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636 Elevated Insulin Can Reduce Effectiveness of PI3K Inhibitors –Rationale for Co-targeting the Insulin Receptor Family and PI3K
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
636 Elevated Insulin Can Reduce Effectiveness of PI3K Inhibitors − Rationale for Co-targeting the Insulin Receptor Family and PI3K 1
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M.J. Blouin , Y. Zhao , E. Birman , M. Pollak . Jewish General Hospital, Lady Davis Institute for Medical Research, Montreal, Canada, 2 Jewish General Hospital McGill University, Lady Davis Institute for Medical Research, Montreal, Canada Background: The phosphatidylinositol 3-kinase (PI3K) signaling pathway is often disrupted in cancer, and there are major drug development programs for PI3K inhibitors. In vivo, as expected, effective PI3K blockade results in hyperinsulinemia and occasionally hyperglycemia, as PI3K signaling is an important aspect of insulin signaling in many tissues. The possibility that hyperinsulinemia may lead to resistance to PI3K targeting is plausible, as in some patients PI3K blockade leads to hyperinsulinemia with normoglycemia, suggesting that elevated insulin was sufficient to restore signaling downstream of the insulin receptor to an extent sufficient to normalize blood glucose. We carried out in vitro work as a first step to determine if high insulin attenuates the consequences of PI3K inhibition and if this could be restored by the use of a small molecule inhibitor of the insulin/IGF-1 receptor kinase. Methods: To determine the response to the drugs, cell lines were treated for 72 hrs with PI3K inhibitors GDC-0941 (a potent and selective inhibitor of class 1 PI3K) alone or in combination with an insulin/IGF-1 receptor inhibitor (BMS-754807) in presence or absence of insulin, with proliferation assessed by MTT as an end point. Western blot analysis was also performed to examine the relevant signaling pathways. Results: Among the cell lines tested with GDC-0941, MCF-7 cell line had the lowest IC50 (~300 nM). However, when cells were treated with GDC-0941 in presence of insulin, the effect of PI3K inhibition was attenuated, and cells were able to proliferate as well as the untreated control. To overcome this, we treated the cells with a combination of GDC-0941 and BMS-754807 in presence of insulin. We observed an additive effect of both drugs eliminating the stimulatory effect of insulin. The western analysis showed that phosphorylation of AKTSer473 was stimulated by insulin and inhibited by both GDC-0941 and BMS-754807. Importantly, in the presence of insulin, AKT activation was less when both inhibitors were used than with GDC-0941 alone. Conclusion: Combining a PI3K inhibitor with insulin/IGF-1 receptor kinase inhibitor may improve antiproliferative efficacy by minimizing the process by which PI3K-induced hyperinsulinemia attenuates the effect of PI3K blockade. Extensions to in vivo experiments and the impact of varying concentrations of insulin and the inhibitors will also be discussed. 637 Role of HEY1 in Osteosarcoma Metastasis T. Setoguchi1 , A. Tsuru1 , Y. Matsunoshita1 , M. Abematsu1 , S. Nagano1 , S. Komiya1 . 1 Graduate School of Medical and Dental Sciences Kagoshima University, Department of Orthopaedic Surgery, Kagoshima, Japan Background: The Notch pathway functions as an organizer in embryonic development. Recent studies have shown constitutive activation of the Notch pathway in various types of malignancies. We previously reported that inhibition of Notch pathway prevents osteosarcoma growth in vitro and in vivo (Tanaka M.et al. British Journal of Cancer 2010). In this report, we examined the function of Notch target gene, HEY1. Materials and Methods: Real-time PCR: To evaluate the expression of Notch pathway molecules, we performed real-time using human osteosarcoma cell lines. Growth inhibition: We evaluate the effect of HEY1 and HEY2 konckdown by MTT assay. Membrane assay: We evaluated osteosarcoma cell invasion by membrane assay following HEY1 and HEY2 knockdown. Xenograft model of osteosarcoma lung metastasis: 143B cells were transfected with GFP lentiviral particles. Stably-GFP-expressing 143B cells (1×106 ) were inoculated into the left knee joint of nude mice. Five weeks after inoculation, metastatic nodules in the lungs were evaluated by direct microscopic visualization. Results: Over-expression of Notch pathway molecules in human osteosarcoma cell lines: We have previously reported that Notch Pathway molecules are up-regulated in osteosarcoma biopsy specimens. Real-time PCR revealed that osteosarcoma cell lines increased the expression of Notch2, Jagged1, Dll1, MAML1, HES1, HES5, HEY1, and HEY2. Knockdown of HEY1 or HEY2 did not prevent osteosarcoma growth in vitro: We examined the function of HEY1 and HEY2 because the expression of HEY1 and HEY2 were up-regulated in the Notch target genes. MTT assay revealed that knockdown of HEY1 or HEY2 did not prevent osteosarcoma growth. Knockdown of HEY1 prevents osteosarcoma cell invasion in vitro: Membrane assay showed that knockdown of HEY1 prevents osteosarcoma invasion. Knockdown of HEY2 did not prevent osteosarcoma cell invasion. Knockdown of HEY1 prevents osteosarcoma metastasis in vivo: Seven of 7 control 143B cell inoculated mice showed lung metastasis. On the other hand, only 1 of 7 HEY1 shRMA transfected cell inoculated mice showed lung metastasis. Knockdown of HEY1 significantly decreased the lung metastasis in vivo.
S151
Knockdown of HEY1 decreased the expression of MMP9: RT-PCR showed that knockdown of HEY1 decreased the expression of MMP9. ELISA assay showed that knockdown of HEY1 decreased the expression of MMP9 protein. These findings suggest that knockdown of HEY1 prevented osteosarcoma cell invasion via down-regulation of MMP9 expression. Our new findings suggest that HEY1promoted osteosarcoma cell metastasis via MMP9 expression. Conclusions: Our finding suggests that inhibition of HEY1 may be regarded as a new effective treatment for patients with osteosarcoma. 638 LKB1 Gene Mutations and Rheb Gene Expression in Renal Cell Cancer N. Dalay1 , N. Buyru2 , Z. Yalniz1 , H. Tigli1 . 1 Oncology Institute, Basic Oncology, Istanbul, Turkey, 2 Cerrahpasa Medical Faculty, Medical Biology, Istanbul, Turkey Introduction: Renal cell carcinoma (RCC) accounts for approximately 3% of all adult malignancies and 90−95% of the kidney neoplasms. Little is known about the signalling pathways and genes associated with RCC. However, there are some evidence indicating the relevance of the PI3K/Akt/mTOR signalling pathway in RCC. Activation of the mTOR signalling pathway has been found to be associated with TSC1 and TSC2 mutations and RCC progression. LKB1 is a serine/threonine kinase which inhibits the mTOR pathway and has the ability to phosphorylate at least 14 downstream proteins. The main canonical target of LKB1 is the energy regulated AMP-activated protein kinase (AMPK). In concert with AMPK, LKB1 restricts cell growth by activation of the TSC1/TSC2 complex and subsequent blocking of Rheb and mTOR. The Rheb (Ras-homolog enriched in brain) protein is a component of PI3K pathway that positively regulates mTOR signaling. There is no report in the literature investigating the LKB1 gene as an upstream activator of the TSC1/TSC2 complex and Rheb in RCC. In this study we investigated LKB1 mutations and Rheb expression in association with tumor progression in RCC patients. Materials and Methods: 62 tumors and matched non-cancerous tissue samples were obtained from patients with renal cell carcinoma. The study was approved by the Istanbul Medical Faculty Ethics Committee. DNA and cellular total RNA were isolated from the matched tissue samples. LKB1 gene mutation analysis was performed on the Applied Biosystem Model 310 automated DNA sequencer. Rheb gene expression levels were analyzed by Quantitative Real Time PCR using the Light Cycler 480. The Glucose 6-Phosphate Dehydrogenase (G6PDH) gene was used as the reference gene to normalize the quantification of mRNA levels. Statistical analysis was performed using the SPSS PASW Statistics software package ver.18.0. Results and Discussion: All coding exons of the LKB1 gene were analyzed by direct sequencing. We detected ten different mutations (51.6%) in the coding sequence of the LKB1 gene in 32 patients. These comprised six missense, three silent and one nonsense mutations. 18 tumor samples harbored single mutations while multiple mutations were present in 14 samples. The most frequent mutations were a nonsense mutation at codon 19 (37.2%) and a single nucleotide substitution resulting in an amino acid change at codon 145 (17.7%). Both mutations were absent in the matched normal tissue samples. The exon 3 codon 145 and exon 1 codon 19 mutations were associated with metastasis and invasion. A significant difference was observed between high and low grade tumors with respect to a polymorphic site (+24 G→T) in exon 2. The median Rheb expression levels were significantly different between the tumors and thr adjacent non-cancerous tissue samples (p = 0.047). Rheb expression levels were significantly upregulated in tumor tissue samples carrying the intron 2 (+24T) variants compared to wild-type (p = 0.002). The level of Rheb expression was not associated with other LKB1 mutations or alterations and with any particular clinicopathological characteristics. Conclusion: Our results indicate that LKB1 mutations may play a major role in renal cell carcinoma by releasing the restrictions on Rheb and mTOR signaling pathway. 639 Mathematical Modeling of Synthetic Lethality With Applications to DNA Repair I. Kuperstein1 , A. Zinovyev1 , E. Barillot1 , W.D. Heyer2 . 1 Institut Curie, Bioinformatics Biostatistics Epidemiology and Computational Systems Biology of Cancer, Paris Cedex 05, France, 2 University of California, Departments of Microbiology and of Molecular and Cellular Biology, Davis CA, USA Background: A synthetic lethal interaction is usually stated when defects in two non-essential genes cause cell death. These two genes are generally assumed functioning in two parallel and mutually compensatory pathways (multi-pathway Synthetic Lethality). However, several examples of synthetic lethal relationships involving genes implicated in the homologous recombination DNA repair pathway extend this paradigm. In this situation defects in two genes which function in the same pathway lead to cell death (single-pathway Synthetic Lethality). Methods: We explored the inherent system properties of such a genetic relationship using mathematical modeling.
european journal of cancer 48, suppl. 5 (2012) S25–S288
636 Elevated Insulin Can Reduce Effectiveness of PI3K Inhibitors − Rationale for Co-targeting the Insulin Receptor Family and PI3K 1
1
1
2
1
M.J. Blouin , Y. Zhao , E. Birman , M. Pollak . Jewish General Hospital, Lady Davis Institute for Medical Research, Montreal, Canada, 2 Jewish General Hospital McGill University, Lady Davis Institute for Medical Research, Montreal, Canada Background: The phosphatidylinositol 3-kinase (PI3K) signaling pathway is often disrupted in cancer, and there are major drug development programs for PI3K inhibitors. In vivo, as expected, effective PI3K blockade results in hyperinsulinemia and occasionally hyperglycemia, as PI3K signaling is an important aspect of insulin signaling in many tissues. The possibility that hyperinsulinemia may lead to resistance to PI3K targeting is plausible, as in some patients PI3K blockade leads to hyperinsulinemia with normoglycemia, suggesting that elevated insulin was sufficient to restore signaling downstream of the insulin receptor to an extent sufficient to normalize blood glucose. We carried out in vitro work as a first step to determine if high insulin attenuates the consequences of PI3K inhibition and if this could be restored by the use of a small molecule inhibitor of the insulin/IGF-1 receptor kinase. Methods: To determine the response to the drugs, cell lines were treated for 72 hrs with PI3K inhibitors GDC-0941 (a potent and selective inhibitor of class 1 PI3K) alone or in combination with an insulin/IGF-1 receptor inhibitor (BMS-754807) in presence or absence of insulin, with proliferation assessed by MTT as an end point. Western blot analysis was also performed to examine the relevant signaling pathways. Results: Among the cell lines tested with GDC-0941, MCF-7 cell line had the lowest IC50 (~300 nM). However, when cells were treated with GDC-0941 in presence of insulin, the effect of PI3K inhibition was attenuated, and cells were able to proliferate as well as the untreated control. To overcome this, we treated the cells with a combination of GDC-0941 and BMS-754807 in presence of insulin. We observed an additive effect of both drugs eliminating the stimulatory effect of insulin. The western analysis showed that phosphorylation of AKTSer473 was stimulated by insulin and inhibited by both GDC-0941 and BMS-754807. Importantly, in the presence of insulin, AKT activation was less when both inhibitors were used than with GDC-0941 alone. Conclusion: Combining a PI3K inhibitor with insulin/IGF-1 receptor kinase inhibitor may improve antiproliferative efficacy by minimizing the process by which PI3K-induced hyperinsulinemia attenuates the effect of PI3K blockade. Extensions to in vivo experiments and the impact of varying concentrations of insulin and the inhibitors will also be discussed. 637 Role of HEY1 in Osteosarcoma Metastasis T. Setoguchi1 , A. Tsuru1 , Y. Matsunoshita1 , M. Abematsu1 , S. Nagano1 , S. Komiya1 . 1 Graduate School of Medical and Dental Sciences Kagoshima University, Department of Orthopaedic Surgery, Kagoshima, Japan Background: The Notch pathway functions as an organizer in embryonic development. Recent studies have shown constitutive activation of the Notch pathway in various types of malignancies. We previously reported that inhibition of Notch pathway prevents osteosarcoma growth in vitro and in vivo (Tanaka M.et al. British Journal of Cancer 2010). In this report, we examined the function of Notch target gene, HEY1. Materials and Methods: Real-time PCR: To evaluate the expression of Notch pathway molecules, we performed real-time using human osteosarcoma cell lines. Growth inhibition: We evaluate the effect of HEY1 and HEY2 konckdown by MTT assay. Membrane assay: We evaluated osteosarcoma cell invasion by membrane assay following HEY1 and HEY2 knockdown. Xenograft model of osteosarcoma lung metastasis: 143B cells were transfected with GFP lentiviral particles. Stably-GFP-expressing 143B cells (1×106 ) were inoculated into the left knee joint of nude mice. Five weeks after inoculation, metastatic nodules in the lungs were evaluated by direct microscopic visualization. Results: Over-expression of Notch pathway molecules in human osteosarcoma cell lines: We have previously reported that Notch Pathway molecules are up-regulated in osteosarcoma biopsy specimens. Real-time PCR revealed that osteosarcoma cell lines increased the expression of Notch2, Jagged1, Dll1, MAML1, HES1, HES5, HEY1, and HEY2. Knockdown of HEY1 or HEY2 did not prevent osteosarcoma growth in vitro: We examined the function of HEY1 and HEY2 because the expression of HEY1 and HEY2 were up-regulated in the Notch target genes. MTT assay revealed that knockdown of HEY1 or HEY2 did not prevent osteosarcoma growth. Knockdown of HEY1 prevents osteosarcoma cell invasion in vitro: Membrane assay showed that knockdown of HEY1 prevents osteosarcoma invasion. Knockdown of HEY2 did not prevent osteosarcoma cell invasion. Knockdown of HEY1 prevents osteosarcoma metastasis in vivo: Seven of 7 control 143B cell inoculated mice showed lung metastasis. On the other hand, only 1 of 7 HEY1 shRMA transfected cell inoculated mice showed lung metastasis. Knockdown of HEY1 significantly decreased the lung metastasis in vivo.
S151
Knockdown of HEY1 decreased the expression of MMP9: RT-PCR showed that knockdown of HEY1 decreased the expression of MMP9. ELISA assay showed that knockdown of HEY1 decreased the expression of MMP9 protein. These findings suggest that knockdown of HEY1 prevented osteosarcoma cell invasion via down-regulation of MMP9 expression. Our new findings suggest that HEY1promoted osteosarcoma cell metastasis via MMP9 expression. Conclusions: Our finding suggests that inhibition of HEY1 may be regarded as a new effective treatment for patients with osteosarcoma. 638 LKB1 Gene Mutations and Rheb Gene Expression in Renal Cell Cancer N. Dalay1 , N. Buyru2 , Z. Yalniz1 , H. Tigli1 . 1 Oncology Institute, Basic Oncology, Istanbul, Turkey, 2 Cerrahpasa Medical Faculty, Medical Biology, Istanbul, Turkey Introduction: Renal cell carcinoma (RCC) accounts for approximately 3% of all adult malignancies and 90−95% of the kidney neoplasms. Little is known about the signalling pathways and genes associated with RCC. However, there are some evidence indicating the relevance of the PI3K/Akt/mTOR signalling pathway in RCC. Activation of the mTOR signalling pathway has been found to be associated with TSC1 and TSC2 mutations and RCC progression. LKB1 is a serine/threonine kinase which inhibits the mTOR pathway and has the ability to phosphorylate at least 14 downstream proteins. The main canonical target of LKB1 is the energy regulated AMP-activated protein kinase (AMPK). In concert with AMPK, LKB1 restricts cell growth by activation of the TSC1/TSC2 complex and subsequent blocking of Rheb and mTOR. The Rheb (Ras-homolog enriched in brain) protein is a component of PI3K pathway that positively regulates mTOR signaling. There is no report in the literature investigating the LKB1 gene as an upstream activator of the TSC1/TSC2 complex and Rheb in RCC. In this study we investigated LKB1 mutations and Rheb expression in association with tumor progression in RCC patients. Materials and Methods: 62 tumors and matched non-cancerous tissue samples were obtained from patients with renal cell carcinoma. The study was approved by the Istanbul Medical Faculty Ethics Committee. DNA and cellular total RNA were isolated from the matched tissue samples. LKB1 gene mutation analysis was performed on the Applied Biosystem Model 310 automated DNA sequencer. Rheb gene expression levels were analyzed by Quantitative Real Time PCR using the Light Cycler 480. The Glucose 6-Phosphate Dehydrogenase (G6PDH) gene was used as the reference gene to normalize the quantification of mRNA levels. Statistical analysis was performed using the SPSS PASW Statistics software package ver.18.0. Results and Discussion: All coding exons of the LKB1 gene were analyzed by direct sequencing. We detected ten different mutations (51.6%) in the coding sequence of the LKB1 gene in 32 patients. These comprised six missense, three silent and one nonsense mutations. 18 tumor samples harbored single mutations while multiple mutations were present in 14 samples. The most frequent mutations were a nonsense mutation at codon 19 (37.2%) and a single nucleotide substitution resulting in an amino acid change at codon 145 (17.7%). Both mutations were absent in the matched normal tissue samples. The exon 3 codon 145 and exon 1 codon 19 mutations were associated with metastasis and invasion. A significant difference was observed between high and low grade tumors with respect to a polymorphic site (+24 G→T) in exon 2. The median Rheb expression levels were significantly different between the tumors and thr adjacent non-cancerous tissue samples (p = 0.047). Rheb expression levels were significantly upregulated in tumor tissue samples carrying the intron 2 (+24T) variants compared to wild-type (p = 0.002). The level of Rheb expression was not associated with other LKB1 mutations or alterations and with any particular clinicopathological characteristics. Conclusion: Our results indicate that LKB1 mutations may play a major role in renal cell carcinoma by releasing the restrictions on Rheb and mTOR signaling pathway. 639 Mathematical Modeling of Synthetic Lethality With Applications to DNA Repair I. Kuperstein1 , A. Zinovyev1 , E. Barillot1 , W.D. Heyer2 . 1 Institut Curie, Bioinformatics Biostatistics Epidemiology and Computational Systems Biology of Cancer, Paris Cedex 05, France, 2 University of California, Departments of Microbiology and of Molecular and Cellular Biology, Davis CA, USA Background: A synthetic lethal interaction is usually stated when defects in two non-essential genes cause cell death. These two genes are generally assumed functioning in two parallel and mutually compensatory pathways (multi-pathway Synthetic Lethality). However, several examples of synthetic lethal relationships involving genes implicated in the homologous recombination DNA repair pathway extend this paradigm. In this situation defects in two genes which function in the same pathway lead to cell death (single-pathway Synthetic Lethality). Methods: We explored the inherent system properties of such a genetic relationship using mathematical modeling.