37 HYPOXIA INDUCES DECREASED EXPRESSION OF BRCA2 IN BREAST CANCER CELL LINES

Abstracts / Cancer Treatment Reviews 36S3 (2010) S95–S119 TSP-1 mRNA and cytosolic and secreted protein. Finally, we did not find any variation of TSP-1 level in cells transfected with let7i. Results were confirmed by transfection with anti-mir21, antimir182 and anti-let7i and, using the same method, we evaluated TSP-1 expression. Conclusions: Data suggest that mir-182 induces degradation of TSP-1 mRNA in HT29 cell line, whereas mir-21 affects probably by blockage of TSP-1 translation. Let-7i does not seem involved in regulation of TSP-1 expression in HT29 cells. Understanding the molecular mechanism by which miRNAs regulate TSP-1 expression could be used to restore TSP-1 expression to contrast angiogenic events in colon cancer. 34 AZD1152 PLUS GEMCITABINE FOR PANCREAS CANCER TREATMENT: IN VITRO AND IN VIVO STUDY A. Azzariti1 , G. Bocci2 , L. Porcelli1 , A.E. Quatrale1 , A. Fioravanti2 , M. Del Tacca2 , A. Paradiso1 . 1 Clinical Experimental Oncology Laboratory, National Cancer Institute, Bari, 2 Division of Pharmacology and Chemotherapy, Department of Internal Medicine, University of Pisa, Pisa, Italy Background: AZD1152 is a prodrug that, after activation in AZD1152-HQPA, impairs cytokinesis by inhibition of the activity of its specific target Aurora B kinase. Aurora B kinase is known to be involved to determining the correct chromosome alignment, kinetochore-microtubule biorientation, and activation of the spindle assembly checkpoint. In this report, we verify the possibility of combine this novel drug with gemcitabine widely used in chemotherapy for pancreas cancer patients. Methods: Pancreatic (MiaPaCa-2) cancer cells were used and the capability of the drug to enhance gemcitabine effectiveness has been evaluated as cell growth inhibition, apoptosis induction and cell cycle perturbation. Results: Our results showed that AZD1152-HQPA strongly modifies cell structure and activity, with an increase in cell size, in polyploidia and chromosome numbers. Its activity was through the inhibition of Histone 3 phosphorylation even if it also seemed to modulate other signal transduction pathways, such as survival one with the implication of p53. Kinetic experiments evidenced that AZD1152-HQPA was an enhancer of gemcitabine effectiveness in MiaPaCA-2 cells and the best schedule was that in which our aurora kinase B inhibitor was given before the chemotherapeutic drug, with a gain of about 20–30% of efficacy. Then, the promising in vitro combination of AZD1152 with gemcitabine has been tested in vivo with MiaPaCa-2 xenografts in CD nu/nu male mice. At the appearance of a measurable subcutaneous tumor (~100 mm3 ), mice were grouped randomly and treated as follows: i) control (vehicle alone), ii) AZD1152 alone (25 mg/kg daily for four days), iii) gemcitabine alone (120 mg/kg four times at 3-day intervals) and iv) the sequential combination of AZD1152 and gemcitabine. AZD1152 and gemcitabine alone significantly inhibit tumour growth in absence of toxicity. When mice were treated sequentially with the two compounds, the tumor growth was delayed and the inhibition of both tumor volumes and weights was markedly enhanced. Conclusions: In conclusion, our results suggest that AZD1152, a novel selective inhibitor of Aurora kinase B, could be a promising therapeutic approach in combination with gemcitabine in pancreas cancer treatment. AZD1152 and AZD1152-HQPA are trademarks of the AstraZeneca group of companies. S105 analyzed Mrna expression of 15 DSB related genes from 20 breast cancers in order to classify them into homogeneous clusters. For genes ATR, G22P1/ku70 and RAD51 was developed a mRNA relative quantification method that was used to analyze additional 55 cases. Methods: RAD51 protein expression was determined by immunohistochemestry on 58 tumours represented on a commercial available tissue microarray. Hierarchical clustering analysis of the DSB repair genes analyzed identified ATR, G22P1/ku70 and RAD51 as differentially expressed among the breast cancer cases. Results: The analysis of the additional 55 tumours for these three genes indicate an association between RAD51 increased mRNA levels and ER-positive/PR-negative breast cancers (P = 0.09). This result was confirmed at protein expression level when a tissue microarray including 58 breast cancers was analyzed by immunohistochemestry (P = 0.003). Conclusions: Our results indicate that the RAD51 gene is differentially expressed in breast cancer characterized by different steroid hormone receptor status and may represent a novel potential breast cancer biomarker. 36 DETECTION OF KRAS MUTATIONS IN COLORECTAL CARCINOMA PATIENTS WITH AN INTEGRATED PCR/SEQUENCING AND REAL TIME PCR APPROACH P. Carotenuto1 , C. Roma1 , A.M. Rachiglio1 , F. Tatangelo2 , C. Pinto3 , F. Ciardiello4 , G. Botti2 , N. Normanno5 . 1 Pharmacogenomic Laboratory, CROM – Centro Ricerche Oncologiche di Mercogliano, Avellino, 2 Surgical Pathology Unit, INT Fondazione “G.Pascale”, Naples, 3 Medical Oncology, S.Orsola-Malpighi Hospital, Bologna, 4 Medical Oncology, Dpt. Experimental and Clinical Medicine and Surgery F. Magrassi and A. Lanzara, Second University of Naples, Naples, 5 Cell Biology and Biotherapy Unit, INT Fondazione “ G.Pascale”, Naples, Italy Background: Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit of treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting for the choice of appropriate therapy. Methods: We developed a cost/effective approach for the determination of KRAS mutations in codons 12 and 13 in clinical practice based on a sensitive PCR/sequencing technique and the commercially available Real-Time PCR-based Therascreen kit (DxS). Results: The PCR/Sequencing test was able to detect 10% mutant DNA in a background of wild-type DNA. By using this assay, we determined the mutational status of KRAS in 527/540 (97.6%) formalin-fixed paraffin-embedded (FFPE) tissues from mCRC patients. PCR/sequencing was not conclusive in 13 cases in which low-intensity peaks suggestive of potential mutations were identified. DxS, which showed a sensitivity of 1%, identified mutations in 11/13 inconclusive cases. Interestingly, 5 of these 11 cases showed high levels of DNA fragmentation. No significant difference was found in the ability of PCR/sequencing and DxS to identify KRAS mutations within 160 cases with >30% tumor cells. However, in 24 samples with ≤30% tumor cells DxS showed an higher sensitivity. Conclusion In conclusion, our findings suggest that PCR/sequencing can be used for mutational analysis of the majority of tumor samples that have >30% tumor cell content, whereas more sensitive and expensive tests should be reserved for inconclusive cases and for samples with a low amount of tumor cells. 35 DNA DOUBLE STRANDS BREAK REPAIR GENES EXPRESSION ANALYSIS REVEAL RAD51 AS A NEW POTENTIAL BIOMARKER IN BREAST CANCER. R. Barbano1 , M. Copetti1 , G. Perrone2 , L.A. Muscarella1 , T. Balsamo1 , M.L. Poeta1 , V.M. Valori1 , T. Latiano1 , E. Maiello1 , M. Carella1 , F. Pellegrini1 , R. Murgo1 , A. Onetti Muda2 , V.M. Fazio1 , P. Parrella1 . 1 IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, FG, 2 University Campus BioMedico, Rome, Italy 37 HYPOXIA INDUCES DECREASED EXPRESSION OF BRCA2 IN BREAST CANCER CELL LINES L.R. Corsini1 , D. Fanale1 , M. Terrasi1 , L. La Paglia1 , N. Margarese1 , V. Amodeo1 , 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: We determined expression for genes that play key roles as sensors, modulators or effectors in this pathway. We Background: The hypoxic tumor microenvironment is a key factor that induces genetic instability. Several studies have demonstrated 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).