Mutated BRAF and NRAS are suspected to contribute to melanomagenesis by activation of extracellular signal-regulated kinase (ERK). To test this notion, we analyzed the presence of phosphorylated ERK1/2 in 170 melanomas with established NRAS/BRAF mutational status and well-documented clinical follow-up by immunohistochemistry. Several notable observations were obtained: (i) phospho-ERK staining was very heterogeneous within the tumor; (ii) in most cases, ERK was phosphorylated in only a minority of tumor cells; (iii) the percentage of phospho-ERK-positive cells was not correlated with the mutational status of NRAS and/or BRAF; (iv) the Raf kinase inhibitor protein (RKIP) was expressed homogeneously in virtually all melanoma samples not reflecting the inhomogeneity of phospho-ERK; and, finally, (v) neither the portion of phospho-ERK-positive tumor cells nor the RKIP staining intensity showed any correlation to the clinical course of the patients. Furthermore, the ability of BRAF mutant melanoma cells to downregulate mitogen-activated protein kinase activation was shown in melanoma cell lines cultured at high densities or under nonadherent conditions. Our findings suggest that mitogen-activated protein kinase (MAPK) activity is subject to regulation even in BRAF/NRAS mutant melanoma cells and that high MAPK pathway signaling may be important only in distinct subsets of tumor cells.
Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin. Recently, we have shown that MCC cells in situ are characterized by a complete absence of mitogen-activated protein kinase (MAPK) pathway signaling, which is preserved in the MCC cell line UISO. Here we present data suggesting that silencing of the MAPK pathway is essential for the survival of MCC cells. Activation of the MAPK pathway could be achieved by inducing a regulatable form of the c-Raf-1 kinase domain in UISO cells. Consequently, MAPK signaling led to morphological changes, loss of actin stress fibers, and induction of apoptosis, which could be prevented by the MAP kinase kinase-specific inhibitor U0126. Hence, despite the fact that activation of the MAPK pathway contributes to oncogenesis in many cancers, it seems to be a negative selection factor for MCC cells. Since ERK phosphorylation was also inducible by the Raf-activating pharmacological agent ZM336372, these results provide new perspectives for potential therapeutics for this highly aggressive tumor.
Due to elaborate control mechanisms, in benign tumors the activation of oncogenes primarily induces senescence, associated with cessation of cellular proliferation; for example, melanocytic nevi expressing mutant B-Raf. These mechanisms include the RB and/or the p53 pathway. The current model of melanomagenesis postulates that progression to immortal melanoma cells requires inactivating aberrations in signaling cascades controlling senescence. Thus, melanoma cells carrying mutant B-Raf should be resistant to mitogen-activated protein kinase (MAPK) pathway-induced senescence. Here, we demonstrate that hyperactivation of the MAPK pathway following activation of an inducible form of oncogenic C-Raf induces a senescence-like proliferation arrest in B-Raf mutant melanoma cells. This Raf-induced senescence is initially strictly dependent on MEK signaling, but seems to be independent of MAPK signaling after prolonged continuance. It is associated with reduced levels of RB phosphorylation and an increase in p21 expression, but is independent of p16(Ink4a) and p53. These data argue against the existence of fundamental changes in melanoma cells completely precluding senescence.
BackgroundThe Raf kinase inhibitor protein (RKIP) has been shown to block MAP kinase pathway as well as NFκB signalling. By means of immunohistochemistry, we previously demonstrated that the MAP kinase pathway is virtually inactive in Merkel cell carcinoma (MCC). Similarly to MCC in situ high RKIP expression accompanies absence of ERK phosphorylation in the MCC cell line UISO suggesting that RKIP might be causative for MAP kinase pathway silencing.MethodsApplying an siRNA approach RKIP expression was knocked down in UISO cells and a possible influence on MAP kinase pathway activity was assessed by Western blot analysis using phospho-specific antibodies. Moreover, a possible effect of RKIP knock down in UISO cells on proliferation as well as chemosensitivity to cisplatin were examined applying the MTS assay.ResultsSurprisingly the absence of phosphorylation of the MAP kinases ERK1 and ERK 2 even following growth factor stimulation was not affected by the RKIP knock down indicating that RKIP is not essential for blocking the MAP kinase pathway in the MCC cell line UISO. Moreover, proliferation as well as chemosensitivity towards cisplatin were not altered upon knock down of RKIP.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.