Cell transformation is the process by which a cell changes its phenotype, or characteristics, in response to an external stimulus. It is a process that is essential for the development and functioning of multicellular organisms. Both cell differentiation and senescence are processes in which cells transform from one state to another. Deregulation of cellular transformation may lead to the formation of malignant cancers. Understanding the signaling basis of transformation can help develop treatments to cancer and other diseases. Proteins are the central players of signaling regulation, and proteomics, the comprehensive evaluation of proteins at a specific time and location is a crucial technology for dissecting cell signaling. The focus of my thesis work was to apply proteomics in dissecting the mechanism of cell transformation.

First, I investigated a novel signaling axis that promotes the multipotent dedifferentiation of adipocytes. I discovered that elevated osmolarity induces mitochondrial stress and prompts the adipocytes to eject mitochondrial components through extracellular vesicles, which in turn enhances the pro-inflammatory TNF-α stress signaling. Functionally, ejected mitochondria and TNF-α mediate the activation of the β-catenin signaling that drives adipocyte dedifferentiation, while alleviating mitochondrial stress inhibited hypertonicity-induced reprogramming. I also observed increased apoptosis of adipocytes as an additional consequence of elevated osmolarity. To circumvent this, I showed that BML-284, a small compound that directly activates the β-catenin signaling could effectively induce multipotent adipocyte dedifferentiation while avoiding the apoptosis prompted by the osmotic stress.

Second, I established a bioorthogonal conjugation-assisted purification (BCAP) workflow that utilizes the Staudinger chemoselective ligation to label and isolate surface-associated proteins while minimizing the binding of endogenous biotin-associated proteins. Label-free quantitative proteomics demonstrated that BCAP is efficient in isolating cell surface proteins with excellent reproducibility. Subsequently, I applied BCAP to compare the surface proteome of proliferating and senescent mouse embryonic fibroblasts (MEFs). Among the results, EHD2 was identified and validated as a novel protein that is enhanced at the cell surface of senescent MEFs.

In conclusion, my thesis work defined a novel mechanism that hypertonicity induces ejection of mitochondria and TNF-α signaling, which activates β-catenin and drives adipocytes dedifferentiation. In addition, I also developed a novel BCAP method to profile the cell surface proteome of transforming cells. Using this method, I found that EHD2 is a protein with an increased presence at the surface of senescent MEFs. This provides a foundation for dissecting the dynamic of proteome in different cell transformation which is essential to identify therapeutic targets for cancer and aging-related diseases.