Assessing the impact of modulating protein degradation pathways in cancer

Proteins are macromolecules, essential constituents of the cells with various functions, such as providing structure to the cells, performing biochemical reactions, transporting molecules. The levels of cellular proteins are in a dynamic equilibrium and regulated by their synthesis and degradation rates. Both processes have an equal importance for regulating protein abundance. A disruption of the balance by dysregulation of degradation pathways has been shown to be linked to a range of diseases including neurodegenerative diseases and cancer.

Oncoproteins play an important role for cancer cell growth, proliferation, invasion, and migration. Therefore, targeting oncoproteins for degradation provides a novel direction for cancer treatment. This thesis aims to investigate the effect of modulating protein degradation pathways to target oncogenic proteins for degradation that offers a new approach for an anticancer strategy.

In paper I, we investigated the role of deubiquitinating enzymes (DUBs) on protein stability of Slug, one of epithelial-to-mesenchymal transition (EMT)-transcription factors (TFs) related to cancer cell invasion and migration. In this study, we identified that the deubiquitinating enzyme USP10 can regulate Slug stability in cancer cells. We found that USP10 has high expression levels in cancer biopsies in line with higher Slug expression. Furthermore, genetic depletion of USP10 results in decreased levels of Slug, as well as the mesenchymal marker, Vimentin. Meanwhile, overexpression of USP10 results in the opposite effect on Slug and Vimentin levels. Overall, our study identifies USP10 as a new regulator of Slug and migratory capacity of cancer cells.

In paper II, we explored the proteome selectively degraded by chaperone-mediated autophagy (CMA) in cancer cells. We identified multiple cellular processes affected by CMA activation, and the components targeted for CMA-mediated degradation. We discovered translation initiation factors as CMA substrates in cancer cells of different origin. Furthermore, we showed that CMA activation significantly reduced cellular translation. This work reveals novel pathways affected by CMA as well as its impact on translational regulation in cancer cells.

In paper III, we studied time-dependent enrichment of proteins in lysosomes upon non- macroautophagy (MA)-activated conditions. To gain more insight into degradation profile when MA is inhibited, we investigated the LAMP2A dependency and importance of KFERQ-like motif for proteins targeted to lysosomes. This study shows that target specificity differs with stress period and may depend on their biological function. Overall, our study provides a better understanding of non-MA-dependent degradome, and requirement of LAMP2A for selective lysosomal targeting of proteins in cancer cells.

In sum, the presented works in this thesis uncovered importance of DUBs for oncogenic protein stability, suggesting their potential targetability, and highlighted the potential of CMA activation as an anticancer strategy through degradation of oncoproteins.

List of scientific papers

I. Amanda Tomie Ouchida*, Merve Kacal*, Adi Zheng*, Gorbatchev Ambroise, Boxi Zhang, Erik Norberg, Helin Vakifahmetoglu-Norberg (2018). USP10 regulates the stability of the EMT-transcription factor Slug/SNAI2. Biochemical and Biophysical Research Communications. 502(4): 429-434. *These authors contributed equally.
https://doi.org/10.1016/j.bbrc.2018.05.156

II. Yuqing Hao*, Merve Kacal*, Amanda Tomie Ouchida, Boxi Zhang, Erik Norberg, Helin Vakifahmetoglu-Norberg (2019). Targetome analysis of chaperone-mediated autophagy in cancer cells. Autophagy. 15:9, 1558-1571. *These authors contributed equally.
https://doi.org/10.1080/15548627.2019.1586255

III. Merve Kacal*, Boxi Zhang*, Yuqing Hao*, Erik Norberg, Helin Vakifahmetoglu- Norberg (2021). Quantitative proteomic analysis of temporal lysosomal proteome and the impact of the KFERQ-like motif and LAMP2A in lysosomal targeting. Autophagy. 17:11, 3865-3874. *These authors contributed equally.
https://doi.org/10.1080/15548627.2021.1876343