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276 Vav1 – the Oncogenic Switch
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
Aim: We aimed to assess the expression of miR-7 and its targets Bcl-2 and EGFR in ovarian cancer and with different TP53 status. Materials and Methods: microRNA and gene expression was assessed by the qRT-PCR in normal human ovarian epithelium and in a panel of primary ovarian cancer samples with a different TP53 mutational status as well as in ovarian cancer cell lines that reflected the clinical samples with regard to their TP53 status. Results: miR-7 was down-regulated in most of the clinical samples without TP53 mutations and overexpressed in the samples with TP53 mutations. We found a significant difference in miR-7 expression with regard to TP53 status (p < 0.01). Bcl-2 was up-regulated, while EGFR was down-regulated in most of the clinical samples and did not relate to the TP53 mutational status. There was no correlation between the expression of miR-7 and of its target genes. EGFR expression was decreased in ovarian cell lines. In ovarian cancer cell lines with TP53 missense mutations there was a reduced Bcl-2 expression, while an enhanced Bcl-2 expression was found in those with the wild-type TP53 and with TP53 null mutation. Conclusions: 1. miR-7 seems to play an important role in ovarian cancer pathogenesis. 2. There seem to be a relationship between TP53 and miR-7. 3. In primary serous ovarian cancer the altered expression of miR-7 does not determine the levels of its targets genes, EGFR and Bcl-2. Supported by the grant of Polish Ministry of Science and Higher Education (NN 401 050 138). 274 Integrin a5b1 Plays a Critical Role in Resistance to Chemotherapy by Interfering With the P53 Pathway in High Grade Glioma H. Janouskova1 , A. Maglott1 , D. Guenot2 , S. Pinel3 , P. Chastagner3 , F. Plenat3 , N. Entz-Werle4 , J. Godet5 , S. Martin5 , M. Dontenwill5 . 1 UMR CNRS 7213, Faculte´ de Pharmacie, Illkirch, France, 2 EA4438, Physiopathologie et Recherche Translationnelle, Strasbourg, France, 3 ´ EA4421, Faculte´ de Medecine, Vandoeuvre Les Nancy, France, 4 Service ´ ˆ de Biochimie et Biologie Moleculaire, Hopital de Hautepierre, Strasbourg, France, 5 CNRS UMR 7213, Faculte´ de Pharmacie, Illkirch, France Introduction: Glioblastoma is the most aggressive and malignant form of brain tumour. Despite advances in standard therapy, including surgical resection followed by radiation and chemotherapy with temozolomide (TMZ), the prognosis for patients with glioblastoma remains unfavourable, with a median survival of 12−18 months. In the recent years, integrins have emerged as a potentially promising anti-cancer target. The aim of this study has been to investigate the role of a5b1 integrin in glioma resistance to TMZ. Material and Method: a5 subunit gene expression was examined by qPCR (95 human brain samples of different pathological grade compared to 20 non tumoural brain samples). To determine the patients’ survival, we analysed the clinical data of grade III and grade IV glioma patients. U87MG (p53wt) cells were modulated to over and downexpress a5 subunit, and were then treated with TMZ and nutlin-3a. p53 activation was determined by western blot analysis with a specific phospho-p53 (ser15) antibody and by qPCR of p53 target genes. The mice survival was studied in subcutaneous model of human brain tumour xenografts treated daily with TMZ (oral gavage, 50 mg/kg/day) for 5 days. Results and Discussion: In vivo study showed that a5b1 integrin was specifically associated with a more aggressive phenotype in brain tumours, and that a high level of a5 integrin gene expression was associated with a decreased patients’ survival with high-grade glioma. TMZ treatment of p53wt cells in vitro caused an increase of p53 activity, but with a lesser extent in a5overexpressing cells. Those cells were also more resistant to TMZ compared to a5-depleted cells. Integrin a5b1 negatively modulated the p53 pathway. Moreover, the analysis in vivo of mice survival showed that the high a5 expressing tumours were more resistant to chemotherapy. We suggest that the inhibition of p53 pathway by a5b1 integrin plays a critical role in the resistance of glioblastomas to chemotherapy. The increase of p53 activity by nutlin-3a sensitized those aggressive tumours in vitro, at least partially, by decreasing a5 integrin expression. Conclusion: Integrin a5b1 is a molecular marker of glioma aggressiveness and a possible therapeutic target for high-grade glioma patients. We showed the existence of an a5b1 integrin-p53 crosstalk with a suggested role in glioma resistance to TMZ. We propose that nutlin-3a alone or in combination with other targeted therapies may be a new therapeutic option in the subset of high-grade glioma expressing a functional p53 protein and a high level of a5b1 integrin. 275 Specific Gene Toxicity of Tumor Cells in Patients With Osteogenic Sarcoma D. Polatova1 , K. Abdikarimov1 , S. Urunbaev1 . 1 National Research Center of Oncology, General Oncology, Tashkent, Uzbekistan Background: In the mechanism of tumor development attend two processes: inhibited (weakened) apoptosis and uncontrolled induction of proliferation.
S67
These processes can be cause of tumor cells development. Expression level of apoptosis in tumor cells of patients with osteogenic sarcoma can be prognostic factor in oncologic clinic and correlates with big lifespan of patients. The aim of the study was comparison of the detecting methods of apoptotic cells of osteogenic sarcoma in patients for assessment of prognosis of life duration and treatment efficacy. Materials and Methods: There were conducted immunohistochemical method of detecting of genes P53 and bcl-2 expression with using of “DAKO” reagents and intravital dyeing of tumor cells with trypan blue. Results: It was found out, that the most quantity of cells, involved in apoptosis was observed while conducting of neoadjuvant chemotherapy by CAP regimen intra-arterial for 4−5 sycles. Low (absent) expression of mutant gene P53 and low expression of bcl-2 were detected. Conclusion: By test results with trypan the most quantity of viable cells were also observed during chemotherapy by CAP regimen. Positive effect of conducted treatment were observed in patients with low expression of mutant gene P53 and antiapoptotic gene bcl-2. In patients with high expression of these genes and low viable of cells treatment efficacy was low. 276 Vav1 − the Oncogenic Switch S. Sebban1 , M. Farago1 , D. Gashai1 , L. Tzadik1 , S. Katzav1 . 1 Hadassah − Hebrew University Medical Center, Developmental Biology and Cancer Research, Jerusalem, Israel Introduction: Since its discovery, research on the signal transducer Vav1 has cycled between understanding its physiological function in the hematopoietic system and understanding how it is dysregulated as a truncated oncogene. Recent reports suggest that it is the wild-type (herein Vav1) form of Vav1, not its oncogenic form (oncVav1), which is involved in human cancers, such as neuroblastoma, pancreatic and lung cancer. In the present study, we focused on understanding the possible role of Vav1 in breast cancer. Material and Methods: Immunohistochemistry, Western blotting, Real-TimePCR, soft agar foci formation, tunnel assay, immunofluoresence, GST-pull down, MTT and gene array analyses. Results and Discussion: Immunohistochemical analysis of human breast cancer tissue array indicated that Vav1 is expressed in 44 of 70 (63%) tumors, mainly estrogen receptor positive cancers. These results highlight the potential importance of Vav1 in breast cancer. Next, we over-expressed Vav1 and oncVav1 in two breast cancer-derived lines, MCF-7 and AU565, which differ in their phenotype and cell-surface receptor expression. In both lines, over-expression of oncVav1 led to an increase in aggressive and enhanced proliferation of the cells. In contrast, over-expression of Vav1 led to opposing phenotypes in these two cell-lines; i.e., Vav1 led to reduced proliferation and enhanced cell death in MCF-7 cells, while it resulted in a similar proliferative effect to that of oncVav1 in AU565 cells. Gene expression microarray showed an increase in mRNA expression of proliferation-related genes in Vav1expressing AU565 cells compared to a significant increase in apoptosisrelated genes in Vav1-expressing MCF-7 cells. Experiments performed to assess whether expression of Vav1 in the transfected cells led to increased apoptosis, verified our genomic microarray analysis. Thus, Vav1-expressing AU565 cells are more aggressive and less prone to death compared to MCF-7 Vav1-expressing cells that are less aggressive and more apoptotic. Since AU565 cells contain a null p53 mutant, while MCF-7 cells express wildtype p53, we hypothesized that the Vav1 effect in these cell lines is p53dependent. Silencing p53 in MCF-7 Vav1-expressing cells led to a marked decrease in apoptosis in these cells, thus erasing the Vav1 effect. These results demonstrate that Vav1 may function upstream to p53 in MCF-7 cells, but not in AU565 cells, in which it promotes proliferation. Conclusion: Our results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer. 277 Identification of Novel Therapeutic Targets Through an Integrated in Vitro and in Vivo Whole Genome ShRNA Screen in Glioma Stem Cells M. Sanzey1 , A. Golebiewska1 , D. Stieber1 , P. Nazarov2 , A. Muller2 , L. Vallar2 , S.P. Niclou1 . 1 Centre de Recherche Public de la Sante (CRP-Sante), NorLux Neuro-Oncology Laboratory, Luxembourg, Luxembourg, 2 Centre de Recherche Public de la Sante (CRP-Sante), Microarray Center, Luxembourg, Luxembourg Anti-angiogenic therapy holds promise for the treatment of Glioblastoma (GBM), an aggressive brain tumor with dismal prognosis. However so far overall patient survival is barely improved and tumors quickly develop resistance mechanisms towards anti-angiogenic treatment. In preclinical GBM models we have recently shown that the anti-angiogenic agent bevacizumab induces a significant reduction in blood flow in treated tumors, which is accompanied by an increase in tumor invasiveness and hypoxia. Based on the hypothesis that GBM contain subpopulations of cells exhibiting stem cell properties (glioma stem cells) and are able to survive and thrive in a
european journal of cancer 48, suppl. 5 (2012) S25–S288
Aim: We aimed to assess the expression of miR-7 and its targets Bcl-2 and EGFR in ovarian cancer and with different TP53 status. Materials and Methods: microRNA and gene expression was assessed by the qRT-PCR in normal human ovarian epithelium and in a panel of primary ovarian cancer samples with a different TP53 mutational status as well as in ovarian cancer cell lines that reflected the clinical samples with regard to their TP53 status. Results: miR-7 was down-regulated in most of the clinical samples without TP53 mutations and overexpressed in the samples with TP53 mutations. We found a significant difference in miR-7 expression with regard to TP53 status (p < 0.01). Bcl-2 was up-regulated, while EGFR was down-regulated in most of the clinical samples and did not relate to the TP53 mutational status. There was no correlation between the expression of miR-7 and of its target genes. EGFR expression was decreased in ovarian cell lines. In ovarian cancer cell lines with TP53 missense mutations there was a reduced Bcl-2 expression, while an enhanced Bcl-2 expression was found in those with the wild-type TP53 and with TP53 null mutation. Conclusions: 1. miR-7 seems to play an important role in ovarian cancer pathogenesis. 2. There seem to be a relationship between TP53 and miR-7. 3. In primary serous ovarian cancer the altered expression of miR-7 does not determine the levels of its targets genes, EGFR and Bcl-2. Supported by the grant of Polish Ministry of Science and Higher Education (NN 401 050 138). 274 Integrin a5b1 Plays a Critical Role in Resistance to Chemotherapy by Interfering With the P53 Pathway in High Grade Glioma H. Janouskova1 , A. Maglott1 , D. Guenot2 , S. Pinel3 , P. Chastagner3 , F. Plenat3 , N. Entz-Werle4 , J. Godet5 , S. Martin5 , M. Dontenwill5 . 1 UMR CNRS 7213, Faculte´ de Pharmacie, Illkirch, France, 2 EA4438, Physiopathologie et Recherche Translationnelle, Strasbourg, France, 3 ´ EA4421, Faculte´ de Medecine, Vandoeuvre Les Nancy, France, 4 Service ´ ˆ de Biochimie et Biologie Moleculaire, Hopital de Hautepierre, Strasbourg, France, 5 CNRS UMR 7213, Faculte´ de Pharmacie, Illkirch, France Introduction: Glioblastoma is the most aggressive and malignant form of brain tumour. Despite advances in standard therapy, including surgical resection followed by radiation and chemotherapy with temozolomide (TMZ), the prognosis for patients with glioblastoma remains unfavourable, with a median survival of 12−18 months. In the recent years, integrins have emerged as a potentially promising anti-cancer target. The aim of this study has been to investigate the role of a5b1 integrin in glioma resistance to TMZ. Material and Method: a5 subunit gene expression was examined by qPCR (95 human brain samples of different pathological grade compared to 20 non tumoural brain samples). To determine the patients’ survival, we analysed the clinical data of grade III and grade IV glioma patients. U87MG (p53wt) cells were modulated to over and downexpress a5 subunit, and were then treated with TMZ and nutlin-3a. p53 activation was determined by western blot analysis with a specific phospho-p53 (ser15) antibody and by qPCR of p53 target genes. The mice survival was studied in subcutaneous model of human brain tumour xenografts treated daily with TMZ (oral gavage, 50 mg/kg/day) for 5 days. Results and Discussion: In vivo study showed that a5b1 integrin was specifically associated with a more aggressive phenotype in brain tumours, and that a high level of a5 integrin gene expression was associated with a decreased patients’ survival with high-grade glioma. TMZ treatment of p53wt cells in vitro caused an increase of p53 activity, but with a lesser extent in a5overexpressing cells. Those cells were also more resistant to TMZ compared to a5-depleted cells. Integrin a5b1 negatively modulated the p53 pathway. Moreover, the analysis in vivo of mice survival showed that the high a5 expressing tumours were more resistant to chemotherapy. We suggest that the inhibition of p53 pathway by a5b1 integrin plays a critical role in the resistance of glioblastomas to chemotherapy. The increase of p53 activity by nutlin-3a sensitized those aggressive tumours in vitro, at least partially, by decreasing a5 integrin expression. Conclusion: Integrin a5b1 is a molecular marker of glioma aggressiveness and a possible therapeutic target for high-grade glioma patients. We showed the existence of an a5b1 integrin-p53 crosstalk with a suggested role in glioma resistance to TMZ. We propose that nutlin-3a alone or in combination with other targeted therapies may be a new therapeutic option in the subset of high-grade glioma expressing a functional p53 protein and a high level of a5b1 integrin. 275 Specific Gene Toxicity of Tumor Cells in Patients With Osteogenic Sarcoma D. Polatova1 , K. Abdikarimov1 , S. Urunbaev1 . 1 National Research Center of Oncology, General Oncology, Tashkent, Uzbekistan Background: In the mechanism of tumor development attend two processes: inhibited (weakened) apoptosis and uncontrolled induction of proliferation.
S67
These processes can be cause of tumor cells development. Expression level of apoptosis in tumor cells of patients with osteogenic sarcoma can be prognostic factor in oncologic clinic and correlates with big lifespan of patients. The aim of the study was comparison of the detecting methods of apoptotic cells of osteogenic sarcoma in patients for assessment of prognosis of life duration and treatment efficacy. Materials and Methods: There were conducted immunohistochemical method of detecting of genes P53 and bcl-2 expression with using of “DAKO” reagents and intravital dyeing of tumor cells with trypan blue. Results: It was found out, that the most quantity of cells, involved in apoptosis was observed while conducting of neoadjuvant chemotherapy by CAP regimen intra-arterial for 4−5 sycles. Low (absent) expression of mutant gene P53 and low expression of bcl-2 were detected. Conclusion: By test results with trypan the most quantity of viable cells were also observed during chemotherapy by CAP regimen. Positive effect of conducted treatment were observed in patients with low expression of mutant gene P53 and antiapoptotic gene bcl-2. In patients with high expression of these genes and low viable of cells treatment efficacy was low. 276 Vav1 − the Oncogenic Switch S. Sebban1 , M. Farago1 , D. Gashai1 , L. Tzadik1 , S. Katzav1 . 1 Hadassah − Hebrew University Medical Center, Developmental Biology and Cancer Research, Jerusalem, Israel Introduction: Since its discovery, research on the signal transducer Vav1 has cycled between understanding its physiological function in the hematopoietic system and understanding how it is dysregulated as a truncated oncogene. Recent reports suggest that it is the wild-type (herein Vav1) form of Vav1, not its oncogenic form (oncVav1), which is involved in human cancers, such as neuroblastoma, pancreatic and lung cancer. In the present study, we focused on understanding the possible role of Vav1 in breast cancer. Material and Methods: Immunohistochemistry, Western blotting, Real-TimePCR, soft agar foci formation, tunnel assay, immunofluoresence, GST-pull down, MTT and gene array analyses. Results and Discussion: Immunohistochemical analysis of human breast cancer tissue array indicated that Vav1 is expressed in 44 of 70 (63%) tumors, mainly estrogen receptor positive cancers. These results highlight the potential importance of Vav1 in breast cancer. Next, we over-expressed Vav1 and oncVav1 in two breast cancer-derived lines, MCF-7 and AU565, which differ in their phenotype and cell-surface receptor expression. In both lines, over-expression of oncVav1 led to an increase in aggressive and enhanced proliferation of the cells. In contrast, over-expression of Vav1 led to opposing phenotypes in these two cell-lines; i.e., Vav1 led to reduced proliferation and enhanced cell death in MCF-7 cells, while it resulted in a similar proliferative effect to that of oncVav1 in AU565 cells. Gene expression microarray showed an increase in mRNA expression of proliferation-related genes in Vav1expressing AU565 cells compared to a significant increase in apoptosisrelated genes in Vav1-expressing MCF-7 cells. Experiments performed to assess whether expression of Vav1 in the transfected cells led to increased apoptosis, verified our genomic microarray analysis. Thus, Vav1-expressing AU565 cells are more aggressive and less prone to death compared to MCF-7 Vav1-expressing cells that are less aggressive and more apoptotic. Since AU565 cells contain a null p53 mutant, while MCF-7 cells express wildtype p53, we hypothesized that the Vav1 effect in these cell lines is p53dependent. Silencing p53 in MCF-7 Vav1-expressing cells led to a marked decrease in apoptosis in these cells, thus erasing the Vav1 effect. These results demonstrate that Vav1 may function upstream to p53 in MCF-7 cells, but not in AU565 cells, in which it promotes proliferation. Conclusion: Our results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer. 277 Identification of Novel Therapeutic Targets Through an Integrated in Vitro and in Vivo Whole Genome ShRNA Screen in Glioma Stem Cells M. Sanzey1 , A. Golebiewska1 , D. Stieber1 , P. Nazarov2 , A. Muller2 , L. Vallar2 , S.P. Niclou1 . 1 Centre de Recherche Public de la Sante (CRP-Sante), NorLux Neuro-Oncology Laboratory, Luxembourg, Luxembourg, 2 Centre de Recherche Public de la Sante (CRP-Sante), Microarray Center, Luxembourg, Luxembourg Anti-angiogenic therapy holds promise for the treatment of Glioblastoma (GBM), an aggressive brain tumor with dismal prognosis. However so far overall patient survival is barely improved and tumors quickly develop resistance mechanisms towards anti-angiogenic treatment. In preclinical GBM models we have recently shown that the anti-angiogenic agent bevacizumab induces a significant reduction in blood flow in treated tumors, which is accompanied by an increase in tumor invasiveness and hypoxia. Based on the hypothesis that GBM contain subpopulations of cells exhibiting stem cell properties (glioma stem cells) and are able to survive and thrive in a