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• W2068420134 abstract "Activation (phosphorylation) of mitogen-activated protein kinase (MAPK) signal transduction through BRAF and RAS causes a variety of functional effects including cell survival and cell death. In this study, we observed high extracellular signal-regulated kinase (ERK)1/2 phosphorylation levels in clinical melanoma metastases and various melanoma cell lines. Treatment of melanoma cell lines with cisplatin, a potent antitumor agent, increased the level of phosphorylated-ERK (P-ERK)1/2 and enhanced chemoresistance through activation of the cell survival protein 90-kDa ribosomal S6 kinase (RSK)1. The mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) was able to block this effect and reduced cell viability and sensitized cells to cisplatin-induced apoptosis, as shown by PARP cleavage, caspase 3 expression, and annexin-V staining. In conclusion, the MAP kinase–ERK pathway is activated in melanoma and reduces the sensitivity of melanoma to cisplatin. Thus, inhibition of ERK1/2 in combination with selected chemotherapeutic agents may hold promise for more effective therapy of melanoma. Activation (phosphorylation) of mitogen-activated protein kinase (MAPK) signal transduction through BRAF and RAS causes a variety of functional effects including cell survival and cell death. In this study, we observed high extracellular signal-regulated kinase (ERK)1/2 phosphorylation levels in clinical melanoma metastases and various melanoma cell lines. Treatment of melanoma cell lines with cisplatin, a potent antitumor agent, increased the level of phosphorylated-ERK (P-ERK)1/2 and enhanced chemoresistance through activation of the cell survival protein 90-kDa ribosomal S6 kinase (RSK)1. The mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) was able to block this effect and reduced cell viability and sensitized cells to cisplatin-induced apoptosis, as shown by PARP cleavage, caspase 3 expression, and annexin-V staining. In conclusion, the MAP kinase–ERK pathway is activated in melanoma and reduces the sensitivity of melanoma to cisplatin. Thus, inhibition of ERK1/2 in combination with selected chemotherapeutic agents may hold promise for more effective therapy of melanoma. 5-bromodeoxyuridine epidermal growth factor extracellular signal-regulated kinase mitogen-activated protein kinase normal human melanocytes phosphorylated-ERK recombinant human EGF 90-kDa ribosomal S6 kinase Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase (MAPK) family, which regulates essential cellular functions like proliferation, differentiation, cell survival, and cell death (Cowley et al., 1994Cowley S. Paterson H. Kemp P. Marshall C.J. Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells.Cell. 1994; 77: 841-852Google Scholar; Hoshino et al., 1999Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. et al.Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors.Oncogene. 1999; 18: 813-822Google Scholar). ERK is activated by numerous extracellular agents such as growth factors, cytokines, hormones, and tumor promotors (Johnson and Vaillancourt, 1994Johnson G.L. Vaillancourt R.R. Sequential protein kinase reactions controlling cell growth and differentiation.Curr Opin Cell Biol. 1994; 6: 230-238Google Scholar; Robinson and Cobb, 1997Robinson M.J. Cobb M.H. Mitogen-activated protein kinase pathways.Curr Opin Cell Biol. 1997; 9: 180-186Google Scholar; He et al., 1999He H. Wang X. Gorospe M. Holbrook N.J. Trush M.A. Phorbol ester-induced mononuclear cell differentiation is blocked by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059.Cell Growth Differ. 1999; 10: 307-315Google Scholar). Constitutive activation of the ERK pathway has been described in cell lines derived from pancreas, colon, lung, ovary, and kidney cancer (Hoshino et al., 1999Hoshino R. Chatani Y. Yamori T. Tsuruo T. Oka H. Yoshida O. et al.Constitutive activation of the 41-/43-kDa mitogen-activated protein kinase signaling pathway in human tumors.Oncogene. 1999; 18: 813-822Google Scholar). In melanoma, activation of ERK1/2 was observed by immunohistochemistry in 54% of primary and 33% of metastastic melanomas, respectively (Jorgensen et al., 2003Jorgensen K. Holm R. Maelandsmo G.M. Florenes V.A. Expression of activated extracellular signal-regulated kinases 1/2 in malignant melanomas: relationship with clinical outcome.Clin Cancer Res. 2003; 9: 5325-5331Google Scholar). One reason for this activation seems to be a somatic missense mutation of the BRAF gene, which was reported to occur within the kinase domain in 66% of malignant melanomas and shown to cause elevated kinase activity from RAS to mitogen-activated protein kinase kinase (MEK)1/2 and ERK1/2 (Davies et al., 2002Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Google Scholar). However, this mutation was also observed in 82% of nevi, suggesting that the RAS/RAF/MAPK pathway is a critical step in the initiation of melanocyte neoplasias but that by itself seems insufficient to cause melanomagenesis (Pollock et al., 2003Pollock P.M. Harper U.L. Hansen K.S. Yudt L.M. Stark M. Robbins C.M. et al.High frequency of BRAF mutations in nevi.Nat Genet. 2003; 33: 19-20Google Scholar). Furthermore, the identification of factors that modulate the dysbalance in favor of ERK may provide additional targets for effective therapy. In melanoma, epidermal growth factor (EGF) is one of the growth factors that can shift the balance in favor of ERK and may eventually cause tumor progression (Mirmohammadsadegh et al., 2005Mirmohammadsadegh A. Hassan M. Gustrau A. Doroudi R. Schmittner N. Nambiar S. et al.Constitutive expression of EGF receptors on normal human melanocytes.J Invest Dermatol. 2005; 125: 392-394Google Scholar). To answer questions related to ERK regulation during chemotherapy against melanoma, we chose cisplatin, one of the most potent antitumor agents, with clinical activity against a variety of solid tumors (Siddik, 2003Siddik Z.H. Cisplatin: mode of cytotoxic action and molecular basis of resistance.Oncogene. 2003; 22: 7265-7267Google Scholar). Cisplatin interacts with DNA to form DNA adducts and activates several signal pathways like MAPK, p53, AKT, and p73 (Siddik, 2003Siddik Z.H. Cisplatin: mode of cytotoxic action and molecular basis of resistance.Oncogene. 2003; 22: 7265-7267Google Scholar). It has been reported that cisplatin is able to activate p38 MAPK in different human cell lines and that its inhibition causes resistance to chemotherapy (Losa et al., 2003Losa J.H. Parada Cobo C. Viniegra J.G. Sanchez-Arevalo Lobo V.J. Ramon y Cajal S. Sanchez-Prieto R. Role of the p38 MAPK pathway in cisplatin-based therapy.Oncogene. 2003; 22: 3998-4006Google Scholar). A recent study demonstrated that cisplatin-induced apoptosis occurred through the p38 MAPK and not through the MEK1/2–ERK1/2 pathway (Wu et al., 2005Wu Y.J. Muldoon L.L. Neuwelt E.A. The chemoprotective agent N-acetylcysteine blocks cisplatin-induced apoptosis through caspase signaling pathway.J Pharmacol Exp Ther. 2005; 312: 424-431Google Scholar). Other studies suggested that ERK1/2 activation by cisplatin induced cell death (Wang et al., 2000Wang X. Martindale J.L. Holbrook N.J. Requirement for ERK activation in cisplatin-induced apoptosis.J Biol Chem. 2000; 275: 39435-39443Google Scholar; Arany et al., 2004Arany I. Megyesi J.K. Kaneto H. Price P.M. Safirstein R.L. Cisplatin-induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells.Am J Physiol Renal Physiol. 2004; 287: F543-F549Google Scholar; Choi et al., 2004Choi B.K. Choi C.H. Oh H.L. Kim Y.K. Role of ERK activation in cisplatin-induced apoptosis in A172 human glioma cells.Neurotoxicology. 2004; 25: 915-924Google Scholar). In contrast, the inhibition of ERK enhanced the sensitivity to cisplatin in ovarian cancer by accumulation of p53 (Persons et al., 1999Persons D.L. Yazlovitskaya E.M. Cui W. Pelling J.C. Cisplatin-induced activation of mitogen-activated protein kinases in ovarian carcinoma cells: inhibition of extracellular signal-regulated kinase activity increases sensitivity to cisplatin.Clin Cancer Res. 1999; 5: 1007-1014Google Scholar). These conflicting findings of pro- and antiapoptotic functions may reflect differences in the cellular context and target proteins regulated by ERK such as p53 and 90-kDa ribosomal S6 kinase (RSK) that are involved in apoptosis and cell survival. The RSK family of serine/threonine kinases regulates gene expression by phosphorylating a number of transcription factors (Chen et al., 1993Chen R.H. Abate C. Blenis J. Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase.Proc Natl Acad Sci USA. 1993; 90: 10952-10956Google Scholar). In humans, the RSK family consists of four isoforms (RSK1–4) and two structurally related proteins, called RSK-like protein kinase (RLPK/MSK1)-A and RSK-B (MSK2). RSK family members are unusual among serine/threonine kinases in that they contain two distinct kinase domains, both of which are catalytically functional (Fisher and Blenis, 1996Fisher T.L. Blenis J. Evidence for two catalytically active kinase domains in pp90rsk.Mol Cell Biol. 1996; 16: 1212-1219Google Scholar). Activated RSK has both cytoplasmic and nuclear substrates. RSK plays an active role in nuclear signaling by phosphorylating the cyclic AMP response element-binding protein (Xing et al., 1996Xing J. Ginty D.D. Greenberg M.E. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase.Science. 1996; 273: 959-963Google Scholar), c-Fos (Chen et al., 1993Chen R.H. Abate C. Blenis J. Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase.Proc Natl Acad Sci USA. 1993; 90: 10952-10956Google Scholar), and IκB (Schouten et al., 1997Schouten G.J. Vertegaal A.C. Whiteside S.T. Israel A. Toebes M. Dorsman J.C. et al.IkappaB alpha is a target for the mitogen-activated 90 kDa ribosomal S6 kinase.EMBO J. 1997; 16: 3133-3144Google Scholar). Phosphorylation of Bad (Bonni et al., 1999Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms.Science. 1999; 286: 1358-1362Google Scholar; Shimamura et al., 2000Shimamura A. Ballif B.A. Richards S.A. Blenis J. Rsk1 mediates a MEK-MAP kinase cell survival signal.Curr Biol. 2000; 10: 127-135Google Scholar) and C/EBPβ (Buck et al., 2001Buck M. Poli V. Hunter T. Chojkier M. C/EBPβ phosphorylation by RSK creates a functional XEXD caspase inhibitory box critical for cell survival.Mol Cell. 2001; 8: 807-816Google Scholar) by RSK can protect cells from apoptosis. Moreover, RSK phosphorylates histone H3 (Sassone-Corsi et al., 1999Sassone-Corsi P. Mizzen C.A. Cheung P. Crosio C. Monaco L. Jacquot S. et al.Requirement of Rsk-2 for epidermal growth factor-activated phosphorylation of histone H3.Science. 1999; 285: 886-891Google Scholar), suggesting that RSK may regulate chromatin remodeling. In this study, we examined the importance of P-ERK in malignant melanoma metastases as well as its modulation after treatment with recombinant human EGF (rEGF), cisplatin, and the MEK inhibitor U0126. The manipulation of ERK activity by specific inhibitors provided evidence that its antiapoptotic function in melanoma is mediated through the activation of the survival protein RSK1. ERK1/2 was constitutively expressed in normal human melanocytes (NHM) and different melanoma cell lines as well as in most (27 of 31; 87%) investigated melanoma metastases of patients before receiving chemotherapy (Figure 1). In contrast, phosphorylated ERK1/2 levels were low in NHM, upregulated in melanoma cell lines, and highly abundant in 25 of 31 (81%) melanoma metastases (Figure 1). Immunohistochemically, ERK and activated ERK could be demonstrated in the cytoplasm and nucleus of melanoma cells, respectively (Figure 2b and d). In a recent study, we have demonstrated high levels of EGFR on NHM as well as on melanoma metastases (Mirmohammadsadegh et al., 2005Mirmohammadsadegh A. Hassan M. Gustrau A. Doroudi R. Schmittner N. Nambiar S. et al.Constitutive expression of EGF receptors on normal human melanocytes.J Invest Dermatol. 2005; 125: 392-394Google Scholar) and have suggested EGF as one important growth factor responsible for ERK phosphorylation in melanoma. Therefore, we confirmed the EGFR expression on the melanoma cell lines used in this study (Figure 3). Western blot experiments demonstrated high EGFR levels on NHM and A375 cells and HaCaT (immortalized epithelial cell line as positive control) and revealed low levels on BLM and MV3 cells, respectively (Figure 3).Figure 2Immunohistochemistry of activated ERK1/2. (a and b) A representative sample of a metastastic melanoma showed, upon higher magnification, predominant cytoplasmatic ERK1/2 staining. (c) A corresponding section showed intense nuclear staining with P-ERK1/2. (*d) Upon higher magnification, most cells contained cytoplasmatic and pronounced nuclear staining. Of note, some tumor cells were negative for P-ERK1/2. The appropriate isotype controls showed no staining (data not shown). Bar=2 μM in (a), 4 μM in (c), and 10 μM in (b) and (d), respectively.View Large Image Figure ViewerDownload (PPT)Figure 3Expression of EGFR. NHM and various melanoma cell lines (A375, BLM, MV3, and M13) were analyzed by Western blot analysis. NHM and A375 cells showed the highest EGFR levels, whereas BLM cells had significantly lower amounts of EGFR. HaCaT cells (immortalized epithelial cell line) were used as positive control.View Large Image Figure ViewerDownload (PPT) Next, we investigated the effect of rEGF on ERK phosphorylation of NHM and A375 cells, known to contain high levels of EGFR. rEGF was able to induce expression and activation of ERK1/2 in NHM and A375 cells as early as after 5 minutes (Figure 4). This finding correlated with the effect of EGF on proliferation of NHM, where proliferation has been shown 24 hours after treatment with rEGF (Mirmohammadsadegh et al., 2005Mirmohammadsadegh A. Hassan M. Gustrau A. Doroudi R. Schmittner N. Nambiar S. et al.Constitutive expression of EGF receptors on normal human melanocytes.J Invest Dermatol. 2005; 125: 392-394Google Scholar). Next we analyzed, if treatment with cisplatin, a commonly used anticancer drug as well as specific inhibition of the MAPK pathway, were able to reduce viability of melanoma cell lines and attempted to explore how these drugs affected ERK activation in vitro (Figure S1). Download .gif (.02 MB) Help with files Figure S1Supplemental Fig 1: Detection of RSK1 and P-RSK1 in melanoma metastases by Western blotting. Both melanoma cell lines and 4 metastasis patient samples showed elevated p-ERK1/2. The BLM melanoma cell line and 2 out of the 4 melanoma metastasis patients that had elevated p-ERK1/2 also showed elevated p-RSK1. Different melanoma cell lines, A375, BLM, MV3, and M13 were used to investigate the effect of cisplatin in combination with the MEK1/2 inhibitor U0126 with regard to cell viability, as measured by conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, and cell proliferation/cells in S-phase, as measured by 5-bromodeoxyuridine (BrdU) incorporation. We observed that most melanoma cell lines treated with cisplatin and U0126 showed a significant decrease in cell survival (Figure 5a) and proliferation (Figure 5b) over cisplatin treatment alone, in particular at lower concentrations (10 and 20 μM), whereas MV3 and BLM cells were less sensitive to different concentrations (Figure 5a and b). Collectively, these results strongly indicate that cisplatin-mediated inhibition of survival and proliferation of melanoma cells is enhanced in the presence of the MEK inhibitor U0126 and that these inhibitory effects are more significant at lower cisplatin concentrations for most melanoma cell lines investigated. Furthermore, following combination therapy, 2.48 times more BLM cells and 1.93 times more A375 cells became annexin-V-positive, respectively (Figure 6a and b), when compared with cells treated with cisplatin only. The MEK inhibitor U0126 alone had no effect on apoptosis (Figure 6a and b). Given that ERK1/2 was phosphorylated in melanoma metastases and cisplatin being a potent anticancer drug, it was important to study the effect of cisplatin with regard to the phosphorylation status on ERK1/2. We observed increasing phosphorylation of ERK1/2 after treatment of different melanoma cell lines with 100 μM cisplatin (Figure 7). This upregulation correlated with a high expression of cleaved poly (ADP-ribose) polymerase (PARP), an apoptosis marker (Figure 7). Next, the role of ERK1/2 in the regulation of apoptosis was investigated in more detail. Next, the time course of cisplatin monotherapy and cisplatin/U0126 combination therapy was analyzed with regard to P-ERK1/2 activation (Figure 8). Cisplatin induced a time-dependent phosphorylation of ERK1/2 in A375 cells with a maximum at 12 hours (Figure 8a). The proapoptotic proteins cleaved PARP and caspase 3 were detected between 12 and 24 hours, which confirmed the occurrence of apoptosis (Figure 8a). Furthermore, the activation of the tumor suppressor protein p53 on different serine residues (P-serine at position 6, 9, 15, 20, 37, and 392) was investigated using phosphorylation-specific anti-serine antibodies. Only serine at position 6 was phosphorylated by cisplatin at 12 hours, being consistent with ongoing apoptosis (Figure 8a). Interestingly, at the same time point, the cell-survival protein RSK1 was also strongly activated by cisplatin in A375 cells (Figure 8a) and BLM cells (Figure 9a), suggesting that cisplatin-treated cells initially tried to avoid apoptosis by activation of cell-survival proteins, but ultimately cell death occurred. Furthermore, the activation of the cell survival protein P-RSK1 was inhibited by U0126 (Figures 8b and 9b).Figure 9Effect of cisplatin and U0126 on expression of ERK1/2, p38, and apoptosis-related proteins in BLM cells. (a) Cisplatin-induced activation of ERK1/2 showed a maximum at 24 hours. P38 was phosphorylated by cisplatin after 24 hours, although weaker than in A375 cells. Cleavage of caspases 8 and 9 occurred after 24 hours. Time-dependent upregulation of Bcl-2 and Bax was detectable. Cleavage of PARP and caspase 3 was also induced by cisplatin with a maximum at 24 hours. Cisplatin-induced activation of RSK1 occurred at 6 hours with a maximum at 24 hours. (b) Upon co-incubation with U0126 (25 μM) and cisplatin (100 μM), the activation of ERK1/2 was inhibited by U0126 and cisplatin was unable to revert this effect. Constant levels of p38 but an upregulation of P-p38 expression were observed at 48 hours. Like in A375 cells, the cleavage of PARP and caspase 3 was strongly induced and persisted between 24 and 48 hours. α-tubulin confirmed equal loading and quality of protein extract.View Large Image Figure ViewerDownload (PPT) To elucidate the molecular mechanism of cisplatin-mediated apoptosis in melanoma cells and its enhancement upon inhibition of MEK/ERK pathway, we compared the regulation of apoptosis-related proteins post-cisplatin treatment to co-treatment of cisplatin and U0126 in A375 and BLM melanoma cells. First, we demonstrated that cisplatin-induced time-dependent phosphorylation of ERK1/2 in A375 and BLM cells was abolished upon co-treatment of cisplatin and U0126 (Figures 8a and b and 9a and b), whereas the pattern of cisplatin-induced phosphorylation of p38 remained unchanged upon co-treatment of cisplatin and U0126 (Figure 8a and b). We also observed an induction of proteins related to apoptosis from both intrinsic (Bcl-2 and Bax) and extrinsic apoptotic pathways (caspases 9, 8, and 3) as well as a more pronounced PARP cleavage upon co-treatment of cisplatin/U0126 in both A375 and BLM cells (Figures 8a and b and 9a and b). These data provide evidence that activated ERK1/2 seems to phosphorylate different antiapoptotic proteins that aim to rescue cells from death conditions. As described in the literature, activated ERK1/2 may exert opposing functions such as cell death and cell survival (Persons et al., 1999Persons D.L. Yazlovitskaya E.M. Cui W. Pelling J.C. Cisplatin-induced activation of mitogen-activated protein kinases in ovarian carcinoma cells: inhibition of extracellular signal-regulated kinase activity increases sensitivity to cisplatin.Clin Cancer Res. 1999; 5: 1007-1014Google Scholar; Wang et al., 2000Wang X. Martindale J.L. Holbrook N.J. Requirement for ERK activation in cisplatin-induced apoptosis.J Biol Chem. 2000; 275: 39435-39443Google Scholar; Arany et al., 2004Arany I. Megyesi J.K. Kaneto H. Price P.M. Safirstein R.L. Cisplatin-induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells.Am J Physiol Renal Physiol. 2004; 287: F543-F549Google Scholar; Choi et al., 2004Choi B.K. Choi C.H. Oh H.L. Kim Y.K. Role of ERK activation in cisplatin-induced apoptosis in A172 human glioma cells.Neurotoxicology. 2004; 25: 915-924Google Scholar; Wei et al., 2004Wei S.Q. Sui L.H. Zheng J.H. Zhang G.M. Kao Y.L. Role of ERK1/2 kinase in cisplatin-induced apoptosis in human ovarian carcinoma cells.Chin Med Sci J. 2004; 19: 125-129Google Scholar). Therefore, to analyze the role of activated ERK1/2 in melanoma was anti- or proapoptotic, A375 and BLM melanoma cell lines were treated with cisplatin alone or in combination with U0126, and the interaction of P-ERK1/2 with either P-p53 at serine position 6 or P-RSK1 was investigated using immunoprecipitation and Western blot techniques (Figure 10). In A375 cells, cisplatin-activated ERK1/2 interacted with p53 as well as with RSK1 (Figure 10a). Inhibition of ERK1/2 phosphorylation by U0126 and treatment with cisplatin prevented this interaction (Figure 10a), suggesting that cisplatin-activated ERK1/2 directly interacts with p53 and RSK1 to phosphorylate these proteins at serine position 6 (p53) and at threonine/serine positions 359 and 363, respectively (Figure 10a). In contrast, there was no significant interaction between P-ERK1/2 and p53 in BLM cells irrespective of treatment with cisplatin or U0126 (Figure 10b). The strong activation of RSK1 under apoptotic conditions in BLM cells may be the reason for their lower sensitivity to cisplatin and the delayed activation of proapoptotic proteins like PARP and caspase 3 in comparison with A375 cells (Figures 8a and b, 9a and b, and 10b). Furthermore, we observed a weaker interaction between total ERK1/2 and RSK1 after cisplatin treatment in comparison with P-ERK1/2 interacting with RSK1 in A375 and BLM cells (Figure 10c). In the same experiment, we could not detect such an interaction with p53 (data not shown). These data demonstrate that for an effective interaction with RSK1 and p53, the activated form of ERK1/2 is imperative. Importantly, melanoma cell lines and four metastasis samples were also analyzed with regard to their P-ERK1/2 and P-RSK1 status (Figure S2). Both melanoma cell lines and four metastasis patient samples showed elevated P-ERK1/2. The BLM melanoma cell line and two out of the four melanoma metastasis patients that had elevated P-ERK1/2 also showed elevated P-RSK1, suggesting a correlation. Download .gif (.02 MB) Help with files Figure S2Supplemental Fig 2: U0126-mediated reduction of P-ERK1/2. The BLM cell line was treated for different concentrations of U0126 for 24 hours. The optimal inhibition of ERK1/2 phosphorylation was observed in a concentration of 25 µM. To corroborate these findings, we used siRNA against ERK1/2 to determine its effects on active RSK1 in the presence of cisplatin (Figure 11). While at least the p44 band of ERK was completely blocked by specific siRNA, P-RSK1 became undetectable in A375 cells and was strongly reduced in BLM cells, demonstrating their regulation by ERK1/2 (Figure 11). To investigate the effect of RSK1 on cell death, the expression of RSK1 was reduced by specific siRNA (Figure 12a) before incubation of the cells with cisplatin for 24 hours (Figure 12b). A375 cells with reduced levels of RSK1 were more sensititve to cisplatin-mediated cell death in comparison with cells exposed to cisplatin only, as demonstrated by cleaved PARP (Figure 12b). In summary, we provide evidence for a crosstalk between proliferation/cell survival pathways activated by the ERK cascade and the growth arrest/apoptosis function of the tumor suppressor protein p53 and the survival protein RSK1. It seemed that cisplatin-mediated activation of ERK1/2 led to phosphorylation of two proteins with opposing functions, that is anti- and proapoptotic; survival was triggered through the activation of RSK1, with cell death eventually emerging through the activation of p53. The MAPK signaling pathway is involved in the regulation of cell growth and development, with a dysbalance leading to abnormal proliferation and eventual tumorigenicity. Activation of ERK appears to be required for many cells to pass the G1 restriction point and to enter the S-phase, during which cellular DNA replication takes place (Lenormand et al., 1993Lenormand P. Sardet C. Pages G. L'Allemain G. Brunet A. Pouyssegur J. Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts.J Cell Biol. 1993; 122: 1079-1088Google Scholar). Our data on ERK1/2 expression in melanoma metastasis samples revealed high ERK activity, which caused disturbed apoptosis and increased tumor cell proliferation. In melanoma, one reason for the high P-ERK1/2 levels may result from the mutation of the BRAF gene as a constitutive activator in RAS–Raf signaling (Davies et al., 2002Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Google Scholar), but another cause seems to be the high production of growth factors like EGF. The expression of the EGFR on NHM and its importance for melanoma development has been discussed controversially (Shahbazi et al., 2002Shahbazi M. Pravica V. Nasreen N. Fakhoury H. Fryer A.A. Strange R.C. et al.Association between functional polymorphism in EGF gene and malignant melanoma.Lancet. 2002; 359: 397-401Google Scholar; Stove et al., 2003Stove C. Stove V. Derycke L. Van Marck V. Mareel M. Bracke M. The heregulin/human epidermal growth factor receptor as a new growth factor system in melanoma with multiple ways of deregulation.J Invest Dermatol. 2003; 121: 802-812Google Scholar; Grahn and Isseroff, 2004Grahn J.C. Isseroff R.R. Human melanocytes do not express EGF receptors.J Invest Dermatol. 2004; 123: 244-246Google Scholar). In a recent study, we showed high EGFR expression in melanoma metastases and NHM (Mirmohammadsadegh et al., 2005Mirmohammadsadegh A. Hassan M. Gustrau A. Doroudi R. Schmittner N. Nambiar S. et al.Constitutive expression of EGF receptors on normal human melanocytes.J Invest Dermatol. 2005; 125: 392-394Google Scholar). This permitted us to investigate whether EGF was responsible for the phosphorylation of ERK1/2 in melanoma. Indeed, rEGF induced JAK-1 and SRC-mediated activation of ERK1/2 in NHM and in melanoma cell lines A375 and BLM, respectively, and was thus likely contributing to the P-ERK1/2 dysbalance in melanoma. In addition, EGF was shown to be involved in melanocyte proliferation through EGFR-mediated activation of STAT3 and STAT5 (Mirmohammadsadegh et al., 2006Mirmohammadsadegh A. Hassan M. Bardenheuer W. Marini A. Gustrau A. Nambiar S. et al.STAT5 phosphorylation in malignant melanoma is mediated through SRC and JAK1 kinases and exerts anti-apoptotic effects.J Invest Dermatol. 2006; 126: 2272-2280Google Scholar). For treatment of malignant melanoma, it is important to know if a reduction in ERK1/2 phosphorylation will permit more successful therapy. Treatment with cisplatin, one of the most potent antitumor agents, did not decrease ERK1/2 phosphorylation in vitro; to the contrary, it increased the activation of ERK1/2. The role of cisplatin-activated ERK1/2 with regard to apoptosis has also been discussed controversially. Depending on the type of cancer, cisplatin-induced ERK1/2 activation has shown both pro- or antiapoptotic functions (Persons et al., 1999Persons D.L. Yazlovitskaya E.M. Cui W. Pelling J.C. Cisplatin-induced activation of mitogen-activated protein kinases in ovarian carcinoma cells: inhibition of extracellular signal-regulated kinase activity increases sensitivity to cisplatin.Clin Cancer Res. 1999; 5: 1007-1014Google Scholar; Wang et al., 2000Wang X. Martindale J.L. Holbrook N.J. Requirement for ERK activation in cisplatin-induced apoptosis.J Biol Chem. 2000; 275: 39435-39443Google Scholar; Wei et al., 2004Wei S.Q. Sui L.H. Zheng J.H. Zhang G.M. Kao Y.L. Role of ERK1/2 kinase in cisplatin-induced apoptosis in human ovarian carcinoma cells.Chin Med Sci J. 2004; 19: 125-129Google Scholar). This discrepancy, however, needs further study to define the role of cisplatin-activated ERK1/2 in apoptosis of melanoma. Inhibition of ERK1/2 activation by specific MEK inhibitors and co-treatment with cisplatin induced strong caspase 3- and PARP-mediated apoptosis in our study as in ovarian cancer (Persons et al., 1999Persons D.L. Yazlovitskaya E.M. Cui W. Pelling J.C. Cisplatin-induced activation of mitogen-activated protein kinases in ovarian carcinoma cells: inhibition of extracellular signal-regulated kinase activity increases sensitivity to cisplatin.Clin Cancer Res. 1999; 5: 1007-1014Google Scholar). To our surprise, cisplatin-activated ERK1/2 was able to activate the cell survival protein RSK1 as well as the tumor suppressor protein p53 in a cell type-specific manner (Woessmann et al., 2002Woessmann W. Chen X. Borkhardt A. Ras-mediated activation of ERK by cisplatin induces cell death independently of p53 in osteosarcoma and neuroblastoma cell lines.Cancer Chenother Pharmacol. 2002; 50: 97-404Google Scholar). ERK1/2 has been shown to phosphorylate the members of the RSK family by interaction through a docking site located near the C-terminus (Roux et al., 2003Roux P.P. Richards S.A. Blenis J. Phosphorylation of p90 ribosomal S6 kinase (RSK) regulates extracellular signal-regulated kinase docking and RSK activity.Mol Cell Biol. 2003; 23: 4796-4804Google Scholar). RSK plays an active role in nuclear signaling by phosphorylating the cyclic AMP response element-binding protein (Xing et al., 1996Xing J. Ginty D.D. Greenberg M.E. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase.Science. 1996; 273: 959-963Google Scholar), cFos (Chen et al., 1993Chen R.H. Abate C. Blenis J. Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase.Proc Natl Acad Sci USA. 1993; 90: 10952-10956Google Scholar), and IκB (Schouten et al., 1997Schouten G.J. Vertegaal A.C. Whiteside S.T. Israel A. Toebes M. Dorsman J.C. et al.IkappaB alpha is a target for the mitogen-activated 90 kDa ribosomal S6 kinase.EMBO J. 1997; 16: 3133-3144Google Scholar), leading to cell survival and proliferation. The role of p53 induced by cytotoxic drugs and ERK signaling has been partially studied in ovarian cancer, where accumulation and phosphorylation of p53 has been demonstrated (Persons et al., 1999Persons D.L. Yazlovitskaya E.M. Cui W. Pelling J.C. Cisplatin-induced activation of mitogen-activated protein kinases in ovarian carcinoma cells: inhibition of extracellular signal-regulated kinase activity increases sensitivity to cisplatin.Clin Cancer Res. 1999; 5: 1007-1014Google Scholar). Phosphorylation of p53 in response to chemotherapy usually occurs at serine 15 and less frequently at other sites (Eisenmann et al., 2003Eisenmann K.M. VanBrocklin M.W. Staffend N.A. Kitchen S.M. Koo H.M. Mitogen-activated protein kinase pathway-dependent tumor-specific survival signaling in melanoma cells through inactivation of the proapoptotic protein bad.Cancer Res. 2003; 63: 8330-8337Google Scholar). Our findings are in concert with a previous study, which supported a role of ERK signaling in activating RSK1 in melanoma, leading to inactivation of the proapoptotic BAD and to increased survival of melanoma cells (Eisenmann et al., 2003Eisenmann K.M. VanBrocklin M.W. Staffend N.A. Kitchen S.M. Koo H.M. Mitogen-activated protein kinase pathway-dependent tumor-specific survival signaling in melanoma cells through inactivation of the proapoptotic protein bad.Cancer Res. 2003; 63: 8330-8337Google Scholar). Under normal conditions, we could not detect RSK1 activation in A375 and BLM cells, which suggests that ERK1/2-activated RSK1 occurs only under cellular stress such as exposure to cytotoxic drugs (e.g., cisplatin). Our study argues that high ERK1/2 phosphorylation in melanoma supports tumor progression and protects tumor cells from apoptosis. Furthermore, these observations indicate that ERK1/2 activation partially protects cells from cisplatin-mediated apoptosis, through the activation of RSK1. Inhibition of ERK1/2 phosphorylation by specific inhibitors and RSK1 activation by RNAi technology led to earlier and stronger apoptosis as detected by the expression of cleaved caspase 3 and PARP. Therefore, simultaneous treatment with cisplatin and U0126 may result in synergistic therapeutic effects at tolerable doses of cisplatin. Further investigation on the mechanism of how the EGF pathway activates ERK and studying its inhibition in combination with anticancer drugs may be helpful in improving melanoma therapy. The significance of phosphorylated RSK1 deserves further study as a therapeutic marker in clinical specimens. NHMs were cultured in PMA-free melanocyte medium (both obtained from PromoCell, Heidelberg, Germany). Cell culture was maintained in a 37°C incubator in a moist atmosphere of 5% CO2. The human melanoma cell lines BLM, MV3, M13, and A375 were cultured in DMEM (Invitrogen-Gibco, Karlsruhe, Germany) supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. As a positive control, the HaCaT epithelial cell line was used. Inhibitors of MEK-1/2 (U0126) and cisplatin were obtained from Sigma-Aldrich, Taufkirchen, Germany. rEGF was obtained from BioSource, Solingen, Germany. The melanoma metastasis samples were extracted from several different patients, who signed informed consent. Samples were snap-frozen in liquid nitrogen and homogenized using a dismembrator (Braun-Melsungen, Melsungen, Germany). The powder was immediately transferred into ice-cold lysis buffer containing 25 mM hydroxyethylpiperazine ethanesulfonic acid pH 7.9, 50 mM NaF, 15 mM Triton X-100, 5 mM EDTA, 100 mM NaCl, and 1 tablet protease inhibitor cocktail per 10 ml buffer (Roche Applied Science, Mannheim, Germany) for Western blot analysis. The study was conducted according to the Declaration of Helsinki Principles and was approved by the Institutional Review Board. Cells were washed twice with ice-cold phosphate buffered saline and lysed in lysis buffer (see above). The samples were pretreated with ultrasound (10 pulses on ice; Sonoplus, Bandelin Electric, Germany) and were centrifuged at 14,000 × g for 20 minutes at 4°C. The protein concentration of the cellular extracts was determined using the advanced protein assay reagent (TEBU, Germany). Twenty micrograms of protein extract were electrophoresed on 4–12% NuPage Bis–Tris–Glycine gels (Invitrogen, Germany) for 2 hours at 120 V. Proteins were blotted onto polyvinylidene fluoride membranes (Roth, Karlsruhe, Germany) at 160 mA for 60 minutes using a tank blot system. The membranes were blocked with 1% non-fat dry milk powder in 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 (TBST buffer) for 1 hour at 4°C and washed three times with TBST. Immunostaining was performed using antibodies against ERK1/2, P-ERK1/2, cleaved PARP, PARP, cleaved caspase 3, caspase 3, caspase 8, caspase 9, p53 and P-RSK1, Bcl-2 (each Cell Signaling, New England Biolabs, Frankfurt, Germany). Bax and RSK1 antibodies were obtained from Becton Dickinson Bioscences, Heidelberg, Germany. α-tubulin antibody was purchased from Oncogene Research Products, Calbiochem, Darmstadt, Germany. Bound antibody was visualized using the chemiluminescence detection system (Pierce, Rockford, IL) following the supplier's instructions. Cells were lysed in Western blot lysis buffer (see above) and incubated with P-ERK1/2, ERK1/2 (Cell Signaling, New England Biolabs, Germany) antibodies overnight at 4°C with constant rotation followed by incubation with immobilized protein A/G (Pierce) for 2 hours. Immunoprecipitates were washed five times with lysis buffer and then resuspended in Laemmli buffer, boiled, and analyzed by Western blotting using anti-p53, anti-P-p53-serine 6, anti-RSK1, and anti-P-RSK1 (threonine/serine positions 359/363). SiRNAs against p42 and p44 (ERK1/2) were provided by Dharmacon (Solingen, Germany). Cells (2 × 105) were incubated with 100 nM siRNA transfection solution without fetal calf serum and antibiotics for 4 hours. At 24 and 48 hours, cells were detached with trypsin and centrifuged for 5 minutes at 500 × g before protein extraction as described above. SiRNA against RSK1 was obtainted from Qiagen (Germany). The transfections were performed in six-well plates according to the manufacturer's recommended protocol. The appearance of phosphatidyleserine on the extracellular side of membrane was evaluated with annexin-V/PI method. The melanoma cell lines A375 and BLM were exposed to U0126 and cisplatin, trypsinized, and washed twice in ice-cold phosphate-buffered saline before resuspension in 1 × binding buffer (Invitrogen). Thereafter, 5 μl of Annexin V-FITC (Vybrant; Invitrogen) and 5 μl propidium iodide (100 μg/ml) were added to 100 μl of cell suspension and incubated for 15 minutes at room temperature protected from light. Finally, 400 μl of binding buffer were added to the samples and handled ice-cold until analysis. The fluorescent signals of FITC and PI was detected by FL1 at 518 nm and FL2 at 620 nm, respectively, on a FACSCalibur (Becton Dickinson Bioscences) and apoptotic cells (annexin-V-positive/PI-negative) were quantified. Melanoma cells were exposed to U0126 and cisplatin in a 96-well plate for 24, 48, and 72 hours cells before incubation with 10 μl of 5 mg/ml stock of MTT (Sigma) for 2 hours at 37°C. Upon solubilization, the number of surviving cells was measured at A540nm. BrdU assays were performed according to the supplier's instructions (Roche Applied Science). All data were analyzed by coefficient of variation. A coefficient of variation value greater than 0.2 was considered statistically significant. The authors declare no conflict of interest. Figure S1. U0126-mediated reduction of P-ERK1/2. Figure S2. Detection of RSK1 and P-RSK1 in melanoma metastases by Western blotting." @default.
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• W2068420134 title "ERK1/2 Is Highly Phosphorylated in Melanoma Metastases and Protects Melanoma Cells from Cisplatin-Mediated Apoptosis" @default.
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