Fibroblast growth factor-2 but not Mel-CAM and/or beta3 integrin promotes progression of melanocytes to melanoma

A variety of melanoma-associated antigens have been identified that mediate adhesion, growth, proteolysis, and modulation of immune response. However, the mechanisms by which human normal melanocytes become malignant are not clearly understood. Among the most consistent observations is the up-regulation of fibroblast growth factor-2 (FGF-2) and of the adhesion molecules beta3 integrin and Mel-CAM during melanoma progression. To evaluate the potential role of FGF-2, beta3 integrin and Mel-CAM in melanoma development we overexpressed FGF-2, beta3 integrin and Mel-CAM in normal human melanocytes using replication-deficient adenoviruses as a gene delivery vehicle. Fibroblast growth factor-2 overexpressing melanocytes in monolayer culture displayed cytological atypia. Furthermore, in human skin reconstructs where the physiological milieu is recreated in vitro, FGF-2-overexpressing melanocytes exhibited marked proliferation, upwards migration, cluster formation and type IV collagen expression within the epidermal compartment, simulating early radial growth phase melanoma. In contrast, overexpression of beta3 integrin and/or Mel-CAM in melanocytes did not affect their biological behaviour in human skin reconstructs. The described results of the current and previous studies emphasise the key role of FGF-2 in melanoma development and progression, underscoring the promise of FGF-2 as a target for therapy.     

miRNA-193b-5p抑制转录调节因子CITED2表达对黑素合成的影响

目的初步探讨miRNA(miR)-193b-5p对黑素合成的影响及可能机制。方法从健康男性包皮环切术后废弃的正常包皮组织中分离培养人原代黑素细胞。分别转染miR-NC模拟物(miR-NC mimics组)和miR-193b-5p模拟物(miR-193b-5p mimics组)至人原代黑素细胞及人黑素瘤MNT1细胞, 转染48 h时实时定量PCR(RT-qPCR)检测miR-193b-5p的过表达效率, 转染72 h时Western印迹检测黑素合成相关蛋白酪氨酸酶和小眼畸形相关转录因子的表达, 转染1周时采用氢氧化钠法测定细胞中黑素含量。通过TargetScan网站预测miR-193b-5p的靶基因并采用双荧光素酶报告实验验证, RT-qPCR及Western印迹检测miR-193b-5p过表达后该靶基因mRNA和蛋白表达水平的变化。两组间比较采用两独立样本t检验。结果人原代黑素细胞及人黑素瘤MNT1细胞中, miR-193b-5p mimics组miR-193b-5p的表达水平均显著高于miR-NC mimics组, t值分别为65.57、22.49, 均P < 0.001, 且...     

Wnt signaling through the beta-catenin pathway is sufficient to maintain, but not restore, anagen-phase characteristics of dermal papilla cells

Dermal papilla cells of the hair follicle can be maintained in an active, hair-inducing state in vitro when cocultured with cells secreting Wnt3a. By inducing cultured dermal papilla cells to secrete Wnt themselves, we demonstrate that this activity is a direct effect of Wnt signaling to dermal papilla cells. We further demonstrate that the effects of Wnt3a are exerted through activation of the beta-catenin signal transduction pathway and do not require alternative Wnt transduction cascades. Once dermal papilla cells have lost hair-inducing properties in vitro, neither treatment with Wnt nor expression of a truncated and activating form of beta-catenin is sufficient to restore these properties to the cultured cells.     

microRNA-21 is upregulated in malignant melanoma and influences apoptosis of melanocytic cells

Overexpression of microRNA-21 (miR-21) has been observed in various cancer types, but little is known about the role of miR-21 in melanoma. In this study, we demonstrate that levels of miR-21 are significantly increased in primary melanoma tissues as compared to benign nevi and in human melanoma cell lines as compared to melanocytic cell preparations. We show that downregulation of miR-21 in melanoma cell lines with high endogenous miR-21 expression induced apoptosis, whereas proliferation was not significantly altered. Upregulation of miR-21 in melanocytes resulted in increased proliferation and decreased apoptosis. However, in the MEWO melanoma cells with low endogenous miR-21 expression, upregulation of miR-21 had no functional effects. These findings indicate a potential pathogenetic role of miR-21 upregulation in a subgroup of melanomas.     

黑素瘤细胞及表皮黑素细胞中酪氨酸激酶c-abl,c-kit和PDGFRα/β的表达

目的观察酪氨酸激酶在多株黑素瘤细胞系及原代表皮黑素细胞中的表达,探讨酪氨酸激酶在黑素瘤发生发展过程中的作用。方法分离并培养人原代表皮黑素细胞,培养浅表垂直生长期黑素瘤细胞株WM793B,转移性黑素瘤细胞株A2058,WM-266-4,Hs294T,SK-MEL-5和SK-MEL-1,用RT-PCR法分别检测7株细胞中酪氨酸激酶c-abl,c-kit和PDGFRα/βmRNA的表达;Western-blot法检测7组细胞中非受体性酪氨酸激酶c-abl蛋白水平的表达。结果与表皮黑素细胞和WM793B细胞相比,5株转移性黑素瘤细胞中的c-ablmRNA及其蛋白水平的表达偏高;而PDGFRα/βmRNA表达明显偏低、c-kitmR-NA在表皮黑素细胞,A2058,WM-266-4和Hs294T中mRNA表达较高,而在WM793B,SK-MEL-5和SK-MEL-1中偏弱。结论酪氨酸激酶c-abl对黑素瘤的发生发展可能有促进作用,而PDGFRα/βmRNA的低表达可能与黑素瘤的转移有关。     

Wnt1 is anti-lymphangiogenic in a melanoma mouse model

Wnt signals contribute to melanoma progression by boosting their proliferation and survival. Initially, we expected that activated Wnt signaling also improves their proficiency to recruit blood and lymph vessels. To assess this, we added cell culture supernatants (SNs) of Wnt1(+) and Wnt1(-) melanoma to endothelial spheroids. Whereas SNs of Wnt1(-) melanoma cells induced lymphatic sprouts, those of Wnt1(+) cells were unable to do so and this was restored by vascular endothelial growth factor C (VEGF-C). Subsequent testing of several human melanoma lines revealed that Wnt1 suppressed their VEGF-C expression. This Wnt1 effect did not depend on glycogen synthase kinase-3β (GSK3β), β-catenin, or activator protein-1, but was blocked by cyclosporine A (CsA). To analyze Wnt1 effects in melanoma in vivo, we selected Wnt1(-) melanoma cell lines, overexpressed Wnt1, and injected them subepidermally into severe combined immunodeficient (SCID) mice. We found reduced VEGF-C expression, reduced lymphangiogenesis, and delayed metastasis to sentinel nodes in Wnt1(+) as compared with Wnt1(-) melanoma (P<0.05). Concomitant overexpression of VEGF-C or feeding of animals with CsA restored lymphangiogenesis and metastasis in Wnt1(+) melanoma. In conclusion, Wnt1 is anti-lymphangiogenic by suppressing melanoma-derived VEGF-C expression.     

The role of RhoC in growth and metastatic capacity of melanoma

Background:  RhoC overexpression in tumor cells promotes invasive and metastatic behavior. RhoC expression levels have been correlated with tumor progression and metastasis in multiple human cancers. In melanoma, RhoC is upregulated in highly metastatic tumors. Induced expression in melanoma cell lines resulted in invasion and metastasis, whereas inhibition of RhoC reversed the metastatic phenotype both in vitro and in vivo .
Methods:  RhoC mRNA and protein expression in two human melanoma cell lines (DX3aza and MeWo) and pooled primary melanocytes were investigated by means of real-time quantitative polymerase chain reaction and western blotting. RhoC protein expression was evaluated in 123 primary cutaneous melanoma samples by the use of immunohistochemistry and correlated with known prognostic features.
Results:  RhoC upregulation was observed in the highly metastatic DX3aza cell line, whereas in MeWo, only low expression levels could be detected. RhoC expression in primary cutaneous melanoma was strongly associated with thicker and ulcerated tumors. RhoC expression was associated with the presence of lymphatic metastases at the time of diagnosis and shorter disease-free and overall survival rates, without being an independent predictor.
Conclusion:  These results further support a role for RhoC in growth and metastasis of 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.     

整合素β8在恶性黑素瘤细胞增殖及侵袭中的作用

目的:明确整合素β8(ITGB8)在恶性黑素瘤侵袭转移中的作用。方法:Transwell侵袭实验在黑素瘤细胞系A375中获得侵袭性癌细胞和相对静止性癌细胞,RT-PCR比较两者整合素家族的表达。流式分选ITGB8细胞,比较其侵袭能力的差异。使用siRNA敲除技术敲低ITGB8,敲低效率分别为:si1组50%,si2组70%,si3组80%,比较其增殖、侵袭能力的变化。Western bolt检测细胞周期蛋白、基质金属蛋白酶及EMT相关分子的表达变化,提取细胞核蛋白比较细胞核内β-catenin的变化。利用TCF4的启动子报告质粒检测Wnt/β-catenin信号通路的激活情况。结果:ITGB8在侵袭性癌细胞中高表达,是静止性癌细胞的5倍(P0.01);ITGB8阳性组侵袭细胞数为516±45,阴性组为120±22,差异有显著性(P0.01);与Mock组细胞相比,si2和si3组细胞的增殖速度明显减弱(P0.01),敲低后细胞阻滞在S期。与Mock细胞相比Cyclin D1、MMP2、MMP9、MMP7、Ncadherin、Vimentin在si2和si3细胞中的表达明显减少,Ecadherin明显增加。si2、si3细胞核内β-catenin相比Mock组明显减少(P0.05)。敲低ITBG8后TCF4的启动子活性明显降低。结论:ITGB8对恶性黑素瘤的增殖及侵袭转移具有重要作用,可能是通过调控Wnt/β-catenin信号通路发挥作用。     

Melanocytes express 3G5 surface antigen

The 3G5-reactive ganglioside antigen (3G5 antigen) is expressed on the surface of various cell types including pericytes, pancreatic islet cells, thyroid follicular cells, and cells of the pituitary and the adrenal medulla. Expression on melanocytes has not yet been reported. We examined 148 5-microm cryosections of 12 normal skin samples and 45 skin tumors (21 melanocytic nevi, 8 malignant melanoma primaries, 4 metastases of malignant melanoma, 3 basal cell carcinomas, and 9 pigmented seborrheic keratoses) by triple fluorescence technique with the monoclonal antibody 3G5, DNA fluorochrome, and the anti-melanocytic antibody A103 (Anti-Melan-A). In normal skin, 3G5 reactivity was detected in epidermal melanocytes of 4 of 12 cases with 14.8 +/- 24.1% positive melanocytes; 20 of 21 nevi (72.2 +/- 29.1% positive nevus cells, mean +/- SD), 8 of 8 primary melanomas (83.9 +/- 12.3% positive melanoma cells), and 4 of 4 melanoma metastases (82.5 +/- 6.5% positive melanoma cells) expressed the 3G5 antigen. All tumor cells of investigated basal cell carcinoma or seborrheic keratosis were 3G5 negative. This is the first report of 3G5 antigen expression in melanocytes. The data demonstrate high expression of this ganglioside in the aggregated melanocytes of malignant or benign tumors but low or absent expression in singular melanocytes (normal epidermis, seborrheic keratoses) reflecting a different biologic state.     

beta-Catenin mutation and its nuclear localization are confirmed to be frequent causes of Wnt signaling pathway activation in pilomatricomas

BACKGROUND: beta-Catenin has been shown to play an important role in the formation of hair follicle-related tumors, including pilomatricomas. Several investigators have shown that beta-catenin gene mutation is observed in pilomatricomas. However, the relationship between the pattern of beta-catenin localization in the cell and beta-catenin gene mutation is still controversial. OBJECTIVES: This work was performed to determine the frequency of beta-catenin nuclear localization in pilomatricoma, the relationship between the pattern of beta-catenin localization and beta-catenin mutation, and the involvement of APC mutation. METHODS: Typical 32 pilomatricomas were examined for beta-catenin expression by immunostaining. Genomic DNA was extracted, amplified and sequenced from 23 pilomaticomas with nuclear beta-catenin staining and 4 pilomaticomas without nuclear beta-catenin staining. Mutations of beta-catenin gene were confirmed by subcloning assay and restriction endonuclease assay. RESULTS: Using immunostaining, we found that 81% (26/32) of pilomatricomas displayed nuclear beta-catenin staining in basophilic cells. Sequence analysis revealed that 61% (14/23) contained mutations in exon 3 of beta-catenin. However, no mutations were detected in 4 pilomaticomas without beta-catenin nuclear staining. Detected mutations were adjacent to or abolished well-known regulatory phosphorylation sites of beta-catenin. APC gene mutations were not detected in 27 pilomatricomas with/without beta-catenin nuclear staining. CONCLUSIONS: These results confirmed that beta-catenin mutation and its nuclear localization are frequent causes of Wnt signaling pathway activation and suggested that beta-catenin activation mutations contribute to tumorigenesis of pilomatricomas.     

β-Catenin expression in the transitional cell zone of pilomatricoma

BACKGROUND: beta-Catenin, a participant in the Wnt pathway, has been shown to play an important role in the morphogenesis of hair follicles and the formation of hair follicle-related tumours, including pilomatricomas. It has been observed that at least 75% of human pilomatricomas possess activating mutations in beta-catenin. These findings suggested that beta-catenin plays an important role in the tumorigenesis of pilomatricomas. However, the pattern of beta-catenin expression in pilomatricoma tissues is still unclear. Objectives To examine the expression of beta-catenin in human pilomatricomas by immunohistochemical staining. METHODS: Twenty-six formalin-fixed and paraffin-embedded samples of pilomatricoma tissue were studied. RESULTS: Most transitional cells of pilomatricoma expressed beta-catenin strongly, but the basophilic cells and shadow cells did not. beta-Catenin showed a prominent membranous immunoreactivity and a small amount of condensed cytoplasmic staining, but there was definitely no evidence of nuclear positivity. CONCLUSIONS: These findings imply that beta-catenin is primarily involved in cell-cell adhesion rather than cellular proliferation during pilomatricoma pathogenesis, and suggest that if beta-catenin is involved in pilomatricoma tumorigenesis and tumour growth, it plays an indirect role.