Armelle Calipel

Extracellular Signal-regulated Kinase-dependent Proliferation Is Mediated through the Protein Kinase A/B-Raf Pathway in Human Uveal Melanoma Cells

Mutated B-Raf-mediated constitutive activation of ERK1/2 is involved in about 66% of cutaneous melanoma. By contrast, activating mutations in B-RAF are rare in ocular melanoma. This study aimed to determine the role of wild-type B-Raf (WTB-Raf) in uveal melanoma cell growth. We used cell lines derived from primary tumors of uveal melanoma to assess the role of WTB-Raf in cell proliferation and to characterize its upstream regulators and downstream effectors. Melanoma cell lines expressing WTB-Raf and WTRas grew with similar proliferation rates, showed constitutive activation of ERK1/2, and had similar levels of B-Raf expression and B-Raf kinase activity as melanoma cell lines expressing the activating V600E mutation (V600EB-Raf). They were equally as sensitive to pharmacological inhibition of MEK1/2 for cell proliferation and transformation as V600EB-Raf cells. siRNA-mediated depletion of Raf-1 did not affect either ERK1/2 activation, whereas siRNA-mediated depletion of B-Raf reduced cell proliferation by up to 65% through the inhibition of ERK1/2 activation, irrespective of the mutational status of B-Raf. Pharmacological inhibition of cAMP-dependent protein kinase (PKA) and siRNA-mediated depletion of PKA greatly reduced B-Raf activity, ERK1/2 activation, and cell proliferation in WTB-Raf cells, whereas it did not affect V600EB-Raf cells, demonstrating a key role of PKA in mediating WTB-Raf/ERK signaling for uveal melanoma cell growth. Moreover, inactivation or depletion of PKA did not affect Rap-1 activity, and Rap-1 depletion did not affect either B-Raf activity or ERK1/2 activation. This ruled out a role for Rap1 in the PKA-mediated B-Raf/ERK activation in WTB-Raf cells. Finally, we demonstrated the importance of cyclin D1 in mediating PKA/WTB-Raf signaling for cell proliferation. Altogether, our results suggest that the PKA/B-Raf pathway is a potential target for therapeutic strategies against WTB-Raf-expressing uveal melanoma.

The PI3K/Akt and mTOR/P70S6K Signaling Pathways in Human Uveal Melanoma Cells: Interaction with B-Raf/ERK

PURPOSE Activated B-Raf alone cannot induce melanoma but must cooperate with other signaling pathways. The phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin (mTOR)/p70S6K pathways are critical for tumorigenesis. The authors investigated the role of these pathways in uveal melanoma cells. METHODS The effects of PI3K and mTOR activation and inhibition on the proliferation of human uveal melanoma cell lines expressing either activated (WT)B-Raf or (V600E)B-Raf were investigated. Interactions among PI3K, mTOR, and B-Raf/ERK were studied. RESULTS Inhibition of PI3K deactivated P70S6 kinase, reduced cell proliferation by 71% to 84%, and increased apoptosis by a factor of 5.0 to 8.4 without reducing ERK1/2 activation, indicating that ERK plays no role in mediating PI3K in these processes. In contrast, rapamycin-induced inhibition of mTOR did not significantly affect cell proliferation because it simultaneously stimulated PI3K/Akt activation and cyclin D1 expression. Regardless of B-Raf mutation status, cotreatment with the PI3K inhibitor effectively sensitized all melanoma cell lines to the B-Raf or ERK1/2 inhibition-induced reduction of cell proliferation. B-Raf/ERK and PI3K signaling, but not mTOR signaling, converged to control cyclin D1 expression. Moreover, p70S6K required the activation of ERK1/2. These data demonstrate that PI3K/Akt and mTOR/P70S6K interact with B-Raf/ERK. CONCLUSIONS Activated PI3K/Akt attenuates the inhibitory effects of rapamycin on cell proliferation and thus serves as a negative feedback mechanism. This finding suggests that rapamycin is unlikely to inhibit uveal melanoma growth. In contrast, targeting PI3K while inhibiting B-Raf/ERK may be a promising approach to reduce the proliferation of uveal melanoma cells.

Activation of the FGF2/FGFR1 autocrine loop for cell proliferation and survival in uveal melanoma cells.

PURPOSE Constitutive activation of ERK1/2 controls proliferation of uveal melanoma cells. Because an autocrine fibroblast growth factor (FGF) activation loop controls ERK1/2 activation in many cancers, this study was conducted to examine the role of the FGF/FGF receptor autocrine loop in the ERK1/2-dependent proliferation and survival of uveal melanoma cells. METHODS Primary tumors and cell lines (OCM-1, MKT-BR, SP6.5, Mel270 and 92.1) were used to define the role of the FGF/FGFR system in human uveal melanoma. Cell proliferation was assessed by MTT-staining, and apoptosis was quantified by flow cytometry. Specific pharmacologic inhibitors of ERK1/2 and FGFR1, an anti-FGF2 neutralizing antibody and an antisense oligonucleotide directed against FGF2 were used to analyze signaling in the FGF/FGFR autocrine loop. RESULTS FGF1, FGF2, and their FGFR1 receptor were strongly expressed in the primary uveal melanomas. All five uveal melanoma cell lines expressed and secreted FGF2. They also expressed FGFR1. Cell proliferation was strongly reduced by the antisense oligonucleotide-mediated depletion of endogenous FGF2, immunoneutralization of secreted FGF2, and pharmacologic inhibition of FGFR1. The FGF2/FGFR1-mediated signaling pathway was identified by showing that inhibition of either FGF2 or FGFR1 reduced ERK1/2 activation, cell proliferation, and survival. CONCLUSIONS The FGF/FGFR/ERK signaling pathway may be a target for therapeutic strategies against uveal melanoma.

Opposite long-term regulation of c-Myc and p27Kip1 through overactivation of Raf-1 and the MEK/ERK module in proliferating human choroidal melanoma cells.

Although there is no current evidence for ras gene mutation in choroidal melanoma, there is an increasing body of evidence indicating that deregulated intracellular signalling pathways are involved in choroidal melanoma pathogenesis. The various components of the linear Raf/MEK/ERK signalling pathway have been implicated in various tumours. We therefore investigated the role of Raf-1 and the MEK/ERK module in the proliferation of human normal choroidal melanocytes (NCM) and cells from the ocular choroidal melanoma (OCM-1) cell line. OCM-1 cells proliferated four times faster than NCM. High basal activation of the MEK/ERK module was observed in unstimulated OCM-1 cells, whereas rapid and persistent activation was detected after serum stimulation, throughout the 24-h period of culture. In contrast, the activation of MEK/ERK was barely detectable in unstimulated NCM and occurred late (6 h) after the stimulation of cell proliferation. Inhibition of Raf-1 and MEK1/2 activation by pharmacological approaches and of the production of Raf-1 and ERK1/2 by antisense oligonucleotide approaches demonstrated that Raf-1 and the MEK/ERK module controlled proliferation in OCM-1 cells, but not in NCM. OCM-1 cells produced very low levels of p27Kip1, whereas NCM produced constant, high levels of p27Kip1. The inhibition of Raf-1 or MEK1/2 induced a large increase in p27Kip1 in OCM-1 cells, associated with an arrest of cell proliferation. Levels of c-Myc production were high and constant in OCM-1 cells and low in NCM, in contrast to what was observed for p27Kip1. The inhibition of both Raf-1 and MEK1/2 induced a decrease in c-Myc production and downregulated c-Myc activity by preventing c-Myc phosphorylation in OCM-1 cells. We conclude that Raf-1 and the MEK/ERK module control the production of both p27Kip1 and c-Myc, and the activation of c-Myc for OCM-1 cell proliferation.

17-AAG and 17-DMAG–Induced Inhibition of Cell Proliferation through B-Raf Downregulation in WTB-Raf–Expressing Uveal Melanoma Cell Lines

PURPOSE The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) has been shown to have promising results in antitumor activity through the degradation of the activated V600E mutant of B-Raf (V600E B-Raf) in cutaneous melanoma cell lines. It has different effects, however, on the wild-type form of B-Raf (WT B-Raf), according to the WT B-Raf activation levels in the tumor cells. Uveal melanoma cells express WT B-Raf and only rarely express V600E B-Raf. This study was conducted to investigate the effects of HSP90 inhibition on uveal melanoma cell lines. METHODS Human uveal melanoma cell lines were treated with the HSP90 inhibitors 17-AAG and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG). Cell proliferation was assessed by MTT staining, and apoptosis was quantified by flow cytometry. Analysis of the expression of HSP90 and activation of the MEK/ERK downstream signaling of B-Raf was performed by Western blot. Effects of the downregulation of the HSP90 cochaperone, cdc37, on cell proliferation and activation of MEK/ERK was investigated by siRNA strategy. RESULTS The inhibition of HSP90 downregulated B-Raf, decreased cell proliferation, and reduced activation of MEK/ERK in uveal melanoma cell lines expressing WT B-Raf. HSP90 inhibition also reduced the expression of Akt, but the inhibition of Akt had no effect on cell proliferation, ruling out a role of Akt in the 17-AAG-induced inhibition of cell proliferation. The downregulation of cdc37 did not affect MEK/ERK signaling and cell proliferation, demonstrating that the cochaperone was not required for HSP90-controlled stability of B-Raf. c-Kit was also downregulated after HSP90 inhibition. The combination of 17-DMAG with imatinib mesylate, the inhibitor of c-kit, had synergistic inhibitory effects on cell proliferation in WT B-Raf uveal melanoma cell lines. CONCLUSIONS These results suggest that targeting HSP90 in tandem with c-Kit inhibition may be a promising therapeutic approach to uveal melanoma.

Status of RASSF1A in uveal melanocytes and melanoma cells.

RASSF1A gene, found at the 3p21.3 locus, is a tumor suppressor gene frequently hypermethylated in human cancers. In this study, we report that compared with melanocytes in normal choroid, RASSF1A is downregulated in uveal melanoma samples and in uveal melanoma cell lines. LOH at 3p21.3 was detected in 50% of uveal melanoma. Moreover, methylation of the RASSF1A promoter was detected in 35 of 42 tumors (83%) and RASSF1A was also weakly expressed at the mRNA level. These data indicate that LOH at the RASSF1A locus or RASSF1A promoter methylation may partly account for the suppression of RASSF1A expression observed in uveal melanoma. Furthermore, following ectopic expression in three RASSF1A-deficient melanoma cell lines (OCM-1, Mel270, and 92.1), RASSF1A weakly reduces cell proliferation and anchorage-independent growth of uveal melanoma cells without effect on ERK1/2 activation, cyclin D1 and p27Kip1 expression. This study explored biological functions and underlying mechanisms of RASSF1A in the ERK1/2 pathway in normal uveal melanocytes. We showed that siRNA-mediated depletion of RASSF1A increased ERK1/2 activation, cyclin D1 expression, and also decreased p27Kip1 expression in normal uveal melanocytes. Moreover, that the depletion of RASSF1A induced senescence-associated β-galactosidase activity and increased p21Cip1 expression suggests that RASSF1A plays a role in the escape of cellular senescence in normal uveal melanocytes. Interestingly, we found that RASSF1A was epigenetically inactivated in long-term culture of uveal melanocytes. Taken together, these data show that depletion of RASSF1A could be an early event observed during senescence of normal uveal melanocytes and that additional alterations are acquired during malignant transformation to uveal melanoma. Mol Cancer Res; 9(9); 1187–98. ©2011 AACR.

Mechanisms of resistance to imatinib mesylate in KIT-positive metastatic uveal melanoma

Imatinib mesylate is used in targeted therapy of cancer to inhibit type III tyrosine kinase receptors, such as KIT and platelet-derived growth factor receptors (PDGFRs). Expression of KIT in uveal melanoma (UM) suggests that this receptor may be the target of imatinib mesylate therapy. However, phase II multicenter clinical studies have shown no effect of imatinib mesylate in patients with unresectable liver metastases of UM. We therefore investigated which molecular mechanisms promote imatinib mesylate-resistance in metastatic UM. Expression of KIT, stem cell factor (SCF), PDGFRα and PDGFRβ, was analyzed by RT-PCR, immunostaining, and Western blot in twenty-four samples of UM liver metastases, as well as UM primary tumor and metastatic cell lines. Soluble SCF was quantified in UM cell lines using enzyme-linked immunosorbent assay. Cell viability of UM cell lines treated with imatinib mesylate and grown in SCF-supplemented medium or in microvascular endothelial cells-conditioned medium was studied by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays. UM liver metastases and cell lines expressed KIT and SCF, but not the PDGFRs. Ninety-five percent of liver metastases expressed KIT at the protein level, but PDGFRs were not detected in these samples. Imatinib mesylate reduced the viability of UM metastatic cell lines in a concentration-dependent manner, but an increased resistance to this drug was observed when cells were incubated in SCF-supplemented or microvascular endothelial cells-conditioned medium. This study provides evidence that tumor microenvironment cytokines such as SCF may promote resistance to imatinib mesylate in metastatic UM.

Differential Radiosensitivity of Uveal Melanoma Cell Lines After X-rays or Carbon Ions Radiation.

PURPOSE We compared the radiosensitivity of uveal melanoma (UM) cell lines after x-ray or carbon-ions radiation (C-ions). METHODS We characterized the radiosensitivity toward x-rays and C-ions of UM cell lines: 92.1, MEL270, SP6.5, MKT-BR, μ2, and TP17. Normal choroidal melanocytes and the retinal pigment epithelial cell line ARPE19 were used as controls for normal cells. X-rays were delivered with an energy of 6 MV at a dose rate of 2 Gy/min. X-rays served as a reference for Relative Biological Effectiveness (RBE) evaluation. Radiation with C-ions was delivered at 75 MeV/u (34 keV/μm) at a dose rate of 2 Gy/min. After single-doses (0-8 Gy) of medical x-rays (6 MV) or C-ions (33 keV/μm), cells sensitivity was measured using standard colony formation assay, and cell growth was examined by counting the cell colonies. The effect of x-rays or C-ions on the expression and activation of ERK1/2 was evaluated by Western Blot. RESULTS C-ions presented with regard to the x-rays a RBE of 1.9 to 2.5 at 10% of UM cells survival. The x-ray sensitivity of UM cells was neither influenced by the synchronization of cells in phase G0/G1 of the cell cycle nor by the level of oxygenation. X-ray and C-ions radiation had the same effects on cell cycle leading to a mitotic catastrophe that appeared earlier after C-ions than x-ray treatment. However, C-ions radiation induced a sustained inhibition of ERK1/2 activation compared to the transitory induction of that signalization pathway after x-ray radiation. CONCLUSIONS This in vitro study shows that C-ions had a better biological effectiveness than x-rays leading to a sustained inhibition of the ERK1/2 pathway.