitative and qualitative analysis of the poly (A)+ transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.

ERBB2 in Cat Mammary Neoplasias Disclosed a Positive Correlation between RNA and Protein Low Expression Levels: A Model for erbB-2 Negative Human Breast Cancer

“…Camp and collaborators (2003) speculated that tumours might overexpress another growth factor receptor that promotes tumour aggression via a ligand-dependent or -independent mechanism [39], [76], [77]. Moreover, comparative analysis showed enrichment in alternative events in ERBB2 over-expressing cells, indicating regulation of alternative splicing mediated by the oncogene [78]. Some authors have illustrated that erbB-2 IHC tests generally reveal a correlation between an erbB-2 positive status and an aggressive phenotype [79], but other authors have proposed that normal levels of the erbB-2 protein could be associated with a similar aggressive phenotype [77], [80].…”

RNA-Seq and human complex diseases: recent accomplishments and future perspectives

“…Moreover, RNA-Seq revealed a valuable resource to identify new ERBB2-mediated events and private fusions in some BRCA1-mutated transcriptomes, novel potential biomarkers for diagnosis and treatment. 60,61 Another main advantage of NGS is the ability to detect ncRNA species, now emerging as potential contributors to different pathogenic mechanisms, also in human cancer. In this regard, a regulatory role of ncRNAs has been suggested by a recent analysis performed in Myelodysplastic syndromes (MDS), in which differences in miRNAs' expression were associated to early and later stages of the disease.…”

NGS-Trex: Next Generation Sequencing Transcriptome profile explorer

“…Table 1 summarizes the comparison between the results shown by Carraro et al [8] and the results obtained with NGS-Trex analysis. It is important to underline that all data reported in the table are readily available from the web interface in the summary table or in the statistical report and show good correlation with data obtained by Carraro et al…”

“…To benchmark our system we processed the sequences obtained by [8] with Titanium 454-Roche-platform. Sequences are available at the NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/Traces/sra) with accession SRA012436.…”