**5. DCT: a novel molecular driver in melanoma progression**

Our most recent studies in two distinct amelanotic melanoma cell lines representing different tumor phenotypes, MJS and SK28, demonstrate a molecular crosstalk, between DCT and caveolin-1 (Cav1), with structural and functional implications [23].

#### **5.1. DCT is associated with Cav1 membranes**

gene-modified B16 cells worked efficiently as a cellular vaccine to protect animals from parental wild-type tumor challenge [101]. The VacciMax® (VM), a liposome-based antigen delivery platform, has been used to deliver DCT 181–188 in combination with p53-derived peptides. A single administration of VM was capable of inducing an effective CTL response to multiple tumor-associated antigens. The responses generated were able to reject 6-day old B16-F10 tumors [102]. Another plasmide liposome DNA vaccine targeting the DCT in combination with chemokine CCL2 as an adjuvant used xenogeneic (human) DCT in a mouse model and resulted in induction of strong anti-DCT cell-mediated immunity after two vaccinations [103]. A novel vaccine system designed from a long TRP2/DCT peptide with a CD8 epitope (TRP2/ DCT 180-88) and a CD4 epitope (TRP2/DCT 88-102) together with α-galactosyl ceramide, a lipid antigen representing a new class of promissing vaccine adjuvants into cationic liposomes tested on mice tumors resulted in the enhanced production of IFN-ϒ and increased cytotoxic T-cell responses [104]. Importantly, the antitumor immune activity involving MDAs as immunotherapeutic targets may have as side effects the damage (depigmentation) of the normal tissues that also express the MDAs [105]. However, in a patient receiving infusion with TIL586 (recognizing the DCT 109–205 peptide), tumor regression was observed, but not depigmentation [20], which demonstrates that immunotherapy directed against some DCT epitopes is specific and does not affect normal tissues. In another study, the inoculation of plasmid DNA encoding murine DCT elicited antigen-specific CTLs that recognized the B16 mouse melanoma and protected the mice from challenge with tumor cells. Moreover, mice that rejected the tumor did not develop generalized vitiligo, indicating that autoimmunity is not automatically triggered by administrating therapeutic MDA-based vaccines [106]. The vaccination with bone marrow-derived dendritic cells loaded with DCT peptide resulted in activation of high avidity CTLs mediating protective antitumor immunity *in vivo* without the development of adverse autoimmunity [107]. In a murine therapeutical model, four of seven mice with preestablished tumor remained tumor-free for 80 days after therapeutic vaccination with mouse DCT gene-modified dendritic cells, using a HIV-1-based lentiviral vector demonstrating again that DCT gene transfer to dendritic cells is a potent therapeutic strategy in melanoma [108]. A very important aspect is DCT immune-based therapy in glioma. DCT is expressed in glioma cells naturally, and DCT-specific CTLs have been detected in patients' peripheral blood mononuclear cells [109]. On the other hand, DCT overexpression is associated with tumor cell resistance to chemo- or radio-therapeutic treatments. The theory that DCT is a key player in the synergy between chemotherapy and immunotherapy was demonstrated in a clinical study in which tumor cells escaped from vaccination against DCT were more sensitive to chemo-

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

therapy with DNA-damaging drugs.

**perspectives**

cancer drugs [110].

**4.3. Anti-melanoma therapies targeting DCT gene or protein: current status and** 

Despite the already acknowledged DCT involvement in melanoma drug-resistance, there are no reports so far, to our knowledge, about melanoma therapies targeting directly the DCT gene or protein. There is, however, a patent claiming the treatment of melanoma cells *in vitro* with antisense nucleotides targeting DCT mRNA in conjunction with DNA-alklylating antiDCT and Cav1 are present in common structures in cytoplasm or decorating segments of PM (**Figure 10A**). Both Cav1 monomers/oligomers and DCT-precursor/mature forms have the same distribution along a density gradient in an ultracentrifugation experiment. Moreover, Cav1 has been identified in western blot and mass spectrometry analysis of the immunoprecipitates obtained with anti-DCT antibody from MJS cell lysates [23]. These experimental data are strongly supported by the structural analysis of DCT and Cav1 and by DCT-Cav1 structural model presented in Section 2.1.

#### **5.2. DCT regulates Cav1 assembly and stability and possibly Cav1-mediated cellular processes**

The transient downregulation of DCT expression (si-DCT) in MJS and SK28 cells increased the amount of Cav1 protein by its redistribution into more stable, insoluble membrane aggregates with altered morphologies [23] (**Figure 10A**). This is the first report about a melanosomal protein that regulates Cav1 assembly. We postulate that DCT may regulate Cav1 and/or lipid raft structures by competing either with different signaling molecules for Cav1 binding or with Cav1 monomers for Cav1 oligomerization domain or for cholesterol binding. Both caveolae and Cav1-scaffolds are associated with lipid rafts, which are membrane domains with a very dynamic structure abundant in cholesterol, sphingolipids recruiting different molecular players of signaling platforms, and controlling numerous and diverse cellular processes [113]. Either directly or indirectly, DCT as a major regulator of Cav1- or cholesterol-membrane architecture is thus expected to impact also different cellular events mediated by Cav1 (**Figure 10C**). For example, the interaction of membrane/lipid rafts, with the cytoskeleton, has impact on trafficking and sorting mechanisms, formation of platforms for cell anchorage to ECM, transduction of signaling cascades across the PM, cell growth and migration, entry of microorganisms (viruses/bacteria), and toxins or nanoparticles [114]. Indeed, we also observed that in MJS cells having downregulated DCT expression, there was an increase in cell volume, a significant redistribution of actin filaments in cell periphery, and a dramatic decrease in cell proliferation by 20 at 48, 60 at 72, and 75% at 96 h coupled with the cell cycle arrest in G1 [unpublished data]. Interestingly, these effects were less prominent in SK28 phenotype that indicates that DCT-mediated processes are tumor phenotype specific. Importantly, our mass spectrometry analysis of immunoprecipitates obtained from MJS cell lysates with anti-DCT antibodies against N- or C-terminus epitopes has identified as potential DCT interactors, regulators of small GTPases (Arf, Rho and Ras) and numerous proteins involved in anti-apoptotic, proliferative, migration, and invasion mechanisms and pathways [unpublished data]. The structural analysis pointed also the possibility that two Ser residues within DCT CYT subdomain to be phosphorylated (Section 2.1). Our theory based on all these data and preliminary information is that DCT, placed in a molecular environment with Cav1, is a key-molecular player acting on one or more signaling pathways involved in tumor cell survival and morphology, either by itself, as a potential target of the phosphorylation cascades, or as modulator of Cav1 or other participants in regulatory processes (**Figure 10C**). The numerous potential interactors present DCT as a possible new molecular scaffold. Further experimental studies are required to validate these interactions and place DCT in the exact pathway(s) where it operates.

#### **5.3. Cav1 controls DCT gene expression, protein processing, and subcellular distribution**

The Cav1 downregulation (si-Cav1) has a dramatic impact on DCT in MJS cells. There is a 20-fold increase over 96 h of Cav1 silencing on DCT mRNA level. Accordingly, there is also a protein increase detected by western blot, and the deglycosylation experiments showed that DCT synthetized in si-Cav1 cells is mainly DCT-precursor. The imagistic studies of confocal immunofluorescence microscopy and Tissue FAXS cytometry quantitative analysis revealed a 7-fold increase in a DCT-population with intense cytoplasmic, but no PM, DCT staining, the "DCT-high clones" (**Figure 10B**). This is the first report about a melanosomal protein/ melanoma antigen-regulated by Cav1 and a novel target gene for Cav1. Cav1 is a regulator of several genes as CyclinD or folate receptor promoters [115] or for survivin, a member of the Inhibitor Apoptosis Protein-family [116]. In melanoma, Cav1 function is still ambiguous. In some studies, Cav1 is associated with tumorigenicity [117], whereas others present Cav1 as a tumor suppressor by inhibiting Wnt-β-catenin-TCF/LEF [118], Src/FAK [119] pathways, or attenuating tumor cell motility by disrupting glycosphingolipid GD3-mediated malignant signaling [120]. In the context of DCT-mediating pro-survival and resistance pathways and

**Figure 10.** The structural and functional relationship between DCT and Cav-1. (A) MJS and SK28 amelanotic melanoma cells immunostained for DCT and Cav1 and analyzed by confocal fluorescence microscopy demonstrate DCT and Cav1 in cytoplasmic and PM common structures; in DCT downregulated cells, the morphologies of Cav1 positive structures are severely altered. The fourth and the sixth panels represent the enlarged details of the indicated insets; (B) the DCT-high clones in MJS having downregulated Cav1 expression analyzed by tissue FAXS. In the upper part of quadrant are shown the cells with high DCT expression; (C) the crosstalk between DCT and Cav1. The impact of si-DCT on Cav1 and of si-Cav1 on DCT is indicated. Possible processes mediated by either DCT or Cav1 are indicated in dotted boxes; (D) DCT, unlike TYR or TRP1 is overexpressed during transition from subconfluent (48 h) to semi-confluent (72 h) and confluent (96 h). Medium was not replenished for 96 h (MR−) or replenished every 24 h (MR+). Autocrine/ paracrine stimulation (starvation, secreted factors by proliferative MJS tumor cells within 48 h) decrease Cav1, increase DCT expressions, and change the cell morphology. The cells at 48 h are polygonal with visible contacts between adjacent

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cells, whereas cells at 96 h are elongated with no cell-cell contacts and form large clusters.

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binding or with Cav1 monomers for Cav1 oligomerization domain or for cholesterol binding. Both caveolae and Cav1-scaffolds are associated with lipid rafts, which are membrane domains with a very dynamic structure abundant in cholesterol, sphingolipids recruiting different molecular players of signaling platforms, and controlling numerous and diverse cellular processes [113]. Either directly or indirectly, DCT as a major regulator of Cav1- or cholesterol-membrane architecture is thus expected to impact also different cellular events mediated by Cav1 (**Figure 10C**). For example, the interaction of membrane/lipid rafts, with the cytoskeleton, has impact on trafficking and sorting mechanisms, formation of platforms for cell anchorage to ECM, transduction of signaling cascades across the PM, cell growth and migration, entry of microorganisms (viruses/bacteria), and toxins or nanoparticles [114]. Indeed, we also observed that in MJS cells having downregulated DCT expression, there was an increase in cell volume, a significant redistribution of actin filaments in cell periphery, and a dramatic decrease in cell proliferation by 20 at 48, 60 at 72, and 75% at 96 h coupled with the cell cycle arrest in G1 [unpublished data]. Interestingly, these effects were less prominent in SK28 phenotype that indicates that DCT-mediated processes are tumor phenotype specific. Importantly, our mass spectrometry analysis of immunoprecipitates obtained from MJS cell lysates with anti-DCT antibodies against N- or C-terminus epitopes has identified as potential DCT interactors, regulators of small GTPases (Arf, Rho and Ras) and numerous proteins involved in anti-apoptotic, proliferative, migration, and invasion mechanisms and pathways [unpublished data]. The structural analysis pointed also the possibility that two Ser residues within DCT CYT subdomain to be phosphorylated (Section 2.1). Our theory based on all these data and preliminary information is that DCT, placed in a molecular environment with Cav1, is a key-molecular player acting on one or more signaling pathways involved in tumor cell survival and morphology, either by itself, as a potential target of the phosphorylation cascades, or as modulator of Cav1 or other participants in regulatory processes (**Figure 10C**). The numerous potential interactors present DCT as a possible new molecular scaffold. Further experimental studies are required to validate these interactions and place DCT in the exact

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

**5.3. Cav1 controls DCT gene expression, protein processing, and subcellular distribution**

The Cav1 downregulation (si-Cav1) has a dramatic impact on DCT in MJS cells. There is a 20-fold increase over 96 h of Cav1 silencing on DCT mRNA level. Accordingly, there is also a protein increase detected by western blot, and the deglycosylation experiments showed that DCT synthetized in si-Cav1 cells is mainly DCT-precursor. The imagistic studies of confocal immunofluorescence microscopy and Tissue FAXS cytometry quantitative analysis revealed a 7-fold increase in a DCT-population with intense cytoplasmic, but no PM, DCT staining, the "DCT-high clones" (**Figure 10B**). This is the first report about a melanosomal protein/ melanoma antigen-regulated by Cav1 and a novel target gene for Cav1. Cav1 is a regulator of several genes as CyclinD or folate receptor promoters [115] or for survivin, a member of the Inhibitor Apoptosis Protein-family [116]. In melanoma, Cav1 function is still ambiguous. In some studies, Cav1 is associated with tumorigenicity [117], whereas others present Cav1 as a tumor suppressor by inhibiting Wnt-β-catenin-TCF/LEF [118], Src/FAK [119] pathways, or attenuating tumor cell motility by disrupting glycosphingolipid GD3-mediated malignant signaling [120]. In the context of DCT-mediating pro-survival and resistance pathways and

pathway(s) where it operates.

**Figure 10.** The structural and functional relationship between DCT and Cav-1. (A) MJS and SK28 amelanotic melanoma cells immunostained for DCT and Cav1 and analyzed by confocal fluorescence microscopy demonstrate DCT and Cav1 in cytoplasmic and PM common structures; in DCT downregulated cells, the morphologies of Cav1 positive structures are severely altered. The fourth and the sixth panels represent the enlarged details of the indicated insets; (B) the DCT-high clones in MJS having downregulated Cav1 expression analyzed by tissue FAXS. In the upper part of quadrant are shown the cells with high DCT expression; (C) the crosstalk between DCT and Cav1. The impact of si-DCT on Cav1 and of si-Cav1 on DCT is indicated. Possible processes mediated by either DCT or Cav1 are indicated in dotted boxes; (D) DCT, unlike TYR or TRP1 is overexpressed during transition from subconfluent (48 h) to semi-confluent (72 h) and confluent (96 h). Medium was not replenished for 96 h (MR−) or replenished every 24 h (MR+). Autocrine/ paracrine stimulation (starvation, secreted factors by proliferative MJS tumor cells within 48 h) decrease Cav1, increase DCT expressions, and change the cell morphology. The cells at 48 h are polygonal with visible contacts between adjacent cells, whereas cells at 96 h are elongated with no cell-cell contacts and form large clusters.

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

whereas DCT was in the deep dermis ones. This is in good correlation with data showing that in MNT-1 cells expressing all TRPs, during autocrine stimulation only DCT expression is increased [23]. The cross talk between DCT and Cav1, DCT as gene target of autocrine/ paracrine stimulation as well as the impact of DCT expression on tumor cell-phenotype proliferation and morphology introduce DCT in the complex signaling pathways and networks

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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 consid-

regulating tumor progression.

exploited for therapeutic purposes.

the malignant cells.

in nonmelanocytic/nonneuronal cell lines or tumors.

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

ering 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

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 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

#### **5.4. DCT and melanoma phenotype switching**

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, whereas DCT was in the deep dermis ones. This is in good correlation with data showing that in MNT-1 cells expressing all TRPs, during autocrine stimulation only DCT expression is increased [23]. The cross talk between DCT and Cav1, DCT as gene target of autocrine/ paracrine stimulation as well as the impact of DCT expression on tumor cell-phenotype proliferation and morphology introduce DCT in the complex signaling pathways and networks regulating tumor progression.
