Distinct Splice Variants and Pathway Enrichment in the Cell-Line Models of Aggressive Human Breast Cancer Subtypes
ABSTRACT This study was conducted as a part of the Chromosome-Centric Human Proteome Project (C-HPP) of the Human Proteome Organization. The United States team of C-HPP is focused on characterizing the protein-coding genes in chromosome 17. Despite its small size, chromosome 17 is rich in protein-coding genes, it contains many cancer-associated genes, including BRCA1, ERBB2 (Her2/neu), and TP53. The goal of this study was to examine the splice variants expressed in three ERBB2 expressed breast cancer cell line models of hormone receptor negative breast cancers by integrating RNA-Seq and proteomic mass spectrometry data. The cell-lines represent distinct phenotypic variations subtype: SKBR3 (ERBB2+ (over-expression)/ ER-/PR-; adenocarcinoma), SUM190 (ERBB2+ (over-expression)/ER-/PR-; inflammatory breast cancer) and SUM149 (ERBB2 (low expression) ER-/PR -; inflammatory breast cancer). We identified more than one splice variant for 1167 genes expressed in at least one of the three cancer cell lines. We found multiple variants of genes that are in the signaling pathways downstream of ERBB2 along with variants specific to one cancer cell line compared to the other two cancer cell lines and to normal mammary cells. The overall transcript profiles based on read counts indicated more similarities between SKBR3 and SUM190. The top-ranking Gene Ontology and BioCarta pathways for the cell-line specific variants pointed to distinct key mechanisms including: amino sugar metabolism, caspase activity, and endocytosis in SKBR3; different aspects of metabolism, especially of lipids in SUM190; cell- to-cell adhesion, integrin and ERK1/ERK2 signaling, and translational control in SUM149. The analyses indicated an enrichment in the electron transport chain processes in the ERBB2 over-expressed cell line models; and an association of nucleotide binding, RNA splicing and translation processes with the IBC models, SUM190 and SUM149. Detailed experimental studies on the distinct variants identified from each of these three breast cancer cell line models may open opportunities for drug target discovery and help unveil their specific roles in cancer progression and metastasis.
- SourceAvailable from: Gong Zhang[Show abstract] [Hide abstract]
ABSTRACT: Chromosome-centric human proteome project (C-HPP) aims at differentiating chromosome-based and tissue-specific protein compositions in terms of protein expression, quantification and modification. We previously found that the analysis of translating mRNA (mRNA attached to ribosome-nascent chain complex, RNC-mRNA) can explain over 94% of mRNA-protein abundance. Therefore, we propose here to use full-length RNC-mRNA information to illustrate protein expression both qualitatively and quantitatively. We performed RNA-seq on RNC-mRNA (RNC-seq) and detected 12,758 and 14,113 translating genes in human normal bronchial epithelial (HBE) cells and human colorectal adenocarcinoma Caco-2 cells, respectively. We found that most of these genes were mapped with greater than 80% of coding sequence coverage. In Caco-2 cells, we provided translating evidence on 4,180 significant single-nucleotide variations. While using RNC-mRNA data as a standard for proteomic data integration, both translating and protein evidence of 7,876 genes can be acquired from 4 inter-laboratory datasets with different MS platforms. In addition, we detected 1,397 non-coding mRNAs that were attached to ribosomes, suggesting potential source of new protein explorations. By comparing the two cell lines, a total of 677 differentially translated genes were found to be non-evenly distributed across chromosomes. In addition, 2,105 genes in Caco-2 and 750 genes in HBE cells are expressed in a cell-specific manner. These genes are significantly and specifically clustered on multiple chromosomes, such as chromosome 19. We conclude that HPP/C-HPP investigations can be considerably improved by integrating RNC-mRNA analysis with MS, bioinformatics and antibody-based verifications.Journal of Proteome Research 11/2013; 13(1). DOI:10.1021/pr4007409 · 5.00 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Many proteins can be modified by multiple types of post-translational modiﬁcations (Mtp-proteins). Although some post-translational modiﬁcations (PTMs) are recently found associated with life-threatening diseases like cancers and neurodegenerative disorders, the underlying mechanisms remain enigmatic to date. In this study, we examined the relationship of human Mtp-proteins and disease and systematically characterized features of these proteins. Our results indicated that Mtp-proteins are significantly more inclined to participate in disease than proteins carrying no known PTM sites. Mtp-proteins were found significantly enriched in protein complexes, having more protein partners and preferred to act as hubs/super-hubs in protein-protein interaction (PPI) networks. They possess a distinct functional focus, such as chromatin assembly or disassembly, and reside in biased, multiple subcellular localizations. Moreover, most Mtp-proteins harbor more intrinsically disordered regions than the others. Mtp-proteins carrying PTM types biased towards locating in the ordered regions were mainly related to protein-DNA complex assembly. Examination of the energetic effects of PTMs on the stability of PPI revealed that only a small fraction of single PTM events influence the binding energy of >2 kcal/mol, whereas the binding energy can change dramatically by combinations of multiple PTM types. Our work not only expands the understanding of Mtp-proteins but also discloses the potential ability of Mtp-proteins to act as key elements in disease development.Journal of Proteome Research 04/2014; 13(6). DOI:10.1021/pr401019d · 5.00 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Combined RNA-Seq and proteomics analyses reveal striking differential expression of splice isoforms of key proteins in important cancer pathways and networks. Even between primary tumor cell lines from histologically-similar inflammatory breast cancers, we find striking differences in hormone receptor-negative cell lines that are ERBB2 (Her2/neu)-amplified versus ERBB1 (EGFR) over-expressed with low ERBB2 activity. We have related these findings to protein-protein interaction networks, signaling and metabolic pathways, and methods for predicting functional variants among multiple alternative isoforms. Understanding the upstream ligands and regulators and the downstream pathways and interaction networks for ERBB receptors is certain to be important for explanation and prediction of the variable levels of expression and therapeutic responses of ERBB+tumors in the breast and in other organ sites. Alternative splicing is a remarkable evolutionary development that increases protein diversity from multi-exonic genes without requiring expansion of the genome. It is no longer sufficient to report up- or down-expression of genes and proteins without dissecting the complexity due to alternative splicing. This article is part of a Special Issue entitled: 20years of Proteomics.Journal of proteomics 05/2014; 107. DOI:10.1016/j.jprot.2014.04.012 · 3.93 Impact Factor