Phd in MORPHOGENESIS AND TISSUE ENGINEERING

Thesis title: Assessing the impact of endolysosomal Two-pore channel 2 (TPC2) on human Malignant Melanoma progression

Background: Two-pore channel 2 (TPC2) is a Ca2+/Na+ ion channel located on the membrane of intracellular acidic organelles, including endo-lysosomes and melanosomes. It plays a crucial role in tumor progression across various cancers, including melanoma, one of the most aggressive and treatment-resistant malignancies, often driven by mutations in the serine/threonine kinase BRAF. Notably, dysregulation of this channel has been shown to impair angiogenesis, metastatization, and autophagy, processes that are established hallmarks of cancer. Aim: This project aims to explore the involvement of TPC2 in melanoma progression, comparing its role in primary and metastatic disease, to determine whether it could serve as a potential therapeutic target for tailored melanoma treatments. Experimental design and methods: Two pairs of human melanoma cell lines (IGR and WM), each derived from the same patient at different stages of tumour progression and genetically characterized by BRAF mutation, were analysed. The primary melanoma cell lines included IGR39 and WM115, while the metastatic counterparts were IGR37 and WM266-4. TPC2 expression levels were compared between the primary and metastatic cell lines. For the characterization of the cell models the expression of EMT markers, including N-cadherin, E-cadherin, and Vimentin, was assessed, also in relation with the EMT transition feature. To investigate the functional role of TPC2, the novel TPC2 inhibitor SG-094 was applied to these cells. Furthermore, TPC2 inhibition was functionally assessed through assays evaluating cell proliferation, migratory and invasive capabilities, adhesion to type I collagen, and β1-integrin recycling. Combination treatments with varying doses of SG-094 and the BRAF inhibitor Dabrafenib were also tested to assess potential synergistic effects on cellular behaviour. Results: We found that IGR and WM human melanoma cell lines exhibit heterogeneous expression of EMT markers and display different behaviours in terms of migratory capability and adhesion to type 1 collagen. Notably, cells derived from the primary tumor site (IGR39 and WM115) have lower TPC2 expression than metastatic cells (IGR37 and WM266-4). Our results demonstrated that pharmacological inhibition of this channel using the TPC2 inhibitor SG-094 significantly reduced the migratory capability of IGR and WM cell lines, as well as the adhesiveness of IGR lines to type 1 collagen and disrupted the β1-itegrin endocytic pathways in WM cell lines, highlighting the role of TPC2-dependent intracellular Ca2+ signaling. Notably, SG-094 treatment also led to a decline in the invasion potential of these cells, in a 3D spheroids model, suggesting an additional impact on tumor progression. Additionally, our studies reveal that the effect of SG-094 may involve the Microphthalmia-associated transcription factor (MITF), a master regulator of melanocytes implicated in metastasis, tumor aggressiveness and survival, in a manner that could involve the MAPK pathway. We also analysed the combinatorial treatment of SG-094 and Dabrafenib (a BRAF V600 inhibitor currently used in therapy) and, notably, we observed a higher sensitivity to the TPC2 inhibitor compared to the BRAF inhibitor. Conclusion: The data demonstrate that TPC2 has a pro-tumorigenic role in BRAF-mutant melanoma cell lines. Our findings show that pharmacological inhibition of TPC2 significantly reduces melanoma cell migration, invasion, adhesion, and proliferation, key processes in tumour progression. The complex regulation of TPC2 in melanoma warrants further investigation, especially considering its involvement with MITF. Furthermore, analysis of combined treatments suggests that targeting TPC2 may offer greater efficacy compared to existing therapies. These results underscore the importance of TPC2-dependent intracellular Ca²⁺/Na⁺ signalling in melanoma progression and the promising potential of TPC2 as a therapeutic target.