SERPINE1 expression discriminates site-specific metastasis in human melanoma

Depth of invasion, a quantifier of vertical growth, is a major cutaneous melanoma staging factor. Stromal penetrance requires pericellular proteolysis regulated by the serine protease and matrix metalloproteinase cascades. The serine protease inhibitor SERPINE1, a poor prognosis biomarker in various cancers, promotes tumor progression likely by titrating the extent and local of plasmin-initiated matrix remodelling. SERPINE1 in human melanoma was assessed using tissue arrays that included primary/metastatic tumors and normal skin. SERPINE1 was basal layer-restricted in the normal epidermis. SERPINE1 immunoreactivity was evident in 27/28 primary (96%) and 24/26 metastatic tumors (92%); cutaneous metastases (80%) had significantly elevated SERPINE1 levels compared with low signals characteristic of lymph node lesions. Moderate SERPINE1 expression was a general finding in primary melanoma, whereas reduced or increased SERPINE1 immunolocalization typified metastatic deposits. The amplitude of SERPINE1 expression may impact melanoma site-specific dissemination, with cutaneous metastases representing a high-SERPINE1 tumor subtype.     

BRG1 expression is increased in human cutaneous melanoma

Summary Background The SWI/SNF chromatin remodelling complex plays important roles in cellular processes including cell differentiation, cell cycle control and DNA repair. Aberrant expression of SWI/SNF subunits is involved in cancer development. The core subunit of the SWI/SNF complex, SNF5, has been shown to be inactivated in malignant rhabdoid tumours and has been defined as a tumour suppressor. However, the role of the catalytic subunit, BRG1, is not well defined in cancer. Objectives To investigate the role of BRG1 in melanoma development, we examined the expression of BRG1 in melanocytic lesions at different stages and analysed the correlation between BRG1 expression and clinicopathological variables and patient survival. Methods Using tissue microarray and immunohistochemistry, we evaluated BRG1 staining in 48 dysplastic naevi, 90 primary melanomas and 47 metastatic melanomas. We studied melanoma cell proliferative ability with reduced BRG1 expression by small interfering RNA using cell proliferation assay and cell cycle analysis. Results We found that BRG1 expression was increased in primary melanoma and metastatic melanoma compared with dysplastic naevi (P < 0·0001). We did not find any correlation between BRG1 expression and melanoma patient survival. In addition, we demonstrated that knockdown of BRG1 in melanoma cell lines resulted in significantly reduced cell proliferative ability. This reduced cell proliferation is due to G₁ phase arrest as cyclin D₁ is downregulated upon BRG1 knockdown. Conclusions Our data indicate that BRG1 is significantly increased in human melanoma and is involved in melanoma initiation.     

XAF1 expression is significantly reduced in human melanoma

Deregulation of apoptotic processes is likely one of the key factors contributing to the malignant nature of melanoma marked by strong chemoresistance. X-linked inhibitor of apoptosis protein (XIAP) suppresses apoptosis through the inhibition of various caspases. Recently, XIAP-associated factor 1 (XAF1) has been identified as a XIAP-binding protein that antagonizes the anti-apoptotic activity of XIAP. In this study, we sought to determine the role of XAF1 in melanoma progression. Analysis of XAF1 mRNA expression in melanoma cell lines revealed that XAF1 mRNA was downregulated in 15 of 16 cell lines examined. We next evaluated XAF1 protein expression on a tissue microarray representing 40 benign nevi and 70 primary melanomas. Our results showed that XAF1 expression in melanoma tissues was significantly reduced compared with benign melanocytic nevi (p<0.05). Our data also demonstrated that the substantial reduction of XAF1 expression occurred in both nucleus and cytoplasm in the tumor cells (p<0.0001 for both). Reduced XAF1 expression, however, was not significantly correlated with tumor thickness and 5-y patient survival. Further studies are required to understand the molecular mechanisms governing the selective loss of XAF1 expression in the tumor tissue.     

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.     

Endothelin-1 induces CXCL1 and CXCL8 secretion in human melanoma cells

The endothelin pathway plays a critical role in melanoma tumor progression by a variety of mechanisms that enhance tumor cell growth, invasion, and metastasis. Here, we investigate the effect of this pathway on CXC chemokine expression in human melanoma cells and melanocytes. As determined by ELISA, endothelin-1 (ET-1) induces CXCL1 and CXCL8 secretion in three human melanoma cell lines in a concentration-dependent fashion. These responses are mediated by the endothelin-B receptor and are sustained over a 40 h time course. ET-1 does not induce CXCL1 secretion in primary human melanocytes but ET-3, an endothelin isoform, induces a low level of CXCL1 secretion in certain cultures. Neither ET-1 nor ET-3 induces secretion of CXCL8 in primary human melanocytes; thus, this response may be specific for melanocytic cells that have undergone malignant transformation. We have previously demonstrated that ET-1 induces changes in the expression of adhesion molecules in melanoma cells such that invasion and metastasis are favored. This study demonstrates that ET-1 additionally induces secretion of CXC chemokines critical for melanoma metastasis and tumor progression.     

Endothelin-1 decreases basic apoptotic rates in human melanoma cell lines

Normal human melanocytes respond to endothelin-1 with induced proliferation and differentiation. Whereas in cultured melanoma cells the predominant endothelin receptor, ET(B)-R, is consistently downregulated, ET(B)-R upregulation was recently reported for melanoma tumors. Contrary to the pro-survival activity described for endothelin in vascular cells, a proapoptotic activity of endothelin-1 has been reported for melanoma cells, in previous studies. We therefore investigated the role of endothelin for melanoma cells with respect to apoptosis and proliferation. Treatment with 10 nM endothelin-1 was a strong mitogenic signal for normal human melanocytes, which responded with a 4-6-fold increase of thymidine incorporation, whereas the response was only 1.2-fold for SK-Mel-19, the melanoma cell line characterized by the highest ET(B)-R expression, and it was even less in other cell lines. Determination of the apoptotic rates revealed that endothelin-1 significantly reduced basic apoptotic rates to 75% both in SK-Mel-19 and in normal melanocytes. After cell synchronization, an antiapoptotic effect of endothelin-1 was seen in five of seven cell lines investigated. In the cell line Bro, which showed no response and which lacks ET(B)-R expression, responsibility could be restored by overexpression of ET(B)-R after stable transfection, indicating that the effectors of the endothelin-1 signal cascade were active in these cells, and that the antiapoptotic effect of endothelin-1 is mediated in a receptor-specific way. This antiapoptotic activity of endothelin for melanoma cells combined with upregulation of endothelin receptors in the tumor may be a crucial step for melanoma progression.     

Polo-like kinase 1 is a potential therapeutic target in human melanoma

Exploration of the human melanoma cell-cycle pathway can lead to identification of new therapeutic targets. By gene set enrichment analysis, we identified the cell-cycle pathway and its member polo-like kinase 1 (Plk-1) to be significantly overexpressed in primary melanomas and in melanoma metastases. In vitro expression of Plk-1 was peaked at the G2/M phase of the cell cycle. Plk-1 knockdown/inhibition led to induction of apoptosis, which was caspase-3/8-dependent and p53-independent, and involved BID and Bcl-2 proteins. Comparative genomic hybridization/single-nucleotide polymorphism arrays showed no genetic alteration in the Plk-1 locus. Previous suggestions and significant enrichment of the mitogen-activated protein kinase (MAPK) signaling pathway pointed to potential regulation of Plk-1 by MAPK signaling. Inhibition of this pathway resulted in decreased Plk-1 expression as a consequence of G1 cell-cycle arrest rather than direct regulation of Plk-1. Inhibition of MAPK and Plk-1 had an additive effect on reduced cell viability. This study shows that in human melanoma, Plk-1 expression is dynamically regulated during the cell cycle, knockdown of Plk-1 leads to apoptotic cell death, and that a combination of Plk-1 and MAPK inhibition has an additive effect on melanoma cell viability. We conclude that combined inhibition of Plk-1 and MAPK could be a potentially attractive strategy in melanoma therapy.     

Matrix metalloproteinases in human melanoma

Cutaneous melanoma is a highly invasive and metastatic tumor. Degradation of basement membranes and extracellular matrix is an essential step in melanoma cell migration, invasion, and metastasis formation. Matrix metalloproteinases and their tissue inhibitors play a crucial role in these complex multistep processes. Melanoma cells may express a number of matrix metalloproteinase family members (MMP-1, MMP-2, MMP-9, MMP-13, and MT1-MMP) as well as their tissue inhibitors (TIMP-1, TIMP-2, and TIMP-3). Numerous studies have examined matrix metalloproteinases, their tissue inhibitors, and the molecules that regulate their expression and/or activation in melanoma cell lines in vitro and in vivo, and in human melanocytic lesions. Recent results have indicated that adhesion molecules such as CD44 and integrin alphavbeta3 are involved in positioning activated matrix metalloproteinase molecules on the cell surface of invasive tumor cells. In this review we evaluate these novel aspects of the role of matrix metalloproteinases and their tissue inhibitors in melanoma progression. We conclude that the balance between levels of activated matrix metalloproteinases and expression levels of their tissue inhibitors, and the coexpression of activated matrix metalloproteinases and adhesion molecules are important factors in determining melanoma cell invasion, tumor growth, and metastasis formation.     

Regulation of pigmentation by substrate elasticity in normal human melanocytes and melanotic MNT1 human melanoma cells

The elasticity of the cellular microenvironment is a key regulator of cellular physiology in many cell types. To investigate the effects of substrate stiffness on the pigmentation process, we cultured normal human melanocytes (NHM) and MNT1 melanoma cells on laminin‐coated polydimethylsiloxane (PDMS) substrates of different stiffness. The dendricity of NHM and MNT1 cells was reduced as the substrate stiffness decreased, and the degree of melanosome transfer from NHM or MNT1 cells to normal human keratinocytes was decreased on softer substrates with the reduced dendricity. Gene and protein expressions of MITF, tyrosinase, TRP2, and gp100/PMEL17 exhibited a consistent decreasing trend with the decreasing stiffness. Because the stiffness sensing is mediated by focal adhesion complex through integrin receptors, we checked laminin specific integrin alpha 6 and p‐FAK for MNT1 cells to observe that the substrate adhesion was weakened as the substrate stiffness decreased. Weaker adhesion on a softer substrate was accompanied by dynamic shape changes in MNT1 cells with higher speed and larger scattering. Dendritic MNT1 cells cultured on a stiffer substrate exhibited lower migration with smaller root mean squared displacement. These results demonstrate the possibility that skin pigmentation can be influenced by mechanical properties of the cellular microenvironment and can increase when the skin becomes stiff.     

Trichochromes in human malignant melanoma.

Decarboxytrichochromes B and C were isolated from a melanoma metastasis in a red-haired man, indicating the presence of trichochromes B and C in the tissue. Trichochromes E and F were not detected.     

Mcl-1 antisense therapy chemosensitizes human melanoma in a SCID mouse xenotransplantation model

It is well established that high expression of the antiapoptotic Bcl-2 family proteins Bcl-2 and Bcl-xL can significantly contribute to chemoresistance in a number of human malignancies. Much less is known about the role the more recently described Bcl-2 family member Mcl-1 might play in tumor biology and resistance to chemotherapy. Using an antisense strategy, we here address this issue in melanoma, a paradigm of a treatment-resistant malignancy. After in vitro proof of principle supporting an antisense mechanism of action with specific reduction of Mcl-1 protein as a consequence of nuclear uptake of the Mcl-1 antisense oligonucleotides employed, antisense and universal control oligonucleotides were administered systemically in combination with dacarbazine in a human melanoma SCID mouse xenotransplantation model. Dacarbazine, available now for more than three decades, still remains the most active single agent for treatment of advanced melanoma. Mcl-1 antisense oligonucleotides specifically reduced target protein expression as well as the apoptotic threshold of melanoma xenotransplants. Combined Mcl-1 antisense oligonucleotide plus dacarbazine treatment resulted in enhanced tumor cell apoptosis and led to a significantly reduced mean tumor weight (mean 0.16 g, 95% confidence interval 0.08-0.26) compared to the tumor weight in universal control oligonucleotide plus dacarbazine treated animals (mean 0.35 g, 95% confidence interval 0.2-0.44) or saline plus dacarbazine treated animals (mean 0.39 g, 95% confidence interval 0.25-0.53). We thus show that Mcl-1 is an important factor contributing to the chemoresistance of human melanoma in vivo. Antisense therapy against the Mcl-1 gene product, possibly in combination with antisense strategies targeting other antiapoptotic Bcl-2 family members, appears to be a rational and promising approach to help overcome treatment resistance of malignant melanoma.     

Steroid hormone receptors in human melanoma

Summary Human epidermis uncontaminated by fibroblasts was isolated by a suction blister method. DNA synthesis in short-time organ cultures of isolated epidermis was strongly inhibited by aphidicolin, suggesting that DNA polymerase is involved in DNA replication in human epidermis. On the basis of their responses to inhibitors, primer-template requirements, and chromatographic properties, DNA polymerases , , and were all identified in epidermal extracts.Supported by the Erna and Olav Aakres Foundation for Cancer Research     

TLR expression in human melanoma cells

There is increasing evidence that melanoma cells express TLR2, -3, and -4. However, the expression of other TLRs by these cells still remains unknown. We investigated the expression patterns of TLR2, -3, -4, -7, -8 and -9 both on melanoma-invaded lymph nodes and on melanoma cell lines. TLR2, -3, -4, -7 and -9 mRNA expression was determined by quantitative RT-PCR. The TLR protein expression level was measured ex vivo by immunohistochemistry and in vitro by flow cytometry. Results: At the mRNA level, melanoma cells in vitro, and possibly ex vivo, expressed TLR2, -3, -4, -7 and -9. TLR2 and -4 protein expressions ex vivo were over 50%, contrasting with an absence of these 2 TLRs in vitro. On the contrary, TLR-3 and -8 proteins had a low expression ex vivo with a high expression in vitro. TLR-7 and -9 proteins were expressed ex vivo and in vitro. Our study demonstrates for the first time that melanoma cells express TLR7 and -8.     

Steroid hormone receptors in human melanoma

Summary Human melanomas were investigated for the presence of highaffinity estrogen-, gestagen-, and glucocorticoid-binding proteins. A statistically significant difference was found for mean estrogen receptor (ER) concentrations in melanomas of male versus female origin: female origin 37.6 (0–107) fmol/mg protein, male origin 3.9 (0–8.3) fmol/mg protein. No significant difference between sexes was found for gestragen receptors: 41.5 (0–194) fmol/mg protein for melanomas of female origin versus 99 (0–362) fmol/mg protein for male.Sucrose density gradient analyses revealed specific binding for both receptor types in the 4–5 S region as well as in the 8 S region. The binding affinities were in the same order of magnitude as reported for receptors found in typical steroid target organs.No significant difference in receptor values depending on sex was found for the glucocorticoid receptor: 19.2 (0–43) fmol/mg protein.     

Membrane type-1 matrix metalloproteinase promotes human melanoma invasion and growth

Membrane type-I metalloproteinase (MT1-MMP) is a transmembrane metalloproteinase that is critical for tumor cell invasion. MT1-MMP can degrade extracellular matrix (ECM) proteins directly and/or indirectly by activating soluble MMPs such as pro-MMP-2. Although MT1-MMP is upregulated in malignant melanoma, the biological consequences of elevated MT1-MMP expression for tumor progression are not entirely understood. In the current study, we have utilized the Bowes melanoma line for evaluating MT1-MMP in invasion and growth. Our studies extend the earlier observations to demonstrate that MT1-MMP expression in Bowes melanoma cells promotes selective invasion into matrigel but not matrices consisting of type-I collagen. Furthermore, MT1-MMP expressing melanoma cells exhibit increased migration in response to laminin 1 but not to type-I or type-IV collagen. MT1-MMP expression results in enhanced 3 dimensional growth in agarose gels and in long-term cultures within matrigel. The hydroxymate inhibitor BB94 inhibits MT1-MMP enhanced invasion and growth in 3 dimensional culture systems, but had no effect on increased motility. We demonstrated that MT1-MMP expression significantly facilitated tumorigenicity and growth by intradermal injection. The results suggest a more general role for elevated MT1-MMP in promoting both the selective invasion and increased growth of malignant melanoma in vivo.     

黑素瘤组织中乙醛脱氢酶1、上皮性钙黏附蛋白及尿激酶型纤溶酶原激活物受体的表达及临床意义

目的研究黑素瘤组织中乙醛脱氢酶1(aldehyde dehydrogenase 1,ALDH1)、上皮性钙黏附蛋白(E-cadherin,E-cad)及尿激酶型纤溶酶原激活物受体(urokinase-type plasminogen activator receptor,uPAR)的表达及与临床病理特征的关系。方法选择50例黑素瘤和50例色素痣患者的病变组织作为研究对象,采用免疫组化法检测病变组织中的ALDH1、E-cad及uPAR的表达情况,分析这3种蛋白表达与患者临床组织病理特征、肿瘤分期的关系。结果黑素瘤组织中ALDH1和uPAR表达阳性率分别为82.00%(41/50)和86.00%(43/50),均显著高于色素痣组(P0.05);黑素瘤组织中E-cad阳性表达率为38.00%(19/50),显著低于色素痣组(P0.05)。ALDH1、uPAR表达阳性率与患者年龄、性别、肿瘤厚度、浸润深度、是否淋巴结转移和组织病理分型均无相关性,但肿瘤厚度≥4 mm、浸润深度Ⅳ~Ⅴ、发生淋巴结转移等患者黑素瘤组织中ALDH1、uPAR表达阳性率均90%。浸润深度Ⅳ~Ⅴ的黑素瘤患者E-cad表达阳性率显著低于浸润深度Ⅰ~Ⅲ的患者(P0.05),黑素瘤患者E-cad表达阳性率与其他临床特征无相关性。结论黑素瘤在发生和进展过程中ALDH1、uPAR表达上调,E-cad表达下调,E-cad表达下调与肿瘤程度有关。     

黑素瘤抑制性活性因子在黑素瘤不同阶段及基底细胞癌中的表达

目的：探讨黑素瘤抑制性活性因子（MIA）在黑素瘤及基底细胞癌中的表达及其作用。方法：应用sP免疫组化技术检测38份黑素瘤石蜡标本、35份基底细胞癌石蜡标本以及32份色素痣石蜡标本中MIA的表达水平。结果：MIA在所有色素痣以及基底细胞癌中均呈阴性表达，而在原位黑素瘤、侵袭性黑素瘤、有淋巴结转移的黑素瘤、无淋巴结转移的黑素瘤阳性表达率分别为21．4％、91．6％、94．1％和42．8％。结论：MIA在黑素瘤的发生发展中起重要作用，MIA有可能成为临床诊断、治疗黑素瘤的靶点。