Tuning circadian rhythm of CAFs against cancer progression

Circadian disruption orchestrates various biological functions associated with carcinogenesis and tumor progression in cancer cells. Yet the influential role in the tumor microenvironment (TME), including cancer-associated fibroblasts (CAFs), remains unveiled. Here, we report an uncovered signaling axis, BMAL1-LHX8, that elicits the transitioning of CAF-like properties via coordinating the tumor-supportive activity in ameloblastoma (AM) stromal. Both in the co-culture system and stroma-rich AM tumoroid model, CAFs augmented tumor growth, cell proliferation, and metabolic activities. Time-series RNA-sequencing analysis indicated the perturbation of circadian rhythm in the AM+CAF group and LHX8 was considerably involved. CRISPR/cas9 system-based loss-of-function study suggested that the BMAL1-LHX8 signaling axis was highly correlated with several tumor-supportive activities, including secretion of growth factors and ECM remodeling molecules. It also contributed to the resistance of BRAF inhibitor (PLX4032), which could be effectively suppressed by the clock regulator (GSK4112, an agonist of REV-ERB which suppresses BMAL1 expression). This phenomenon confirmed that GSK4112 elevated the anti-tumor efficacy of PLX4032 in the xenograft model. Collectively, these data underscore the role of the BMAL1-LHX8 axis in exacerbating the CAF-like transitioning in AM stromal and tuning the circadian rhythm via clock regulators. This research offering the experimental evidence for the development of chrono-chemotherapy and offering new possibilities for the cancer treatment in the future. Overall design: Comparative gene expression profiling analysis of RNA-seq data among stroma-rich ameloblastoma tumoroids (ameloblastoma tumoroids enriched with normal fibroblasts or ameloblastoma tumoroids enriched with cancer-associated fibroblasts derived from ameloblastoma patients) at time points of 0,8,16,24,32,40,48 h after synchronized with dexamethasone.