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Beta-arrestin1, which regulates many aspects of seven transmembrane receptor (7TMR) biology, has also been shown to serve as an adaptor, which brings Mdm2, an E3 ubiquitin ligase to the insulin-like growth factor-1 receptor (IGF-1R), leading to its proteasome-dependent destruction. Here we demonstrate that IGF-1R stimulation also leads to ubiquitination of beta-arrestin1, which regulates vesicular trafficking and activation of ERK1/2. This beta-arrestin1-dependent ERK activity can occur even when the classical tyrosine kinase signaling is impaired. siRNA-mediated suppression of beta-arrestin1 in human melanoma cells ablates IGF-1-stimulated ERK and prolongs the G1 phase of the cell cycle. These data suggest that beta-arrestin-dependent ERK signaling by the IGF-1R regulates cell cycle progression and may thus be an important regulator of the growth of normal and malignant cells.

Insulin-like growth factor 1 receptor (IGF-1R) plays many crucial roles in cancer, like anti-apoptotic activity and necessity for transformation. IGF-1R knockout cells (R−) represent a useful tool for molecular mapping of biological properties of the receptor. R− cells have been shown to be refractory to transformation by viral and cellular oncogenes, highlighting the necessity of this receptor for transformation. Surprisingly, more recent studies have shown that these cells can undergo spontaneous transformation. This observation raises the question as whether R− cells over the years have acquired some properties mimicking those of IGF-1R. Using an IGF-1R inhibitor (cyclolignan PPP) we have identified clones of R− (R−s) that are sensitive to this compound. Since, PPP is closely related to podophyllotoxin, which is an efficient microtubule inhibitor, we first investigated if such a mechanism could explain the sensitivity to PPP. However, highly purified PPP showed no or very slight tubulin binding. Further analysis of R−s revealed expression of a 90 kDa protein being reactive to IGF-1R β-subunit antibodies. This protein was weakly but constitutively tyrosine phosphorylated and was downregulated by siRNA targeting IGF-1R. This downregulation was paralleled by decreased R−s survival. Taken together, our study suggests that clones of R− express IGF-1R activity and dependency, which in turn may explain that R− can undergo spontaneous transformation.

The insulin-like growth factor-1 receptor (IGF-1R) plays important roles in physiological growth and aging as well as promoting several crucial functions in cancer cells. However, the molecular mechanisms involved in expression and down-regulation of IGF-1R are still poorly understood. Here we provide evidence that beta-arrestin, otherwise known to be involved in the regulation of G protein-coupled receptors, serves as an adaptor to bring the oncoprotein E3 ubiquitin ligase MDM2 to the IGF-1R. In this way, beta-arrestin acts as a crucial component in the ubiquitination and down-regulation of the receptor. Both MDM2 and beta-arrestin co-immunoprecipitated with the IGF-1R. The beta-arrestin isoform 1 appeared to be more strongly associated with the receptor than isoform 2, and in a molecular context it was 4-fold more efficient in inducing polyubiquitination of IGF-1R, a reaction that required the presence of beta-arrestin and MDM2. Ligand stimulation accelerated IGF-1R ubiquitination. In mouse P6 cells (overexpressing human IGF-1R) absence of beta-arrestin 1, but not of beta-arrestin 2, blocked ubiquitination of IGF-1R. Conversely, in the two studied human melanoma cell lines both beta-arrestin isoforms seemed to be involved in IGF-1R ubiquitination. However, because depletion of beta-arrestin 1 almost completely eliminated degradation, and IGF-1 induced down-regulation of the receptor in these cells, whereas beta-arrestin 2 only had a partial effect, beta-arrestin 1 seems to the more important isoform in affecting the expression of IGF-1R. To our knowledge this is the first study demonstrating a defined molecular role of beta-arrestin with direct relevance to cell growth and cancer.

The insulin-like growth factor-1 receptor (IGF-1R) plays a pivotal role in transformation, growth, and survival of malignant cells, and has emerged as a general and promising target for cancer treatment. However, no fully selective IGF-1R inhibitors have thus far been found. This is explained by the fact that IGF-1R is highly homologous to the insulin receptor, coinhibition of which may cause diabetic response. The receptors are both tyrosine kinases, and their ATP binding sites are identical, implying that ATP inhibitors cannot discriminate between them. Therefore, the current strategy has been to identify compounds interfering with receptor autophosphorylation at the substrate level. In this study we investigated the effects of cyclolignans and related molecules on IGF-1R activity. We report that certain cyclolignans are potent and selective inhibitors of tyrosine phosphorylation of the IGF-1R. Of particular interest was picropodophyllin (PPP), which is almost nontoxic (LD50 >500 mg/kg in rodents). PPP efficiently blocked IGF-1R activity, reduced pAkt and phosphorylated extracellular signal regulated kinase 1 and 2 (pErk1/2), induced apoptosis in cultured IGF-1R-positive tumor cells, and caused complete tumor regression in xenografted and allografted mice. PPP did not affect the insulin receptor or compete with ATP in an in vitro kinase assay, suggesting that it may inhibit IGF-1R autophosphorylation at the substrate level. This is also in agreement with our molecular model of how the cyclolignans may act on the IGF-1R kinase. Our results open the possibility to use PPP or related compounds with inhibitory effects on IGF-1R as lead compounds in development of anticancer agents.

The prime position of the insulin-like growth factor 1 receptor (IGF-1R), at the head of the principle mitogenic and anti-apoptotic signalling cascades, along with the resilience to transformation of IGF-1R deficient cells fuelled great excitement for its anti-cancer targeting. Yet its potential has not been fulfilled, as clinical trial results fell far short of expectations. Advancements in understanding of other receptors’ function have now begun to shed light on this incongruity, with the now apparent parallels highlighting the immaturity of our understanding of IGF-1R biology, with the model used for drug development now recognised as having been too simplistic. Gathering together the many advancements of the field of IGF-1R research over the past decade, alongside those in the GPCR field, advocates for a major paradigm shift in our appreciation of the subtle workings of this receptor. This review will emphasise the updating of the IGF-1R’s classification from an RTK, to an RTK/GPCR functional hybrid, which integrates both canonical kinase signalling with many functions characteristic of a GPCR. Recognition of the shortcomings of IGF-1R inhibitor drug development programs and the models used not only allows us to reignite the initial interest in the IGF-1R as an anti-cancer therapeutic target, but also points to the possibility of biased ligand therapeutics, which together may hold a very powerful key to unlocking the true potential of IGF-1R modulation.

Insulin-like growth factor-1 receptor (IGF-1R) has been shown to be important for melanoma cell growth and survival. In this study we first show, using immunohistochemistry, that progression from benign nevi to malignant melanoma is paralleled by an increased expression of IGF-1R and a down-regulation of the cyclin-dependent kinase inhibitor p27Kip1. Even though the expression of p27Kip1 was drastically reduced compared to benign tumors, detectable amounts of it could be assayed by Western blotting in cultured melanoma cells. To analyze whether there is a causative relationship between the IGF-1 pathway and p27Kip1 expression, melanoma cells were treated with alpha IR-3, an antibody blocking the IGF-1 binding to IGF-1R, or Tunicamycin, which inhibits the translocation of IGF-1R to the cell surface. From these studies we could conclude that the overall expression of p27Kip1 is independent of the IGF-1 pathway. In contrast, the association of p27Kip1 with the different cyclins was drastically affected. Both TM and alpha IR-3 decreased the binding of p27Kip1 to cyclin D1, whose expression was drastically reduced. On the other hand there was an increased binding of p27Kip1 to cyclin E and cyclin A. This redistribution of p27Kip1 may be a mechanism for growth arrest and induction of apoptosis following interruption of the IGF-1 pathway in melanoma cells.

The Ewing's sarcoma cell line RD-ES, which carries the EWS/FLI-1 fusion gene, responded to the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin with growth arrest. Replenishment of mevalonate (MVA) to the arrested cells restored cell growth. However, if tunicamycin (TM), which is an inhibitor of N-linked glycosylation, was present together with MVA the cells remained arrested, indicating that N-linked glycosylation is of importance for growth of Ewing's sarcoma cells. Inhibition of the biosynthesis of EWS/FLI-1 fusion protein by treatment with antisense oligonucleotides also led to growth arrest, suggesting that this protein is of importance for cell growth. We investigated whether MVA synthesis and N-linked glycosylation could be involved in regulation of the expression of the EWS/FLI-1 fusion protein, which in fact contains four potential sites for N-linked glycosylation. We found that inhibition of both HMG-CoA reductase and N-linked glycosylation drastically decreased the expression of the fusion protein, which mainly appears in the cell nuclei. There was no significant difference in the inhibitory effect on the fusion protein between the cytoplasm and the cell nuclei, indicating that the transport of the fusion protein to the cell nucleus is not affected. The fusion protein did not exhibit any gel electrophoretic mobility shift after treatment of the cells with lovastatin or TM, and it did not incorporate [3H]glucosamine. Therefore we can conclude that the fusion protein is not a glycoprotein. The decreased expression of the fusion protein following lovastatin or TM treatment was found to be due to a lowered stability ofde novo-synthesized fusion protein. The down-regulation of the fusion protein was correlated to growth arrest. Furthermore, the kinetics between the expression of EWS/FLI-1 fusion protein and the initiation of DNA synthesis in MVA-stimulated cells were similar. Taken together, our data suggest that the regulatory role of N-linked glycosylation in the expression of the EWS/FLI-1 fusion protein is important for growth of Ewing's sarcoma cells. Possible mechanisms underlying TM-induced decrease in EWS/FLI-1 expression may involve the breaking of growth factor receptor pathways.

The IGF-1R pathway is essential for the initiation and progression of many cancers. In contrast to other receptor tyrosine kinases involved in cancer, it is not frequently mutated or amplified. The classical model of signaling through the IGF-1R centers on ligand initiated kinase activation, allowing binding of adaptor molecules and downstream activation of the MAPK and PI3K pathways. The signaling is terminated through receptor ubiquitination and subsequent degradation. To date, therapies targeting IGF-1R have been designed solely aiming to block phosphorylation mediated signaling by preventing receptor-ligand interaction or by limiting kinase activation. Yet, the classical model is insufficient to explain receptor behavior induced by some IGF-1R inhibitors. This review advocates an updated model of IGF-1R signaling, accommodating the "classical" kinase signaling and the IGF-1R-kinase independent signaling thus providing the theoretical background for receptor downregulation induced by IGF-1R inhibitors. This model should be considered for future design of effective therapies targeting the IGF-1R pathway.

β-Arrestins are multifunctional proteins that play central roles in G protein-coupled receptor (GPCR) trafficking and signaling. β-Arrestin1 is also recruited to the insulin-like growth factor-1 receptor (IGF-1R), a receptor tyrosine kinase (RTK), mediating receptor degradation and signaling. Because GPCR phosphorylation by GPCR-kinases (GRKs) governs interactions of the receptors with β-arrestins, we investigated the regulatory roles of the four widely expressed GRKs on IGF-1R signaling/degradation. By suppressing GRK expression with siRNA, we demonstrated that lowering GRK5/6 abolishes IGF1-mediated ERK and AKT activation, whereas GRK2 inhibition increases ERK activation and partially inhibits AKT signaling. Conversely, β-arrestin–mediated ERK signaling is enhanced by overexpression of GRK6 and diminished by GRK2. Similarly, we demonstrated opposing effects of GRK2 and -6 on IGF-1R degradation: GRK2 decreases whereas GRK6 enhances ligand-induced degradation. GRK2 and GRK6 coimmunoprecipitate with IGF-1R and increase IGF-1R serine phosphorylation, promoting β-arrestin1 association. Using immunoprecipitation, confocal microscopy, and FRET analysis, we demonstrated β-arrestin/IGF-1R association to be transient for GRK2 and stable for GRK6. Using bioinformatic studies we identified serines 1248 and 1291 as the major serine phosphorylation sites of the IGF-1R, and subsequent mutation analysis demonstrated clear effects on IGF-1R signaling and degradation, mirroring alterations by GRKs. Targeted mutation of S1248 recapitulates GRK2 modulation, whereas S1291 mutation resembles GRK6 effects on IGF-1R signaling/degradation, consistent with GRK isoform-specific serine phosphorylation. This study demonstrates distinct roles for GRK isoforms in IGF-1R signaling through β-arrestin binding with divergent functional outcomes.

PURPOSE: Uveal melanoma has a high mortality rate due to a high incidence of metastasis (up to 50%), which preferentially occurs in the liver. Conventional chemotherapy, being the only therapeutic option today against metastatic uveal melanoma, has not proved to be effective. Therefore, new molecular targets important for malignant phenotype of uveal melanoma have to be found to design efficient pharmacologic agents. EXPERIMENTAL DESIGN: We previously reported data indicating that the insulin-like growth factor-1 receptor (IGF-IR) is a metastasis predictor as well as a therapeutic target for uveal melanoma. In the present study, we made use of the cyclolignan picropodophyllin (PPP), which is an inhibitor of the IGF-IR. RESULTS: We showed that PPP efficiently blocks growth and viability of uveal melanoma cells in cultures and causes tumor regression in xenografted mice. In addition, treatment with PPP inhibited several mechanisms involved in metastasis, including tumor cell adhesion to extracellular matrix proteins, activity and expression of matrix metalloproteinase 2, and cell migration as well as invasion through basement membranes and endothelial cell layers. Furthermore, PPP significantly delayed establishment of uveal melanoma tumors and drastically reduced the incidence of liver metastasis in mice. CONCLUSIONS: Our data suggest that IGF-IR is crucial for growth and survival as well as invasion and metastasis of uveal melanoma cells. Targeting this receptor may therefore comprise a strategy to treat ongoing disease (today incurable) as well as a strategy to prevent development of metastases in patients with primary disease.

Insulin-like growth factor 1 receptor (IGF-1R) is important for transformation of cells with cellular and viral oncogenes. This knowledge is mainly based on experiments on IGF-1R knockout mouse fibroblasts, which mostly are unable to transform after introduction of various oncogenes. Recently, we observed two variants of R- cells, one of which (R-s) surprisingly expresses the beta-subunit of IGF-1R whereas the other one (R-r) does not. Here we show that the beta-subunit is localized intracellularly and forms perinuclear aggregates. It expresses tyrosine kinase activity and appears to be crucial for cell survival since knockdown of it kills the R-s cells. H-RasV12 and/or polyoma middle T-antigen fail to transform R-r, whereas R- cells expressing the beta-subunit were transformed as assessed by formation of colonies in soft agar. The oncogenic transformation of R-s cells was, however, abrogated when the aberrant beta-subunit was knockdown by siRNA. The occurrence of intracellular IGF-1R, especially in tumor cells, has been widely reported but its function has not been understood. Our study provides evidence that it may be important for cell survival and transformation.

The initial event upon binding of insulin-like growth factor 1 to the insulin-like growth factor type-I receptor (IGF-1R) is auto-phosphorylation of tyrosine residues within the activation loop of the kinase domain followed by phosphorylation of other receptor tyrosine residues and the subsequent activation of the intracellular signaling cascades. We found recently that the cyclolignan picropodophyllin (PPP) inhibits phosphorylation of IGF-1R and phosphatidyl-3 kinase/Akt (protein kinase B) signaling molecules without interfering with the highly homologous insulin receptor. Furthermore, PPP causes regression of tumor grafts and substantially prolongs the survival of animals with systemic tumor disease. It is of interest that we show here that short treatments with PPP activate the intracellular extracellular signal-regulated kinase (ERK) signaling. Our data suggest that PPP induces IGF-1R ubiquitination and in turn activates ERK1/2. The PPP-induced ERK activation requires IGF-1R because PPP is not able to induce ERK phosphorylation in IGF-1R-negative cells or in cells in which the receptor is knocked down by small interfering RNA. Moreover, in the absence of Mdm2, an E3 ligase that has been shown previously to be involved in IGF-1R ubiquitination, the phosphorylation of ERK did not occur. Thus, apart from inhibiting the receptor activity, PPP can induce IGF-1R ubiquitination and stimulate ERK in an Mdm2-dependent manner. This response could contribute to the apoptotic effect of PPP.

Insulin-like growth factor 1 (IGF-1) plays a pleiotropic role in multiple myeloma (MM), that is, in survival, proliferation, chemotaxis, and angiogenesis. Strategies targeting the IGF-1 receptor (IGF-1R) may therefore be important to develop efficient anti-MM agents. In this work we investigated the effect of an IGF-1R tyrosine kinase (IGF-1RTK) inhibitor (picropodophyllin or PPP) in the 5T33MM mouse model. In vitro data showed that PPP reduced IGF-1R autophosphorylation and downstream ERK activation, leading to inhibition of IGF-1–stimulated proliferation and vascular endothelial growth factor (VEGF) secretion of MM cells. In an in vivo study, PPP reduced the bone marrow tumor burden and serum paraprotein in 5T33MM mice by 77% and 90%, respectively, compared to vehicle-treated animals. Angiogenesis was assessed by quantifying the microvessel density on CD31-stained paraffin sections and this was reduced by 60% in the PPP-treated group. In a separate survival experiment, Kaplan-Meier analysis demonstrated a significant increase in survival in PPP-treated 5T33MM animals compared to the vehicle controls (28 versus 18 days). These data suggest that the IGF-1RTK inhibitor PPP possesses a marked antitumor activity and strongly points to the possibility of using IGF-1R inhibitors in the treatment of MM.

Emerging evidence suggests the insulin-like growth factor-1 receptor (IGF-1R) to be an important mediator of tumor-cell survival and resistance to cytotoxic therapy in multiple myeloma (MM). Recently, members of the cyclolignan family have been shown to selectively inhibit the receptor tyrosine kinase (RTK) activity of the IGF-1R beta-chain. The effects of the cyclolignan picropodophyllin (PPP) were studied in vitro using a panel of 13 MM cell lines and freshly purified tumor cells from 10 patients with MM. PPP clearly inhibited growth in all MM cell lines and primary MM samples cultured in the presence or absence of bone marrow stromal cells. PPP induced a profound accumulation of cells in the G(2)/M-phase and an increased apoptosis. Importantly, IGF-1, IGF-2, insulin, or IL-6 did not reduce the inhibitory effects of PPP. As demonstrated by in vitro kinase assays, PPP down-regulated the IGF-1 RTK activity without inhibiting the insulin RTK activity. This conferred decreased phosphorylation of Erk1/2 and reduced cyclin dependent kinase (CDK1) activity. In addition, the expression of mcl-1 and survivin was reduced. Taken together, we suggest that interfering with the IGF-1 RTK by using the cyclolignan PPP offers a novel and selective therapeutic strategy for MM.

Craniopharyngioma is a rare benign intracranial epithelial tumor that, however, often recurs and sometimes kills the affected patients, one-third of which are children. In many cases, the patients acquire growth hormone deficiency and postoperatively need substitution. Generally, growth hormone promotes local release of insulin-like growth factor I (IGF-I), which in turn activates the IGF-I receptor (IGF-IR) if present. Together, these circumstances raise the question whether IGF-IR may be involved in craniopharyngioma growth. To address this issue, we analyzed phenotypically well-characterized primary low-passage craniopharyngioma cell lines from nine different patients for IGF-IR expression and IGF-I dependency. Two of the cell lines showed no/very low expression of the receptor and was independent on IGF-I, whereas five cell lines exhibited a strong expression and was clearly contingent on IGF-I. The two remaining cell lines had low receptor expression and IGF-I dependency. Upon treatment with an IGF-IR inhibitor, cells with high IGF-IR expression responded promptly with decreased Akt phosphorylation followed by growth arrest. These responses were not seen in cells with no/very low receptor expression. Growth of cell lines with low IGF-IR expression was only slightly affected by IGF-IR inhibition. Taken together, our data suggest that IGF-IR may be involved in the growth of a subset of craniopharyngiomas and points to the possibility of the involvement of IGF-IR inhibitors as a treatment modality to obtain complete tumor-free conditions before growth hormone substitution.

Purpose: To evaluate known and suggested prognostic markers, especially insulin-like growth factor type 1 receptor (IGF-1R), in highly malignant soft tissue sarcomas (STS). Experimental Design: A cohort of 101 patients with primary STS of high malignancy grade was studied with respect to development of metastasis, local recurrence, and survival during a minimum of 5 years follow-up. All tumors were analyzed by immunohistochemistry for expression of Ki-67, p53, p27, Bcl-2, IGF-1R, and microvessel density. The traditional clinical variables size, malignancy grade (3 or 4), necrosis, mitotic frequency, infiltrative tumor growth, vascular invasion, depth, and surgical margins were also evaluated. Results: A significant association was shown between high expression of IGF-1R and favorable outcome. Among STS with positive IGF-1R immunoreactivity, cases with high expression (76-100% positive cells) had the best outcome, whereas cases with the lowest expression (1-25% positive cells) had the worst. As expected, large tumor size (>11 cm), presence of necrosis, high mitotic count, intralesional surgery, and deep location were all significantly associated with poor outcome, both in univariate and multivariate analyses. No difference in outcome was observed between cases of malignancy grade 3 versus 4, whereas the included and more objective variables necrosis and mitotic count were found to be reliable prognostic markers. Conclusion: IGF-1R expression is a common feature of highly malignant STS. Further elucidation of the role of IGF-1R and the IGF system in STS may both provide a basis for development of new prognostic tools in STS, as well as shed light on the basic mechanisms of the STS development.

The insulin-like growth factor-1 receptor (IGF-1R) is crucial for many functions in neoplastic cells, for example, antiapoptosis. Recently, we demonstrated that the cyclolignan PPP efficiently inhibited phosphorylation of IGF-1R without interfering with insulin receptor activity. PPP preferentially reduced phosphorylated Akt, as compared to phosphorylated Erk1/2, and caused apoptosis. Now, we aimed to investigate how PPP inhibits the IGF-1R tyrosine kinase (IGF-1RTK) and the PI3K/Akt apoptotic pathway. Using a baculovirus driven IGF-1RTK we found that PPP interfered with tyrosine phosphorylation in the activation loop of the kinase domain. Specifically, it blocked phosphorylation of tyrosine (Y) 1136, while sparing the two others (Y1131 and Y1135). To explore the impact of inhibition of Y1136 on Akt phosphorylation we transfected P6 cells (overexpressing IGF-1R) and malignant melanoma cells with different IGF-1R mutants, including Y1136F (tyrosine replaced by phenylalanine). Y1136F was found to strongly decrease IGF-1 stimulated phosphorylation of Akt. Conversely, Akt phosphorylation was weakly affected in the Y1131F transfectant. Taken together, our data suggest that the preferential inhibition of phosphorylated Akt, after PPP treatment, may be due to specific inhibition of Y1136. PPP was proven not to interfere directly with Akt or any of its downstream molecules in the apoptotic pathway.

Adhesion between the CD44s receptor and hyaluronic acid plays an important role in cell migration, tumour growth and progression. Although the alternative splicing of CD44 variant exons represents the principal regulatory mechanism of CD44-mediated functions, CD44v spliced variants are scantily expressed in melanoma cells. For this reason, we have investigated the possibility that post-translational modifications of the CD44 standard receptor could play a pivotal role in regulating CD44-mediated functions in melanoma. Using metabolic inhibitors of N- and O-glycosylation, as well as melanoma transfectants expressing CD44s O-glycosylation site-specific mutants, we performed structural and functional analysis of N- and O-deglycosylated CD44s molecules expressed in melanoma cells. We discovered that complete N- and O-glycosylation is not required by CD44s to be correctly expressed on the melanoma cell surface. Indeed, variably glycosylated and functionally different CD44s molecules were constitutively expressed in primary and metastatic lesions. Furthermore, we observed that changes in N- and O-glycosylation of CD44s could modulate its cleavage. In fact, spontaneous CD44s shedding was dependent on the presence of partial or complete O-glycosylation of four serine-glycine motifs localized in the membrane-proximal CD44 ectodomain. Mutation of these serine residues, as well as an extensive metabolic O-deglycosylation, strongly impaired spontaneous CD44 shedding. Furthermore, an O-glycosylation-independent mechanism of CD44 cleavage has been identified. This alternative mechanism of receptor cleavage is phorbol 12-myristate-13-acetate (PMA) inducible, mediated by metalloproteinase and requires the presence of N-linked sugar residues. Our findings demonstrate that the post-translational modification of CD44s represents the principal regulatory mechanism of CD44s-mediated functions in melanoma.

Recently, p53 was demonstrated to affect the expression of the insulin-like growth factor 1 receptor (IGF-1R), a receptor tyrosine kinase that plays a crucial role in growth and survival of cancer cells. However, the underlying mechanisms for interaction between p53 and IGF-1R are still not fully understood. One of the challenging questions remaining to be answered is why the wild-type p53, which per se represses the transcription of the IGF-1R gene, in overexpressed form is necessary for a high IGF-1R expression. In this study, we show that inhibition of p53 causes ubiquitination and down-regulation, through increased degradation, of the IGF-1R in human malignant melanoma cells. This effect, which was independent of the p53 status (i.e., wild type or mutated), was prevented if Mdm2 was coinhibited. Similar results were obtained in UV-irradiated human melanocytes (harboring wild-type p53), in which level of the IGF-1R increased after up-regulation of p53. Interestingly, the basal ubiquitination of the IGF-1R in untreated cells also depended on Mdm2. We could prove that Mdm2 physically associates with IGF-1R and that Mdm2 causes IGF-1R ubiquitination in an in vitro assay. Taken together our data provide evidence that Mdm2 serves as a ligase in ubiquitination of the IGF-1R and thereby causes its degradation by the proteasome system. Consequently, by sequestering Mdm2 in the cell nuclei, the level of p53 may indirectly influence the expression of IGF-1R. This role of Mdm2 and p53 represents an unexpected mechanism for the regulation of IGF-1R and cell growth.

To investigate the expression of the insulin-like growth factor-1 receptor (IGF-1R) with special focus on its role in cell growth in uveal melanoma.Paraffin material from 36 clinicopathologically well characterized cases of primary uveal melanomas (18 of which had metastasized to the liver) with more than 15 years' follow-up was used for immunohistochemical analysis. In the experimental studies, three uveal melanoma cell lines (OCM-1, OCM-3, and 92-1) were used. The expression level of IGF-1R in the cell lines was modulated by glycosylation inhibitors, and the IGF-1R was neutralized with the antibody alphaIR-3. Expression of IGF-1R was assayed by Western blot analysis and immunohistochemistry. Cell growth and survival were analyzed by cell counting, thymidine incorporation, and viability assays.Western blot analysis and immunohistochemistry confirmed that IGF-1R is expressed in uveal melanoma. Although 10 of 18 patients who died of metastasizing disease showed high IGF-1R expression, only 5 of 18 tumors from patients who survived for 15 years or more after enucleation exhibited a high IGF-1R expression. Kaplan-Meier analysis showed a significant association (P = 0.035) between a high IGF-1R expression and death due to metastatic uveal melanoma. Using in vitro experimental models, we found that inhibition of the IGF-1R activity (tyrosine phosphorylation) was associated with a drastic decrease in uveal melanoma cell viability.These data suggest an important role of IGF-1R in uveal melanoma. The significant association between high IGF-1R expression and death due to metastatic disease may be explained by the fact that IGF-1 is mainly produced in the liver, which is the preferential site for uveal melanoma metastases. These data also point to the possibility of therapeutically interfering with IGF-1R, which appears to be expressed preferentially in uveal melanomas that appear to follow an aggressive clinical course.

The EWS/FLI-1 fusion gene is characteristic of most cases of Ewing's sarcoma and has been shown to be crucial for tumor transformation and cell growth. In this study we demonstrate a drastic down-regulation of the EWS/FLI-1 protein, and a growth arrest, following serum depletion of Ewing's sarcoma cells. This indicates that growth factor circuits may be involved in regulation of the fusion gene product. Of four different growth factors tested, basic fibroblast growth factor (bFGF) was found to be of particular significance. In fact, upon treatment of serum-depleted cells with bFGF, expression of the EWS/FLI-1 protein and growth of the Ewing's sarcoma cells were restored. In addition, a bFGF-neutralizing antibody, which was confirmed to inhibit FGF receptor (FGFR) phosphorylation, caused down-regulation of EWS/FLI-1. Experiments using specific cell cycle blockers (thymidine and colcemide) suggest that EWS/FLI-1 is directly linked to bFGF stimulation, and not indirectly to cell proliferation. We also demonstrated expression of FGFRs in several tumor samples of Ewing's sarcoma. Taken together, our data suggest that expression of FGFR is a common feature of Ewing's sarcoma and, in particular, that the bFGF pathway may be important for the maintenance of a malignant phenotype of Ewing's sarcoma cells through up-regulating the EWS/FLI-1 protein. Oncogene (2000) 19, 4298 - 4301

Purpose: Uveal melanoma disseminates preferentially to the liver. The mechanism for this homing is largely unknown, but growth factors synthesized in the liver may be involved. The present study was undertaken to investigate the possible relationship between cell surface receptors for two such growth factors: the c-Met proto-oncogene, which constitutes the receptor for hepatocyte growth factor/scatter factor (HGF/SF), and the insulin-like growth factor 1 receptor (IGF-1R). Their role as a prognostic factor was also clarified. Methods: Paraffin-embedded tumor specimens from 132 patients with primary uveal melanoma were analyzed by using well-established specific antibodies against c-Met and IGF-1R. The intercorrelation of receptor expression and association with melanoma-related survival of patients were determined by univariate and multivariate analyses. Results: Whereas the expression of both IGF-1R and c-Met was significantly associated with melanoma-specific mortality by univariate analysis (p = 0.004 and p = 0.007, respectively) only IGF-1R showed independent prognostic value by multivariate analysis, p = 0.004. The prognostic value of IGF-1R was stronger than such currently used prognostic parameters as tumor cell type and tumor diameter (p = 0.021 and p = 0.026, respectively). The expression patterns of the two growth factors receptors were weakly intercorrelated. Conclusions: In conclusion, the data suggest that the receptors for IGF-1 and HGF/SF may play a role in the spread of uveal melanoma and its affinity to the liver. The strong correlation between IGF-1R expression and melanoma-specific mortality points to the use of IGF-1R as a prognostic tool [Economou MA, All-Ericsson C, Bykov V, Girnita L, Bartolazzi A, Larsson O & Seregard S (2005): Receptors for the liver synthesized growth factors IGF-1 and HGF/SF in uveal melanoma: intercorrelation and prognostic implications. Invest Ophthalmol Vis Sci46: 4372–4375].

In multiple myeloma (MM) emerging evidence suggest the IGF-1R as an important mediator of tumor cell survival and thus resistance to cytotoxic therapy. Since IGF-1R is not an absolute requirement for maintenance of normal cell homeostasis, interfering with IGF-1R signaling at the receptor tyrosine kinase (RTK) level represents an attractive strategy to improve anti-cancer treatment. However, most IGF-1 RTK inhibitors do not fully discriminate between the IGF-1 RTK and the insulin RTK, i.e. are diabetogenic. Recently, members of the cyclolignan family have been shown to selectively inhibit the phosphorylation of the IGF-1R β-chain without down regulating the RTK activity of the insulin R1. The effect of two of these compounds, picropodophyllin (PPP) and deoxypodophyllotoxin (DPPT), was studied in vitro using a panel of nine MM cell lines and primary tumor cells from MM patients, and in vivo using the 5TMM mouse MM model2. Both IGF-1 RTK inhibitors effectively inhibited growth in all MM cell lines providing increased apoptosis and cell cycle arrest in the G2/M-phase. Notably, the two drug-resistant subclones of the MM cell line RPMI 8226/Dox40 (doxorubicin) and RPMI 8226/LR5 (melphalan), were highly sensitive to the IGF-1 RTK inhibitors. In addition, PPP and DPPT showed inhibitory effects on primary MM cells cultured in vitro with bone marrow stromal cells. Inhibition of the IGF-1 RTK with PPP has been shown to be non-competitive with ATP suggesting interference with the IGF-1R at the substrate level1. To identify tyrosine phosphorylation site(s) on the IGF-1R β-chain potentially regulated by the IGF-1 RTK inhibitors, IGF-1R extracted from RPMI 8226 cells was analyzed by tryptic phosphopeptide mapping following 32P-labeling and treatment with PPP or DPPT. Cleaved phosphopeptides were separated by thin layer chromatography and analyzed by a modified radio-Edman degradation. The results show that the IGF-1 RTK inhibitors do not down regulate any specific tyrosine phosphorylation site of the IGF-1R in MM cells. In vitro kinase assay suggests that PPP/DPPT-induced inhibition of the IGF-1R instead is conducted via a general down regulation of the tyrosine kinase activity of the receptor. Extraction of tyrosine phosphorylated proteins followed by electrophoresis and mass spectrometric analysis revealed additional signaling molecules affected by treatment with the IGF-1 RTK inhibitors e.g. impaired tyrosine phosphorylation of CDK1/cdc2, a cell cycle associated protein known as a potent regulator of apoptosis. Taken together, we show that treatment of MM cells with selective IGF-1 RTK inhibitors decreases survival/proliferation and affects the function of crucial intracellular signaling proteins, thus emphasizing the pivotal role for IGF-1R signaling in MM.

Purpose This study was conducted to investigate the expression and functional impact of the proto-oncogene c-kit in uveal melanoma. Methods Based on immunohistochemical (IHC) study of paraffin-embedded specimens from 134 uveal melanomas and Western blot analysis on eight fresh-frozen samples the expression of c-kit in uveal melanoma was studied. Furthermore, the phosphorylation of c-kit and the impact of the tyrosine kinase inhibitor STI571 was examined in the three uveal melanoma cell lines OCM-1, OCM-3, and 92-1. Results Eighty-four of 134 paraffin-embedded samples and six of eight fresh-frozen samples expressed c-kit. c-Kit was strongly expressed and tyrosine phosphorylated in cultured uveal melanoma cells compared with cutaneous melanoma cells. Moreover, in contrast to cutaneous melanoma cell lines c-kit maintained a high phosphorylation level in serum-depleted uveal melanoma cells. No activation-related mutations in exon 11 of the KIT gene were found. On the contrary, expression of the stem cell growth factor (c-kit ligand) was detected in all three uveal melanoma cell lines, suggesting the presence of autocrine (paracrine) stimulation pathways. Treatment of uveal melanoma cell lines with STI571, which blocks c-kit autophosphorylation, resulted in cell death. The IC(50) of the inhibitory effects on c-kit phosphorylation and cell proliferation was of equal size and less than 2.5 microM. Conclusions The results confirm that c-kit is vastly expressed in uveal melanoma, suggest that the c-kit molecular pathway may be important in uveal melanoma growth, and point to its use as a target for therapy with STI571.

The insulin-like growth factor type 1 receptor (IGF-1R) plays a key role in the development and progression of cancer; however, therapeutics targeting it have had disappointing results in the clinic. As a receptor tyrosine kinase (RTK), IGF-1R is traditionally described as an ON/OFF system, with ligand stabilizing the ON state and exclusive kinase-dependent signaling activation. Newly added to the traditional model, ubiquitin-mediated receptor downregulation and degradation was originally described as a response to ligand/receptor interaction and thus inseparable from kinase signaling activation. Yet, the classical model has proven over-simplified and insufficient to explain experimental evidence accumulated over the last decade, including kinase-independent signaling, unbalanced signaling, or dissociation between signaling and receptor downregulation. Based on the recent findings that IGF-1R "borrows" components of G-protein coupled receptor (GPCR) signaling, including β-arrestins and G-protein-related kinases, we discuss the emerging paradigm for the IGF-1R as a functional RTK/GPCR hybrid, which integrates the kinase signaling with the IGF-1R canonical GPCR characteristics. The contradictions to the classical IGF-1R signaling concept as well as the design of anti-IGF-1R therapeutics treatment are considered in the light of this paradigm shift and we advocate recognition of IGF-1R as a valid target for cancer treatment.

Recent data indicate that the estrogen receptor (ER) blocker tamoxifen (TAM) can induce cell death in malignant melanoma cells. However, as shown in the present study and several other studies melanoma cells usually do not express classical ERs. In the present study we investigated whether the cytotoxic effect of TAM on melanoma cells could depend on interference with the expression or function of the insulin-like growth factor-1 receptor (IGF-1R), a plasma membrane receptor important for cell survival in this tumor cell type. Several melanoma cell lines were included in the analysis. Administration of TAM at a concentration of 15 microm or more resulted in cell death of the melanoma cells within 48 h. TAM treatment was correlated to a slight to moderate inhibition of IGF-1 binding to IGF-1R. Since it has been reported that TAM can increase the release of IGF binding proteins (IGFBPs) we then investigated whether this mechanism could underly the decreased IGF-1 binding. However, we could demonstrate that the amount of released IGFBPs were unchanged or decreased in TAM-treated cells. Whereas TAM did not have any strong effect on IGF-1 binding and the expression of IGF-1R at the cell surface, it was was found to efficently block tyrosine phosphorylation of IGF-1R beta-subunit. Taken together, our data suggest that TAM-induced cytotoxicity of malignant melanoma cells can be due to inactivation of IGF-1R.