Human TIMELESS, a potential circadian clock regulator, plays an essential role in survival and reawakening of metastasis-initiating cells in bone

Bone metastasis is becoming increasingly common globally. In such cases, cancer cells disseminate from the primary site to bone, subsequently entering a dormant state. Following a certain incubation period, the cells reawaken and grow, forming a metastatic mass. These dormant cells are called metastasis-initiating cells (MICs), but their survival and reawakening are poorly understood. Here, we established an in vivo MIC-reawakening mouse model in bone. Microarray analysis demonstrated enrichment for mitochondrial oxidative phosphorylation (OXPHOS) and fatty acid synthesis in isolated MICs. In addition, transcriptional regulator TIMELESS was identified as an independent poor prognostic factor by using clinical transcriptomic datasets, which was validated by immunostaining on 209 breast cancer cases. TIMELESS-deficient breast, prostate, and bladder cancer cell lines exhibited reduced viability and MIC reawakening in bone. Single-cell analysis on the epigenetic landscape of MICs revealed that motif activities of CLOCK and BMAL1/ARNTL were enhanced in a cluster with increased TIMELESS accessibility scores. As TIMELESS regulates stemness through the OXPHOS metabolic state, an inhibitor targeting the metabolic pathway, MP-A08, was identified to suppress MIC reawakening and growth in bone more efficiently than cisplatin. These results suggest that TIMELESS regulates survival and reawakening in MICs and MP-A08 could contribute to adjuvant therapeutic strategies.