Combination of MEK and SRC inhibition suppresses melanoma cell growth and invasion.

The RAS-RAF-MEK-ERK pathway is deregulated in over 90% of malignant melanomas, and targeting MEK as a central kinase of this pathway is currently tested in clinical trials. However, dose-limiting side effects are observed, and MEK inhibitors that sufficiently reduce ERK activation in patients show a low clinical response. Apart from dose limitations, a reason for the low response to MEK targeting drugs is thought to be the upregulation of counteracting signalling cascades as a direct response to MEK inhibition. Therefore, understanding the biology of melanoma cells and the effects of MEK inhibition on these cells will help to identify new combinatorial approaches that are more potent and allow for lower concentrations of the drug being used. We have discovered that in melanoma cells MEK inhibition by selumetinib (AZD6244, ARRY-142886) or PD184352, while efficiently suppressing proliferation, stimulates increased invasiveness. Inhibition of MEK suppresses actin-cortex contraction and increases integrin-mediated adhesion. Most importantly, and surprisingly, MEK inhibition results in a significant increase in matrix metalloproteases (MMP)-2 and membrane-type 1-MMP expression. All together, MEK inhibition in melanoma cells induces a 'mesenchymal' phenotype that is characterised by protease-driven invasion. This mode of invasion is dependent on integrin-mediated adhesion, and because SRC kinases are the main regulators of this process, the SRC kinase inhibitor, saracatinib (AZD0530), completely abolished the MEK inhibitor-induced invasion. Moreover, the combination of saracatinib and selumetinib effectively suppressed the growth and invasion of melanoma cells in a 3D environment, suggesting that combined inhibition of MEK and SRC is a promising approach to improve the efficacy of targeting the ERK/MAP kinase pathway in melanoma.

In melanoma, beta-catenin is a suppressor of invasion.

Cell-type-specific signalling determines cell fate under physiological conditions, but it is increasingly apparent that also in cancer development the impact of any given oncogenic pathway on the individual cancer pathology is dependent on cell-lineage-specific molecular traits. For instance in colon and liver cancer canonical Wnt signalling produces increased cytoplasmic and nuclear localised beta-catenin, which correlates with invasion and poor prognosis. In contrast, in melanoma increased cytoplasmic and nuclear beta-catenin is currently emerging as a marker for good prognosis, and thus seems to have a different function compared with other cancer types; however, this function is unknown. We discovered that in contrast to its function in other cancers, in melanoma, beta-catenin blocks invasion. We demonstrate that this opposing role of nuclear beta-catenin in melanoma is mediated through MITF, a melanoma-specific protein that defines the lineage background of this cancer type. Downstream of beta-catenin MITF not only suppresses the Rho-GTPase-regulated cell morphology of invading melanoma cells, but also interferes with beta-catenin-induced expression of the essential collagenase MT1-MMP, thus affecting all aspects of an invasive phenotype. Importantly, overexpression of MITF in invasive colon cancer cells modifies beta-catenin-directed signalling and induces a 'melanoma phenotype'. In summary, the cell-type-specific presence of MITF in melanoma affects beta-catenin's pro-invasive properties otherwise active in colon or liver cancer. Thus our study reveals the general importance of considering cell-type-specific signalling for the accurate interpretation of tumour markers and ultimately for the design of rational therapies.

Apoptosis suppression by Raf-1 and MEK1 requires MEK- and phosphatidylinositol 3-kinase-dependent signals.

Two Ras effector pathways leading to the activation of Raf-1 and phosphatidylinositol 3-kinase (PI3K) have been implicated in the survival signaling by the interleukin 3 (IL-3) receptor. Analysis of apoptosis suppression by Raf-1 demonstrated the requirement for mitochondrial translocation of the kinase in this process. This could be achieved either by overexpression of the antiapoptotic protein Bcl-2 or by targeting Raf-1 to the mitochondria via fusion to the mitochondrial protein Mas p70. Mitochondrially active Raf-1 is unable to activate extracellular signal-related kinase 1 (ERK1) and ERK2 but suppresses cell death by inactivating the proapoptotic Bcl-2 family member BAD. However, genetic and biochemical data also have suggested a role for the Raf-1 effector module MEK-ERK in apoptosis suppression. We thus tested for MEK requirement in cell survival signaling using the interleukin 3 (IL-3)-dependent cell line 32D. MEK is essential for survival and growth in the presence of IL-3. Upon growth factor withdrawal the expression of constitutively active MEK1 mutants significantly delays the onset of apoptosis, whereas the presence of a dominant negative mutant accelerates cell death. Survival signaling by MEK most likely results from the activation of ERKs since expression of a constitutively active form of ERK2 was as effective in protecting NIH 3T3 fibroblasts against doxorubicin-induced cell death as oncogenic MEK. The survival effect of activated MEK in 32D cells is achieved by both MEK- and PI3K-dependent mechanisms and results in the activation of PI3K and in the phosphorylation of AKT. MEK and PI3K dependence is also observed in 32D cells protected from apoptosis by oncogenic Raf-1. Additionally, we also could extend these findings to the IL-3-dependent pro-B-cell line BaF3, suggesting that recruitment of MEK is a common mechanism for survival signaling by activated Raf. Requirement for the PI3K effector AKT in this process is further demonstrated by the inhibitory effect of a dominant negative AKT mutant on Raf-1-induced cell survival. Moreover, a constitutively active form of AKT synergizes with Raf-1 in apoptosis suppression. In summary these data strongly suggest a Raf effector pathway for cell survival that is mediated by MEK and AKT.

Ligand-independent dimerization and activation of the oncogenic Xmrk receptor by two mutations in the extracellular domain.

Overexpression of the oncogenic receptor tyrosine kinase ONC-Xmrk is the first step in the development of hereditary malignant melanoma in the fish Xiphophorus. However, overexpression of its proto-oncogene counterpart (INV-Xmrk) is not sufficient for the oncogenic function of the receptor. Compared with INV-Xmrk, the ONC-Xmrk receptor displays 14 amino acid changes, suggesting the presence of activating mutations. To identify such activating mutations, a series of chimeric and mutant receptors were studied. None of the mutations present in the intracellular domain was found to be involved in receptor activation. In the extracellular domain, we found two mutations responsible for activation of the receptor. One is the substitution of a conserved cysteine (C578S) involved in intramolecular disulfide bonding. The other is a glycine to arginine exchange (G359R) in subdomain III. Either mutation leads to constitutive dimer formation and thereby to activation of the ONC-Xmrk receptor. Besides, the presence of these mutations slows down the processing of the Xmrk receptor in the endoplasmic reticulum, which is apparent as an incomplete glycosylation.

Activation of phosphatidylinositol 3-kinase by a complex of p59fyn and the receptor tyrosine kinase Xmrk is involved in malignant transformation of pigment cells.

Malignant melanoma in the fish Xiphophorus is induced by overexpression of the Xmrk-oncogene, encoding a subclass I receptor tyrosine kinase. The mutationally activated Xmrk protein triggers constitutive mitogenic signalling in fish melanoma cells. In recent studies we showed that in melanoma cells phosphatidylinositol (PtdIns) 3-kinase, as well as p59fyn, has elevated levels of kinase activity. Both bind directly to different phosphotyrosine residues in the Xmrk receptor C-terminus through their SH2 domains. To analyse the mechanism of regulation of these Xmrk-associated kinases in melanoma we characterized the protein-protein interactions between PtdIns 3-kinase, p59fyn and the Xmrk receptor in detail. A ternary complex in which the p85 subunit of PtdIns 3-kinase is associated with p59fyn as well as with Xmrk was identified. Contrary to complexes described for other receptors, the adaptor protein p120Cbl was not involved in these interactions. Thus, we describe here a new mechanism of activation of PtdIns 3-kinase by a receptor of the epidermal growth factor receptor family in which p59fyn acts as an adaptor as well as an activator of PtdIns 3-kinase. Activation of PtdIns 3-kinase activity by fyn was also found in vivo. The fact that this was only detectable in highly transformed Xmrk overexpressing melanomas but not in benign lesions points to the essential role of the Xmrk receptor in this mechanism of regulation.

PI3-kinase is involved in mitogenic signaling by the oncogenic receptor tyrosine kinase Xiphophorus melanoma receptor kinase in fish melanoma.

Overexpression of the mutationally activated receptor tyrosine kinase Xiphophorus melanoma receptor kinase (Xmrk) initiates formation of hereditary malignant melanoma in the fish Xiphophorus. In melanoma as well as in a melanoma-derived cell line (PSM) this receptor is highly activated resulting in constitutive Xmrk-mediated mitogenic signaling. In order to analyze mitogenic signaling triggered by Xmrk a possible involvement of phosphatidylinositol 3 (PI3)-kinase in Xmrk signal transduction was examined. Constitutive binding of the p85 adapter subunit of PI3-kinase to the Xmrk receptor was detected in PSM melanoma cells. Further analyses in BHK cells expressing a Xmrk chimera (HER-mrk) showed that p85 association with the intracellular part of Xmrk was dependent on autophosphorylation of the receptor. In vitro binding studies revealed that the interaction is mediated mainly through the N-terminal SH2 domain of p85 which directly binds to a sequence motif around phosphorylated Tyr-983 in the Xmrk carboxy-terminus. In accordance with recruitment of p85 by Xmrk in PSM cells, the PI3-kinase downstream target Akt was found to be highly phosphorylated on Ser-473, indicating efficient PI3-kinase signaling in melanoma cells. PI3-kinase activation was also detected in Xiphophorus melanoma. Moreover, malignant melanomas exhibited an increased level of PI3-kinase activity which was about three times higher than that in benign pigmented lesions. Inhibition of PI3-kinase activity in PSM melanoma cells by both Wortmannin and LY294002 blocked entry into S-phase. Together these data demonstrate that PI3-kinase is a substrate of the oncogenic Xmrk receptor and plays a significant role in mitogenic signaling of melanoma cells and the formation of malignant melanoma in Xiphophorus.

Multiple binding sites in the growth factor receptor Xmrk mediate binding to p59fyn, GRB2 and Shc.

Melanoma formation in Xiphoporus is initiated by overexpression of the EGFR-related receptor tyrosine kinase Xmrk (Xiphoporus melanoma receptor kinase). This receptor is activated in fish melanoma as well as in a melanoma-derived cell line (PSM) resulting in constitutive Xmrk-mediated mitogenic signaling. In order to define the underlying signaling pathway(s), triggered by the activated Xmrk receptor, we attempted to identify its physiological substrates. Examination of the Xmrk carboxyterminus for putative tyrosine autophosphorylation sites revealed the presence of potential binding motifs for GRB2 as well as for Shc. Binding of these adaptor proteins to the Xmrk receptor was detected in vitro and in cells expressing the mrk kinase. The GRB2 and Shc interactions with the receptor could be disrupted individually by phosphotyrosine peptides containing putative Xmrk autophosphorylation sites, indicating direct binding of both proteins. Recruitment of GRB2 by the constitutively activated Xmrk receptor led to strong MAP kinase activation in Xiphoporus melanoma cells. We also identified a high-affinity binding site for src-kinases (pYEDL) in the Xmrk carboxyterminus. Competition experiments with phosphopeptides comprising this site confirmed that it is used for high-affinity binding of Xiphoporus fyn (Xfyn) to Xmrk in melanoma cells. Thus, Xmrk can initiate different signaling pathways by using multiple substrate-binding sites to trigger proliferation of pigment cells.

A two-step selection approach for the identification of ligand-binding determinants in cytokine receptors.

We have developed a novel cell-based method for the isolation and selection of mutant cytokine receptors with defects in ligand binding and applied it to the human interleukin-4 receptor. The experimental procedure is based upon the functional heterologous expression of receptor mutants in eukaryotic cells followed by a two-step selection procedure. Positive selection for cells that express receptor variants is achieved by means of an agonistic antibody that mediates cell survival through receptor dimerization. An IL-4-coupled toxin is subsequently used to select against cells expressing wild-type receptors. Cells expressing mutant receptors that are unable to bind the cytotoxic ligand survive and can be amplified. The procedure allows the isolation of rare receptor variants from cell pools containing predominantly wild-type cells. This method, which should be equally applicable to similar receptor systems, was used to demonstrate the importance of a critical charged amino acid residue in the human IL-4 receptor alpha-subunit for IL-4-induced receptor activation.

Signalling by the oncogenic receptor tyrosine kinase Xmrk leads to activation of STAT5 in Xiphophorus melanoma.

Overexpression of the mutationally activated Xmrk receptor initiates the formation of hereditary malignant melanoma in the fish Xiphophorus. In addition to transcriptional overexpression a cell-type specific signal transduction is essential for Xmrk mediated tumor formation. To elucidate the consequence of Xmrk signalling and to identify target proteins that characterize the tumor phenotype, we analysed proteins that are strongly tyrosine phosphorylated in the fish melanoma cell line PSM. One of the most prominent phosphotyrosine proteins was found to be the signal transducer and activator of transcription STAT5. In a heterologous cell system (murine pro B-cells), activation of the Xmrk kinase in a chimeric receptor induced tyrosine phosphorylation, nuclear translocation and DNA binding of STAT5. Following receptor stimulation, expression of the STAT5 specific target genes cis, osm and pim-1 was induced. In Xiphophorus PSM cells STAT5 was found to be preferentially localized in the nucleus, but treatment with tyrphostin AG555, a specific Xmrk kinase-inhibitor, blocked nuclear localization. In these cells as well as in Xiphophorus melanoma expression of pim-1 and constitutive DNA-binding activity of STAT5 was detectable. This constitutive activity was higher in malignant than in benign melanomas, indicating that STAT5 activation is correlated with the malignancy of these tumors.

Activation of the Xmrk proto-oncogene of Xiphophorus by overexpression and mutational alterations.

Xmrk is a receptor tyrosine kinase closely related to the human EGF receptor. In the teleost fish Xiphophorus two versions of the Xmrk gene exist, an oncogene (ONC) and a proto-oncogene (INV). While ONC-Xmrk is the melanoma-inducing gene, INV-Xmrk appears not to be involved in transformation of pigment cells. To elucidate the mechanism that converts the proto-oncogene into a transforming oncogene a comparative analysis of the structure, expression and function of both versions of the gene was performed. In contrast to ONC-Xmrk which is expressed at high levels in melanoma cells, the proto-oncogene INV-Xmrk is ubiquitously expressed at very low levels indicating overexpression as one possible reason for tumorigenicity by ONC-Xmrk. As sequence comparison of the proto-oncogene and the oncogene revealed a number of amino acid changes, a possible effect of these mutations on the activation of the ONC-Xmrk receptor was determined. A constitutive activation of the oncogenic receptor was found and ectopic expression of INV-Xmrk after microinjection into medakafish embryos did not lead to the high tumour rate in transgenic fish as observed for the oncogene. Our data therefore suggest that overexpression of the receptor alone is not sufficient for melanoma induction, but that in addition activating mutations in ONC-Xmrk are responsible for its full tumorigenic potential.

Receptor tyrosine kinase Xmrk mediates proliferation in Xiphophorus melanoma cells.

Over-expression of the oncogenic receptor tyrosine kinase (RTK) Xmrk is sufficient to induce formation of hereditary malignant melanoma in the fish Xiphophorus. In the melanoma tissue as well as in a melanoma-derived cell line (PSM), the Xmrk protein shows strong tyrosine phosphorylation, indicating either ligand-independent or autocrine activation of its kinase domain. However, it is unknown whether the constitutively activated Xmrk receptor itself directly triggers the proliferative signals, thus leading to uncontrolled growth of the pigment cells. In order to evaluate the role of Xmrk in proliferation of melanoma cells, we inhibited its kinase activity by using a Xmrk specific tyrphostin. At a concentration of 10 microM, tyrphostin AG555 led to a decrease of the Xmrk-induced DNA synthesis to 10% in NIH 3T3 Hm cells, whereas serum dependent 3H-thymidine incorporation was unaffected. In fish melanoma cells, the drug efficiently blocked DNA synthesis and cellular growth. This anti-proliferative activity correlated with the potency of AG555 to inhibit Xmrk autophosphorylation, indicating that the Xmrk receptor is the major determinant of mitogenic signaling in Xiphophorus melanoma cells.

Signal transduction by the oncogenic receptor tyrosine kinase Xmrk in melanoma formation of Xiphophorus.

Melanoma formation in Xiphophorus is initiated by overexpression of an oncogenic version of the EGFR-related receptor tyrosine kinase Xmrk (Xiphophorus melanoma receptor kinase). High steady-state levels of Xmrk oncogene mRNA are found in malignant melanoma; however, this overabundance of transcripts appears to be not sufficient for manifestation of the full oncogenic potential of Xmrk. In addition, several amino acid exchanges cause the oncogenic receptor to be highly active, resulting in a strong tyrosine phosphorylation even without growth factor stimulation. Besides the receptor itself a Xmrk-specific signal transduction seems to be a critical part of the transformation machinery. Expression experiments in transgenic fish indicate that the Xmrk-mediated intracellular signalling is contributing to the cell-type specificity in development of hereditary melanoma in Xiphophorus.

Association between the melanoma-inducing receptor tyrosine kinase Xmrk and src family tyrosine kinases in Xiphophorus.

Melanoma formation in the fish Xiphophorus is an in vivo model for the function of receptor tyrosine kinases (RTKs) in tumor development. The overexpression and high activity of the RTK Xmrk (Xiphophorus melanoma receptor kinase) is responsible for the formation of hereditary malignant melanoma in this fish, but the mechanism by which Xmrk signals cell proliferation has not been elucidated. Remarkably, in earlier experiments an elevated level of a pp60c-src related kinase activity was found in the melanomas. In order to evaluate the significance of src family SH2 domain interactions in the intracellular signalling of Xmrk, we determined its relative binding affinity to the ubiquitous general RTK substrate, PLC gamma, and to the Xiphophorus cytoplasmic kinases Xsrc, Xfyn and Xyes. Recombinant Xmrk purified from baculovirus infected Sf9 cells bound with high affinity to the SH2 domains of PLC gamma and Xfyn in vitro. The affinity of Xmrk to Xsrc and Xyes SH2 domains was 5- to 10-fold lower. Coprecipitation experiments revealed that the Xmrk/Xfyn interaction occurred also in melanoma cells. Moreover, stimulation of the Xmrk kinase activity was paralleled by an increase in Xfyn activity. These results suggest that in malignant melanoma of Xiphophorus the highly activated Xmrk may enhance the activity of Xfyn through direct interaction and that both kinases are linked in a signal transduction pathway.