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P-12 IGF-1R as therapeutic target in melanoma
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Growth Hormone & IGF Research 18 (2008) Suppl S1
insulin receptor (IR). However, these regions appear to have an important role in determining ligand affinity towards the IGF-I receptor (IGF-IR), a transmembrane receptor with important roles in tumor biology. Our aim was to test whether long-acting insulin analogues Glargine (Lantus®, Sanofi Aventis) and Detemir (Levemir®, Novo Nordisk) exhibit IGF-I-like activities, including enhanced mitogenic and antiapoptotic effects, and to examine the mechanisms that mediate the actions of these analogues in comparison to regular human insulin (rhINS) and IGF-I in several colon cancer derived cell lines. Proliferation rates were measured with hemocytometer cell counting or with MTT assay. Potential antiapoptotic activities were evaluated with Annexin V/FITC kit and PARP immunoblotting. The ability of the analogues to activate the IGF-IR and/or IR was assessed by immunoprecipitation. Expression levels of the receptors and the ability of the analogues to activate specific signaling pathways were measured by western immunoblotting. Results obtained showed that both analogues but not rhINS stimulated cell proliferation in a similar fashion to IGF-I. Apoptosis measurements demonstrated that the analogues and IGF-I exhibited an antiapoptotic effect, while rhINS did not exhibit such effect. Immunoprecipitation assays showed that Glargine and Detemir can activate both IGF-IR and IR. Furthermore, the ability of the analogues to activate PI3K and MAPK signaling pathways, in terms of kinetics and intensity, are essentially different from those of insulin. In conclusion, Glargine and Detemir exhibit potent mitogenic and antiapoptotic activities, which resemble IGF-I actions. They also exhibit different binding characteristics to the IGF-IR, which may lead to differences in their activation of signaling pathways and in end-point biological activities. The clinical implications of these findings remain to be established. P-11 Activation of cAMP responsive element binding protein 1 (CREB1) by stimulation of the Src/ERK pathway is a common mechanism underlying IGF-I receptor upregulation by androgens and estrogens in prostate cancer cells M. Genua1 , G. Pandini1 ° , R. Vigneri1 , A. Belfiore2 . 1 University of Catania, Catania, Italy, 2 University of Catanzaro, Catanzaro, Italy Various lines of evidence suggest a role for IGF-IR overexpression in prostate cancer progression. We have previously shown that both androgens and estrogens markedly upregulate IGF-IR in prostate cancer cells by activating a nongenomic pathway after binding to the cognate receptors (AR and ER). We now show that stimulation of prostate cancer cells with either androgens (dihydrotestosterone and the synthetic androgen R1881), or estrogens (17beta-estradiol (E2) and the synthetic steroid Estren) upregulates IGF-IR by inducing CREB activation. Both R1881 and E2 were able to phosphorylate CREB at Ser133 in a dose-dependent manner in AR-positive LNCaP cells, whereas only E2 to phosphorylated CREB in AR-negative PC3 cells. This activation did not require PKA, PKC or CamKII but involved AR and ER binding to c-Src and subsequent activation of ERK1/2. CREB phosphorylation also occurred in cells transfected with an AR mutant that does not bind DNA, and by an ER mutant that does not localize to the nucleus, confirming that receptor binding to specific DNA response elements is not required. Inhibition of c-Src or ERK1/2 activity completely blocked IGF-IR upregulation and CREB phosphorylation, whereas inhibition of the PI3-K partially blocked CREB phosphorylation. Moreover, both CREB mRNA interference and inhibition by a dominant negative mutant completely blocked IGF-IR up-regulation by sex steroids. Finally, to further clarify the role of CREB we localized CREB binding sites in a specific region of the human IGF-IR promoter and demonstrated CREB binding to this promoter region by chromatin immuno-precipitation (ChIP). In conclusion, we have elucidated a novel mechanism by which androgens and estrogens induce IGF-IR upregulation and promoter activity by the nongenotropic activation of the Src-ERK-CREB pathway in
Abstracts, 4th Int. Congress of GRS & IGF Society prostate cancer cells. The identification of this mechanism suggests that inhibition of CREB and/or of the Src-ERK pathway may be useful in halting prostate cancer progression. P-12 IGF-1R as therapeutic target in melanoma A. Girnita ° , D. Vasilcanu, L. Girnita. Karolinska Institute, Stockholm, Sweden Melanoma is a malignant tumor of melanocytes which are found predominantly in skin but also in the bowel and the eye. Skin melanoma causes the majority of skin cancer related deaths. Despite many years of intensive laboratory and clinical research, the sole effective cure is surgical resection of the primary tumor before it achieves a thickness greater than 1 mm. When there is distant metastasis, the cancer is generally considered incurable. The five year survival rate is less than 10%. The insulin-like growth factor 1 receptor (IGF-1R) is crucial for growth and survival of melanoma cells. IGF-1R downregulation required expression of the MDM2 E3 ligase, which recently was found to ubiquitinate and cause degradation of the IGF-1R. Here we compare two different approaches of targeting IGF-1R in melanoma cells both, in cell cultures and animal models: inhibition of IGF-1R kinase activity versus IGF-1R dowregulation. We used dominant negative mutants to inhibit IGF-1R kinase activity and RNA interference strategies to downregulate IGF-1R. As alternative experimental model of downregulation we overexpressed MDM-2, the ubiquitine ligase responsabile for IGF-1R degradation. Our results suggest that IGF-1R degradation, although partial, is very important to the final outcome, while downregulation of IGF-1R is necessary for induction of apoptosis in melanoma cells. An inhibition of IGF-1R phosphorylation, without accompanying downregulation, leads only to decreased proliferation but not to apoptosis. Targeting IGF-1R 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 P-13 The type 1 insulin-like growth factor receptor (IGF1R) as a therapeutic target in renal cancer Y. Wang1 ° , J.S.P. Yuen1 , M. Sullivan2 , A. Protheroe2 , V. Macaulay1 . Weatherall Institute of Molecular Medicine, Oxford, UK, 2 Department of Urology, Churchill Hospital, Oxford, UK 1
The IGF1R mediates many key aspects of the malignant phenotype, including growth, transformation, motility and protection from apoptosis. We recently showed that IGF1R expression is inhibited at the level of Sp1-mediated transcription and mRNA stability by the protein product of the von Hippel Lindau (VHL) tumour suppressor. Furthermore the IGF1R is upregulated in sporadic clear cell renal cell cancers (CC-RCC), which frequently harbour inactivating VHL mutations. We hypothesise that IGF1R upregulation may be a significant driver for renal tumorigenesis, and also for resistance to cancer therapy. We explored this hypothesis by silencing the IGF1R gene, using siRNAs that are specific for the IGF1R, without influencing insulin receptor expression. Transfection with IGF1R siRNA induced profound IGF1R depletion, and also suppressed expression of the hypoxia-inducible factor HIF-1?, consistent with known post-transcriptional regulation of HIF-1? expression by IGF signaling. IGF1R depletion blocked survival of CC-RCC cells expressing either mutant (MT) or wildtype (WT) VHL, but induced sensitisation to 5-fluorouracil only in CC-RCC cells expressing MT but not WT VHL. These results support the hypothesis that the IGF1R upregulation that follows VHL inactivation contributes to chemoresistance. We also evaluated effects of IGF1R depletion on sensitivity to mTOR inhibition, since rapamycin analogues have proven clinical activity in metastatic RCC. Rapamycin treatment of CC-RCC cells suppressed activation of the
Growth Hormone & IGF Research 18 (2008) Suppl S1
insulin receptor (IR). However, these regions appear to have an important role in determining ligand affinity towards the IGF-I receptor (IGF-IR), a transmembrane receptor with important roles in tumor biology. Our aim was to test whether long-acting insulin analogues Glargine (Lantus®, Sanofi Aventis) and Detemir (Levemir®, Novo Nordisk) exhibit IGF-I-like activities, including enhanced mitogenic and antiapoptotic effects, and to examine the mechanisms that mediate the actions of these analogues in comparison to regular human insulin (rhINS) and IGF-I in several colon cancer derived cell lines. Proliferation rates were measured with hemocytometer cell counting or with MTT assay. Potential antiapoptotic activities were evaluated with Annexin V/FITC kit and PARP immunoblotting. The ability of the analogues to activate the IGF-IR and/or IR was assessed by immunoprecipitation. Expression levels of the receptors and the ability of the analogues to activate specific signaling pathways were measured by western immunoblotting. Results obtained showed that both analogues but not rhINS stimulated cell proliferation in a similar fashion to IGF-I. Apoptosis measurements demonstrated that the analogues and IGF-I exhibited an antiapoptotic effect, while rhINS did not exhibit such effect. Immunoprecipitation assays showed that Glargine and Detemir can activate both IGF-IR and IR. Furthermore, the ability of the analogues to activate PI3K and MAPK signaling pathways, in terms of kinetics and intensity, are essentially different from those of insulin. In conclusion, Glargine and Detemir exhibit potent mitogenic and antiapoptotic activities, which resemble IGF-I actions. They also exhibit different binding characteristics to the IGF-IR, which may lead to differences in their activation of signaling pathways and in end-point biological activities. The clinical implications of these findings remain to be established. P-11 Activation of cAMP responsive element binding protein 1 (CREB1) by stimulation of the Src/ERK pathway is a common mechanism underlying IGF-I receptor upregulation by androgens and estrogens in prostate cancer cells M. Genua1 , G. Pandini1 ° , R. Vigneri1 , A. Belfiore2 . 1 University of Catania, Catania, Italy, 2 University of Catanzaro, Catanzaro, Italy Various lines of evidence suggest a role for IGF-IR overexpression in prostate cancer progression. We have previously shown that both androgens and estrogens markedly upregulate IGF-IR in prostate cancer cells by activating a nongenomic pathway after binding to the cognate receptors (AR and ER). We now show that stimulation of prostate cancer cells with either androgens (dihydrotestosterone and the synthetic androgen R1881), or estrogens (17beta-estradiol (E2) and the synthetic steroid Estren) upregulates IGF-IR by inducing CREB activation. Both R1881 and E2 were able to phosphorylate CREB at Ser133 in a dose-dependent manner in AR-positive LNCaP cells, whereas only E2 to phosphorylated CREB in AR-negative PC3 cells. This activation did not require PKA, PKC or CamKII but involved AR and ER binding to c-Src and subsequent activation of ERK1/2. CREB phosphorylation also occurred in cells transfected with an AR mutant that does not bind DNA, and by an ER mutant that does not localize to the nucleus, confirming that receptor binding to specific DNA response elements is not required. Inhibition of c-Src or ERK1/2 activity completely blocked IGF-IR upregulation and CREB phosphorylation, whereas inhibition of the PI3-K partially blocked CREB phosphorylation. Moreover, both CREB mRNA interference and inhibition by a dominant negative mutant completely blocked IGF-IR up-regulation by sex steroids. Finally, to further clarify the role of CREB we localized CREB binding sites in a specific region of the human IGF-IR promoter and demonstrated CREB binding to this promoter region by chromatin immuno-precipitation (ChIP). In conclusion, we have elucidated a novel mechanism by which androgens and estrogens induce IGF-IR upregulation and promoter activity by the nongenotropic activation of the Src-ERK-CREB pathway in
Abstracts, 4th Int. Congress of GRS & IGF Society prostate cancer cells. The identification of this mechanism suggests that inhibition of CREB and/or of the Src-ERK pathway may be useful in halting prostate cancer progression. P-12 IGF-1R as therapeutic target in melanoma A. Girnita ° , D. Vasilcanu, L. Girnita. Karolinska Institute, Stockholm, Sweden Melanoma is a malignant tumor of melanocytes which are found predominantly in skin but also in the bowel and the eye. Skin melanoma causes the majority of skin cancer related deaths. Despite many years of intensive laboratory and clinical research, the sole effective cure is surgical resection of the primary tumor before it achieves a thickness greater than 1 mm. When there is distant metastasis, the cancer is generally considered incurable. The five year survival rate is less than 10%. The insulin-like growth factor 1 receptor (IGF-1R) is crucial for growth and survival of melanoma cells. IGF-1R downregulation required expression of the MDM2 E3 ligase, which recently was found to ubiquitinate and cause degradation of the IGF-1R. Here we compare two different approaches of targeting IGF-1R in melanoma cells both, in cell cultures and animal models: inhibition of IGF-1R kinase activity versus IGF-1R dowregulation. We used dominant negative mutants to inhibit IGF-1R kinase activity and RNA interference strategies to downregulate IGF-1R. As alternative experimental model of downregulation we overexpressed MDM-2, the ubiquitine ligase responsabile for IGF-1R degradation. Our results suggest that IGF-1R degradation, although partial, is very important to the final outcome, while downregulation of IGF-1R is necessary for induction of apoptosis in melanoma cells. An inhibition of IGF-1R phosphorylation, without accompanying downregulation, leads only to decreased proliferation but not to apoptosis. Targeting IGF-1R 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 P-13 The type 1 insulin-like growth factor receptor (IGF1R) as a therapeutic target in renal cancer Y. Wang1 ° , J.S.P. Yuen1 , M. Sullivan2 , A. Protheroe2 , V. Macaulay1 . Weatherall Institute of Molecular Medicine, Oxford, UK, 2 Department of Urology, Churchill Hospital, Oxford, UK 1
The IGF1R mediates many key aspects of the malignant phenotype, including growth, transformation, motility and protection from apoptosis. We recently showed that IGF1R expression is inhibited at the level of Sp1-mediated transcription and mRNA stability by the protein product of the von Hippel Lindau (VHL) tumour suppressor. Furthermore the IGF1R is upregulated in sporadic clear cell renal cell cancers (CC-RCC), which frequently harbour inactivating VHL mutations. We hypothesise that IGF1R upregulation may be a significant driver for renal tumorigenesis, and also for resistance to cancer therapy. We explored this hypothesis by silencing the IGF1R gene, using siRNAs that are specific for the IGF1R, without influencing insulin receptor expression. Transfection with IGF1R siRNA induced profound IGF1R depletion, and also suppressed expression of the hypoxia-inducible factor HIF-1?, consistent with known post-transcriptional regulation of HIF-1? expression by IGF signaling. IGF1R depletion blocked survival of CC-RCC cells expressing either mutant (MT) or wildtype (WT) VHL, but induced sensitisation to 5-fluorouracil only in CC-RCC cells expressing MT but not WT VHL. These results support the hypothesis that the IGF1R upregulation that follows VHL inactivation contributes to chemoresistance. We also evaluated effects of IGF1R depletion on sensitivity to mTOR inhibition, since rapamycin analogues have proven clinical activity in metastatic RCC. Rapamycin treatment of CC-RCC cells suppressed activation of the