Proteomics of Downstream Responses to Growth Signals in Proliferating Cells

Abstract

Some of the most profound changes elicited by cell growth stimuli influence dramatic rewiring of metabolism. Intriguingly, rapidly dividing cells with aberrant growth factor signalling, such as cancer cells, tend to rely on glycolysis to generate an adequate supply of building blocks required for cell proliferation and invasion. In this study, we observed that in response to stimulation with insulin-like growth factor 1 (IGF-1), MCF-7 breast adenocarcinoma cells show increased levels of the key glycolysis proteins pyruvate kinase M2 and lactate dehydrogenase A. We then developed targeted multiple reaction monitoring (MRM) mass spectrometry assays to conduct quantitative analysis of glycolysis proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs), the latter implicated in pyruvate kinase splicing and many other aspects of cell proliferation. Application of the glycolysis MRM assay to examine IGF-1 stimulated MCF-7 cells revealed increased levels of all sequential proteins from phosphoglycerate mutase 1 to lactate dehydrogenase A in the glycolysis pathway. An extension of this study to cell lines of varying invasiveness, suggest a relation between glycolysis and metastasis. The clinical applicability of glycolysis MRM assay was also shown by its successful application to lung cancer biopsy analysis. Success with the targeted analysis of glycolysis proteins led to a similar approach for the hnRNP family. Our results showed evidence that a poorly characterized hnRNP (A/B) may be regulated by the c-Myc transcription factor but does not evidently influence pyruvate kinase splicing. Our approach using MRM to examine small subsets of proteins downstream of cellular growth signals is relatively novel. Our results demonstrate the potential for such targeted MS strategies because of their high selectivity and multiplexing capabilities. Further, the findings from our analyses provide novel insights into the downstream changes elicited by growth signals such as IGF-1 and c-Myc.