Disease progression and patient survival are significantly influenced by BRAF protein expression in primary melanoma.

BACKGROUND: Mutation of BRAF is a prevalent event in melanoma. Despite much attention to the role of BRAF mutation in melanoma, the status of BRAF protein expression and its significance in melanoma progression are unknown. OBJECTIVES: We investigated the BRAF expression level in different stages of melanocytic lesions and evaluated its correlation with clinicopathological features and patient survival. METHODS: Using tissue microarray, BRAF expression and its correlation with patient outcome was evaluated in 49 naevi samples and 370 patients with melanoma. We also evaluated the correlation of BRAF protein expression and V600E mutation using direct sequencing. RESULTS: Compared with naevi samples, BRAF expression was remarkably increased in primary melanomas and further increased in metastatic melanomas (P = 1·8 × 10(-11) ). High BRAF expression was significantly correlated with thicker tumours, ulceration and higher American Joint Committee on Cancer stages (P = 1·5 × 10(-7) , 1·5 × 10(-5) and 3·6 × 10(-13) , respectively). In cases of primary melanoma, patients with high BRAF expression had significantly worse overall (P = 0·009) and disease-specific 5-year survival (P = 0·007). While there was a trend for higher prevalence of BRAF V600E mutation in patients with high BRAF protein expression, no significant correlation was observed between protein expression and BRAF mutation. Furthermore, univariate Cox regression analysis confirmed high BRAF protein expression as a strong risk factor for poor patient survival in primary melanoma [hazard ratio (HR) 2·08 for overall survival; HR 2·39 for disease-specific survival]. CONCLUSIONS: Our data demonstrate that BRAF protein expression is significantly increased during melanoma progression. In addition, we revealed a novel prognostic value for BRAF protein expression in primary melanoma as it is significantly correlated with poor patient survival.

Sox4-mediated Dicer expression is critical for suppression of melanoma cell invasion.

We previously reported reduced expression of Sox4 in metastatic melanoma and its role in suppression of cell migration and invasion through inhibition of nuclear factor (NF)-κB p50. Sox4 can also bind to the promoter sequence of Dicer, a microRNA (miRNA) biogenesis factor. Interestingly, altered expression of Dicer was also observed in cancers. However, the potential mechanisms that regulate Dicer expression and its potential significance in melanoma progression are unknown. Here, we studied the regulation of Dicer expression by Sox4 and its role in suppression of melanoma invasion. Our data showed that Sox4 positively regulates Dicer expression by binding to its promoter sequences and enhancing its activity. We found that knockdown of Dicer enhances the matrigel invasion of melanoma cells by at least twofold. In addition, we revealed that overexpression of exogenous Dicer reverts the enhanced melanoma cell invasion upon Sox4 knockdown. Furthermore, we examined the expression of Dicer protein in a large set of melanocytic lesions (n=514) at different stages by tissue microarray and found that Dicer expression is inversely correlated with melanoma progression (P<0.0001). Consistently, reduced Dicer expression was correlated with a poorer overall and disease-specific 5-year survival of patients (P=0.015 and 0.0029, respectively). In addition, we found a significant correlation between expression of Sox4 and Dicer proteins in melanoma biopsies (P=0.009), further indicating the regulation of Dicer expression by Sox4. Finally, we revealed that knockdown of Sox4 induces a major change in the expression pattern of miRNAs in melanoma cells, mainly due to reduced expression of Dicer. Our results pinpoint the regulation of Dicer expression by Sox4 in melanoma and the critical role of Dicer in suppression of melanoma invasion. Our findings on Sox4-regulated miRNA biogenesis pathway may aid toward the development of novel targeted therapeutic approaches for melanoma.

Integrin-linked kinase regulates melanoma angiogenesis by activating NF-κB/interleukin-6 signaling pathway.

Integrin-linked kinase (ILK) is a highly conserved serine-threonine protein kinase involved in cell-extracellular matrix interactions, cytoskeletal organization and cell signaling. Overexpression of ILK in epithelial cells leads to anchorage-independent growth with increased cell cycle progression. Previously, we have shown that ILK upregulation strongly correlates with melanoma progression, invasion and inversely correlates with 5-year survival of melanoma patients. However, the molecular mechanism by which ILK enhances melanoma progression is currently unknown. In the present study, we found that proangiogenic molecule interleukin-6 (IL-6) is the downstream target of ILK in melanoma cells. ILK overexpression increased IL-6, whereas silencing of ILK suppressed IL-6 expression at both messenger RNA and protein levels. ILK also altered the activity and subcellular localization of nuclear factor-kappaB (NF-κB) subunit p65. We further found that ILK enhanced the IL-6 gene transcription by promoting the binding of NF-κB p65 to IL-6 promoter. Moreover, ILK overexpression in melanoma cells enhanced the tube-forming ability of endothelial cells in vitro and microvessel formation in vivo. ILK-induced tube and blood vessel formation of endothelial cells was significantly reduced upon IL-6 inhibition in ILK-overexpressing melanoma cells. To delineate the mechanism by which ILK-induced IL-6 production can enhance angiogenesis, further analysis of the downstream targets of IL-6 signaling showed an increased activity of the signal transducer and activator of transcription 3 (STAT3) in ILK-overexpressing cells. As STAT3 binds to vascular endothelial growth factor (VEGF) promoter, we found that VEGF levels were elevated in ILK-overexpressing cells and declined upon transfection of IL-6 small interfering RNA, suggesting that ILK may regulate VEGF expression through IL-6 pathway by activating STAT3.

Differential effects of Nucleostemin suppression on cell cycle arrest and apoptosis in the bladder cancer cell lines 5637 and SW1710.

OBJECTIVES: The Nucleostemin (NS) gene encodes a nucleolar protein enriched in adult and embryonic stem cells. NS is thought to regulate cancer cell proliferation, but the mechanisms involved are poorly understood. In this study, we have investigated the role of NS in bladder cancer. MATERIALS AND METHODS: Expression of NS was determined by quantitative reverse transcription-polymerase chain reaction in bladder carcinoma cell lines and in normal uro-epithelial cell cultures. We used an RNAi strategy to investigate the function of NS in two selected carcinoma cell lines. RESULTS: High NS expression was found in most bladder carcinoma cell lines and normal uro-epithelial cells. Knockdown of NS expression induced a severe decline in cell proliferation in 5637 and SW1710 cell lines, both with mutant p53. Apoptosis was more strongly enhanced in 5637 cells lacking RB1 than in SW1710 cells lacking p16(INK4A). Moreover, NS-siRNA-treated 5637 cells accumulated mainly in G(2)/M, whereas SW1710 cells arrested in G(0)/G(1). CONCLUSION: Our data indicate that NS expression is necessary for cell proliferation and evasion of apoptosis in bladder cancer cells, independent of its effect on p53. Also, we speculate that the precise effect of NS on cell cycle regulation may relate to functional status of RB1 and CDKN2A/p16(INK4A).

Knocking-down the expression of nucleostemin significantly decreases rate of proliferation of rat bone marrow stromal stem cells in an apparently p53-independent manner.

OBJECTIVES: Nucleostemin (NS) is a recently identified GTP-binding protein, predominantly expressed in embryonic and adult stem cells but not in terminally differentiated cells. NS is expressed in bone marrow-derived mesenchymal stem cells, and its expression ceases upon induction of neural differentiation. The major aim of this study was to determine whether down-regulation of NS expression acts as a promoter, or otherwise as a by-product of differentiation and senescence processes. MATERIALS AND METHODS: We used RNA interference protocols to specifically knock down NS in rat bone marrow-derived stromal stem cells. Changes in rate of proliferation and cell cycle profile after knocking-down of NS were measured. In addition, changes in expression of associated genes were studied by semiquantitative RT-PCR, Western blotting and immunocytochemistery. RESULTS: Knocked-down expression of NS caused a significant decrease in the rate of cell proliferation with concomitant shutting off of expression of cyclin D1 and survivin, two other well-known regulators of cell proliferation. Interestingly, we noticed no obvious changes in expression level of p21, the main effector of p53 for its cell cycle repressing function. CONCLUSION: Our findings revealed a master role for NS in promoting proliferation of rat bone marrow-derived stromal stem cells. Moreover, we suggest that despite previous proposals, the cell cycle arrest/inhibitory role of NS is unlikely to be related to its proposed property of interaction with p53.