The role of two-pore channels 2 (TPC2) in melanoma tumourigenesis and metastasis

Thesis

Melanoma is an exceedingly aggressive and resistant to treatment form of cancer. Therefore, the identification of novel therapeutic approaches continues to be a crucial task. Understanding the pathophysiological role of potential drug targets for this disease enables the discovery of new therapeutic strategies. The two-pore channel 2, TPC2, is located on late endosomes, lysosomes, and melanosomes. Here, we evaluated the effect of TPC2 knockout (KO) on human metastatic melanoma (CHL-1) and murine primary melanoma (B16) cells. TPC2- depleted melanoma cell lines were created using CRISPR-Cas9 gene editing. CHL-1, an amelanotic metastatic melanoma cell line, exhibited in vitro induced migratory traits after TPC2 depletion. In contrast, B16 TPC2 KO cells displayed a diminished ability to migrate in vitro. TPC2 KO activated genes regulated by YAP/TAZ, which are important regulators of tumorigenesis and metastasis, in CHL-1. TPC2 KO and RNA interference knockdown decreased the expression levels of ORAI1, a component of store-operated Ca2+ entry (SOCE), and PKC-II, a component of the HIPPO pathway that adversely affects YAP/TAZ activity. These findings are explained by a cellular mechanism mediated by ORAI1/ Ca2+/PKC-II.

Furthermore, CHL-1 and B16 TPC2 KO proteomic signatures were examined to WT cells. The differential expression of proteins between WT and KO cells enables us to evaluate TPC2's role in various crucial pathways and processes. Accordingly, the bioenergetic phenotype of TPC2-deficient cells was assessed. TPC2 depletion boosted ATP synthesis in CHL-1 and B16 TPC2 KO cells relative to WT cells, but increased lactate generation in B16 cells. TPC2 deficiency enhanced anaerobic respiration in B16 cells but reduced it in CHL-1 cells. OCR/ECAR analysis confirmed TPC2-KO cells' metabolic shift. CHL-1 TPC2 KO cells are more aerobic due to a reduced OCR/ECAR ratio. OCR/ECAR was elevated in B16 KO cells in complete media. B16 KO cells increased oxygen utilisation and lactate production without glucose. In addition, the cytoskeleton and synthesis and release of melanin were altered in TPC2 KO cells.

In vivo evaluation of B16 TPC2 KO melanoma cell lines following subcutaneous injection revealed a significantly reduced tumour volume compared to WT cells. However, histopathological examination of mice injected with KO cells revealed that the hepatic and splenic structures were drastically changed.

Clinical association is vital to determining correlations between molecular discoveries and cancer patient survival. TPC2 high-expression patients had a shorter overall survival time than low-expression patients. Furthermore, high TPC2 expression is associated with lower survival in BRAF mutant melanoma. In summary, the work presented in this thesis indicates that TPC2 plays vital functions in melanoma's progression, invasion, and metastasis.

Authors: Hanbashi, AA

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: TPC2; melanoma; CRISPR-Cas9; Two-Pore Channel 2 (TPC2)
• Subjects: The role of two-pore channels 2 (TPC2) in melanoma