Titolo della tesi: Uncovering the impact of endo-lysosomal Two-Pore Channel 2 (TPC2) in melanoma malignant traits and microenvironment remodelling
The ion channel TPC2, localized on the membranes of acidic organelles such as endo-lysosomes and melanosomes, is engaged in pigmentation and is known to play a role in relevant pathologies, e.g. tumor metastasis and viral infection reviewed in [1]. Our laboratory has demonstrated that NAADP/TPC2/Ca2+ signalling is involved in melanoma progression and metastasis [2, 3]. To date, it is well known that tumour microenvironment has a key role in cancer progression and it was shown that cancer cells could release exosomes and melanosomes with pro-metastatic factors and modify the tumor microenvironment to their advantage [4]. In addition, increasing attention in oncology is paid to the cell to cell interactions within solid tumors, therefore we analysed the impact of TPC2 in the cross-talk between tumor cells and tumor microenvironment, an interaction known to regulate tumor aggressiveness.
Our project has pursued two aims. In the first we explored whether TPC2 plays a different role in primary malignant melanoma (MM) and in metastatic melanoma, as suggested by its differential expression in human MM clinical samples [3]. We explored which hallmarks of the malignant phenotype are traits regulated by TPC2 and, in a parallel series of experiments, we observed the impact of TPC2 expression in MM cells on autophagy and secretion, relevant in the tumor microenvironment. As for the latter aim we observed the response of macrophages to MM secretome of tumor cells, and their M1-like or M2-like polarization, because this can determine the switch of the tumor microenvironment from anti-tumor immune microenvironment to immune suppressive microenvironment and it could promote and increase tumor growth [5, 6]. Our experimental model is represented by murine melanoma B16F0 and B16F10 cell lines and human metastatic melanoma CHL1 cell line, with different levels of aggressiveness, in which we have characterized the relevance of TPC2-regulated signaling. Data showing that TPC2 participates in the regulation of autophagy and secretion suggest focusing on these functions and their interrelations. We have demonstrated the impact of TPC2-KD and TPC2 genetic and pharmacological inhibition on the autophagic process and on the increase of the aggressiveness of the metastatic melanoma cells, compared with the primary melanoma. We have observed an increase in the autophagic flux in B16F10 melanoma sub-lines, compared to the primitive melanoma (B16F0), both with genetic and pharmacological TPC2 inhibitions. We have observed an increase in migration, a decrease in the apoptosis pathway, an increase in the epithelial-to-mesenchymal transition in the metastatic cells, compared with the primary one, following TPC2 silencing. We have discovered that TPC2 can influence the ability of metastatic cells to bind the collagen type I matrix, in that TPC2-KD cells increased the expression levels of Vimentin, considered as mesenchymal marker. We also uncovered the role of secretoma of melanoma cell lines, silenced for TPC2, in macrophage polarization as well as the impact of TPC2 loss on acquired immunity mediated by macrophages. In particular, we found a correlation between the downregulation of TPC2 and the polarization of macrophages to M2-like phenotype, with the increase of the expression of IL-10, cytokine with a immune-suppressive role, and with a melanoma cell lysis operated by CD8+ cells, compared to the control.
In conclusion, our research shows a novel role of TPC2 in melanoma, that could be different at the late stages of cancer development, compared to the initial carcinogenesis; furthermore, we found a new correlation between the modulation of TPC2 and the polarization of the innate immune cells in the tumor microenvironment. Taken together, these data support the hypothesis that Two-Pore Channel 2 (TPC2) could have a new role in MM and it could represent a new therapeutic target for experimental and clinical research purposes.
1. Barbonari, S., et al., Relevance of lysosomal Ca(2+) signalling machinery in cancer. Cell Calcium, 2022. 102: p. 102539.
2. Favia, A., et al., NAADP-Dependent Ca(2+) Signaling Controls Melanoma Progression, Metastatic Dissemination and Neoangiogenesis. Sci Rep, 2016.
3. D'Amore, A., et al., Loss of Two-Pore Channel 2 (TPC2) Expression Increases the Metastatic Traits of Melanoma Cells by a Mechanism Involving the Hippo Signalling Pathway and Store-Operated Calcium Entry. Cancers (Basel), 2020. 12(9).
4. Tan, Y., et al., Tumor-derived exosomes: the emerging orchestrators in melanoma. Biomed Pharmacother, 2022. 149: p. 112832.
5. Marzagalli, M., N.D. Ebelt, and E.R. Manuel, Unraveling the crosstalk between melanoma and immune cells in the tumor microenvironment. Semin Cancer Biol, 2019. 59: p. 236-250.
6. Anderson, N.M. and M.C. Simon, The tumor microenvironment. Curr Biol, 2020. 30(16): p. R921-r925.