Interleukin-8 in Melanoma Pathogenesis, Prognosis and Therapy-An Integrated View into Other Neoplasms and Chemokine Networks.

Cutaneous melanoma accounts for only about 7% of skin cancers but is causing almost 90% of deaths. Melanoma cells have a distinct repertoire of mutations from other cancers, a high plasticity and degree of mimicry toward vascular phenotype, stemness markers, versatility in evading and suppress host immune control. They exert a significant influence on immune, endothelial and various stromal cells which form tumor microenvironment. The metastatic stage, the leading cause of mortality in this neoplasm, is the outcome of a complex, still poorly understood, cross-talk between tumor and other cell phenotypes. There is accumulating evidence that Interleukin-8 (IL-8) is emblematic for advanced melanomas. This work aimed to present an updated status of IL-8 in melanoma tumor cellular complexity, through a comprehensive analysis including data from other chemokines and neoplasms. The multiple processes and mechanisms surveyed here demonstrate that IL-8 operates following orchestrated programs within signaling webs in melanoma, stromal and vascular cells. Importantly, the yet unknown molecularity regulating IL-8 impact on cells of the immune system could be exploited to overturn tumor fate. The molecular and cellular targets of IL-8 should be brought into the attention of even more intense scientific exploration and valorization in the therapeutical management of melanoma.

Interaction Analysis of Commercial Graphene Oxide Nanoparticles with Unicellular Systems and Biomolecules.

The ability of commercial monolayer graphene oxide (GO) and graphene oxide nanocolloids (GOC) to interact with different unicellular systems and biomolecules was studied by analyzing the response of human alveolar carcinoma epithelial cells, the yeast Saccharomyces cerevisiae and the bacteria Vibrio fischeri to the presence of different nanoparticle concentrations, and by studying the binding affinity of different microbial enzymes, like the α-l-rhamnosidase enzyme RhaB1 from the bacteria Lactobacillus plantarum and the AbG ß-d-glucosidase from Agrobacterium sp. (strain ATCC 21400). An analysis of cytotoxicity on human epithelial cell line A549, S. cerevisiae (colony forming units, ROS induction, genotoxicity) and V. fischeri (luminescence inhibition) cells determined the potential of both nanoparticle types to damage the selected unicellular systems. Also, the protein binding affinity of the graphene derivatives at different oxidation levels was analyzed. The reported results highlight the variability that can exist in terms of toxicological potential and binding affinity depending on the target organism or protein and the selected nanomaterial.

Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression.

l-Dopachrome tautomerase (l-DCT), also called tyrosinase-related protein-2 (TRP-2), is a melanoma antigen overexpressed in most chemo-/radiotherapeutic stress-resistant tumor clones, and caveolin-1 (CAV1) is a main regulator of numerous signaling processes. A structural and functional relationship between DCT and CAV1 is first presented here in two human amelanotic melanoma cell lines, derived from vertical growth phase (MelJuSo) and metastatic (SKMel28) melanomas. DCT co-localizes at the plasma membrane with CAV1 and Cavin-1, another molecular marker for caveolae in both cell phenotypes. Our novel structural model proposed for the DCT-CAV1 complex, in addition to co-immunoprecipitation and mass spectrometry data, indicates a possible direct interaction between DCT and CAV1. The CAV1 control on DCT gene expression, DCT post-translational processing, and subcellular distribution is cell phenotype-dependent. DCT is a modulator of CAV1 stability and supramolecular assembly in both cell phenotypes. During autocrine stimulation, the expressions of DCT and CAV1 are oppositely regulated; DCT increases while CAV1 decreases. Sub-confluent MelJuSo clones DCT(high)/CAV1(low) are proliferating and acquire fibroblast-like morphology, forming massive, confluent clusters as demonstrated by immunofluorescent staining and TissueFAXS quantitative image cytometry analysis. CAV1 down-regulation directly contributes to the expansion of MelJuSo DCT(high) subtype. CAV1 involved in the perpetuation of cell phenotype-overexpressing anti-stress DCT molecule supports the concept that CAV1 functions as a tumor suppressor in early stages of melanoma. DCT is a regulator of the CAV1-associated structures and is possibly a new molecular player in CAV1-mediated processes in melanoma.

Variations in the expression of TIMP1, TIMP2 and TIMP3 in cutaneous melanoma with regression and their possible function as prognostic predictors.

Regression in melanoma is a frequent biological event of uncertain prognostic value as the lesion exhibits heterogeneous phenotypical features, both at the morphological and immunohistochemical level. In the present study, we examined the expression of tissue inhibitors of metalloproteinases (TIMP1, TIMP2 and TIMP3) in melanoma with regression. We specifically examined the expression levels of these TIMPs in regressed components (RC) and non-regressed components (NRC) of the tumor and compared their expression levels with those in non-regressed melanomas. We found that TIMP1 was overexpressed in the NRC of melanomas with partial regression (PR) compared with the NRC in melanomas with segmental regression (SR) (P=0.011). TIMP2 was overexpressed in the NRC of melanomas with PR compared with the NRC in melanomas with SR (PR/SR, P=0.009); or compared with the NRC in melanomas with simultaneous SR-PR (P=0.002); or compared with melanomas without regression (absence of regression) (P=0.037). Moreover, TIMP3 was overexpressed in the NRC of all melanomas with SR as compared to the RC component (P=0.007). Our findings on the differential expression of TIMP1, TIMP2 and TIMP3 in melanomas with regression support the hypothesis that the morphological differences identified in the melanoma regression spectrum may have a correlation with prognosis. This may explain the controversial findings within the literature concerning the biological and prognostic role of regression in melanoma.

Value of dopachrome tautomerase detection in the assessment of melanocytic tumors.

Dopachrome tautomerase (DCT) and tyrosinase (Tyr) are melanogenic enzymes and structurally related melanosomal proteins. The present study investigates DCT expression comparatively with Tyr, the most tested melanoma biomarker, aiming to evaluate DCT potential in the assessment of melanocytic tumors and gain insights into the molecular and pathological characterization of DCT-phenotype in tumor progression. DCT and Tyr are simultaneously analyzed in melanoma cell lines by semiquantitative RT-PCR, western blot, and N-glycan analysis, and in cell populations of melanocytic tumors by immunohistofluorescence using a novel anti-hDCT antibody against an extended sequence within DCT luminal domain. DCT, unlike Tyr, is fully processed along the secretory pathway in both pigmented and amelanotic melanoma cells. In 53 nevi and 116 primary malignant melanomas, 81% and 52%, respectively, are DCT+/Tyr+, showing that DCT is a stable antigen, retained by most tumors and partially expressed in Tyr-negative cell populations. The DCT/Tyr disjunction is a process correlated with melanocyte neoplastic transformation and malignant progression. A tumor architecture--DCT-phenotype-containing DCT+/Tyr- cell populations selected into the innermost dermis from double-positive cells is detected in 35% of DCT+/Tyr+ specimens. The DCT-phenotype is associated with enhanced neurotization in benign nevi and with ulceration in thin malignant melanomas. The intradermal DCT+/Tyr- clones in superficial melanomas acquire the expression and specific subcellular distribution of unfavorable prognostic markers. DCT assessment shows specific antigen patterns with potential significance in the outcome of melanocytic lesions, connecting DCT, a mediator of a melanoma stress-resistant pathway, and an antiapoptotic molecule to DCT- phenotypes that are possibly more stable and stress resistant.

Spectrum of morphologic alterations of regression in cutaneous melanoma--potential for improving disease prognosis.

INTRODUCTION: Regression occurs as a complex interaction between tumor cells and host's immune response; neither biologic mechanisms, nor regression prognostic significance are deciphered to date but promising anti-cancer vaccine strategies were thus developed. METHODS: We analyzed 127 superficial spreading melanomas identifying melanoma with regression (segmentary (SR), partial (PR) and segmentary & partial (SR-PR)) or without regression (AR). Several histopathologic parameters were registered; statistical analysis was performed (level of significance P < 0.05). RESULTS: Regression was present in 52% cases, less frequently in pT4 melanomas. Ulceration and vascular invasion were similarly present in pT2-pT4 melanomas with regression and significantly less in pT1 ones; their incidence increased with stage in AR (P < 0.001). SR and SR-PR melanomas showed significantly more tumor infiltrating lymphocytes within the non-regressed tumor than AR melanomas (P < 0.05). SR melanomas presented significantly less frequent mitoses than PR (P = 0.04), SR-PR (P = 0.04) or AR ones (P = 0.03). Marked inflammation and more numerous melanophages were present regressed areas advanced stage melanomas. More numerous plasma cells were identified in advanced stages; in SR and SR-PR melanomas less numerous plasma cells were present in pT1 than in advanced stages. Vascular hyperplasia was significantly higher in SR than SR-PR cases. CONCLUSIONS: Differences in perception of regression might be the result of labeling with similar name of various processes comprising inflammation and tumor cells destruction; at least in thin melanomas, PR and SR seem to belong to different spectrum of alteration, SR bearing a more favorable potential. Further studies will be performed in order to further elucidate the mechanisms involved in regression in melanoma.

Cutaneous metastases of malignant melanoma--how difficult can it be?

Malignant melanoma is one of the most aggressive skin neoplasms with histopathological-based therapeutic approach. Unfortunately, in some cases, even the elementary issue of dealing with a primary or metastatic lesion may be sometimes incredible difficult to settle. We studied 11 cases of malignant melanomas that required careful histopathological and immunohistochemical analysis to differentiate between primary and secondary tumor. We evaluated epidermotropism of primary MM including synchronous tumors, local recurrences and metastases.

Tyrosinase-related protein-2 and -1 are trafficked on distinct routes in B16 melanoma cells.

Tyrosinase related protein (TRP)-1 and -2 regulate the main steps in melanin synthesis and are immune targets in skin cancer or autoimmune pigmentary disorders. We found that ionophore monensin (Mon) and the quaternary amine chloroquine (CQ) discriminate between the traffic routes of TRP-2 and TRP-1. TRP-2 N-glycan processing is interrupted by Mon between ER and trans-Golgi, whereas this process continues for TRP-1. Mature TRP-2 is diverted by CQ treatment to a degradation pathway which depends on functional vacuolar ATPases. Conversely, the subcellular distribution and stability of TRP-1 were not affected by CQ. We propose that TRP-2 is sorted and trafficked in the early secretory pathway with a cargo which does not include TRP-1; post Golgi, TRP-2 intersects the endocytic pathway following a route via early endosomes, possibly by rapid recycling from the plasma membrane. These data show that highly structural homologous glycoproteins use distinct trafficking pathways in the same cell.

Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells.

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.

The inhibition of early N-glycan processing targets TRP-2 to degradation in B16 melanoma cells.

Tyrosinase-related protein-2 (TRP-2) is a DOPAchrome tautomerase catalyzing a distal step in the melanin synthesis pathway. Similar to the other two melanogenic enzymes belonging to the TRP gene family, tyrosinase and TRP-1, TRP-2 is expressed in melanocytes and melanoma cells. Despite the increasing evidence of its efficiency as a melanoma antigen, little is known about the maturation and intracellular trafficking of TRP-2. Here we show that TRP-2 is mainly distributed in the TGN of melanoma cells instead of being confined solely to melanosomes. This, together with the plasma membrane occasional localization observed by immunofluorescence, suggest the TRP-2 participation in a recycling pathway, which could include or not the melanosomes. Using pulse-chase experiments we show that the TRP-2 polypeptide folds in the endoplasmic reticulum (ER) in the presence of calnexin, until it reaches a dithiothreitol-resistant conformation enabling its ER exit to the Golgi. If N-glycosylation inhibitors prevent the association with calnexin, the TRP-2 nascent chain undergoes an accelerated degradation process. This process is delayed in the presence of proteasomal inhibitors, indicating that the misfolded chain is retro-translocated from the ER into the cytosol and degraded in proteasomes. This is a rare example in which calnexin although indispensable for the nascent chain folding is not required for its targeting to degradation. Therefore TRP-2 may prove to be a good model to document the calnexin-independent retro-translocation process of proteasomally degraded proteins. Clearly, TRP-2 has a distinct maturation pathway from tyrosinase and TRP-1 and possibly a second regulatory function within the cell.