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Melanoma is one of the deadliest human cancers with limited therapeutic options. MicroRNAs are a class of short noncoding RNAs regulating gene expression at the post-transcriptional level. To identify important miRNAs in melanoma, we compared the miRNome of primary and metastatic melanomas in The Cancer Genome Atlas dataset and found lower miR-203 abundance in metastatic melanoma. Lower level of miR-203 was associated with poor overall survival in metastatic disease. We found that the methylation levels of several CpGs in the MIR203 promoter negatively correlated with miR-203 expression and that treatment with the demethylating agent 5-aza-2-deoxycytidine induced miR-203 expression, which was associated with demethylation of the promoter CpGs, in melanoma cell lines. In vitro, there was a decreased expression of miR-203 in melanoma cell lines in comparison with primary melanocytes. Ectopic overexpression of miR-203 suppressed cell motility, colony formation, and sphere formation as well as the angiogenesis-inducing capacity of melanoma cells. In vivo, miR-203 inhibited xenograft tumor growth and reduced lymph node and lung metastasis. SLUG was shown as a target of miR-203, and knockdown of SLUG recapitulated the effects of miR-203, whereas its restoration was able to reverse the miR-203-mediated suppression of cell motility. These results establish a role for miR-203 as a tumor suppressor in melanoma which suppresses both early and late steps of metastasis. Hence, restoration of miR-203 has therapeutic potential in melanoma.

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 tow 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. © 2005 American Association for Cancer Research.

Estrogen receptor alpha (ER alpha) is a key regulator of breast growth and breast cancer development. Here, we report how ER alpha impacts these processes by reprogramming metabolism in malignant breast cells. We employed an integrated approach, combining genome-wide mapping of chromatin-bound ER alpha with estrogeninduced transcript and metabolic profiling, to demonstrate that ER alpha reprograms metabolism upon estrogen stimulation, including changes in aerobic glycolysis, nucleotide and amino acid synthesis, and choline (Cho) metabolism. Cho phosphotransfse CHPT1, identified as a direct era-regulated gene, was required for estrogen- induced effects on Cho metabolism, including increased phosphatidylcholine synthesis. CHPT1 silencing inhibited anchorage- independent growth and cell proliferation, also suppressing early-stage metastasis of tamoxifen-resistant breast cancer cells in a zebrafish xenograft model. Our results showed that era promotes metabolic alterations in breast cancer cells mediated by its target CHPT1, which this study implicates as a candidate therapeutic target. (C) 2016 AACR.

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 over expressed 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 p21(Waf1) 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., QC 20100525

Background: Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment. Methodology/Principal Findings: Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines. Conclusions: Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions.

BACKGROUND. Antibody preconditioning with tacrolimus monotherapy has allowed many renal allograft recipients to be maintained on spaced weaning. METHODS. Of 279 renal allograft recipients transplanted between March 2003 and December 2004, 222 (80%) had spaced weaning (i.e., reduction of tacrolimus monotherapy dosing to every other day, three times a week, twice a week, or once a week) attempted. Routine monitoring for donor-specific antibody (DSA) was begun in September 2004. Mean follow-up is 34±6.5 months after transplantation and 26±8.1 months after the initiation of spaced weaning. RESULTS. One hundred and twenty-two (44%) patients remained on spaced weaning. One- and 2-year actual patient/graft survival was 99%/99%, and 97%/96%. Fifty-six (20%) patients experienced acute rejection after initiation of spaced weaning. One- and 2-year actual patient/graft survival was 100%/98%, and 94%/78%. Forty-two (15%) patients with stable renal function had spaced weaning stopped because of the development of DSA, which disappeared in 17 (40%). One- and 2-year actual patient and graft survival was 100% and 100%. CONCLUSION. Adult renal transplant recipients who are able to be maintained on spaced weaning have excellent outcomes. Patients with stable renal function who have reversal of weaning because of the development of DSA also have excellent outcomes. Routine monitoring for DSA may allow patients to avoid late rejection after spaced weaning. © 2008 Lippincott Williams & Wilkins, Inc.

BACKGROUND. Antibody preconditioning with tacrolimus monotherapy has allowed many renal allograft recipients to be maintained on spaced weaning. METHODS. Of 279 renal allograft recipients transplanted between March 2003 and December 2004, 222 (80%) had spaced weaning (i.e., reduction of tacrolimus monotherapy dosing to every other day, three times a week, twice a week, or once a week) attempted. Routine monitoring for donor-specific antibody (DSA) was begun in September 2004. Mean follow-up is 34±6.5 months after transplantation and 26±8.1 months after the initiation of spaced weaning. RESULTS. One hundred and twenty-two (44%) patients remained on spaced weaning. One- and 2-year actual patient/graft survival was 99%/99%, and 97%/96%. Fifty-six (20%) patients experienced acute rejection after initiation of spaced weaning. One- and 2-year actual patient/graft survival was 100%/98%, and 94%/78%. Forty-two (15%) patients with stable renal function had spaced weaning stopped because of the development of DSA, which disappeared in 17 (40%). One- and 2-year actual patient and graft survival was 100% and 100%. CONCLUSION. Adult renal transplant recipients who are able to be maintained on spaced weaning have excellent outcomes. Patients with stable renal function who have reversal of weaning because of the development of DSA also have excellent outcomes. Routine monitoring for DSA may allow patients to avoid late rejection after spaced weaning. © 2008 Lippincott Williams & Wilkins, Inc.

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.

BACKGROUND: Multiple drug immunosuppression has allowed the near elimination of rejection, but without commensurate improvements in longterm graft survival and at the cost of quality of life. We have suggested that transplantation outcomes can be improved by modifying the timing and dosage of immunosuppression to facilitate natural mechanisms of alloengraftment and acquired tolerance. STUDY DESIGN: Two therapeutic principles were applied for kidney transplantation: pretransplant recipient conditioning with antilymphoid antibody preparations (Thymoglobulin [Sangstat] or Campath [ILEX Pharmaceuticals]), and minimal posttransplant immunosuppression with tacrolimus monotherapy including "spaced weaning" of maintenance doses when possible. The results in Thymoglobulin- (n = 101) and Campath-pretreated renal transplantation recipients (n = 90) were compared with those in 152 conventionally immunosuppressed recipients in the immediately preceding era. RESULTS: Spaced weaning was attempted in more than 90% of the kidney transplant recipients after pretreatment with both lymphoid-depleting agents, and is currently in effect in two-thirds of the survivors. Although there was a much higher rate of acute rejection in the Thymoglobulin-pretreated recipients than in either the Campath-pretreated or historic control recipients, patient and graft survival in both lymphoid depletion groups is at least equivalent to that of historic control patients. In the Thymoglobulin-conditioned patients for whom followups are now 24 to 40 months, chronic allograft nephropathy (CAN) progressed at the same rate as in historic control patients. Selected patients on weaning developed donor-specific nonreactivity. CONCLUSIONS: After lymphoid depletion, kidney transplantation can be readily accomplished under minimal immunosuppression with less dependence on late maintenance immunosuppression and a better quality of life. Campath was the more effective agent for pretreatment. Guidelines for spaced wea

Objectives: Acute and chronic rejection remain unresolved problems after lung transplantation, despite heavy multidrug immunosuppression. In turn, the strong immunosuppression has been responsible for mortality and pervasive morbidity. It also has been postulated to interdict potential mechanisms of alloengraftment. Methods: In 48 lung recipients we applied 2 therapeutic principles: (1) recipient pretreatment with antilymphoid antibody preparations (Thymoglobulin [SangStat, Fremont, Calif] or Campath [alemtuzumab; manufactured by ILEX Pharmaceuticals, LP, San Antonio, Tex; distributed by Berlex Laboratories, Richmond, Calif]) and (2) minimal posttransplant immunosuppression with tacrolimus monotherapy or near-monotherapy. Our principal analysis was of the events during the critical first 6 posttransplant months of highest immunologic and infectious disease risk. Results were compared with those of 28 historical lung recipients treated with daclizumab induction and triple immunosuppression (tacrolimus-prednisone-azathioprine). Results: Recipient pretreatment with both antilymphoid preparations allowed the use of postoperative tacrolimus monotherapy with prevention or control of acute rejection. Freedom from rejection was significantly greater with Campath than with Thymoglobulin (P = .03) or daclizumab (P = .05). After lymphoid depletion with Thymoglobulin or Campath, patient and graft survival at 6 months was 90% or greater. Patient and graft survival after 9 to 24 months is 84.2% in the Thymoglobulin cohort, and after 10 to 12 months, it is 90% in the Campath cohort. There has been a subjective improvement in quality of life relative to our historical experience. Conclusion: Our results suggest that improvements in lung transplantation can be accomplished by altering the timing, dosage, and approach to immunosuppression in ways that might allow natural mechanisms of alloengraftment and diminish the magnitude of required maintenance immunosuppression. Copyright © 20

Objectives: Acute and chronic rejection remain unresolved problems after lung transplantation, despite heavy multidrug immunosuppression. In turn, the strong immunosuppression has been responsible for mortality and pervasive morbidity. It also has been postulated to interdict potential mechanisms of alloengraftment. Methods: In 48 lung recipients we applied 2 therapeutic principles: (1) recipient pretreatment with antilymphoid antibody preparations (Thymoglobulin [SangStat, Fremont, Calif] or Campath [alemtuzumab; manufactured by ILEX Pharmaceuticals, LP, San Antonio, Tex; distributed by Berlex Laboratories, Richmond, Calif]) and (2) minimal posttransplant immunosuppression with tacrolimus monotherapy or near-monotherapy. Our principal analysis was of the events during the critical first 6 posttransplant months of highest immunologic and infectious disease risk. Results were compared with those of 28 historical lung recipients treated with daclizumab induction and triple immunosuppression (tacrolimus-prednisone-azathioprine). Results: Recipient pretreatment with both antilymphoid preparations allowed the use of postoperative tacrolimus monotherapy with prevention or control of acute rejection. Freedom from rejection was significantly greater with Campath than with Thymoglobulin (P = .03) or daclizumab (P = .05). After lymphoid depletion with Thymoglobulin or Campath, patient and graft survival at 6 months was 90% or greater. Patient and graft survival after 9 to 24 months is 84.2% in the Thymoglobulin cohort, and after 10 to 12 months, it is 90% in the Campath cohort. There has been a subjective improvement in quality of life relative to our historical experience. Conclusion: Our results suggest that improvements in lung transplantation can be accomplished by altering the timing, dosage, and approach to immunosuppression in ways that might allow natural mechanisms of alloengraftment and diminish the magnitude of required maintenance immunosuppression. Copyright © 20

BACKGROUND: Multiple drug immunosuppression has allowed the near elimination of rejection, but without commensurate improvements in longterm graft survival and at the cost of quality of life. We have suggested that transplantation outcomes can be improved by modifying the timing and dosage of immunosuppression to facilitate natural mechanisms of alloengraftment and acquired tolerance. STUDY DESIGN: Two therapeutic principles were applied for kidney transplantation: pretransplant recipient conditioning with antilymphoid antibody preparations (Thymoglobulin [Sangstat] or Campath [ILEX Pharmaceuticals]), and minimal posttransplant immunosuppression with tacrolimus monotherapy including "spaced weaning" of maintenance doses when possible. The results in Thymoglobulin- (n = 101) and Campath-pretreated renal transplantation recipients (n = 90) were compared with those in 152 conventionally immunosuppressed recipients in the immediately preceding era. RESULTS: Spaced weaning was attempted in more than 90% of the kidney transplant recipients after pretreatment with both lymphoid-depleting agents, and is currently in effect in two-thirds of the survivors. Although there was a much higher rate of acute rejection in the Thymoglobulin-pretreated recipients than in either the Campath-pretreated or historic control recipients, patient and graft survival in both lymphoid depletion groups is at least equivalent to that of historic control patients. In the Thymoglobulin-conditioned patients for whom followups are now 24 to 40 months, chronic allograft nephropathy (CAN) progressed at the same rate as in historic control patients. Selected patients on weaning developed donor-specific nonreactivity. CONCLUSIONS: After lymphoid depletion, kidney transplantation can be readily accomplished under minimal immunosuppression with less dependence on late maintenance immunosuppression and a better quality of life. Campath was the more effective agent for pretreatment. Guidelines for spaced wea

Cancer is a multi-step process where the accumulation of several genetic and epigenetic alterations drives the transformation of a normal cell to a malignant cell, skipping the normal control of defense, repair and apoptosis. During the selection process towards a malignant phenotype, the transforming cells make use of normal (physiological) extracellular signaling pathways to create growth advantage over normal cells. In this context, insulin-like growth factor 1 receptor (IGF-1R) emerges as an important factor responsible for the transformation and proliferation of malignant cells. IGF-1R confers protection against apoptosis, maintains the malignant phenotype, and defends against antitumor therapy. In contrast, the IGF-1R is not an absolute requirement for normal cell growth. The multiple functions of IGF-1R in cancer development coupled with its redundancy in maintaining cell growth make this receptor an attractive target for cancer treatment. Until recently there were multiple strategies to block the IGF-1R pathway in order to prevent growth and to increase apoptosis of malignant cells. However, none of them has so far presented the necessary requirements for clinical use. In this context, co-inhibition of the highly homologous insulin receptor cannot be accepted for a pharmaceutical agent. This study presents two different principles to specifically target the IGF-1R One is to modulate physiological mechanisms to degrade and therewith inactivate the receptor. This approach is still at a basic research level. The other principle is a small molecule strategy to reduce the catalytic activity of IGF-1R. Relating to the first principle, we recently showed that the oncoprotein MDM2 serves as a ligase (E3) in ubiquitination of IGF-1R. We demonstrated a physical association of IGF-1R to MDM2 and that a mixture of MDM2 and IGF-1R in an in vitro assay resulted in ubiquitination of IGF-1R. IGF-1R. In cultured cells, an increase in MDM2-mediated IGF-1R ubiquitination cause

Cancer is a multi-step process where the accumulation of several genetic and epigenetic alterations drives the transformation of a normal cell to a malignant cell, skipping the normal control of defense, repair and apoptosis. During the selection process towards a malignant phenotype, the transforming cells make use of normal (physiological) extracellular signaling pathways to create growth advantage over normal cells. In this context, insulin-like growth factor 1 receptor (IGF-1R) emerges as an important factor responsible for the transformation and proliferation of malignant cells. IGF-1R confers protection against apoptosis, maintains the malignant phenotype, and defends against antitumor therapy. In contrast, the IGF-1R is not an absolute requirement for normal cell growth. The multiple functions of IGF-1R in cancer development coupled with its redundancy in maintaining cell growth make this receptor an attractive target for cancer treatment. Until recently there were multiple strategies to block the IGF-1R pathway in order to prevent growth and to increase apoptosis of malignant cells. However, none of them has so far presented the necessary requirements for clinical use. In this context, co-inhibition of the highly homologous insulin receptor cannot be accepted for a pharmaceutical agent. This study presents two different principles to specifically target the IGF-1R One is to modulate physiological mechanisms to degrade and therewith inactivate the receptor. This approach is still at a basic research level. The other principle is a small molecule strategy to reduce the catalytic activity of IGF-1R. Relating to the first principle, we recently showed that the oncoprotein MDM2 serves as a ligase (E3) in ubiquitination of IGF-1R. We demonstrated a physical association of IGF-1R to MDM2 and that a mixture of MDM2 and IGF-1R in an in vitro assay resulted in ubiquitination of IGF-1R. IGF-1R. In cultured cells, an increase in MDM2-mediated IGF-1R ubiquitination cause

Cancer is a multi-step process where the accumulation of several genetic and epigenetic alterations drives the transformation of a normal cell to a malignant cell, skipping the normal control of defense, repair and apoptosis. During the selection process towards a malignant phenotype, the transforming cells make use of normal (physiological) extracellular signaling pathways to create growth advantage over normal cells. In this context, insulin-like growth factor 1 receptor (IGF-1R) emerges as an important factor responsible for the transformation and proliferation of malignant cells. IGF-1R confers protection against apoptosis, maintains the malignant phenotype, and defends against antitumor therapy. In contrast, the IGF-1R is not an absolute requirement for normal cell growth. The multiple functions of IGF-1R in cancer development coupled with its redundancy in maintaining cell growth make this receptor an attractive target for cancer treatment. Until recently there were multiple strategies to block the IGF-1R pathway in order to prevent growth and to increase apoptosis of malignant cells. However, none of them has so far presented the necessary requirements for clinical use. In this context, co-inhibition of the highly homologous insulin receptor cannot be accepted for a pharmaceutical agent. This study presents two different principles to specifically target the IGF-1R One is to modulate physiological mechanisms to degrade and therewith inactivate the receptor. This approach is still at a basic research level. The other principle is a small molecule strategy to reduce the catalytic activity of IGF-1R. Relating to the first principle, we recently showed that the oncoprotein MDM2 serves as a ligase (E3) in ubiquitination of IGF-1R. We demonstrated a physical association of IGF-1R to MDM2 and that a mixture of MDM2 and IGF-1R in an in vitro assay resulted in ubiquitination of IGF-1R. IGF-1R. In cultured cells, an increase in MDM2-mediated IGF-1R ubiquitination cause

Cancer is a multi-step process where the accumulation of several genetic and epigenetic alterations drives the transformation of a normal cell to a malignant cell, skipping the normal control of defense, repair and apoptosis. During the selection process towards a malignant phenotype, the transforming cells make use of normal (physiological) extracellular signaling pathways to create growth advantage over normal cells. In this context, insulin-like growth factor 1 receptor (IGF-1R) emerges as an important factor responsible for the transformation and proliferation of malignant cells. IGF-1R confers protection against apoptosis, maintains the malignant phenotype, and defends against antitumor therapy. In contrast, the IGF-1R is not an absolute requirement for normal cell growth. The multiple functions of IGF-1R in cancer development coupled with its redundancy in maintaining cell growth make this receptor an attractive target for cancer treatment. Until recently there were multiple strategies to block the IGF-1R pathway in order to prevent growth and to increase apoptosis of malignant cells. However, none of them has so far presented the necessary requirements for clinical use. In this context, co-inhibition of the highly homologous insulin receptor cannot be accepted for a pharmaceutical agent. This study presents two different principles to specifically target the IGF-1R One is to modulate physiological mechanisms to degrade and therewith inactivate the receptor. This approach is still at a basic research level. The other principle is a small molecule strategy to reduce the catalytic activity of IGF-1R. Relating to the first principle, we recently showed that the oncoprotein MDM2 serves as a ligase (E3) in ubiquitination of IGF-1R. We demonstrated a physical association of IGF-1R to MDM2 and that a mixture of MDM2 and IGF-1R in an in vitro assay resulted in ubiquitination of IGF-1R. IGF-1R. In cultured cells, an increase in MDM2-mediated IGF-1R ubiquitination cause

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.

Cancer is a multi-step process where the accumulation of several genetic and epigenetic alterations drives the transformation of a normal cell to a malignant cell, skipping the normal control of defense, repair and apoptosis. During the selection process towards a malignant phenotype, the transforming cells make use of normal (physiological) extracellular signaling pathways to create growth advantage over normal cells. In this context, insulin-like growth factor 1 receptor (IGF-1R) emerges as an important factor responsible for the transformation and proliferation of malignant cells. IGF-1R confers protection against apoptosis, maintains the malignant phenotype, and defends against antitumor therapy. In contrast, the IGF-1R is not an absolute requirement for normal cell growth. The multiple functions of IGF-1R in cancer development coupled with its redundancy in maintaining cell growth make this receptor an attractive target for cancer treatment. Until recently there were multiple strategies to block the IGF-1R pathway in order to prevent growth and to increase apoptosis of malignant cells. However, none of them has so far presented the necessary requirements for clinical use. In this context, co-inhibition of the highly homologous insulin receptor cannot be accepted for a pharmaceutical agent. This study presents two different principles to specifically target the IGF-1R One is to modulate physiological mechanisms to degrade and therewith inactivate the receptor. This approach is still at a basic research level. The other principle is a small molecule strategy to reduce the catalytic activity of IGF-1R. Relating to the first principle, we recently showed that the oncoprotein MDM2 serves as a ligase (E3) in ubiquitination of IGF-1R. We demonstrated a physical association of IGF-1R to MDM2 and that a mixture of MDM2 and IGF-1R in an in vitro assay resulted in ubiquitination of IGF-1R. IGF-1R. In cultured cells, an increase in MDM2-mediated IGF-1R ubiquitination cause

Cancer is a multi-step process where the accumulation of several genetic and epigenetic alterations drives the transformation of a normal cell to a malignant cell, skipping the normal control of defense, repair and apoptosis. During the selection process towards a malignant phenotype, the transforming cells make use of normal (physiological) extracellular signaling pathways to create growth advantage over normal cells. In this context, insulin-like growth factor 1 receptor (IGF-1R) emerges as an important factor responsible for the transformation and proliferation of malignant cells. IGF-1R confers protection against apoptosis, maintains the malignant phenotype, and defends against antitumor therapy. In contrast, the IGF-1R is not an absolute requirement for normal cell growth. The multiple functions of IGF-1R in cancer development coupled with its redundancy in maintaining cell growth make this receptor an attractive target for cancer treatment. Until recently there were multiple strategies to block the IGF-1R pathway in order to prevent growth and to increase apoptosis of malignant cells. However, none of them has so far presented the necessary requirements for clinical use. In this context, co-inhibition of the highly homologous insulin receptor cannot be accepted for a pharmaceutical agent. This study presents two different principles to specifically target the IGF-1R One is to modulate physiological mechanisms to degrade and therewith inactivate the receptor. This approach is still at a basic research level. The other principle is a small molecule strategy to reduce the catalytic activity of IGF-1R. Relating to the first principle, we recently showed that the oncoprotein MDM2 serves as a ligase (E3) in ubiquitination of IGF-1R. We demonstrated a physical association of IGF-1R to MDM2 and that a mixture of MDM2 and IGF-1R in an in vitro assay resulted in ubiquitination of IGF-1R. IGF-1R. In cultured cells, an increase in MDM2-mediated IGF-1R ubiquitination cause

Cancer is a multi-step process where the accumulation of several genetic and epigenetic alterations drives the transformation of a normal cell to a malignant cell, skipping the normal control of defense, repair and apoptosis. During the selection process towards a malignant phenotype, the transforming cells make use of normal (physiological) extracellular signaling pathways to create growth advantage over normal cells. In this context, insulin-like growth factor 1 receptor (IGF-1R) emerges as an important factor responsible for the transformation and proliferation of malignant cells. IGF-1R confers protection against apoptosis, maintains the malignant phenotype, and defends against antitumor therapy. In contrast, the IGF-1R is not an absolute requirement for normal cell growth. The multiple functions of IGF-1R in cancer development coupled with its redundancy in maintaining cell growth make this receptor an attractive target for cancer treatment. Until recently there were multiple strategies to block the IGF-1R pathway in order to prevent growth and to increase apoptosis of malignant cells. However, none of them has so far presented the necessary requirements for clinical use. In this context, co-inhibition of the highly homologous insulin receptor cannot be accepted for a pharmaceutical agent. This study presents two different principles to specifically target the IGF-1R One is to modulate physiological mechanisms to degrade and therewith inactivate the receptor. This approach is still at a basic research level. The other principle is a small molecule strategy to reduce the catalytic activity of IGF-1R. Relating to the first principle, we recently showed that the oncoprotein MDM2 serves as a ligase (E3) in ubiquitination of IGF-1R. We demonstrated a physical association of IGF-1R to MDM2 and that a mixture of MDM2 and IGF-1R in an in vitro assay resulted in ubiquitination of IGF-1R. IGF-1R. In cultured cells, an increase in MDM2-mediated IGF-1R ubiquitination cause

Cancer is a multi-step process where the accumulation of several genetic and epigenetic alterations drives the transformation of a normal cell to a malignant cell, skipping the normal control of defense, repair and apoptosis. During the selection process towards a malignant phenotype, the transforming cells make use of normal (physiological) extracellular signaling pathways to create growth advantage over normal cells. In this context, insulin-like growth factor 1 receptor (IGF-1R) emerges as an important factor responsible for the transformation and proliferation of malignant cells. IGF-1R confers protection against apoptosis, maintains the malignant phenotype, and defends against antitumor therapy. In contrast, the IGF-1R is not an absolute requirement for normal cell growth. The multiple functions of IGF-1R in cancer development coupled with its redundancy in maintaining cell growth make this receptor an attractive target for cancer treatment. Until recently there were multiple strategies to block the IGF-1R pathway in order to prevent growth and to increase apoptosis of malignant cells. However, none of them has so far presented the necessary requirements for clinical use. In this context, co-inhibition of the highly homologous insulin receptor cannot be accepted for a pharmaceutical agent. This study presents two different principles to specifically target the IGF-1R One is to modulate physiological mechanisms to degrade and therewith inactivate the receptor. This approach is still at a basic research level. The other principle is a small molecule strategy to reduce the catalytic activity of IGF-1R. Relating to the first principle, we recently showed that the oncoprotein MDM2 serves as a ligase (E3) in ubiquitination of IGF-1R. We demonstrated a physical association of IGF-1R to MDM2 and that a mixture of MDM2 and IGF-1R in an in vitro assay resulted in ubiquitination of IGF-1R. IGF-1R. In cultured cells, an increase in MDM2-mediated IGF-1R ubiquitination cause

The multi-step development of tumors involves numerous changes at genomic level such as oncogene activation, loss of function of tumor suppressor genes and translocations resulting in fusion genes that encodes for chimeric proteins with tumorigenic functions etc. However, in the selection leading to cancer in somatic tissues it is likely that the cancer cells make use of the normal extracellular signaling for proliferation and/or anti-apoptosis to create growth advantage over the normal cells. These signals are, in part, mediated by the growth factor receptors. This thesis aims to explore the mechanisms involved in expression and function of these receptors with special focus on insulin-like growth factor-1 receptor IGF-1R. The final goal is to identify some "Achilles' heel" in the growth factor pathways as a possible target in cancer therapy. N-linked glycosylation is crucial for expression of growth factor receptors at the cell surface. In Ewing's sarcoma cells, which carry the EWS-FLI-1 fusion gene, we found that inhibition of N-linked glycoproteins suppressed the EWS-FLI-1 protein leading to growth arrest. Since the fusion protein was demonstrated to not be a glycoprotein, we conclude that some other glycoproteins may be involved in regulation of EWS-FLI-1. Since growth factor receptors are N-linked glycoproteins and most N-linked glycoproteins are confined to the plasma membrane, the possibility of a link between cell surface expression of growth factor receptors and EWS-FLI-1 expression may be raised. We therefore tested different specific growth factor pathways regarding their potential influence on the EWS-FLI-1 protein. Our data indicate that the basic fibroblast growth factor (bFGF) pathway is important for up-regulation the EWS-FLI-1 protein. Other investigated growth factors pathways (e.g. IGF-1) seemed not to regulate the fusion protein. We investigated the functional impact of p53 for IGF-1R expression in malignant cells. Using three different system-(

The multi-step development of tumors involves numerous changes at genomic level such as oncogene activation, loss of function of tumor suppressor genes and translocations resulting in fusion genes that encodes for chimeric proteins with tumorigenic functions etc. However, in the selection leading to cancer in somatic tissues it is likely that the cancer cells make use of the normal extracellular signaling for proliferation and/or anti-apoptosis to create growth advantage over the normal cells. These signals are, in part, mediated by the growth factor receptors. This thesis aims to explore the mechanisms involved in expression and function of these receptors with special focus on insulin-like growth factor-1 receptor IGF-1R. The final goal is to identify some "Achilles' heel" in the growth factor pathways as a possible target in cancer therapy. N-linked glycosylation is crucial for expression of growth factor receptors at the cell surface. In Ewing's sarcoma cells, which carry the EWS-FLI-1 fusion gene, we found that inhibition of N-linked glycoproteins suppressed the EWS-FLI-1 protein leading to growth arrest. Since the fusion protein was demonstrated to not be a glycoprotein, we conclude that some other glycoproteins may be involved in regulation of EWS-FLI-1. Since growth factor receptors are N-linked glycoproteins and most N-linked glycoproteins are confined to the plasma membrane, the possibility of a link between cell surface expression of growth factor receptors and EWS-FLI-1 expression may be raised. We therefore tested different specific growth factor pathways regarding their potential influence on the EWS-FLI-1 protein. Our data indicate that the basic fibroblast growth factor (bFGF) pathway is important for up-regulation the EWS-FLI-1 protein. Other investigated growth factors pathways (e.g. IGF-1) seemed not to regulate the fusion protein. We investigated the functional impact of p53 for IGF-1R expression in malignant cells. Using three different system-(

The multi-step development of tumors involves numerous changes at genomic level such as oncogene activation, loss of function of tumor suppressor genes and translocations resulting in fusion genes that encodes for chimeric proteins with tumorigenic functions etc. However, in the selection leading to cancer in somatic tissues it is likely that the cancer cells make use of the normal extracellular signaling for proliferation and/or anti-apoptosis to create growth advantage over the normal cells. These signals are, in part, mediated by the growth factor receptors. This thesis aims to explore the mechanisms involved in expression and function of these receptors with special focus on insulin-like growth factor-1 receptor IGF-1R. The final goal is to identify some "Achilles' heel" in the growth factor pathways as a possible target in cancer therapy. N-linked glycosylation is crucial for expression of growth factor receptors at the cell surface. In Ewing's sarcoma cells, which carry the EWS-FLI-1 fusion gene, we found that inhibition of N-linked glycoproteins suppressed the EWS-FLI-1 protein leading to growth arrest. Since the fusion protein was demonstrated to not be a glycoprotein, we conclude that some other glycoproteins may be involved in regulation of EWS-FLI-1. Since growth factor receptors are N-linked glycoproteins and most N-linked glycoproteins are confined to the plasma membrane, the possibility of a link between cell surface expression of growth factor receptors and EWS-FLI-1 expression may be raised. We therefore tested different specific growth factor pathways regarding their potential influence on the EWS-FLI-1 protein. Our data indicate that the basic fibroblast growth factor (bFGF) pathway is important for up-regulation the EWS-FLI-1 protein. Other investigated growth factors pathways (e.g. IGF-1) seemed not to regulate the fusion protein. We investigated the functional impact of p53 for IGF-1R expression in malignant cells. Using three different system-(

The multi-step development of tumors involves numerous changes at genomic level such as oncogene activation, loss of function of tumor suppressor genes and translocations resulting in fusion genes that encodes for chimeric proteins with tumorigenic functions etc. However, in the selection leading to cancer in somatic tissues it is likely that the cancer cells make use of the normal extracellular signaling for proliferation and/or anti-apoptosis to create growth advantage over the normal cells. These signals are, in part, mediated by the growth factor receptors. This thesis aims to explore the mechanisms involved in expression and function of these receptors with special focus on insulin-like growth factor-1 receptor IGF-1R. The final goal is to identify some "Achilles' heel" in the growth factor pathways as a possible target in cancer therapy. N-linked glycosylation is crucial for expression of growth factor receptors at the cell surface. In Ewing's sarcoma cells, which carry the EWS-FLI-1 fusion gene, we found that inhibition of N-linked glycoproteins suppressed the EWS-FLI-1 protein leading to growth arrest. Since the fusion protein was demonstrated to not be a glycoprotein, we conclude that some other glycoproteins may be involved in regulation of EWS-FLI-1. Since growth factor receptors are N-linked glycoproteins and most N-linked glycoproteins are confined to the plasma membrane, the possibility of a link between cell surface expression of growth factor receptors and EWS-FLI-1 expression may be raised. We therefore tested different specific growth factor pathways regarding their potential influence on the EWS-FLI-1 protein. Our data indicate that the basic fibroblast growth factor (bFGF) pathway is important for up-regulation the EWS-FLI-1 protein. Other investigated growth factors pathways (e.g. IGF-1) seemed not to regulate the fusion protein. We investigated the functional impact of p53 for IGF-1R expression in malignant cells. Using three different system-(

The multi-step development of tumors involves numerous changes at genomic level such as oncogene activation, loss of function of tumor suppressor genes and translocations resulting in fusion genes that encodes for chimeric proteins with tumorigenic functions etc. However, in the selection leading to cancer in somatic tissues it is likely that the cancer cells make use of the normal extracellular signaling for proliferation and/or anti-apoptosis to create growth advantage over the normal cells. These signals are, in part, mediated by the growth factor receptors. This thesis aims to explore the mechanisms involved in expression and function of these receptors with special focus on insulin-like growth factor-1 receptor IGF-1R. The final goal is to identify some "Achilles' heel" in the growth factor pathways as a possible target in cancer therapy. N-linked glycosylation is crucial for expression of growth factor receptors at the cell surface. In Ewing's sarcoma cells, which carry the EWS-FLI-1 fusion gene, we found that inhibition of N-linked glycoproteins suppressed the EWS-FLI-1 protein leading to growth arrest. Since the fusion protein was demonstrated to not be a glycoprotein, we conclude that some other glycoproteins may be involved in regulation of EWS-FLI-1. Since growth factor receptors are N-linked glycoproteins and most N-linked glycoproteins are confined to the plasma membrane, the possibility of a link between cell surface expression of growth factor receptors and EWS-FLI-1 expression may be raised. We therefore tested different specific growth factor pathways regarding their potential influence on the EWS-FLI-1 protein. Our data indicate that the basic fibroblast growth factor (bFGF) pathway is important for up-regulation the EWS-FLI-1 protein. Other investigated growth factors pathways (e.g. IGF-1) seemed not to regulate the fusion protein. We investigated the functional impact of p53 for IGF-1R expression in malignant cells. Using three different system-(

The multi-step development of tumors involves numerous changes at genomic level such as oncogene activation, loss of function of tumor suppressor genes and translocations resulting in fusion genes that encodes for chimeric proteins with tumorigenic functions etc. However, in the selection leading to cancer in somatic tissues it is likely that the cancer cells make use of the normal extracellular signaling for proliferation and/or anti-apoptosis to create growth advantage over the normal cells. These signals are, in part, mediated by the growth factor receptors. This thesis aims to explore the mechanisms involved in expression and function of these receptors with special focus on insulin-like growth factor-1 receptor IGF-1R. The final goal is to identify some "Achilles' heel" in the growth factor pathways as a possible target in cancer therapy. N-linked glycosylation is crucial for expression of growth factor receptors at the cell surface. In Ewing's sarcoma cells, which carry the EWS-FLI-1 fusion gene, we found that inhibition of N-linked glycoproteins suppressed the EWS-FLI-1 protein leading to growth arrest. Since the fusion protein was demonstrated to not be a glycoprotein, we conclude that some other glycoproteins may be involved in regulation of EWS-FLI-1. Since growth factor receptors are N-linked glycoproteins and most N-linked glycoproteins are confined to the plasma membrane, the possibility of a link between cell surface expression of growth factor receptors and EWS-FLI-1 expression may be raised. We therefore tested different specific growth factor pathways regarding their potential influence on the EWS-FLI-1 protein. Our data indicate that the basic fibroblast growth factor (bFGF) pathway is important for up-regulation the EWS-FLI-1 protein. Other investigated growth factors pathways (e.g. IGF-1) seemed not to regulate the fusion protein. We investigated the functional impact of p53 for IGF-1R expression in malignant cells. Using three different system-(

The multi-step development of tumors involves numerous changes at genomic level such as oncogene activation, loss of function of tumor suppressor genes and translocations resulting in fusion genes that encodes for chimeric proteins with tumorigenic functions etc. However, in the selection leading to cancer in somatic tissues it is likely that the cancer cells make use of the normal extracellular signaling for proliferation and/or anti-apoptosis to create growth advantage over the normal cells. These signals are, in part, mediated by the growth factor receptors. This thesis aims to explore the mechanisms involved in expression and function of these receptors with special focus on insulin-like growth factor-1 receptor IGF-1R. The final goal is to identify some "Achilles' heel" in the growth factor pathways as a possible target in cancer therapy. N-linked glycosylation is crucial for expression of growth factor receptors at the cell surface. In Ewing's sarcoma cells, which carry the EWS-FLI-1 fusion gene, we found that inhibition of N-linked glycoproteins suppressed the EWS-FLI-1 protein leading to growth arrest. Since the fusion protein was demonstrated to not be a glycoprotein, we conclude that some other glycoproteins may be involved in regulation of EWS-FLI-1. Since growth factor receptors are N-linked glycoproteins and most N-linked glycoproteins are confined to the plasma membrane, the possibility of a link between cell surface expression of growth factor receptors and EWS-FLI-1 expression may be raised. We therefore tested different specific growth factor pathways regarding their potential influence on the EWS-FLI-1 protein. Our data indicate that the basic fibroblast growth factor (bFGF) pathway is important for up-regulation the EWS-FLI-1 protein. Other investigated growth factors pathways (e.g. IGF-1) seemed not to regulate the fusion protein. We investigated the functional impact of p53 for IGF-1R expression in malignant cells. Using three different system-(

The multi-step development of tumors involves numerous changes at genomic level such as oncogene activation, loss of function of tumor suppressor genes and translocations resulting in fusion genes that encodes for chimeric proteins with tumorigenic functions etc. However, in the selection leading to cancer in somatic tissues it is likely that the cancer cells make use of the normal extracellular signaling for proliferation and/or anti-apoptosis to create growth advantage over the normal cells. These signals are, in part, mediated by the growth factor receptors. This thesis aims to explore the mechanisms involved in expression and function of these receptors with special focus on insulin-like growth factor-1 receptor IGF-1R. The final goal is to identify some "Achilles' heel" in the growth factor pathways as a possible target in cancer therapy. N-linked glycosylation is crucial for expression of growth factor receptors at the cell surface. In Ewing's sarcoma cells, which carry the EWS-FLI-1 fusion gene, we found that inhibition of N-linked glycoproteins suppressed the EWS-FLI-1 protein leading to growth arrest. Since the fusion protein was demonstrated to not be a glycoprotein, we conclude that some other glycoproteins may be involved in regulation of EWS-FLI-1. Since growth factor receptors are N-linked glycoproteins and most N-linked glycoproteins are confined to the plasma membrane, the possibility of a link between cell surface expression of growth factor receptors and EWS-FLI-1 expression may be raised. We therefore tested different specific growth factor pathways regarding their potential influence on the EWS-FLI-1 protein. Our data indicate that the basic fibroblast growth factor (bFGF) pathway is important for up-regulation the EWS-FLI-1 protein. Other investigated growth factors pathways (e.g. IGF-1) seemed not to regulate the fusion protein. We investigated the functional impact of p53 for IGF-1R expression in malignant cells. Using three different system-(

QC 20190603