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.

Splice variants in the proteome: a promising and challenging field to targeted drug discovery

“…The article by Menon et al [23] summarizes how the discovery of alternative splice variants can be explored using RNA-Seq/MS experiments. As part of C-HPP, the group focused on proteincoding genes of chromosome 17, which include cancer-associated genes, such as breast cancer 1/2, early onset (BRCA1/2) and tumor protein 53 (TP53).…”

Haplotype-phased synthetic long reads from short-read sequencing

“…However, the loss of linkage information during the generation of short reads limits their utility. In particular, short reads are insufficient to phase the haplotypes of individuals within mixtures of similar sequences, including homeologous and homologous chromosomes in polyploids [3,4], viral quasispecies [5], multiply or alternatively spliced mRNA [6], genes from metagenomic samples containing related organisms [7,8], and immune antibody gene repertoires [9]. In these cases, additional information is required to determine whether mutations separated by distances longer than the read length are present in the same individual.…”

GenomewidePDB 2.0: A Newly Upgraded Versatile Proteogenomic Database for the Chromosome-Centric Human Proteome Project

“…Thus, the identities of most peptides from an MS analysis of the representative proteins can usually be shared by their own ASPs. 20 Identification of ASPs using shotgun proteomics requires extreme care in handling false-positives, which should be distinguished from ASP-specific peptides. 20,21 Targeted proteomics can complement shotgun (or bottomup) proteomics with top-down proteomics, selected reaction monitoring (SRM), or multiple reaction monitoring (S/MRM) assays.…”

“…20 Identification of ASPs using shotgun proteomics requires extreme care in handling false-positives, which should be distinguished from ASP-specific peptides. 20,21 Targeted proteomics can complement shotgun (or bottomup) proteomics with top-down proteomics, selected reaction monitoring (SRM), or multiple reaction monitoring (S/MRM) assays. 22−24 The top-down approach may be a good way to detect large polypeptides spanning long regions of a given protein, 25 maximizing the sequence coverage of identified proteins.…”