BRD9 status is a major contributor for cysteine metabolic remodeling through MST and EAAT3 modulation in malignant melanoma.

Cutaneous melanoma (CM) is the most aggressive skin cancer, showing globally increasing incidence. Hereditary CM accounts for a significant percentage (5-15 %) of all CM cases. However, most familial cases remain without a known genetic cause. Even though, BRD9 has been associated to CM as a susceptibility gene. The molecular events following BRD9 mutagenesis are still not completely understood. In this study, we disclosed BRD9 as a key regulator in cysteine metabolism and associated altered BRD9 to increased cell proliferation, migration and invasiveness, as well as to altered melanin levels, inducing higher susceptibility to melanomagenesis. It is evident that BRD9 WT and mutated BRD9 (c.183G>C) have a different impact on cysteine metabolism, respectively by inhibiting and activating MPST expression in the metastatic A375 cell line. The effect of the mutated BRD9 variant was more evident in A375 cells than in the less invasive WM115 line. Our data point out novel molecular and metabolic mechanisms dependent on BRD9 status that potentially account for the increased risk of developing CM and enhancing CM aggressiveness. Moreover, our findings emphasize the role of cysteine metabolism remodeling in melanoma progression and open new queues to follow to explore the role of BRD9 as a melanoma susceptibility or cancer-related gene.

Melanin's Journey from Melanocytes to Keratinocytes: Uncovering the Molecular Mechanisms of Melanin Transfer and Processing.

Skin pigmentation ensures efficient photoprotection and relies on the pigment melanin, which is produced by epidermal melanocytes and transferred to surrounding keratinocytes. While the molecular mechanisms of melanin synthesis and transport in melanocytes are now well characterized, much less is known about melanin transfer and processing within keratinocytes. Over the past few decades, distinct models have been proposed to explain how melanin transfer occurs at the cellular and molecular levels. However, this remains a debated topic, as up to four different models have been proposed, with evidence presented supporting each. Here, we review the current knowledge on the regulation of melanin exocytosis, internalization, processing, and polarization. Regarding the different transfer models, we discuss how these might co-exist to regulate skin pigmentation under different conditions, i.e., constitutive and facultative skin pigmentation or physiological and pathological conditions. Moreover, we discuss recent evidence that sheds light on the regulation of melanin exocytosis by melanocytes and internalization by keratinocytes, as well as how melanin is stored within these cells in a compartment that we propose be named the melanokerasome. Finally, we review the state of the art on the molecular mechanisms that lead to melanokerasome positioning above the nuclei of keratinocytes, forming supranuclear caps that shield the nuclear DNA from UV radiation. Thus, we provide a comprehensive overview of the current knowledge on the molecular mechanisms regulating skin pigmentation, from melanin exocytosis by melanocytes and internalization by keratinocytes to processing and polarization within keratinocytes. A better knowledge of these molecular mechanisms will clarify long-lasting questions in the field that are crucial for the understanding of skin pigmentation and can shed light on fundamental aspects of organelle biology. Ultimately, this knowledge can lead to novel therapeutic strategies to treat hypo- or hyper-pigmentation disorders, which have a high socio-economic burden on patients and healthcare systems worldwide, as well as cosmetic applications.

RAB3A Regulates Melanin Exocytosis and Transfer Induced by Keratinocyte-Conditioned Medium.

Skin pigmentation is imparted by melanin and is crucial for photoprotection against UVR. Melanin is synthesized and packaged into melanosomes within melanocytes and is then transferred to keratinocytes (KCs). Although the molecular players involved in melanogenesis have been extensively studied, those underlying melanin transfer remain unclear. Previously, our group proposed that coupled exocytosis/phagocytosis is the predominant mechanism of melanin transfer in human skin and showed an essential role for RAB11B and the exocyst tethering complex in this process. In this study, we show that soluble factors present in KC-conditioned medium stimulate melanin exocytosis from melanocytes and transfer to KCs. Moreover, we found that these factors are released by differentiated KCs but not by basal layer KCs. Furthermore, we found that RAB3A regulates melanin exocytosis and transfer stimulated by KC-conditioned medium. Indeed, KC-conditioned medium enhances the recruitment of RAB3A to melanosomes in melanocyte dendrites. Therefore, our results suggest the existence of two distinct routes of melanin exocytosis: a basal route controlled by RAB11B and a RAB3A-dependent route, stimulated by KC-conditioned medium. Thus, this study provides evidence that soluble factors released by differentiated KCs control skin pigmentation by promoting the accumulation of RAB3A-positive melanosomes in melanocyte dendrites and their release and subsequent transfer to KCs.

The Dark Side of Melanin Secretion in Cutaneous Melanoma Aggressiveness.

Skin cancers are among the most common cancers worldwide and are increasingly prevalent. Cutaneous melanoma (CM) is characterized by the malignant transformation of melanocytes in the epidermis. Although CM shows lower incidence than other skin cancers, it is the most aggressive and responsible for the vast majority of skin cancer-related deaths. Indeed, 75% of patients present with invasive or metastatic tumors, even after surgical excision. In CM, the photoprotective pigment melanin, which is produced by melanocytes, plays a central role in the pathology of the disease. Melanin absorbs ultraviolet radiation and scavenges reactive oxygen/nitrogen species (ROS/RNS) resulting from the radiation exposure. However, the scavenged ROS/RNS modify melanin and lead to the induction of signature DNA damage in CM cells, namely cyclobutane pyrimidine dimers, which are known to promote CM immortalization and carcinogenesis. Despite triggering the malignant transformation of melanocytes and promoting initial tumor growth, the presence of melanin inside CM cells is described to negatively regulate their invasiveness by increasing cell stiffness and reducing elasticity. Emerging evidence also indicates that melanin secreted from CM cells is required for the immunomodulation of tumor microenvironment. Indeed, melanin transforms dermal fibroblasts in cancer-associated fibroblasts, suppresses the immune system and promotes tumor angiogenesis, thus sustaining CM progression and metastasis. Here, we review the current knowledge on the role of melanin secretion in CM aggressiveness and the molecular machinery involved, as well as the impact in tumor microenvironment and immune responses. A better understanding of this role and the molecular players involved could enable the modulation of melanin secretion to become a therapeutic strategy to impair CM invasion and metastasis and, hence, reduce the burden of CM-associated deaths.

Melanocore uptake by keratinocytes occurs through phagocytosis and involves protease-activated receptor-2 internalization.

In the skin epidermis, melanin is produced and stored within melanosomes in melanocytes, and then transferred to keratinocytes. Different models have been proposed to explain the melanin transfer mechanism, which differ essentially in how melanin is transferred-either in a membrane-bound melanosome or as a melanosome core, that is, melanocore. Here, we investigated the endocytic route followed by melanocores and melanosomes during internalization by keratinocytes, by comparing the uptake of melanocores isolated from the supernatant of melanocyte cultures, with melanosomes isolated from melanocytes. We show that inhibition of actin dynamics impairs the uptake of both melanocores and melanosomes. Moreover, depletion of critical proteins involved in actin-dependent uptake mechanisms, namely Rac1, CtBP1/BARS, Cdc42 or RhoA, together with inhibition of Rac1-dependent signaling pathways or macropinocytosis suggest that melanocores are internalized by phagocytosis, whereas melanosomes are internalized by macropinocytosis. Interestingly, we found that Rac1, Cdc42 and RhoA are differently activated by melanocore or melanosome stimulation, supporting the existence of two distinct routes of melanin internalization. Furthermore, we show that melanocore uptake induces protease-activated receptor-2 (PAR-2) internalization by keratinocytes to a higher extent than melanosomes. Because skin pigmentation was shown to be regulated by PAR-2 activation, our results further support the melanocore-based mechanism of melanin transfer and further refine this model, which can now be described as coupled melanocore exo/phagocytosis.