TMEM165-CLOCK fusion protein drives metastasis through enhanced lysosomal exocytosis

TMEM165-CLOCK is a chimeric protein identified in osteosarcoma, where its expression correlates with poor prognosis. The fusion incorporates CLOCK in reverse orientation without clear functional contribution but truncates the C-terminal transmembrane domain of TMEM165, potentially altering its subcellular localization and function. We investigated its clinical impact and underlying mechanisms. TMEM165-CLOCK expression was quantified by RT-qPCR in UPS and NSCLC cohorts and correlated with clinical outcomes. Subcellular localization was assessed by colocalization imaging. Calcium flux, lysosomal exocytosis, and cathepsin S secretion were compared between parental KHOS/NP cells and TMEM165-CLOCK-overexpressing KHOS/NP cells. Tumor aggressiveness was evaluated by migration, invasion, and a rat metastasis model. RT-qPCR analysis in the UPS cohort showed that higher TMEM165-CLOCK expression correlated with worse progression-free survival (HR: 2.71, p=0.040). In the lung cancer cohort, TMEM165-CLOCK expression significantly correlated with advanced tumor stage (p= 0.038) and nodal stage (p=0.014) at presentation, suggesting cross-cancer relevance. Colocalization imaging revealed a marked shift from Golgi localization to increased presence at lysosomes and the plasma membrane. His-tag imaging confirmed C-terminal exposure on the cell surface, rendering the fusion protein as a druggable target. Despite this redistribution, TMEM165-CLOCK did not alter Golgi glycosylation, as glycosylated LAMP2 levels remained unchanged. In contrast, TMEM165-CLOCK-overexpressing KHOS/NP cells displayed impaired ATP-induced calcium dynamics, with reduced peak amplitude and delayed recovery to baseline, consistent with disrupted calcium homeostasis. These cells showed enhanced lysosomal exocytosis, evidenced by increased surface LAMP1 exposure, and significantly elevated cathepsin S secretion. Functionally, TMEM165-CLOCK overexpression increased cell migration and invasion. siRNA-mediated knockdown of the fusion protein rescued the invasive phenotype, establishing causality. In vivo, rats injected with TMEM165-CLOCK-expressing UPS cells developed markedly greater metastatic burden compared to controls, confirming the fusion's pro-metastatic activity. TMEM165-CLOCK promotes tumor progression through mechanism involving subcellular relocalization, disrupted calcium homeostasis, and enhanced lysosomal exocytosis with cathepsin S release. Its association with aggressive disease across sarcoma and lung cancer identifies TMEM165-CLOCK as a driver of metastasis and potential therapeutic target.