Ultraviolet-B irradiation enhances melanoma cell motility via induction of autocrine interleukin 8 secretion

Ultraviolet radiation (UVR) is known to be involved in the initiation and progression of malignant melanoma. Many studies have focused on the initiation of melanoma, but less is known about the effect of UVR on established tumor cells. Here, we show that after ultraviolet-B (UVB) irradiation, melanoma cells (MM) are able to secrete autocrine factors that enhance their motility. Time-lapse videomicroscopy of UVB irradiated (15 or 30 mJ/cm(2)) MM showed an initial decrease in MM cell motility one hour after irradiation, with subsequent increase 24 h after UV-B treatment. Conditioned media harvested from MM 24 h following UV-B irradiation specifically enhanced the motility of un-irradiated MM, suggesting that a newly synthesized soluble factor released by UVB MM is involved. As interleukin 8 (IL-8) is known to be up-regulated by different cell types after UV-B irradiation, we investigated IL-8 expression after UVB exposure. Quantitative RT-PCR and ELISA demonstrated an induction of IL-8 in MM by UVB (15 or 30 mJ/cm(2)), and addition of recombinant IL-8 to cell cultures enhanced cell motility to a similar degree than UVB. Importantly, blocking IL-8 activity by a neutralizing anti IL-8 antibody inhibited the up-regulation of MM motility after UVB treatment. We conclude that UVB enhances MM motility and that this effect is mediated at least in part by IL-8 released by MM in an autocrine fashion. Our findings are consistent with the hypothesis that UVB is not only involved in the initiation of melanoma, but may also be important for some aspects of tumor progression.     

Role of Ets‐1 in fibronectin‐derived heparin‐binding domain polypeptides alleviating melanoma cell invasiveness and chemoresistance

In this study, we observed that rhFNHN29 and rhFNHC36, two recombinant heparin‐binding domain polypeptides of fibronectin, suppressed adhesion and invasion of B16F10 and A375 melanoma cells mediated by integrin αv and α2 in a dose‐dependent manner. Combined with low‐concentration epirubicin (EPI), rhFNHN29 or rhFNHC36 exhibited a synergistic inhibition on the viability and metastasis of B16F10 cells. Moreover, in the presence of high‐concentration rhFNHN29 or rhFNHC36, the Ets‐1 activity and the expression of p‐FAK, p‐Erk1/2 and Ets‐1 were notably downregulated in B16F10 cells. Ets‐1 is one of the central regulatory links for rhFNHN29 and rhFNHC36 to suppress the adhesion and invasion of melanoma cells. Combining rhFNHN29 or rhFNHC36 with EPI may be a good way to alleviate invasiveness or chemoresistance in melanoma.     

Different patterns of fibronectin and tenascin-C splice variants expression in primary and metastatic melanoma lesions

We have investigated the staining patterns of primary and metastatic melanoma lesions using F8, L19 and F16. These three clinical-stage antibodies are currently being studied in clinical trials for the pharmacodelivery of cytokines or therapeutic radionuclides to neoplastic sites in patients with cancer. Frozen sections of 24 primary and 29 metastatic melanoma lesions were stained, using immunofluorescence procedures, with biotinylated preparations of the F8, L19 and F16 antibodies, which are specific to the alternatively spliced extra domain A and extra domain B domains of fibronectin and A1 domain of tenascin-C, respectively. Blood vessels were costained using von Willebrand factor-specific antibodies. In primary cutaneous melanoma lesions, F16 and F8 (but not L19) strongly stained the basal lamina at the interface between epidermis and dermis, with a strikingly complementary pattern. By contrast, metastatic melanoma lesions displayed a strong and diffuse pattern of immunoreactivity with all three antibodies. It was found that the extracellular matrix in melanoma undergoes extensive remodelling during the transition from primary to metastatic lesions. The intense staining of metastatic melanoma lesions by the F8, L19 and F16 antibodies provides a strong rationale for the use of these antibodies and their derivatives for the treatment of melanoma patients and possibly for the personalized choice of the best performing antibody in individual patients.     

PD‐L1 expression by tumor cell lines: A predictive marker in melanoma

Prognostic biomarkers for patients with melanoma after lymph node resection are of clinical relevance and could thus enable the identification of patients who therefore would most benefit from adjuvant treatment. The aim of this work was to determine, using an in vitro model, whether immune‐related biomarkers, such as MHC‐class I and II, melanoma‐associated antigens, IDO1 and PD‐L1, could also be relevant to predict the risk of relapse of patients with stage III melanoma after lymph node resection. We established tumor cell lines from metastatic lymph nodes of 50 patients with melanoma. The expression of investigated biomarkers was determined on untreated and IFN‐γ treated melanoma cell lines using flow cytometry. Among the selected biomarkers, the IFN‐γ‐induced expression of PD‐L1 and IDO1 was associated with an increased risk of relapse (P = .0001 and P = .013, respectively) and was also associated with death for IDO1 (P = .0005). In the future, this immunologic signature could permit the identification of patients at higher risk of relapse and justifying an adjuvant treatment using immunotherapy.     

Oncogenic BRAF signalling increases Mcl‐1 expression in cutaneous metastatic melanoma

The Bcl‐2 family member Mcl‐1 is essential for melanoma survival; however, the influence of oncogenic BRAF signalling remains elusive. In this study, Mcl‐1 splice variant expression was determined in a panel of melanoma cell lines in relation to BRAF mutational status. Mcl‐1L mRNA expression was increased in melanoma cells compared with primary melanocytes with significantly increased mRNA and protein expression observed in BRAF^V600E mutant melanoma cells. Although no change in Mcl‐1S mRNA was observed, Mcl‐1S protein expression also increased in BRAF mutant melanoma cells. Additionally, while over‐expression of mutant BRAF^V600E increased both Mcl‐1L and Mcl‐1S expression, inhibition of hyperactive BRAF signalling resulted in decreased Mcl‐1L expression. These studies suggest that the regulation of Mcl‐1 expression by BRAF signalling is increased by oncogenic activation of BRAF, revealing a mechanism of apoptotic resistance which may be overcome by the use of more specifically targeted Mcl‐1 inhibitors.     

The novel tumour suppressor gene ING1 is overexpressed in human melanoma cell lines

BACKGROUND: Epidemiological evidence indicates that exposure to ultraviolet (UV) radiation is directly linked to the increase of both incidence and mortality of melanoma. However, the genetic changes caused by UV radiation that lead to melanoma formation remain poorly understood. Recently, a potential tumour suppressor gene ING1 (inhibitor of growth 1) was shown to inhibit cell growth and induce apoptosis in the presence of p53. We have demonstrated that the expression of ING1 is induced after UV irradiation and that ING1 enhances the repair of UV-damaged DNA. OBJECTIVES: To investigate if ING1 plays a role in melanoma formation. METHODS: We examined p33ING1 expression levels in 14 melanoma cell lines. RESULTS: We found that p33ING1 is overexpressed at both mRNA and protein levels in melanoma cell lines compared with normal melanocytes. Single-strand conformation polymorphism (SSCP) analysis showed band shifting in two melanoma cell lines. DNA sequencing confirmed that there were nucleotide alterations in the ING1 gene in Sk-mel-24 and Sk-mel-110 cell lines. Two silent nucleotide alterations in exon 1a were detected in Sk-mel-110. In Sk-mel-24, the A-->G nucleotide alteration at codon 260 resulted in an amino acid change from Asn to Ser, while seven other nucleotide alterations were silent. To determine if the silent nucleotide alterations in these two melanoma cell lines were due to polymorphism, SSCP analysis of ING1 gene was performed in 25 healthy volunteers. No band shift was observed in the SSCP analysis, suggesting that the nucleotide alterations in the melanoma cell lines are unlikely to be due to polymorphism. CONCLUSIONS: Taken together, our data demonstrate that ING1 is overexpressed, but infrequently mutated, in melanoma cell lines.     

Analysis of APAF-1 expression in human cutaneous melanoma progression

APAF-1 plays a pivotal role in mitochondria-dependent apoptosis, binding to cytochrome c and favoring activation of caspase-9. It has been shown that epigenetic silencing of the APAF-1 gene is a common event in several metastatic melanoma cells in vitro. We determined, by Western blot, variation in the level of expression of APAF-1 in several human melanoma cell lines and, by immunohistochemistry, in a group of 106 histological samples including benign and malignant melanocytic lesions. We observed APAF-1 down-regulation or loss of expression in two metastatic melanoma cell lines, compared to primary melanoma cell lines. The immunohistochemical analysis revealed a significant difference in APAF-1 staining between nevi and melanomas. In addition, we found a significant negative correlation between APAF-1 expression level and tumor thickness and between primary melanomas and metastases. We conclude that loss of APAF-1 expression can be considered as an indicator of malignant transformation in melanoma.     

Switch in syndecan-1 and syndecan-4 expression controls maturation associated dendritic cell motility

Dendritic cells (DCs) need to mobilize within the extracellular matrix (ECM) during their maturation and concomitant migration from peripheral sites to lymphoid organs. Syndecans are cell surface proteoglycans that mediate the interaction of DCs with the ECM. Here we investigated the influence of syndecans on dendritic cell motility and morphology. Langerhans cells of the epidermis and monocyte-derived DCs were found to undergo a switch in syndecan expression during maturation. Syndecan-1 was downregulated and syndecan-4 was strongly upregulated within the first hours of lipopolysaccharide-induced dendritic cell maturation and during Langerhans cell emigration from human skin, as shown by flow cytometry and qRT-PCR. Syndecan-1 downregulation was inhibited by syndecan-4 siRNA knock-down, indicating a functional interconnection between enhanced syndecan-4 expression and syndecan-1 downregulation. Syndecan-4 upregulation is functionally involved in dendritic cell motility, as inhibition of syndecan-4 function by means of blocking antibodies or through siRNA knock-down decreased dendritic cell motility. In other experiments, the cytoskeletal component a-actinin was observed to be upregulated in DCs as a consequence of the induction of maturation, and was found to colocalize with syndecan-4. Furthermore, lammellopodial spreading by DCs on fibronectin (FN)-coated surfaces was dependent on syndecan-4. This binding of syndecan-4 to FN and its association with the cytoskeleton may be relevant for syndecan-4-dependent dendritic cell motility. We conclude that the switch in syndecan expression during dendritic cell maturation controls the motility of DCs in a way that appears to be crucial for their mobilization from peripheral sites and subsequent migration to lymphoid tissues.     

Efficacy of combined radiotherapy and anti‐programmed death 1 therapy in acral and mucosal melanoma

Some studies showed that clinical response to immune check point inhibitors is lower in acral and mucosal melanoma than in cutaneous melanoma. Although the synergistic effect of radiotherapy (RT) and ipilimumab has been reported in patients with brain metastasis, the efficacy of combined RT and anti‐programmed death 1 (PD‐1) therapy for acral and mucosal melanoma is unclear. The present study aimed to evaluate the efficacy of combined RT and anti‐PD‐1 therapy for acral and mucosal melanoma. We retrospectively analyzed patients with acral or mucosal melanoma who were treated with anti‐PD‐1 and RT at Sapporo Medical University Hospital. In 10 patients (acral, 3; mucosal, 7), the response rate (RR) and the disease control rate (DCR) were 40% and 60%, respectively. As regards mucosal melanoma, four of the seven patients had achieved complete response + partial response, and three had progressive disease (RR = 57.1%). Meanwhile, two of the three patients with acral melanoma had stable disease and one had progressive disease (RR and DCR were 0% and 66.6%, respectively). Except for the patients treated with palliative RT for bone metastasis in the present study, the RR was 50% (4/8 patients), and the DCR was 75% (6/8 patients). Vitiligo developed after RT in five (50%) patients at a median duration of 2 months after RT. The clinical response and the high occurrence of vitiligo suggest that the combination of RT and anti‐PD‐1 therapy could be effective in some patients with mucosal melanoma.     

Highly pigmented Tg(Grm1) mouse melanoma develops non‐pigmented melanoma cells in distant metastases

Murine model systems are critically required tools for the investigation of unknown mechanisms of melanoma development and metastasis and for developing more efficient therapies. The Tg(Grm1)EPv melanoma mouse model is characterized by spontaneous development of pigmented cutaneous melanomas at hairless skin regions, with a short latency and 100% penetrance. Local metastasis was described in initial analyses; however, melanoma cells were not observed in distant organs. Here, we demonstrate that the established Tg(Grm1)EPv melanoma mouse model exhibits more extensive metastasis into distant organs than previously described. Disseminated cells undergo phenotypic changes, as we observed high numbers of non‐pigmented Grm1‐expressing melanoma cells within distant organs. As such changes during metastasis are common in human melanoma, our findings demonstrate that this mouse model represents an even more useful tool to study unknown mechanisms of metastasis in human melanoma than previously assumed.     

Mutation of the tumour suppressor p33ING1b is rare in melanoma

BACKGROUND: The p33ING1b gene is involved in the p53-dependent response to DNA damage following exposure to ultraviolet radiation, and has recently been reported to be mutated in 20% of melanoma tumours. OBJECTIVES: We sought to assess the p33ING1b mutation rate in our large panels of fresh melanomas and melanoma cell lines. METHODS: We screened 83 primary melanomas and 55 melanoma cell lines for mutations in p33ING1b by single-strand conformational polymorphism analysis and by direct sequencing. RESULTS: In contrast to previous reports, we found no somatic p33ING1b mutations in our panel of melanomas. We found that some of the discrepancy between our results and previously published studies may be due to inadvertent amplification of the ING1 pseudogene (INGX), and/or contamination of some samples with murine Ing1. CONCLUSIONS: p33ING1b mutations in melanoma are rare. We have highlighted the importance of allele-specific primer design to avoid pseudogene amplification, and also the necessity to confirm the genetic identity and species of origin of individual cell lines. Further studies are needed to clarify the possible role of p33ING1b in melanoma tumorigenesis.     

A diagnostically‐challenging case of melanoma ex blue nevus with comprehensive molecular analysis,including the 23‐gene expression signature (myPath melanoma)

Melanoma ex blue nevus (MEBN) is a rare, aggressive, and potentially lethal neoplasm. Distinguishing MEBN from an atypical cellular blue nevus can be very challenging. We report a diagnostically difficult case of MEBN with lymph node metastases, in which single nucleotide polymorphism array and fluorescence in situ hybridization were used to arrive at the correct diagnosis. It was also analyzed by the recently‐introduced proprietary 23‐gene expression signature test. To the best of our knowledge, this is the second reported case of MEBN analyzed by the 23‐gene expression signature, which provided a false‐negative result. More studies are needed to assess the sensitivity and specificity of this test in various melanocytic proliferations.     

Fibroblasts surrounding melanoma express elevated levels of matrix metalloproteinase-1 (MMP-1) and intercellular adhesion molecule-1 (ICAM-1) in vitro

Tumour growth and metastasis involve the degradation of extracellular matrix components by matrix degrading enzymes produced by tumour cells and stromal fibroblasts. In this study, fibroblasts were obtained from biopsies on the border (TB) and 1 cm distant from the melanoma (TD) and cultured separately. Similar studies were performed with fibroblasts surrounding melanocytic nevi as control. The expression of matrix metalloproteinase-1 (MMP-1) mRNA and tissue matrix metalloproteinase inhibitor 1 (TIMP-1) were studied by Northern blot analysis. The activation antigen intercellular adhesion molecule-1 (ICAM-1) in TB-and TD-fibroblasts was investigated by flow cytometry. In melanoma, TB-fibroblasts showed an increased expression of MMP-1 mRNA mainly in fibroblasts obtained from tumours with extended invasive growth demonstrated by Clark level whereas the expression of the major specific inhibitor TIMP-1 was unaltered. In contrast, fibroblasts surrounding benign melanocytic nevi did not express elevated levels of MMP-1. The upregulation of MMP-1 in TB-fibroblasts compared to TD-fibroblasts was maintained during cultivation. Furthermore, MMP-1 mRNA expression and MMP-1 total protein amount in normal fibroblasts were increased by melanoma cell conditioned medium. We demonstrated an increased expression of ICAM-1 in TB-fibroblasts compared to TD-fibroblasts in vitro depending on the amount of inflammatory infiltrate in situ. The differences of ICAM expression disappeared during continued cell culture. These results support the idea that fibroblasts surrounding melanoma are activated and are possibly involved in the degradation of matrix proteins surrounding the tumour.     

Clinical significance of serum levels of soluble intercellular adhesion molecule-1 and soluble L-selectin in malignant melanoma

Although several molecules have been evaluated as tumor markers of malignant melanoma (MM) progression, there are few available markers sensitive enough to detect recurrence or metastasis. The objective of the present study was to determine clinical significance of serum soluble adhesion molecules in the monitoring of progression in patients with MM. Serum levels of soluble L-selectin (sL-selectin), sE-selectin, sP-selectin, and soluble intercellular adhesion molecule-1 (sICAM-1) were determined by ELISA in 57 MM patients. In a retrospective longitudinal study, nine serum samples from two MM patients were analyzed during a follow-up period of 4.0 and 4.3 years, respectively. Serum sICAM-1 levels in MM patients were significantly higher than those in healthy controls and tended to be elevated as the disease stage progressed. In contrast, serum sL-selectin levels were significantly lower in MM patients compared to healthy controls and tended to decrease as the disease stage progressed. There was no significant difference in serum sE-selectin and sP-selectin levels between MM patients and normal control. In a longitudinal study, increased sICAM-1 and decreased sL-selectin levels were generally associated with the progression of MM. These results suggest that monitoring both sICAM-1 and sL-selectin is available to evaluate the progression of MM.     

Loss of cell invasiveness through PKC‐mediated syndecan‐1 downregulation in melanoma cells under anchorage independency

Anchorage‐independent survival is one of the key features for malignant tumor cells. Whether specific gene alterations contributed by anchorage independency would further affect metastatic phenotypes of melanoma cells was unclear. We adapted suspension culture of melanoma cells to establish anchorage independency. The suspended melanoma cells lost their invasive abilities in vitro. Specific loss of laminin‐binding ability in suspended melanoma cells was observed, which was correlated with downregulation of syndecan‐1 as revealed by microarray and validated by qPCR and Western blot. Modulation of syndecan‐1 expression level affected laminin binding, transwell migration and matrix metalloproteinase‐2 secretion in melanoma cells. SDC1 expression and transwell migration were correlated with activity or level of protein kinase Cδ as evidence by specific inhibitors and shRNA transfection. In this study, we compared metastatic phenotypes and gene expressions of adherent and suspended melanoma cells. The anchorage independency led to protein kinase Cδ‐mediated syndecan‐1 downregulation, which contributed to loss of laminin‐binding ability, reduced metalloproteinase‐2 secretion and loss of invasiveness.