Melanoma is the most lethal type of skin cancer, with increasing incidence and mortality rates worldwide. Multiple studies have demonstrated a link between cancer development/progression and circadian disruption; however, the complex role of tumor-autonomous molecular clocks remains poorly understood. With that in mind, we investigated the pathophysiological relevance of clock gene expression in metastatic melanoma. Methods: We analyzed gene expression, somatic mutation, and clinical data from 340 metastatic melanomas from The Cancer Genome Atlas, as well as gene expression data from 234 normal skin samples from Genotype-Tissue Expression. Findings were confirmed in two independent datasets. Results: In melanomas, the expression of most clock genes was remarkably reduced and displayed a disrupted pattern of co-expression compared to the normal skins, indicating a dysfunctional circadian clock. Importantly, we demonstrate that the expression of the clock gene BMAL1 positively correlates with patient overall survival and with the expression of T-cell activity and exhaustion markers in the tumor bulk. Accordingly, high BMAL1 expression in pre-treatment samples was significantly associated with clinical benefit from immune checkpoint inhibitors. The robust intratumoral T-cell infiltration/activation observed in patients with high BMAL1 expression was associated with a decreased expression of key DNA repair enzymes, and with an increased mutational/neoantigen load. Conclusions: Overall, our data corroborate previous reports regarding the impact of BMAL1 expression on the cellular DNA repair capacity and indicate that alterations in the tumor-autonomous molecular clock could influence the cellular composition of the surrounding microenvironment. Moreover, we revealed the potential of BMAL1 as a clinically relevant prognostic factor and biomarker for T-cell-based immunotherapies.

Circadian rhythm may be understood as a temporal organization that works to orchestrate physiological processes and behavior in a period of approximately 24 h. Because such temporal organization has evolved in the presence of predictable environmental clues, such as day length, tides, seasons, and temperature, the organism has confronted the natural selection in highly precise intervals of opportunities and risks, generating temporal programs and resetting mechanisms, which are well conserved among different taxa of animals. The present review brings some evidence of how these programs may have co-evolved in systems able to deal with 2 or more environmental clues, and how they similarly function in different group of animals, stressing how important temperature and light were to establish the temporal organizations. For example, melanopsin and rhodopsin, photopigments present respectively in circadian and visual photoreceptors, are required for temperature discrimination in Drosophila melanogaster. These pigments may signal light and temperature via activation of cationic membrane channel, named transient-receptor potential channel (TRP). In fact, TRPs have been suggested to function as thermal sensor for various groups of animals. Another example is the clock machinery at the molecular level. A set of very-well conserved proteins, known as clock proteins, function as transcription factors in positive and negative auto-regulatory loops generating circadian changes of their expression, and of clock-controlled genes. Similar molecular machinery is present in organisms as diverse as cyanobacteria (Synechococcus), fungi (Neurospora), insects (Drosophila), and vertebrates including humans.

Melanopsin has been implicated in the mammalian photoentrainment by blue light. This photopigment, which maximally absorbs light at wavelengths between 470 and 480 nm depending on the species, is found in the retina of all classes of vertebrates so far studied. In mammals, melanopsin activation triggers a signaling pathway which resets the circadian clock in the suprachiasmatic nucleus (SCN). Unlike mammals, Drosophila melanogaster and Danio rerio do not rely only on their eyes to perceive light, in fact their whole body may be capable of detecting light and entraining their circadian clock. Melanopsin, teleost multiple tissue (tmt) opsin and others such as neuropsin and va-opsin, are found in the peripheral tissues of Danio rerio, however, there are limited data concerning the photopigment/s or the signaling pathway/s directly involved in light detection. Here, we demonstrate that melanopsin is a strong candidate to mediate synchronization of zebrafish cells. The deduced amino acid sequence of melanopsin, although being a vertebrate opsin, is more similar to invertebrate than vertebrate photopigments, and melanopsin photostimulation triggers the phosphoinositide pathway through activation of a G_q/11-type G protein. We stimulated cultured ZEM-2S cells with blue light at wavelengths consistent with melanopsin maximal absorption, and evaluated the time course expression of per1b, cry1b, per2 and cry1a. Using quantitative PCR, we showed that blue light is capable of slightly modulating per1b and cry1b genes, and drastically increasing per2 and cry1a expression. Pharmacological assays indicated that per2 and cry1a responses to blue light are evoked through the activation of the phosphoinositide pathway, which crosstalks with nitric oxide (NO) and mitogen activated protein MAP kinase (MAPK) to activate the clock genes. Our results suggest that melanopsin may be important in mediating the photoresponse in Danio rerio ZEM-2S cells, and provide new insights about the modulation of clock genes in peripheral clocks.