Core Molecular Clock Factors Regulate Osteosarcoma Stem Cell Survival and Behavior via CSC/EMT Pathways and Lipid Droplet Biogenesis

The circadian clock, an intrinsic 24 h cellular timekeeping system, regulates fundamental biological processes, including tumor physiology and metabolism. Cancer stem cells (CSCs), a subpopulation of cancer cells with self-renewal and tumorigenic capacities, are implicated in tumor initiation, recurrence, and metastasis. Despite growing evidence for the circadian clock's involvement in regulating CSC functions, its precise regulatory mechanisms remain largely unknown. Here, using a human osteosarcoma (OS) model (143B), we have shown that core molecular clock factors are critical for OS stem cell survival and behavior via direct modulation of CSC and lipid metabolic pathways. In single-cell-derived spheroid formation assays, 143B OS cells exhibited robust spheroid-forming capacity under 3D culture conditions. Furthermore, siRNA-mediated depletion of core clock components (i.e., <i>BMAL1</i>, <i>CLOCK</i>, <i>CRY1/2</i>, <i>PER1/2</i>)-essential positive and negative elements of the circadian clock feedback loop-significantly reduced spheroid formation in 143B CSCs isolated from in vivo OS xenografts. In contrast, knockdown of the secondary clock-stabilizing factor genes <i>NR1D1</i> and <i>NR1D2</i> had little effect. We also found that knockdown of <i>BMAL1, CLOCK</i>, or <i>CRY1/2</i> markedly impaired the migration and invasion capacities of 143B CSCs. At the molecular level, silencing of <i>BMAL1, CLOCK,</i> or <i>CRY1/2</i> distinctly altered the expression of genes associated with stem cell properties and the epithelial-mesenchymal transition (EMT) in 143B CSCs. In addition, disruption of <i>BMAL1</i>, <i>CLOCK</i>, or <i>CRY1/2</i> expression significantly reduced lipid droplet formation by downregulating the expression of genes involved in lipogenesis (e.g., <i>DGAT1</i>, <i>FASN</i>, <i>ACSL4</i>, <i>PKM2</i>, <i>CHKA</i>, <i>SREBP1</i>), which are closely linked to CSC/EMT processes. Furthermore, transcriptomic analysis of human OS patient samples revealed that compared with other core clock genes, <i>CRY1</i> was highly expressed in OS tumors relative to controls, and its expression exhibited strong positive correlations with patient prognosis, survival, and LD biogenesis gene expression. These findings highlight the critical role of the molecular circadian clock in regulating CSC properties and metabolism, underscoring the therapeutic potential of targeting the core clock machinery to enhance OS treatment outcomes.