Abstract

Genomic tools in the last few decades have made it possible to produce proteins for pharmaceutical use in mammalian cells such as Chinese Hamster Ovary cells (CHO). As such there is an ever increasing demand protein biopharmaceuticals. By identifying key proteins involved in the growth patterns of mammalian cells we hope to be able to manipulate CHO cell for biopharmaceutical production. We used two different methods to manipulate cell growth and then identified the proteins involved using quantitative label free LC-mass spectrometry. Previous studies in our lab showed up-regulation of microRNA-7 (miR-7) reduces proliferation in CHO-K1-SEAP cells but increases productivity over time. A similar phenotype is observed in temperature shifted (31 oC) CHO cells and is often used in industry for increased productivity. The mechanism of both these phenotypes in CHO are largely unknown at the protein level. Using label-free LC-MS/MS we identified catalase and stathmin as potential targets of miR-7, the potential role of glutathione metabolism up-regulation and the potential role of structural process inhibition in causing this phenotype. Using the same techniques combined with subcellular fractionation to analyse the temperature shift phenotype in CHO-K1-SEAP cells we were able to double the number of protein identifications from 960 with no fractionation to 2298 using fractionation. Two differentially regulated proteins, cyclon and lamin A/C, were identified as significantly reducing cell proliferation and cell size and may have potential as targets to induce an industrially relevant phenotype in CHO cells. Breast cancer is the leading cause of cancer death in women, there is an urgent need to identify new molecular targets for certain aggressive breast cancer subtypes to lead to improved treatments for patients. We used bioinformatics profiling of publicly available data-sets to compare gene expression across breast cancer sub-types compared to normal breast tissue to identify a panel of differentially expressed membrane candidate targets. Candidate target expression was validated in membrane enriched breast cancer cell line extracts and extensive immunohistochemical (IHC) analysis of target expression in breast cancer subtypes, normal breast tissue and highly proliferating tissues was carried out. Two proteins, IGSF9 and KLRG2, not previously associated with breast cancer, were demonstrated to show significantly higher expression in triple negative (TNBC) and HER-2 positive breast cancers than in normal breast tissue and also have very low presence in other normal (and highly proliferating) tissues. These two protein targets may have potential to be further investigated as ADC molecular targets for these aggressive breast cancer subtypes.