PROTEASE ACTIVATED RECEPTOR 2 AND SERPINB3 AS NOVEL BIOMARKERS AND THERAPEUTIC TARGETS IN HEPATIC CANCER

Cancer is the second leading cause of death worldwide, responsible for nearly 10 million deaths annually, with rising diagnosis in both Western and emerging economies. Mortality rate depends on a multiplicity of factors such as diagnosis stage and access to treatment. Aggressive cancers such as lung, breast, prostate, and liver cancers are correlated with a lower 5-year survival. These aggressive tumors often arise from cancer stem cells (CSCs), which drive tumor growth and adaptability. Given the key role of CSCs, in recent decades, they have become an interesting target for the development of novel strategies for the treatment of these malignancies. The understanding of underlying molecular pathways is of paramount importance to identify novel targets that could be relevant for early detection or pharmacological treatment. Despite advances in early detection, several cancers still remain difficult to detect due to a lack of specific biomarkers, highlighting the need for better diagnostic tools and therapeutic strategies. The present work focuses on two novel molecular markers, identified as a key player in the survival and proliferation of CSCs: protease activated receptor 2 (PAR2) and SERPINB3. Both proteins are involved in the action of proteases, but have very different roles. In particular, PAR2 is used for extracellular translation and matrix remodeling through proteases activity, in inflammatory and migration processes; while SERPINB3 is responsible for inhibiting intracellular proteases released from lysosomal rupture, thus acting as an antiapoptotic and pro-proliferative factor. In the first part, PAR2 will be taken into consideration as a pharmacological target. A small compound, 1-piperidine propionic acid, has been described and confirmed by computational and biochemical assays as a PAR2 inhibitor. Since 1-PPA was able to inhibit the PAR2 signaling cascade by reducing Erk1/2 phosphorylation, this small molecule was used to treat and reduce NASH development in mice and cellular models. The efficacy of this compound in mice models showed not only a reduction in fibrosis and inflammation, but also a reduction in SERPINB3 synthesis. Therefore, it is hypothesized that an axis PAR2-SERPINB3 supports the progression of NASH. The second part of the dissertation revolves around the use of yeast surface display to develop highly specific nanobodies against SERPINB3. Because a whole immunization library was subcloned in yeast display system for the first time, control screening was performed against maltose binding protein, resulting in the identification of a picomolar affine nanobody with unique properties described with structural investigations. SERPINB3 was then explored as an antigen against the library, and due to its conformational flexibility, the selection led to the discovery of two nanobodies with low micromolar affinity. By affinity maturation and the use of chimeric constructs, it was possible to mature these antibody fragments into stronger binders that are going to be tested in the diagnostic procedure for the antigen detection in solid and liquid biopsies. Studies reported in this work pave the way for the development of novel bioactive small and macromolecules that could implement the already available tools for early diagnosis of cancer. The importance of using complementary approaches that could be integrated into the pharmacological setting is also important. Lastly, but not least, noteworthy is the intrinsic potential of yeast surface display as a platform for the development of recombinant antibody against complex target, allowing the discovery of low-affine binders and their maturation towards bioactive macromolecules.