| [Background of RASSF1A]Malignant melanoma (MM) accounts for 80 percent of deaths from skin cancer, only 14 percent of patients with metastatic melanoma survive for five years. Numerous molecular events, many of them revealed by genomic and proteomic methods, have been associated with the development of MM. Several group reported that high frequency of Ras association domain family 1, isoform A (RASSF1A) gene promoter hypermethylation was detected in MM cell lines, tissues and serum of MM patients, which suggested that aberrant alteration of RASSFIA gene is also involved in melanoma progression. RASSFIA is a member of a new group of RAS effectors thought to regulate cell proliferation and apoptosis. It has been shown that introduction of RASSF1A into lung cancer cell lines lacking RASSF1A gene expression can reduce colony formation, growth in soft agar, and tumorigenesis in nude mice. At present, understanding the physiological role of RASSFIA is still in its early stages. The effect of RASSFIA expression on melanoma tumorigenic potential in vitro and in vivo has not been reported.[Downexpression of RASSF1A protein in melanoma cell lines and tissues]In this study, we analyzed the expression level of RASSFIA protein in MM tissues and cell lines. Firstly, we have screened the expression levels of RASSFIA in MM biopsies, navus pigmentosus and normal skins. By immunohistochemical analysis,10 of 10 (100%) paraffin-embedded archival normal skins showed positive to strong cytoplasmic staining of RASSFIA in most of the melanocyte cells.8 of 9 (88.9%) of paraffin-embedded navus pigmentosus showed strong cytoplasmic staining of RASSFIA in pigmentosus nest. Only 8 of 23 (34.8%) showed weak positive to moderate staining of RASSFIA in MM samples. Thus the percentage of MM tissues with a positive to strong staining intensity was significantly lower than that observed in normal or benign melanocytes(P<0.01). The relative importance of RASSF1A expression levels and clinical parameters of the patients were analyzed. The positive expression rate of RASSFIA was 57.1%(8/14) in the group of 14 MM patients without metastasis, while it was 0%(0/9) in the group of 9 MM patients with metastasis (P=0.007). Thus the positive ratio of RASSF1A expression was higher in the non-metastasis group than that in the metastasis group.[Exogenous expression of RASSFIA suppresses melanoma tumorigenecity in vitro and in vivo]We have screened the expression levels of RASSFIA in several melanoma cell lines, it shows a absent pattern in metastatic melanoma cell lines(1205Lu, MeWo, A375SM, M14 and A375 cell lines), while a weak expression pattern in non-metastatic melanoma cells(WM1552C, WM1341D, WM793, WM164). To identify the effect of RASSFIA expression on melanoma tumorigenic potential in vitro and in vivo, the wild-type RASSF1A was stablely introducted into melanoma A375 cells. By quantitative RT-PCR, variable levels of RASSFIA expression were observed in RASSF1A-expressing clones. In contrast, undetectable level of RASSFIA expression was found in pIRESneo3 transfected cells. RASSFIA protein expression of the pooled clones was further confirmed by Western blotting. To investigate the effect of RASSFIA in cell growth, we then examined the proliferation rate of the RASSF1A-expressing cells. Expression of wild-type RASSFIA in the stable transfectants significantly reduced the number of colonies formed. The number of colonies formed in the RASSF1A-expressing clones was decreased markedly (-43%) (P=0.005). The proliferation rate of the RASSF1A-transfected clones were retarded when we compared to the vector control. The in vivo tumorigenicity potential of transfectants was then tested in athymic nude mice. Monitoring the injected mice showed that RASSF1A-expressing clones resulted in a drastic reduction in tumor sizes when compared to vector control. The wet weight of tumors in RASSF1A-expressing cells was also decreased significantly by the time of sacrifice (P-0.005).[Exogenous expression of RASSFIA induces apoptosis and cell cycle G1-S phase arrest in melanoma cells]RASSFIA may function as a mediator of apoptosis. To characterize the putative antioncogenic properties of RASSFIA on melanoma cells, we then examined whether inhibition of tumorigenicity was through RASSF1A-induced apoptosis. We used Hoechst33258 staining to measure the apoptosis of RASSF1A expressing A375 cell. RASSF1A-expressing cells showed a increase in apoptosis relative to control cells. Samilar result was obtained from FCAs analysis. We also used propidium iodide incorporation to investigate the effect of RASSF1A on cell cycle progression in the melanoma cell line A375. RASSF1A expression resulted in an increase of cells in the G1 phase while a decrease of cells in the S phase of the cell cycle compared with the mocked-transfected cells. This was correlated to decreased levels of cyclin Dl and phosphorylated Rb protein level in RASSF1A-expressing cells.[Analysis of RASSF1A target genes in melanoma cell lines]In order to identify genes modulated by RASSF1A in melanoma, the expression profile of RASSF1A expressing melanoma cells and controls were investigated. High-density oligonucleotide microarray was performed on well-characterized RASSF1A-expressing pooled clones and the vector control clones. Change of gene expression between transfected clones and control cells with a minimum of two-fold was scored as significant. Using these selection criteria, we identified 210 differentially expressed genes in the microarray analysis. Totally,26 genes were downregulated whereas 184 were upregulated. Microarray analysis of melanoma cells stably expressing RASSF1A identified expression changes consistent with the observed phenotypic effects such as increased apoptosis modifiers (ASK1, MST1 and RASSF2), reduced cell cycle effectors (cyclins D2 and GAS1), and alterations in the levels of a number of cell adhesion molecules.[RASSF1A mediates mitochondrion apoptosis pathway through upregulation of ASK1 in melanoma cells]Quantitative RT-PCR analysis confirmed changes in the mRNA levels of apoptosis signal-regulating kinase 1(ASK1) and RASSF2 in accordance with the microarray data. RASSF1A mediated up-regulation of ASK1 mRNA levels was also reflected by increases in the level of ASK1 protein. ASK1 activated stress-activated protein kinase (SAPK, also known as JNK; c-Jun amino-terminal kinase) and P38 subgroups of MAP kinases, respectively. Hence we questioned whether up-regulation ASK1 by overexpression of RASSF1A would promote activation of JNK and P38 MAP kinase. Indeed, although basal expression of P38 was unaffected in RASSF1A overexpressing cells, phosphorylation of P38 was found to be increased in cells overexpressing RASSF1A. However, activation of JNK was undetectable both in cultured RASSF1A overexpressing A375 cells and vector control cells. RASSF1A also induced a decrease of Bcl2 protein. Cleaved caspase 3, a marker of active effector caspase activity, appeared at elevated levels in RASSF1A expressing cells. RASSF1A expression also promoted precocious cytochrome c release, as determined by subcellular fraction and western blot. To assess the relevance of ASK1 for the RASSF1A-mediated apoptotic cell death, we tested the effect of reducing ASK1 expression on RASSF1A-induced apoptosis. To that end, A375 cells expressing RASSF1A were transfected with either ASK 1-specific siRNA or irrelevant siRNA. Two distinct ASK1-specific siRNAs were pooled and used, and expression of ASK1-specific siRNAs significantly impaired ASK1 expression. As expected, silencing ASK1 with siRNAs partially rescued cells from RASSF1A induced apoptosis, as revealed by FCAs analysis. These data indicate that up-regulation of ASK1 contributes to RASSF1A-induced apoptosis in MM cells.[Exogenous expression of RASSF1A blocks the AKT-p70S6K-eIF4E pathway]To identify the pathway upstream of RASSF1A-induced apoptosis and cell cycle arrest, we did Western blot analysis. It is interesting that phosphorylated AKT was downregulated in the RASSF1A expressing A375 cells. This result is consistent with a previous report, which showed that the exogenous expression of RASSF1A down-regulated the expression of phosphorylated AKT. The downstream mediators of AKT were also examined by Western blot analysis. The exogenous expression of RASSF1A inhibited the phosphorylation of p70S6K and eIF4E. To analyze whether overexpression of AKT in RASSF1A transfected cells could restore PI3K/AKT/eIF4E signaling, we exogenously expressed a myc-tagged AKT in RASSF1A stable transfected A375 cells. Exogenous expression of myc-AKT1 increased the phosphorylation of eIF4E and expression level of Bcl2 and cyclin D1, while the expression level of phosphorylated P38 MAPK decreased. Thus these result indicates that RASSF1A inhibits AKT-mTOR (p70S6K and eIF4E) signal pathway in melanoma A375 cells, which at least partially contribute to mediate the expression level of apoptosis regulator Bcl2 and cell cycle molecules such as cyclin D1, which in turn induced apoptosis and cell cycle G1-S arrest. |
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