Abstract: 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.

Abstract: PURPOSE: We aimed to report a case of orbital solitary fibrous tumour (SFT) in a child and to review the relevant literature. METHODS: We describe an SFT in a 13-year-old boy with a 1-month history of painless proptosis in the left eye. RESULTS: Magnetic resonance imaging revealed a well circumscribed mass filling most of the left intraconal orbit. The lesion was excised and histopathological examination revealed a malignant SFT. Postoperative follow-up for 18 months was uneventful. CONCLUSIONS: Malignant SFT of the orbit should be included in the differential diagnosis of paediatric orbital tumours. Complete surgical excision remains the preferred method of management and the longterm prognosis is guarded.

Abstract: INTRODUCTION: Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration (AMD) and a leading cause of vision loss. Along with other angiogenic factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and its receptor, IGF-1R, have been implicated in CNV. PURPOSE: A prior study has shown that the cyclolignan picropodophyllin (PPP) efficiently blocks the insulin-like growth factor-1 receptor (IGF-1R) activity and causes cell death in uveal melanoma cell lines and in an in vivo model. In this study we investigated the effect of PPP on VEGF expression, both in vitro and in vivo, and whether this effect has antiangiogenic consequences in a murine CNV model. METHODS: C57BL/6J mice with laser-induced CNVs were treated with PPP. Effects on CNV area were assayed by image analysis. VEGF levels in the choroid and retinal pigment epithelial cells (ARPE-19) were measured by Western blot or ELISA. Transcriptional activation of the VEGF promoter was determined by luciferase reporter gene assay. RESULTS: Mice treated with PPP, administered intraperitoneally or orally, showed a 22% to 32% (P = 0.002) decrease in CNV area. Furthermore, VEGF levels in the choroid were significantly reduced. In cultured ARPE-19 cells, IGF-1 was shown to increase VEGF secretion. This increase was completely blocked by PPP. PPP reduced the level of transcriptional activity of the VEGF promoter. CONCLUSIONS: PPP reduces IGF-1-dependent VEGF expression and CNV in vivo. Accordingly, IGF-1R inhibitors may be useful tools in the treatment of conditions associated with CNV, including neovascular AMD.

Abstract: Recently we demonstrated that the synovial sarcoma specific fusion gene SS18-SSX is crucial for cyclin D1 expression and is linked to cell proliferation. In this report we explore the role of SS18-SSX and IGF-1R for their potential functions in cellular proliferation and survival in cultured synovial sarcoma cells. We found that targeting of SS18-SSX mRNA by antisense oligonucleotide treatment drastically and rapidly decreased cell proliferation but caused only a slight increase of apoptosis. The synovial sarcoma cells were confirmed to express IGF-1R, and treatment with an IGF-1R inhibitor resulted in substantially reduced cell viability by inducing apoptosis in these cells. Conversely, inhibition of the IGF-1R resulted only in a slight to moderate decrease in DNA synthesis. In conclusion, SS18-SSX and IGF-1R seem to play important but different roles in maintaining malignant growth of synovial sarcoma cells. Whereas SS18-SSX maintains cyclin D1 and cell proliferation, IGF-1R protects from apoptosis.

Abstract: The insulin-like growth factor 1 receptor (IGF-1R) is crucial for growth and survival of malignant cells. Experience in targeting IGF-1R in cancer models has shown that strategies promoting downregulation of the receptor are much more efficient in inducing apoptosis than those inhibiting the IGF-1R activity. Recently, we found that the cyclolignan picropodophyllin (PPP) inhibits phosphorylation of IGF-1R and activation of downstream signaling without interfering with the highly homologous insulin receptor (IR). Furthermore, PPP treatment caused strong regression of tumor grafts and prolonged survival of animals with systemic tumor disease. Here we demonstrate that PPP also downregulates the IGF-1R, whereas the IR and several other receptors were not affected. PPP-induced IGF-1R downregulation required expression of the MDM2 E3 ligase, which recently was found to ubiquitinate and cause degradation of the IGF-1R. In addition knockdown of beta-arrestin1, the adaptor molecule known to bridges MDM2 and IGF-1R, prevented downregulation of the receptor and significantly decreased PPP-induced cell death. All together these data suggest that PPP downregulates IGF-1R by interfering with the action of beta-arrestin1/MDM2 as well as the achieved receptor downregulation contributes to the apoptotic effect of PPP.

Abstract: 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.

Abstract: Unsaturated malonyl esters underwent Pd-catalyzed intramolecular allylic alkylation to give 4-vinyl-substituted gamma-lactones. In contrast to the formerly studied cyclization of malonamides, this reaction could be achieved only with a substrate incorporating a suitably positioned silicon moiety, which directs the ionization toward the desired eta(3)-allylpalladium complex. The resulting 4-[dimethyl-(2-thienyl)silylvinyl]lactone could be subsequently engaged into Hiyama couplings with various iodoarenes, to give the corresponding 4-(alpha-styryl)-gamma-lactones. The use of a specifically substituted iodoarene generated an advanced tetracyclic lactone intermediate incorporating rings A-D of lignans belonging to the podophyllotoxin family. Subsequent electrophilic aromatic substitution with a variety of electron-rich arenes afforded the target picropodophyllin analogs.

Abstract: PURPOSE: The cyclolignan picropodophyllin (PPP) efficiently blocks the activity of insulinlike growth factor-1 receptor (IGF-1R) and inhibits the growth of uveal melanoma cells in vitro and in vivo. In this study, the authors investigated the efficiency of orally administered PPP on the growth of uveal melanoma xenografts. In addition, they focused on the effect of PPP on vascular endothelial growth factor (VEGF) in vivo and evaluated its effects in combination with other established antitumor agents in vitro. METHODS: Four different uveal melanoma cell lines (OCM-1, OCM-3, OCM-8, 92-1) were treated with PPP alone and in combination with imatinib mesylate, cisplatin, 5-fluorouracil, and doxorubicin. Cell viability was determined by XTT assay. SCID mice that underwent xenografting with uveal melanoma cells were used to determine antitumor efficacy of oral PPP in vivo. Five mice were used per group. Tumor samples obtained from the in vivo experiments were analyzed for VEGF and IGF-1R expression by Western blotting. RESULTS: PPP was found to be superior to the other antitumor agents in killing uveal melanoma cells in all four cell lines (IC50 < 0.05 microM). Oral PPP inhibited uveal melanoma growth in vivo in OCM-3 (P = 0.03) and OCM-8 (P = 0.01) xenografts and was well tolerated by the animals. PPP decreased VEGF expression in the OCM-1 (P = 0.006) and OCM-8 (P = 0.01) tumors. CONCLUSIONS: Oral PPP was well tolerated in vivo, caused total growth inhibition of uveal melanoma xenografts, and decreased VEGF levels in the tumors.

Abstract: The insulin-like growth factor receptor (IGF-IR) plays several pivotal roles in cancer. Although most studies on the function of the IGF-IR have been attributed to kinase-dependent signaling, recent findings by our group and others have implicated biological roles mediated by ubiquitination of the receptor. As previously reported, the E3 ligases Mdm2 and Nedd4 mediate IGF-IR ubiquitination. Here we show that c-Cbl is a novel E3 ligase for IGF-IR. On ligand stimulation, both Mdm2 and c-Cbl associate with IGF-IR and mediate receptor polyubiquitination. Whereas Mdm2 catalyzed lysine 63 (K63) chain ubiquitination, c-Cbl modified IGF-IR through K48 chains. Mdm2-mediated ubiquitination occurred when cells were stimulated with a low concentration (5 ng/mL) of IGF-I, whereas c-Cbl required high concentrations (50-100 ng/mL). Mdm2-ubiquitinated IGF-IR was internalized through the clathrin endocytic pathway whereas c-Cbl-ubiquitinated receptors were endocytosed via the caveolin route. Taken together, our results show that c-Cbl constitutes a new ligase responsible for the ubiquitination of IGF-IR and that it complements the action of Mdm2 on ubiquitin lysine residue specificity, responsiveness to IGF-I, and type of endocytic pathway used. The actions and interactions of Mdm2 and c-Cbl in the ubiquitination and endocytosis of IGF-IR may have implications in cancer. In addition, identification and functional characterization of new E3 ligases are important in itself because therapeutic targeting of substrate-specific E3 ligases is likely to represent a critical strategy in future cancer treatment.

Abstract: Introduction: Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration (AMD) and a leading cause of vision loss. Along with other angiogenic factors like vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1) and its receptor, IGF-1R, have been implicated in CNV. Purpose: We have previously shown that the cyclolignan picropodophyllin (PPP) efficiently blocks the insulin-like growth factor-1 receptor (IGF-1R) activity and causes cell death in uveal melanoma cell lines and in an in-vivo model. In this study we investigated the effect of PPP on VEGF expression both in vitro and in vivo and whether this effect has anti-angiogenic consequences in a murine CNV model. Materials and Methods: C57BL/6J mice with laser-induced CNVs were treated with PPP. Effects on CNV area were assayed by image analysis. VEGF levels in choroids and retinal pigment epithelial cells (APRE-19) were measured by Western blot or ELISA. Transcriptional activation of the VEGF promoter was determined by luciferase reporter gene assay. Results: Mice treated with PPP, administered intraperitoneally or orally, showed 22-32% (p = 0.002) decrease in CNV area. Furthermore, VEGF levels in the choroids were significantly reduced. In cultured APRE-19 cells, IGF-1 was shown to increase VEGF secretion. This increase was completely blocked by PPP. We could confirm that PPP reduced the level of transcriptional activity of VEGF promoter. Conclusions: PPP reduces IGF-1 dependent VEGF expression and CNV in vivo. Accordingly, IGF-1R inhibitors may be useful tools in the therapy of conditions associated with CNV including neovascular AMD.

Abstract: Purpose: The cyclolignan picropodophyllin (PPP) efficiently blocks the activity of insulin-like growth factor-1 receptor (IGF-1R) and inhibits growth of uveal melanoma cells in vitro and in vivo. In this study, we aimed to investigate the efficiency of orally administered PPP on growth of uveal melanoma xenografts. Further, we focused on the effect of PPP on vascular endothelial growth factor (VEGF) in vivo and evaluated its effects in combination with other established anti-tumor agents in vitro. Methods: Four different uveal melanoma cell lines (OCM-1, OCM-3, OCM-8, 92-1) were treated with PPP alone and in combination with imatinib mesylate, cisplatin, 5-FU and doxorubicin. Cell viability was determined by XTT assay. SCID mice xenografted with uveal melanoma cells were used to determine anti-tumor efficacy of oral PPP in vivo. Tumor samples obtained from the in vivo experiments were analyzed for VEGF and IGF-1R expression by western blotting. Results: PPP was found to be superior to the other anti-tumor agents in killing uveal melanoma cells. Oral PPP inhibited uveal melanoma growth in vivo and was well tolerated by the animals. PPP decreased VEGF expression in the tumors. Conclusions: Oral PPP is well tolerated in vivo and caused total growth inhibition of uveal melanoma xenografts as well as it decreased the levels of VEGF in the tumors.

Abstract: 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.

Abstract: 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. RESULT: We showed that PPP efficiently block growth and viability of uveal melanoma cells in cultures and causes tumor regression in xenografted mice. In addition, treatment with PPP inhibited several mechanism involved in metastasis, including tumor cells 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 layer. Furthermore, PPP significantly delayed established of uveal melanoma tumor and drastically reduced the incidence of liver metastasis in mice. CONCLUSIONS: Our data suggest that IGR-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.

Abstract: 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.

Abstract: The insulin-like growth factor (IGF-1) signalling is highly implicated in cancer. In this signalling the IGF-1 receptor (IGF-1R) is unquestionable, the predominating single factor. IGF-1R is crucial for tumour transformation and survival of malignant cell, but is only partially involved in normal cell growth. This is in part due to the interactions with oncogenes. Recent findings suggest a close interplay with the p53/MDM2 pathway. Disturbances in components in the p53/MDM2/IGF-1R network may cause IGF-1R upregulation and growth advantage for the cancer cell. Targeting of IGF-1R is more and more seen as a promising option for future cancer therapy. Single chain antibodies and small molecules with selective effects on IGF-1R dependent malignant growth are of particular interest. Forthcoming clinical trials are welcome and will indeed be the only way to evaluate the impact of IGF-1R targeting in human cancer.

Abstract: OBJECTIVE: Smooth muscle cell proliferation (SMC) is a pivotal factor in the development of intimal hyperplasia after vascular injury. A number of growth factors, including insulin-like growth factor-1 (IGF-1), have been shown to be involved in SMC proliferation. We evaluated the effect of picropodophyllin (PPP), a new IGF-1 receptor inhibitor, in the prevention of SMC proliferation and development of intimal hyperplasia after vascular injury. METHODS: The effects of systemic administration of PPP on intimal hyperplasia were studied in a balloon rat carotid injury model. Lesions were quantified by morphometry and SMC proliferation and apoptosis was studied by immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and activated caspase 3, respectively. The effect of PPP on rat aortic SMC proliferation and apoptosis was studied in vitro by using cell counting, 3[H]-thymidine incorporation, and a flow cytometry assay for annexin V. Phosphorylation of the IGF-1 receptor, protein kinase B (Akt), and extracellular signal-regulated kinase 1/2 (ERK1/2) in vitro and in vivo were analyzed by using Western blotting. RESULTS: PPP inhibited IGF-1-mediated SMC proliferation in vitro but no significant increase in apoptosis was detected. In rats treated with PPP, a more than a twofold reduction in carotid intima area was observed 2 weeks after balloon injury, a significant decrease in PCNA staining was demonstrated in early lesions, but activated caspase 3 was not detected. In addition, PPP attenuated phosphorylation of the IGF-1 receptor, Akt, and ERK1/2 in IGF-1-stimulated SMCs in vitro, and a reduced phosphorylation of the IGF-1 receptor and Akt was found in balloon-injured carotid arteries in rats treated with PPP. CONCLUSION: These results show that PPP potently blocks IGF-1-mediated phosphorylation of the IGF-1 receptor in SMCs, decreases downstream Akt and ERK1/2 activation, inhibits SMC replication, and subsequently attenuates intimal hyperplasia after balloon injury of rat carotid arteries.

Abstract: BACKGROUND: The insulin-like growth factor 1 receptor (IGF-1R) plays numerous crucial roles in cancer biology. The majority of knowledge on IGF-1R signaling is concerned with its role in the activation of the canonical phosphatidyl inositol-3 kinase (PI3K)/Akt and MAPK/ERK pathways. However, the role of IGF-1R ubiquitination in modulating IGF-1R function is an area of current research. In light of this we sought to determine the relationship between IGF-1R phosphorylation, ubiquitination, and modulation of growth signals. METHODOLOGY: Wild type and mutant constructs of IGF-1R were transfected into IGF-1R null fibroblasts. IGF-1R autophosphorylation and ubiquitination were determined by immunoprecipitation and western blotting. IGF-1R degradation and stability was determined by cyclohexamide-chase assay in combination with lysosome and proteasome inhibitors. PRINCIPAL FINDINGS: IGF-1R autophosphorylation was found to be an absolute requirement for receptor ubiquitination. Deletion of C-terminal domain had minimal effect on IGF-1 induced receptor autophosphorylation, however, ubiquitination and ERK activation were completely abolished. Cells expressing kinase impaired IGF-1R, exhibited both receptor ubiquitination and ERK phosphorylation, however failed to activate Akt. While IGF-1R mutants with impaired PI3K/Akt signaling were degraded mainly by the proteasomes, the C-terminal truncated one was exclusively degraded through the lysosomal pathway. CONCLUSIONS: Our data suggest important roles of ubiquitination in mediating IGF-1R signaling and degradation. Ubiquitination of IGF-1R requires receptor tyrosine kinase activity, but is not involved in Akt activation. In addition we show that the C-terminal domain of IGF-1R is a necessary requisite for ubiquitination and ERK phosphorylation as well as for proteasomal degradation of the receptor.

Abstract: 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.

Abstract: 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.

Abstract: The cyclolignan PPP was recently demonstrated to inhibit the activity of insulin-like growth factor-1 receptor (IGF-1R), without affecting the highly homologous insulin receptor. In addition, PPP caused complete regression of xenografts derived from various types of cancer. These data highlight the use of this compound in cancer treatment. However, a general concern with antitumor agents is development of resistance. In light of this problem, we aimed to investigate whether malignant cells may develop serious resistance to PPP. After trying to select 10 malignant cell lines, with documented IGF-1R expression and apoptotic responsiveness to PPP treatment (IC50s less than 0.1 microM), only two survived an 80-week selection but could only tolerate maximal PPP doses of 0.2 and 0.5 microM, respectively. Any further increase in the PPP dose resulted in massive cell death. These two cell lines were demonstrated not to acquire any essential alteration in responsiveness to PPP regarding IGF-1-induced IGF-1R phosphorylation. Neither did they exhibit any increase in expression of the multidrug resistance proteins MDR1 or MRP1. Consistently, they did not exhibit decreased sensitivity to conventional cytostatic drugs. Rather, the sensitivity was increased. During the first half of the selection period, both cell lines responded with a temporary and moderate increase in IGF-1R expression, which appeared to be because of an increased transcription of the IGF-1R gene. This increase in IGF-1R might be necessary to make cells competent for further selection but only up to a PPP concentration of 0.2 and 0.5 microM. In conclusion, malignant cells develop no or remarkably weak resistance to the IGF-1R inhibitor PPP.

Abstract: 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 beta-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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: The insulin-like growth factor (IGF-1) signalling is highly implicated in cancer. In this signalling the IGF-1 receptor (IGF-1R) is unquestionable, the predominating single factor. IGF-1R is crucial for tumour transformation and survival of malignant cell, but is only partially involved in normal cell growth. This is in part due to the interactions with oncogenes. Recent findings suggest a close interplay with the p53/MDM2 pathway. Disturbances in components in the p53/MDM2/IGF-1R network may cause IGF-1R upregulation and growth advantage for the cancer cell. Targeting of IGF-1R is more and more seen as a promising option for future cancer therapy. Single chain antibodies and small molecules with selective effects on IGF-1R dependent malignant growth are of particular interest. Forthcoming clinical trials are welcome and will indeed be the only way to evaluate the impact of IGF-1R targeting in human cancer.

Abstract: 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.

Abstract: 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 (LD(50) >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.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: Increasing evidence suggests that the SYT-SSX fusion gene plays an important role in synovial sarcoma development and progression. However, very little is known about the downstream targets of SYT-SSX. In this study, we used antisense oligonucleotides to block the expression of the SYT-SSX fusion gene in synovial sarcoma cells. By comparing SYT-SSX inhibited cells with noninhibited cells, the gene expression profile was analysed using cDNA microarray and established by real-time PCR. Herewith, using a filter containing 1176 cancer-relevant genes, we found that the DNA repair gene XRCC4 and the DNA mismatch repair gene MSH2 were downregulated, whereas the gene encoding for the serine/threonine protein kinase PRK (also known as CNK), and the macrophage inhibitory cytokine MICI (also known as PLAB) were upregulated after the inhibition of SYT-SSX. In comparison, expression of the XRCC4 gene was undergoing the strongest alteration. Consistently, the protein expression of XRCC4 was found to be decreased after SYT-SSX inhibition, whereas there were no detectable changes for the other gene products. Our study provides some clues to elucidate the signaling pathways of the SYT-SSX fusion gene, as well as it demonstrates a valuable model system for search for other SYT-SSX targets.

Abstract: 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.

Abstract: PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Abstract: The insulin-like growth factor-1 receptor (IGF-1R) has been shown to be of critical importance for tumor development and tumor cell survival of various types of malignancies. We have previously demonstrated that an adequate N-linked glycosylation of IGF-1R is required for its translocation to the cell surface in melanoma cells. This raises the possibility of using glycosylation inhibitors as therapeutic agents against IGF-1R-dependent malignancies. In this study we show that inhibition of N-linked glycosylation using tunicamycin or the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin resulted in down-regulation of IGF-1R at the cell surface in Ewing's sarcoma cell lines (RD-ES and ES-1 cells). The down-regulation of plasma membrane-bound IGF-1R was correlated with a drastic decrease in IGF-1R autophosphorylation, suggesting biochemical inactivation of the receptor. Whereas RD-ES and ES-1 cells responded differently with regard to DNA synthesis, the decrease in IGF-1R expression was accompanied by a rapid and substantial decrease in survival of both cell lines. Our data suggest that relatively untoxic HMG-CoA reductase inhibitors (e.g. lovastatin) could have therapeutic significance in IGF-1R-dependent neoplasms like Ewing's sarcoma.

Abstract: 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.

Abstract: 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.

Abstract: 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

Abstract: We investigated the functional impact of p53 on insulin-like growth factor I receptor (IGF-IR) expression in malignant cells. Using the BL-41tsp53-2 cell line, a transfectant carrying temperature-sensitive (ts) p53 and endogenous mutant p53 (codon 248), we demonstrated a drastic down-regulation of plasma membrane-bound IGF-IRs on induction of wild-type p53. However, a similar response was obtained by treatment of BL-41tsp53-2 cells expressing mutant ts p53 with a p53 antisense oligonucleotide. Thus, even if the negative effect of wild-type p53 predominates under a competitive condition, these data indicate that mutant p53 may be important for up-regulation of IGF-IR. To further elucidate this issue, three melanoma cell lines (BE, SK-MEL-5, and SK-MEL-28) that overexpressed p53 were investigated. The BE cell line has a "hot spot" mutation (codon 248) and expresses only codon 248-mutant p53. SK-MEL-28 has a point mutation at codon 145. SK-MEL-5 cells did not exhibit any p53 mutations, but the absence of p21Waf1 expression suggested functionally aberrant p53. Our data suggest that interaction with Mdm-2 may underlie p53 inactivation in these cells. Using p53 antisense oligonucleotides, we demonstrated a substantial down-regulation of cell surface expression of IGF-IR proteins in all melanoma cell lines after 24 h. This was paralleled by decreased tyrosine phosphorylation of IGF-IR and growth arrest, and, subsequently, massive cell death was observed (this was also seen in BL-41tsp53-2 cells with mutant conformation of ts p53). Taken together, our results suggest that up-regulation of IGF-IR as a result of expression of aberrant p53 may be important for the growth and survival of malignant cells.

Abstract: We present a study made during 4 years (1992-1996), which pursued the underlining of the atypical intraductal hiperplasias (A.I.D.H.) lesions, met isolated or in the association with mamar carcinoma. Our study included a 188 number of the breast tumors, among: in the 23 cases we established the existence of the modification by the A.I.D.H, type at the fibrocystics disease associated or not with the other benign diseases of the breast (fibroadenosis, intraductal papiloma) and in the 63 cases there were the modification by the AIDH associated with in situ or invasiv carcinoma. Epithelial hyperplasia is frequently associated with the fibrocystic changes, being included in the category of fibrocystic or proliferating modifications. The synonymous terms used for the epithelial hyperplasia are the hiperplazia ductala, or the epitelioza, or the papilomatosis The last two are suggested by the proliferation possibility (papillary or linear) of the epithelial or the mio-epithelial cells. Regardless of the microscopic aspect of the lesion, that should be acknowledged and treated as it is, due to the increased risk of the development of a carcinoma later on, and also due to the ratio of association between the modification and the mammary carcinoma. The risk of occurrence of subsequent carcinoma is augmented in the presence of the epithelial atipii and also increases in the presence of a mammary carcinoma at the relatives of the first rank (1.3). In this context, the importance of the differential diagnosis between the simple intraductal hyperplasia and the atypical one, the difficulty of differentiation from intraductal carcinoma in some cases, and finally the association with an increased risk of subsequent occurrence of carcinoma, constitute into sufficient arguments to consider this topic separately.