Characterizing cancer cell signaling at the protein level : from targeted to proteome and phosphoproteome­‐wide analyses

Proteins are the effectors of cellular functions and the constituting elements of cellular signaling cascades. The ability to analyze the abundances and the dynamics of proteins is central to dissect cellular signaling and its effects on cell physiology. The aim of this thesis is to gain insight into protein level regulatory mechanisms that contribute to the development of cancer, by optimizing and employing targeted and largescale methods. Specifically, to examine mechanisms regulating protein stability, localization, protein-protein interactions, and to characterize targets of a protein phosphatase enzymatic activity. Additionally, to optimize a workflow for quantitative phosphoproteomics analysis with the goal of improving the sensitivity and lower the requirement in terms of sample quantity of current methods.

Study I elucidated a mechanism by which S100A4 interacts with p53 in the nucleus thereby promoting its degradation, and the effects of this interaction on the growth and survival of lung and cervix adenocarcinoma cell lines, by employing targeted methods for the analyses of protein stability, protein localization and protein-protein interactions. Study II elucidated a mechanism by which TRAP promotes metastasis-related cell properties in breast cancer cells via the TGFβ-pathway and CD44, by a combination of proteomics and phosphoproteomics analyses with targeted methods. Furthermore, a moderate-depth phosphoproteomic profiling of TRAP overexpressing cells was achieved by peptide fractionation by high-resolution isoelectric focusing (HiRIEF) on IPG strips pH range 2.5-3.7, and provided a list of putative targets of TRAP phosphatase activity. Study III developed a workflow for in-depth quantitative phosphoproteomics analysis based on high-resolution isoelectric focusing (HiRIEF) fractionation on a wide pH range (2.5-10). The workflow employs phospho-enrichment by titanium dioxide coupled with isobaric labeling by TMT, and provides for good analytical depth and sensitivity, requiring a low amount of starting material. Application of this workflow for the analysis of cervix adenocarcinoma cells HeLa revealed 1,264 novel phosphorylation sites, of which 165 phospho-sites that are suggested to have a regulatory function during the mitotic phase, based on kinase-association analysis.

In summary, the work presented in this thesis contributes to the collective effort of improving and applying targeted and large-scale methods for the analysis of protein level regulatory mechanisms, particularly by focusing on the optimization of a workflow for phosphoproteomics analysis. Development of these methods and improvements in integrating discovery and validation efforts, will be central in the coming years and offer unprecedented opportunities for increasing our understanding of life and to discover new treatments and cures for diseases.

List of scientific papers

I. Lukas M. Orre, Elena Panizza, V.O. Kaminskyy, E. Vernet, T. Gräslund, Boris Zhivotovsky and Janne Lehtiö. S100A4 interacts with p53 in the nucleus and promotes p53 degradation. Oncogene. (2013), 32, 5531–5540.
https://doi.org/10.1038/onc.2013.213

II. Anja Reithmeie, Elena Panizza, Michael Krumpel, Lukas M Orre, Rui MM Branca, Janne Lehtiö, Barbro Ek-Rylander, Göran Andersson. Tartrate-resistant acid phosphatase (TRAP/ACP5) promotes metastasisrelated properties via TGFβ2/TβR and CD44 in MDA-MB-231 breast cancer cells. [Manuscript]

III. Elena Panizza, Rui MM Branca, Peter Oliviusson, Lukas M Orre, and Janne Lehtiö. Isoelectric point-based fractionation by HiRIEF coupled to LC-MS allows for in-depth quantitative analysis of the phosphoproteome. Scientific Reports. 2017 Jul 3;7(1):4513.
https://doi.org/10.1038/s41598-017-04798-z