**6. Conclusions, open questions, and perspectives**

the upregulation of DCT in si-Cav1 cells, we consider that Cav1 acts as a tumor suppressor gene, at least in this early malignant phenotype. The exact mechanism of how Cav1 controls DCT gene expression and how this intersects DCT-mediated processes (**Figure 10C**) needs to

The oncogenic epithelial-mesenchymal transition (EMT) is a multistep process by which epithelial cells acquire invasive mesenchymal phenotype characteristics essential in metastatic spread [121]. EMT is regulated and characterized by molecular mechanisms involving specific transcription factors, signaling pathways, and biomarkers. In melanoma cells which do not have epithelial origin, there is a phenotype switching, with similitudes between the EMT program from development, and this EMT-like switch is a major determinant in tumor metastasis [122]. The role of Cav1 in the oncogenic EMT process is significant but controversial and depends on the type of cancer. In bladder cancer cells, Cav1 promotes invasive phenotypes by inducing EMT [123] in A431 human epidermoid carcinoma cells, the Cav1 downregulation by EGF (an EMT inducer) results in E-cadherin loss, and increased tumor cell invasion [124], whereas in primary tumors of head and neck, squamous cell carcinoma increases EMT and prometastatic properties [125]. During transition from subconfluent (48 h) to confluent (96 h) cultures in MJS, SK28, or MNT-1 cell lines, there is an increase in DCT expression, not observed for either TYR or TRP1 and more abrupt in MJS (VGP) than in MNT or SK28 (metastatic) cells (**Figure 10D**). Oppositely, in the same MJS culture, Cav1 was severely downregulated, in the same cells highly expressing DCT. The most stimulating agent for DCT overexpression is the culture medium exhausted in nutrients but rich in cytokines and growth factors secreted by the tumor cells during 96 h proliferation, whereas changing medium every 24 h has a lower impact on DCT increase (**Figure 10D**). EMT can result from multiple extracellular stimuli; for instance, a synergistic effect on EMT has been observed with combined stimulation of EGF and TGF-β [126]. Interestingly, the cell morphology of MJS, but not SK28 cells was dramatically changed during transition from subconfluent to confluent stage from a polygonal, low-expressing DCT/high-Cav1 to an elongated phenotype high-DCT/low- or negative Cav1 (**Figure 10D**). The same phenotype switching has been observed in si-Cav1 cells highly expressing cytoplasmic DCT. Oppositely, si-DCT cells adopt a wider morphology. We consider that, in MJS phenotype, the DCT and Cav1 crosstalk is a possible part of the EMT program. In subconfluent MJS culture (48 h), groups of 2–4 polygonal cells are interconnected via fine filaments and express low DCT and high Cav1. In confluent culture (96 h), the environmental signals trigger probably, independently, the DCT increase and Cav1 decrease. Furthermore, Cav1 downregulation itself sustains even more the DCT increase. The dynamic analysis of tumor cell populations with Tissue FAXS system demonstrates the perpetuation of a subset of DCT-high/Cav1-low, elongated fibroblast-like cells with long extensions, and forming large clusters (**Figure 10D**). This metamorphosis is an *in vitro* recapitulation of an *in vivo* situation encountered during our analysis of the molecular signature of the DCT+ cells in tumor components of human specimens [38]. The tumor cells in subepidermal layer are DCT+/Cav1+, whereas the ones in deep dermis, a more hostile environment, are DCT+/Cav1- (**Figure 8**). In DCT-phenotype, TYR was always in cells from superficial tumor components,

be deciphered and validated in one or more melanoma cell line(s) in addition to MJS.

**5.4. DCT and melanoma phenotype switching**

72 Human Skin Cancers - Pathways, Mechanisms, Targets and Treatments

TRP2/L-DCT is, undoubtedly, a benefit for the cell expressing it. In melanocytes, the detoxification processes involve the conversion of DCT natural substrate, DHICA into less toxic products. In nonmelanocytic cells, exogenous DCT is able to decrease the effects of oxidative stress acting on substrate analogs. In melanoma, the "preservation" of the expression of certain melanosomal antigens able to ensure tumor cell viability prevails over that of the key-enzymes for pigment production, and TRP2/L-DCT qualifies for this selection. For this prosurvival molecule, the tumor cells reserve complex transcriptional and post-translational mechanisms distinct from the other TRPs. DCT functions as a sensor in case of the autocrine stimulation/stressful conditions when its expression is highly increased, no matter whether the melanogenic pathway is active or not. There is a molecular crosstalk between DCT and Cav1, a master regulator of numerous cellular processes. The members of signaling platforms identified by mass-spectrometry analysis as potential DCT interactors, as well as the impact of DCT expression on cell proliferation, morphology, and cytoskeleton remodeling are strong proofs that DCT is a key player in cellular processes, acting, in our opinion, as a molecular scaffold within one or more signaling hubs. The recent findings about DCT expression pattern in the tumor architecture in correlation with a stable, longlasting/"die-hard" phenotype in benign lesions and with bad prognostic parameters in malignant lesions advocate for considering DCT as a warning indicative of possibly tumor unfavorable outcome.

On the other hand, TRP2/L-DCT has its own vulnerabilities in terms of stability that can be exploited for therapeutic purposes.

In spite of all these information, the role of DCT in melanoma is far from being elucidated or fully exploited and several issues still need clarification: the molecularity behind DCT regulation by Cav1 and DCT impact on Cav1 structural organization; the decipherment of the signaling pathways in which DCT activates, in amelanotic versus pigmented phenotypes in different stages of tumor progression; how are the DCT structural subdomains involved in DCT tumor cell regulatory mechanisms; the DCT role in tumor cell phenotype switching process; the value of DCT phenotype as prognostic indicative; the efficiency of NBDNJ, CQ, as possible adjuvants in melanoma therapeutic strategies; the clarification of DCT expression in nonmelanocytic/nonneuronal cell lines or tumors.

In melanoma, DCT is a double-edged sword, a lethal weapon for cancer cells serving the tumor progression or an exploitable molecular tool for scientists and clinicians to eradicate the malignant cells.
