Proteomic approaches for quantitative cancer cell signaling

Abstract

Cancer is a genetic disease and historically the discovery of underlying genetic alterations has been critical to our understanding of disease and past treatment successes. However, cancer still poses an important health threat and most available drugs are not capable of providing complete remission. Drug targets are typically proteins, but are based on genetic findings. Thus studying cancer systems at the level of proteins and their signaling can provide the additional level of data needed for the development of effective drugs. This thesis summarizes the work undertaken during my doctoral studies in an effort to contribute to the study of signaling dynamics in cancer systems. This thesis is divided into two parts. Part I begins with a brief introduction in the use of omics in systems cancer research with a focus on mass spectrometry as a means to quantitatively measure protein and signaling dynamics in the identified protein networks (Chapter 1). Gene fusions are portrayed in-depth as an example of a major genetic alteration found to occur in a variety of cancers, the most infamous of which has lead to the development of the specific tyrosine kinase inhibitor imatinib and a major success in the treatment of chronic myelogenous leukemia. However, this is the exception rather than the norm as most drugs are developed based on genetic findings while designed to act on the protein level, and might contribute to explaining the paucity of specific effective cancer therapeutics available. Furthermore, this underlines the importance of proteomic studies and the conclusions drawn for the high-throughput data generated in the latter. Chapter 2 gives a temporal overview of precision gene-editing in the context of systems biology. Following the past successes of methods such as zinc-finger nucleases and TALEs, the novel CRISPR-Cas technology has rapidly become an extremely popular gene-editing tool. Its mechanism of action, several applications and potential shortcomings are discussed. The Chapter is concluded with a final application: chromosomal translocations can be generated in vitro or in vivo using nuclease-based targeted geneediting. Part II illustrates the use of mass spectrometry-based proteomics and phospho-proteomics in studying the effects of perturbations at the cellular level. Chapter three captures the very early signaling dynamics related o cell migration following wounding in triple negative breast cancer cells, and their potential role as novel targets for therapies aimed at reducing the metastases. Chapter four describes the induction of the oncogenic chimeric gene PRKAR1A-RET in thyroid cells. Its transformative potential is shown and the ensuing changes are measured at the protein and signaling levels. This study demonstrates the use of the novel CRISPR-Cas technology for the generation of chromosomal rearrangements in vitro and investigates the effects of this important genetic aberration in a physiologically relevant cellular setting. Part III concludes the thesis by providing a global discussion and future perspectives for the studies presented in part II. Overall, the work presented herein aims to underscore the importance of studying cancer systems at the protein level, the dynamics of which define phenotypic outcome. The effects of cellular and genetic perturbations at the protein network level were studied using mass spectrometry-based proteomics and, the results whereof suggest interesting avenues for future development of cancer therapies.