Investigating the interplay between circadian rhythms and lipid metabolism in the acidic tumor microenvironment

While disruption of circadian rhythms has been largely associated with an increased risk of metabolic disorders including cancer, fewer studies have determined that alterations in clock genes influence the progression of established tumors. The interplay between circadian rhythms and metabolism makes this question highly relevant considering the growing interest for tumor bioenergetics. The host laboratory previously highlighted that a critical shift from glucose to lipid metabolism was observed in cancer cells from the most acidic tumor areas, and that this preference for fatty acids was associated with more aggressive phenotypes. Impacting on circadian rhythms may therefore offer new therapeutic options to target lipid metabolism in cancer cells. The main goals of my Master thesis were to characterize the clock machinery in cancer cells and to evaluate the pharmacological effects of circadian drugs alone or in combination with modulators of lipid metabolism. For this purpose, we used colorectal cancer (CRC) cell lines adapted to acidic pH as well as 3D spheroids that spontaneously recapitulate pH gradients observed in tumors. To assess real-time oscillations, we used cancer cells transduced with lentiviral vectors reporting the activity of two master clock protein BMAL1 and PER2. The link between fatty acid metabolism and circadian rhythms was addressed via drugs targeting the Peroxisome Proliferator-Activated Receptor (PPARs) axis, with a focus on PPARα, a transcription factor supporting fatty acid oxidation and PPARγ known for its critical role in adipogenesis. Our results identified the oscillatory expression of core clock components in CRC cells and revealed different profiles of expression of clock proteins according to the pH condition. We also documented the “druggability” of the circadian machinery. Treatment with KL001, an activator of CRY, a protein known to dimerize with PER2 to co-repress BMAL1, led to a dose-dependent lengthening of oscillation periods. KL001 also induced cell line-dependent growth inhibitory effects together with a reoxygenation of the center of tumor spheroids. Additive and synergistic effects were further observed in 3D spheroids when combining KL001 with GW9662, a PPARγ antagonist or GW6471, a PPARα antagonist. We also evaluated the effects of GO289 and CX4945, two inhibitors of Casein Kinase 2, a protein known to phosphorylate several circadian clock regulators. Both compounds led to a stabilized expression of PER2 associated with a lengthening of the period and a reduced amplitude of the BMAL1 oscillations. Consistent growth inhibitory effects were observed with CX4945 which also displayed a higher affinity for acidic cancer cells. Altogether, our data shed light on the tumor expression of clock genes and the possibility to impact cancer cell growth, tumor microenvironment and lipid metabolism by pharmacologically modulating the activity of major regulators of circadian rhythms.