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39 EXPRESSION ANALYSIS OF AURKA UNDER HYPOXIA IN BREAST CANCER CELL LINES
S106
Abstracts / Cancer Treatment Reviews 36S3 (2010) S95–S119
that hypoxia inhibits the DNA repair process and promotes genomic instability in human cancers. Very little is known regarding the functional consequences of hypoxia in the expression of proteins involved in DNA double-strand break repair in human breast cancer. Therefore the aim of our studies is to evaluate the effects of hypoxia on genomic stability in breast cancer cell lines to obtain new insights on role of the hypoxic tumor microenvironment on DNA repair and on genetic instability. Methods: A microarray analysis, using Affymetrix platform, was performed in MCF7, MDA-MB-231 and SKBr3 breast cancer cell lines, cultured under normoxia and hypoxia for 24 and 48 hours, to identify genes showing a differential gene expression profile in the examined conditions. Among all the genes, we selected those involved in DNA repair mechanisms to obtain new knowledge about the process that regulate genomic instability in response to hypoxia. Results: MCF-7, MDA-MB-231 and SKBr3 breast cancer cell lines have shown a downregulated expression of BRCA2 and other genes involved in DNA repair process. By focusing our attention on BRCA2, our results were confirmed evaluating the reduction of mRNA levels and the related protein by Real-Time PCR and Western Blotting. In the three breast cancer cell lines there was a reduction of the protein levels after 48 hours, but no particular difference after 24 hours. Conclusions: Our data suggest that the hypoxia, decreasing the DNA repair capacity by downregulated expression of BRCA2 and other genes involved in the same pathway, could be responsible for the continuous changes that affect the DNA during the process of tumorigenesis favoring the progression to stage more advanced of breast cancer. 38 ANTIANGIOGENIC PROPERTIES OF IMMUNOMODULATORY DRUG LENALIDOMIDE IN ENDOTHELIAL CELLS OF PATIENTS WITH ACTIVE MULTIPLE MYELOMA A. De Luisi1,4 , A. Ferrucci1 , G. Di Pietro1 , S. Berardi1 , A. Basile1 , R. Ria1 , D. Ribatti2 , A.M.L. Coluccia3 , M. Maffia3 , G. Ranieri4 , A. Paradiso5 , A. Guarini6 , A. Vacca1 . 1 Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, 2 Department of Human Anatomy, Histology and Embryology, University of Bari Medical School, Bari, 3 Hematology and Clinical Proteomics Research Unit, “Vito Fazzi” Hospital, University of Salento, Lecce, 4 Interventional Radiology Unit with Integrated Section of Medical Oncology, National Cancer Institute Giovanni Paolo II, Bari, 5 Clinical Experimental Oncology Lab, National Cancer Institute Giovanni Paolo II, Bari, 6 Hematology Unit, National Cancer Institute Giovanni Paolo II, Bari, Italy Background: The immunomodulatory drug lenalidomide (Revlimid® ) belongs to a novel class of small molecules, structurally related to thalidomide, with more potent and less toxic antiinflammatory and anti-tumor activities, successfully used for the treatment of hematological cancers. It has shown impressive response rates in patients with relapsed/refractory multiple myeloma (MM), resulting in improved disease-free survival and overall survival. Its anti-tumor activity in MM is due to a dual mechanism: i) direct cytotoxic effect on MM plasma cells, through inhibition of plasma cell growth and induction of apoptosis, ii) indirect effect on their survival, by interfering with several components of the bone marrow microenvironment. Lenalidomide, indeed, inhibits the support of bone marrow stromal cells to plasma cells, by impairing cell adhesion, as well as the expression and secretion of the pro-angiogenic factors (VEGF and bFGF), and of other growth signals (TNF-a and IL-6) that promote bone marrow angiogenesis. It also stimulates T-cell and NK cell activities to plasma cells. However, its role in bone marrow endothelial cells of patients with MM (MMECs), remain still undefined. Here we investigated whether lenalidomide can directly inhibit angiogenesis of bone marrow ECs of patients with MM in active phase, and sought to elucidate the molecular mechanisms involved. Methods: We evaluated by in vivo experiment the angiogenic pathway through the chorioallantoic membrane (CAM) assay, in the interstitial fluid of patients daily treated with lenalidomide. The
evaluation of angiogenic pathway was performed also by in vitro experiments. Real-Time PCR was performed to evaluate the drug effect on the expression of key genes closely related to angiogenesis, and western blotting and comparative proteomic analysis were performed to confirm the obtained data. Results: We showed that 1.75 mM lenalidomide, i.e. the concentration reached in the interstitial fluid of patients daily treated with 25 mg, induces a significant inhibition of angiogenesis in vivo in the chorioallantoic membrane (CAM) assay. In vitro, lenalidomide inhibited angiogenesis and migration of MMECs, but not of ECs of patients with monoclonal gammopathies of undetermined significance (MGECs), while had no effect on MMECs proliferation, apoptosis and adhesion. Real-Time RT-PCR revealed that the drug strongly down-regulates the expression of key genes closely related to angiogenesis (VEGF, bFGF, CCL2, CXCL12, BNIP3, IER3, SEPW1). Finally, western blotting and comparative proteomic analysis showed that lenalidomide markedly affects VEGF/VEGFR2-mediated downstream signaling pathways involved in the motility process, such as mitogen activated protein kinase (MAPK) extracellular signal regulated kinase-1/2 (Erk-1/2), Src kinase, vascular endothelial (VE)-cadherin and NF-úB, and several other proteins controlling ECs invasiveness, cell-shape, cytoskeleton remodelling and energy metabolism as well. Conclusions: Overall data provide evidence that lenalidomide exerts an antiangiogenic activity in vivo and in vitro on MMECs, and earmark new avenues for enhancing therapeutic activity in MM patients. 39 EXPRESSION ANALYSIS OF AURKA UNDER HYPOXIA IN BREAST CANCER CELL LINES D. Fanale1 , L.R. Corsini1 , M. Terrasi1 , V. Amodeo1 , L. La Paglia1 , N. Margarese1 , L. Insalaco1 , L. Napoli1 , G.B. Damiani1 , M. Castiglia1 , F. Di Piazza1 , M.C. Miraglia1 , V. Bazan1 , A. Russo1 . 1 Department of Surgery and Oncology, University of Palermo, Italy Background: AURKA is an oncogenic serine/treonine kinase that is highly misregulated in several types of human tumors, including breast cancer. Its overexpression inducing aneuploidy and centrosome amplification has been correlated with chromosomal instability and clinically aggressive disease. Since hypoxia is a typical tumoral condition which influences the expression of various proteins involved in proliferation and cell cycle progression, aim of our study is to identify the mechanisms involved in AURKA expression, evaluating the possible HIF-1 role in its transcriptional control. Methods: A microarray analysis, using Affymetrix platform, was performed in MCF7, MDA-MB-231 and SKBr3 breast cancer cell lines cultured under normoxia and hypoxia in order to compare the differential gene expression profile in response to hypoxia. A set of genes involved in cell cycle progression, angiogenesis and tumor pathogenesis was selected. Results: We found a reduced expression of AURKA in all breast cancer cell lines analyzed and we confirmed this results showing a reduction of both mRNA levels and related protein, by RealTime PCR and Western Blotting. The involvement of HIF-1 in the transcriptional control of AURKA expression was demonstrated by ChIP assay. Conclusions: Our data suggest a new mechanism of AURKA regulation and, in discordance with previous reports, we hypothesize that this specific downregulation of AURKA might be able to suppress the proliferation and lead to the apoptosis of breast cancer cell lines. 40 ThinPrep® CYTOLOGICAL SPECIMENS ARE OFTEN MORE SUITABLE THAN HISTOLOGICAL SPECIMENS TO DETECT EGFR AND K-RAS MUTATIONS IN NSCLC AND COLORECTAL CARCINOMA D. Galetta1 , G. Simone1 , D. Petriella1 , V. Rubini1 , R. Pinto1 , R. Daprile1 , A. Paradiso1 , N. Silvestris1 , G. Colucci1 , S. Tommasi1 . 1 Cancer Institute “Giovanni Paolo II”, Bari, Italy Background: KRAS (exon 2) and EGFR (exons 19–21) mutations have to be investigated before setting a target therapy in colorectal and lung cancer, respectively (NCCN guidelines v2.0, 2010).
Abstracts / Cancer Treatment Reviews 36S3 (2010) S95–S119
that hypoxia inhibits the DNA repair process and promotes genomic instability in human cancers. Very little is known regarding the functional consequences of hypoxia in the expression of proteins involved in DNA double-strand break repair in human breast cancer. Therefore the aim of our studies is to evaluate the effects of hypoxia on genomic stability in breast cancer cell lines to obtain new insights on role of the hypoxic tumor microenvironment on DNA repair and on genetic instability. Methods: A microarray analysis, using Affymetrix platform, was performed in MCF7, MDA-MB-231 and SKBr3 breast cancer cell lines, cultured under normoxia and hypoxia for 24 and 48 hours, to identify genes showing a differential gene expression profile in the examined conditions. Among all the genes, we selected those involved in DNA repair mechanisms to obtain new knowledge about the process that regulate genomic instability in response to hypoxia. Results: MCF-7, MDA-MB-231 and SKBr3 breast cancer cell lines have shown a downregulated expression of BRCA2 and other genes involved in DNA repair process. By focusing our attention on BRCA2, our results were confirmed evaluating the reduction of mRNA levels and the related protein by Real-Time PCR and Western Blotting. In the three breast cancer cell lines there was a reduction of the protein levels after 48 hours, but no particular difference after 24 hours. Conclusions: Our data suggest that the hypoxia, decreasing the DNA repair capacity by downregulated expression of BRCA2 and other genes involved in the same pathway, could be responsible for the continuous changes that affect the DNA during the process of tumorigenesis favoring the progression to stage more advanced of breast cancer. 38 ANTIANGIOGENIC PROPERTIES OF IMMUNOMODULATORY DRUG LENALIDOMIDE IN ENDOTHELIAL CELLS OF PATIENTS WITH ACTIVE MULTIPLE MYELOMA A. De Luisi1,4 , A. Ferrucci1 , G. Di Pietro1 , S. Berardi1 , A. Basile1 , R. Ria1 , D. Ribatti2 , A.M.L. Coluccia3 , M. Maffia3 , G. Ranieri4 , A. Paradiso5 , A. Guarini6 , A. Vacca1 . 1 Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, 2 Department of Human Anatomy, Histology and Embryology, University of Bari Medical School, Bari, 3 Hematology and Clinical Proteomics Research Unit, “Vito Fazzi” Hospital, University of Salento, Lecce, 4 Interventional Radiology Unit with Integrated Section of Medical Oncology, National Cancer Institute Giovanni Paolo II, Bari, 5 Clinical Experimental Oncology Lab, National Cancer Institute Giovanni Paolo II, Bari, 6 Hematology Unit, National Cancer Institute Giovanni Paolo II, Bari, Italy Background: The immunomodulatory drug lenalidomide (Revlimid® ) belongs to a novel class of small molecules, structurally related to thalidomide, with more potent and less toxic antiinflammatory and anti-tumor activities, successfully used for the treatment of hematological cancers. It has shown impressive response rates in patients with relapsed/refractory multiple myeloma (MM), resulting in improved disease-free survival and overall survival. Its anti-tumor activity in MM is due to a dual mechanism: i) direct cytotoxic effect on MM plasma cells, through inhibition of plasma cell growth and induction of apoptosis, ii) indirect effect on their survival, by interfering with several components of the bone marrow microenvironment. Lenalidomide, indeed, inhibits the support of bone marrow stromal cells to plasma cells, by impairing cell adhesion, as well as the expression and secretion of the pro-angiogenic factors (VEGF and bFGF), and of other growth signals (TNF-a and IL-6) that promote bone marrow angiogenesis. It also stimulates T-cell and NK cell activities to plasma cells. However, its role in bone marrow endothelial cells of patients with MM (MMECs), remain still undefined. Here we investigated whether lenalidomide can directly inhibit angiogenesis of bone marrow ECs of patients with MM in active phase, and sought to elucidate the molecular mechanisms involved. Methods: We evaluated by in vivo experiment the angiogenic pathway through the chorioallantoic membrane (CAM) assay, in the interstitial fluid of patients daily treated with lenalidomide. The
evaluation of angiogenic pathway was performed also by in vitro experiments. Real-Time PCR was performed to evaluate the drug effect on the expression of key genes closely related to angiogenesis, and western blotting and comparative proteomic analysis were performed to confirm the obtained data. Results: We showed that 1.75 mM lenalidomide, i.e. the concentration reached in the interstitial fluid of patients daily treated with 25 mg, induces a significant inhibition of angiogenesis in vivo in the chorioallantoic membrane (CAM) assay. In vitro, lenalidomide inhibited angiogenesis and migration of MMECs, but not of ECs of patients with monoclonal gammopathies of undetermined significance (MGECs), while had no effect on MMECs proliferation, apoptosis and adhesion. Real-Time RT-PCR revealed that the drug strongly down-regulates the expression of key genes closely related to angiogenesis (VEGF, bFGF, CCL2, CXCL12, BNIP3, IER3, SEPW1). Finally, western blotting and comparative proteomic analysis showed that lenalidomide markedly affects VEGF/VEGFR2-mediated downstream signaling pathways involved in the motility process, such as mitogen activated protein kinase (MAPK) extracellular signal regulated kinase-1/2 (Erk-1/2), Src kinase, vascular endothelial (VE)-cadherin and NF-úB, and several other proteins controlling ECs invasiveness, cell-shape, cytoskeleton remodelling and energy metabolism as well. Conclusions: Overall data provide evidence that lenalidomide exerts an antiangiogenic activity in vivo and in vitro on MMECs, and earmark new avenues for enhancing therapeutic activity in MM patients. 39 EXPRESSION ANALYSIS OF AURKA UNDER HYPOXIA IN BREAST CANCER CELL LINES D. Fanale1 , L.R. Corsini1 , M. Terrasi1 , V. Amodeo1 , L. La Paglia1 , N. Margarese1 , L. Insalaco1 , L. Napoli1 , G.B. Damiani1 , M. Castiglia1 , F. Di Piazza1 , M.C. Miraglia1 , V. Bazan1 , A. Russo1 . 1 Department of Surgery and Oncology, University of Palermo, Italy Background: AURKA is an oncogenic serine/treonine kinase that is highly misregulated in several types of human tumors, including breast cancer. Its overexpression inducing aneuploidy and centrosome amplification has been correlated with chromosomal instability and clinically aggressive disease. Since hypoxia is a typical tumoral condition which influences the expression of various proteins involved in proliferation and cell cycle progression, aim of our study is to identify the mechanisms involved in AURKA expression, evaluating the possible HIF-1 role in its transcriptional control. Methods: A microarray analysis, using Affymetrix platform, was performed in MCF7, MDA-MB-231 and SKBr3 breast cancer cell lines cultured under normoxia and hypoxia in order to compare the differential gene expression profile in response to hypoxia. A set of genes involved in cell cycle progression, angiogenesis and tumor pathogenesis was selected. Results: We found a reduced expression of AURKA in all breast cancer cell lines analyzed and we confirmed this results showing a reduction of both mRNA levels and related protein, by RealTime PCR and Western Blotting. The involvement of HIF-1 in the transcriptional control of AURKA expression was demonstrated by ChIP assay. Conclusions: Our data suggest a new mechanism of AURKA regulation and, in discordance with previous reports, we hypothesize that this specific downregulation of AURKA might be able to suppress the proliferation and lead to the apoptosis of breast cancer cell lines. 40 ThinPrep® CYTOLOGICAL SPECIMENS ARE OFTEN MORE SUITABLE THAN HISTOLOGICAL SPECIMENS TO DETECT EGFR AND K-RAS MUTATIONS IN NSCLC AND COLORECTAL CARCINOMA D. Galetta1 , G. Simone1 , D. Petriella1 , V. Rubini1 , R. Pinto1 , R. Daprile1 , A. Paradiso1 , N. Silvestris1 , G. Colucci1 , S. Tommasi1 . 1 Cancer Institute “Giovanni Paolo II”, Bari, Italy Background: KRAS (exon 2) and EGFR (exons 19–21) mutations have to be investigated before setting a target therapy in colorectal and lung cancer, respectively (NCCN guidelines v2.0, 2010).