**4. DCT as target in melanoma therapy**

#### **4.1. Anti-melanoma therapies**

The surgical removal is the only cure for melanoma with the condition that the excised lesion be in an early stage. However, the micrometastases cannot be addressed exclusively by the surgery and therefore, combinatorial therapeutical strategies are applied in the attempt to extend survival rates. The treatment options in melanoma are continuously revised, and there are several excellent reviews about this topic [98–100]. The schematic representation of the treatment of metastatic melanoma including different approaches is shown in **Figure 9**.

#### **4.2. Anti-melanoma immunotherapeutic strategies involving DCT**

The identification of different T-cell clones in melanoma patients recognizing peptides derived from DCT (**Figure 2**) raised the interest for this antigen in the development of anti-melanoma immunotherapeutical strategies. The cellular vaccine engineered to co-express a DCT epitope, with IFN-γ in the same gene by replacing the IFN-γ signal peptide with a DCT epitope-expressing signal peptide, resulted in decreased B16 tumorigenicity and enhanced immunogenicity after gene transfer. More importantly, irradiated transiently, TRP-2 epitope-expressing, IFN-c

**Figure 9.** The therapies in melanoma. The different anti-melanoma treatment strategies are presented. DCT-based therapies are integrated part of the targeted therapies. The solid lines indicate the already existing therapies, whereas the dotted lines are proposed as possible adjuvant therapies based on the molecular studies about DCT intracellular processing and stability in melanoma cells. The melanoma specimen is an ulcerated nodular melanoma of a 26-yearold man, from lumbar region (by courtesy of Dr. S. Zurac, Department of Pathology, Colentina University Hospital,

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Bucharest, Romania).

in benign specimens with high neurotization and also in some early malignant ones having low Breslow/Clark indexes but with ulceration. Our theory is that DCT-phenotype is emblematic for a long-lasting, "die-hard" phenotype. The DCT-intense expression is observed in large areas of compound neurotized nevi contributing probably to the well-acknowledged enhanced stability and low proliferation rate of these nevus cells [95] and may not represent a life-threatening problem in benign tumors. However, the superficial malignant melanomas, with low indexes Clark or Breslow but having DCT-phenotype could be a warning signal for considering those specimens as ones of high risk with a possible unfavorable prognostic. The DCT-clones selected in inner dermis of early malignant lesions acquire the expression and subcellular distribution of molecular markers reported to be associated with different types of neoplasms, including melanoma, with extended migratory capacities (caveolin-1-), survival in stressful conditions (cytoplasmic Hif-1α+), activated anti-apoptotic mechanisms (cytoplasmic cyclin D+ and Bcl-1+), angiogenic, and metastatic potential (cytoplasmic cyclin E+) (**Figure 8B**). Several ALMs or ACMs advanced melanomas diagnosed by anatomopathological analysis with bad prognostic detected DCT as the unique melanosomal antigen. The ALMs distinguish themselves from other melanoma types in terms of a worse prognosis, enhanced aggressiveness, and by a more advanced stage at diagnosis [96], whereas some ACMs are characterized by a peculiar and aggressive evolution [97]. It is very possible that DCT expression in ALMs and ACMs mediates tumor stress resistance pathways and contributes to the malignant characteristics of these melanoma categories. DCT could be an useful adjunct marker increasing sensitivity of tumor cell detection in specimens having downregulated other melanoma antigens, and the DCT-phenotype could represent a parameter associ-

The surgical removal is the only cure for melanoma with the condition that the excised lesion be in an early stage. However, the micrometastases cannot be addressed exclusively by the surgery and therefore, combinatorial therapeutical strategies are applied in the attempt to extend survival rates. The treatment options in melanoma are continuously revised, and there are several excellent reviews about this topic [98–100]. The schematic representation of the treatment of metastatic melanoma including different approaches is shown in **Figure 9**.

The identification of different T-cell clones in melanoma patients recognizing peptides derived from DCT (**Figure 2**) raised the interest for this antigen in the development of anti-melanoma immunotherapeutical strategies. The cellular vaccine engineered to co-express a DCT epitope, with IFN-γ in the same gene by replacing the IFN-γ signal peptide with a DCT epitope-expressing signal peptide, resulted in decreased B16 tumorigenicity and enhanced immunogenicity after gene transfer. More importantly, irradiated transiently, TRP-2 epitope-expressing, IFN-c

ated with high-risk for bad disease outcome.

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

**4. DCT as target in melanoma therapy**

**4.2. Anti-melanoma immunotherapeutic strategies involving DCT**

**4.1. Anti-melanoma therapies**

**Figure 9.** The therapies in melanoma. The different anti-melanoma treatment strategies are presented. DCT-based therapies are integrated part of the targeted therapies. The solid lines indicate the already existing therapies, whereas the dotted lines are proposed as possible adjuvant therapies based on the molecular studies about DCT intracellular processing and stability in melanoma cells. The melanoma specimen is an ulcerated nodular melanoma of a 26-yearold man, from lumbar region (by courtesy of Dr. S. Zurac, Department of Pathology, Colentina University Hospital, Bucharest, Romania).

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 chemotherapy with DNA-damaging drugs.

Our data about the intracellular processing and the main checkpoints in DCT fate in tumor cells (Section 2.3) indicate that pharmacological agents that impact DCT stability could represent also potential adjuvants in melanoma therapy. For example, NBDNJ or A3B5 produce specific DCT proteasomal degradation possibly sensitizing tumor cells to therapeutic stress and could also generate DCT-peptides suitable for MHCI presentation and immune response. The selective premature DCT degradation induced in melanoma cells following CQ treatment is another possible way to decrease tumor cell resistance to therapies. CQ has been found to strongly potentiate the inhibitory effect of radiation on tumor cell proliferation [111], to be effective in eliminating chemotherapy-resistant cancer cells and to significantly improve the median survival in glioblastomamultiformis patients [112]. Moreover, the DCT detection at PM by us in melanoma cells [23] and by others in glioma cells [65] introduces DCT as a suitable molecule for targeting tumor cells with specific antibodies. If studies will confirm that DCT is internalized from the PM, this will open interesting perspectives of coupling anti-DCT antibodies with nanocarriers loaded with various antitumor agents. And finally by downregulating DCT (by siRNA or CRISPR/Cas9 system), it can be targeted the Cav1 stability and architecture and possibly some Cav1-mediated pathways including ones involved in tumor progression. The DCT-mediated therapeutic strategies are presented as integrated part

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of anti-melanoma treatments in **Figure 9**.

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

structural model presented in Section 2.1.

**processes**

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

caveolin-1 (Cav1), with structural and functional implications [23].

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

DCT 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

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

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

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

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 anticancer drugs [110].

Our data about the intracellular processing and the main checkpoints in DCT fate in tumor cells (Section 2.3) indicate that pharmacological agents that impact DCT stability could represent also potential adjuvants in melanoma therapy. For example, NBDNJ or A3B5 produce specific DCT proteasomal degradation possibly sensitizing tumor cells to therapeutic stress and could also generate DCT-peptides suitable for MHCI presentation and immune response. The selective premature DCT degradation induced in melanoma cells following CQ treatment is another possible way to decrease tumor cell resistance to therapies. CQ has been found to strongly potentiate the inhibitory effect of radiation on tumor cell proliferation [111], to be effective in eliminating chemotherapy-resistant cancer cells and to significantly improve the median survival in glioblastomamultiformis patients [112]. Moreover, the DCT detection at PM by us in melanoma cells [23] and by others in glioma cells [65] introduces DCT as a suitable molecule for targeting tumor cells with specific antibodies. If studies will confirm that DCT is internalized from the PM, this will open interesting perspectives of coupling anti-DCT antibodies with nanocarriers loaded with various antitumor agents. And finally by downregulating DCT (by siRNA or CRISPR/Cas9 system), it can be targeted the Cav1 stability and architecture and possibly some Cav1-mediated pathways including ones involved in tumor progression. The DCT-mediated therapeutic strategies are presented as integrated part of anti-melanoma treatments in **Figure 9**.
