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101 Inhibition of survivin and aurora B kinase radiosensitizes mesothelioma cells by enhancing mitotic arrests
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Friday, October 20, 2006 / Poster discussion session: Tumor biology
intervals at single cell resolution. BCL-2, MCL-1 and BCL-XL expression was not significantly altered at this timepoint. Conclusions: Bortezomib potentiates direct activation of BAX and BAK in MMP mesothelioma cells suggesting priming of these proapoptotic proteins.
97
Cisplatin-induced apoptosis in malignant pleural mesothelioma cells involves p53 but not p14ARF
S. Hopkins-Donaldson 1 , T.M. Marti 1 , L.L. Belyanskaya 1 , A.P. Simões-Wüst 1 , B. Sigrist 1 , S. Kurtz 1 , U. Zangemeister-Wittke 2 , R. Stahel 1 . 1 University Hospital Zurich, Zurich, Switzerland; 2 University of Bern, Bern, Switzerland Background: Malignant pleural mesothelioma (MPM) tumours often contain homozygous deletions in the INK4A locus that encodes p14ARF, a tumour suppressor protein that promotes p53 stability by blocking the p53 inhibitor MDM2. It has been suggested that p53 is inactive in MPM due to the absence of p14ARF expression. Methods and Results: We investigated p53 activation in p14ARF negative MPM cells or primary cultures after cisplatin treatment and found increased levels of phospho-p53 (serine-15), total p53 protein and p53 specific DNAbinding activity in the majority of cell lines. In addition, the expression levels of p53-positively controlled genes p21WAF, Bax, pig3 and MDM2 increased on incubation with cisplatin, whereas levels of the negatively regulated p53 target gene survivin decreased. Cisplatin-induced apoptosis was detected in p14ARF-deficient cells expressing wild type p53 but not in a p53 mutant cell line. Knockdown of wild type p53 with siRNA rendered MPM cells more resistant to cisplatin-induced apoptosis. Over-expression of p14ARF in p53 wild type MPM cells by transient transfection did not increase their sensitivity to cisplatininduced apoptosis, however, pre-incubation with antisense oligonucleotides targeting the inhibitor of apoptosis protein survivin lead to p53 activation and increased apoptosis sensitivity to cisplatin. Conclusions: These results suggest that p53 is functional in MPM in the absence of p14ARF, and that therapies that target the inhibitor of apoptosis protein survivin are worthy of investigation in MPM.
98
Anoikis resistance in malignant pleural mesothelioma cells
J. Daubriac, J. Fleury-Feith, J.M.C. Jaurand. INSERM U674, Paris, France Background: Pleural fluid (PF) accumulation is a frequent clinical observation in diffuse malignant pleural mesothelioma (MM). The cytological analysis of PF often revealed the presence of spherical clusters of malignant cells, addressing the question of how non-adherent tumor cells survive and are able to develop new tumor foci in the pleural cavity. As anoikis is the mechanism whereby cells should die following loss of matricial anchorage, the aim of the present work was to determine whether MM cells are resistant to anoïkis. Methods: To study anoikis resistance of mesothelioma cells, 4 MM cell lines were cultured on poly-2-hydroxyethyl methacrylate (polyHEMA)-coated plates. The effect of the specific PI3K/Akt pathway inhibitor, LY294002, on cell death was determined by assessing DNA fragmentation in situ by flow cytometry. Apoptosis mechanism was also investigated by the measurement of caspase-3 activation, and the percentage of cycling cells was evaluated by studying Ki-67 antigen expression by flow cytometry. Activation of Akt, PAK and Rac proteins was estimated by western-immunoblot using phospho-specific antibodies. Results: MM cells are regularly cultured in adherent conditions, but loss of anchorage results in the formation of spherical clusters, able to survive more than one week in a cycling state. The percentage of Ki-67 positive cells decreases as the size and the compactness of the clusters increase, but cells remain able to reattach and grow on regular tissue culture plates. While LY294002 can reduce apoptosis resistance of MM cells growing on a substratum, the drug has a poor effect on three cell lines cultured in anchorage-independent conditions. Besides, phospho-Akt, phospho-PAK and phospho-Rac proteins were found to be downregulated following the lost of anchorage. Conclusion: Human malignant pleural MM cells are resistant to anoikis, in a uncommon PI3K/Akt independent pathway. Loss of anchorage can reduce the sensitivity of the MM cells to inhibitors of survival. Hence, it is of importance to better investigate signaling pathways in both non-adherent and adherent MM cells.
99
Role of notch signaling in human malignant mesothelioma
I. Graziani 1 , M.A. De Marco 1 , Y. Chen 1 , R.M. DeMay 2 , H.I. Pass 3 , M. Bocchetta 1 . 1 Loyola University Chicago, USA; 2 University of Chicago, Chicago, USA; 3 New York University, New York, USA Notch signaling regulates key cell fate decisions during development and postnatal life. Increasingly, Notch receptors, their ligands and downstream effectors have been implicated in carcinogenesis. The biological effects of Notch signaling appear highly context-dependent. Therefore, both tumor suppressor
and oncogenic activities of Notch have been reported in different tumor models. Here we analyzed a panel of human malignant mesothelioma cell lines, SV40-transformed human mesothelial cell lines and primary human mesothelial cell cultures for Notch expression, both at the protein and at the mRNA level. Additionally, we analyzed a panel of 21 matched biopsies of frozen lung and malignant mesothelioma. We found that Notch receptors and downstream effectors are deregulated in mesotheliomas and their derived cell lines compared to normal tissues. SV40-transformed mesothelial cell lines displayed a specific pattern of Notch receptors contrasting that of SV40 Large T antigen negative mesothelioma cell lines. One common feature of all malignant cells was a marked downregulation of Notch-2. Reintroduction of constitutively active Notch-2 caused apoptosis in both SV40-transformed mesothelial cells and mesothelioma cell lines. The putative mechanisms of Notch-2 functions in mesothelial cells are discussed. In conclusion, it appears that Notch-2 may have tumor suppressor activities in the human mesothelium.
100
Bax and Bak immunoreactivity in malignant pleural mesothelioma (MPM)
A. Klabatsa 1 , S. Bax 1 , J.P.C. Steele 1 , R.M. Rudd 1 , M.T. Sheaff 1 , D.A. Fennell 2 . 1 St Bartholomew’s Hospital, London, UK; 2 Belfast City Hospital, Belfast, Northern Ireland Background: Two proteins of the Bcl-2 family, Bax and Bak, are known to have a key role in mediating apoptosis via the mitochondrial pathway. Recent double knockout studies have shown that BAX and BAK knockout confers resistance to chemotherapy. In this study we therefore examined the co-expression of BAX and BAK and its relationship to survival. Methods: A tissue microarray (TMA) of 100 cases of MPM was used (epithelioid 58%, sarcomatoid 8%, biphasic 29%, reactive mesothelium 3%, normal mesothelium 2%). Immunohistochemistry was semi-quantitatively scored (0, +, ++, +++) according to the number of cells stained and intensity of staining. Results: Seventy-two cases were suitable for analysis; Bax and Bak positivity was seen in 65.3% and 63.4% of cases respectively. BAX/BAK double immunonegativity was seen in 31% of cases. BAX/BAK expression was not influenced by histology. Overall median survival (MS) for the population was 8 months (mo). MS for BAX- was 3 mo versus 11 for BAX+ (Log Rank statistic= 10.34, p= 0.0013), and 3 mo for BAK- versus 9 mo for BAK+ (Log Rank statistic= 8.12, p= 0.0044). Conclusion: BAX/BAK double immunonegativity is a potential prognostic factor in mesothelioma and suggests an important role for these proteins as tumour suppressors.
101
Inhibition of survivin and aurora B kinase radiosensitizes mesothelioma cells by enhancing mitotic arrests
K.W. Kim, B. Lu. Vanderbilt University, Nashville, USA Survivin, a member of the inhibitor of apoptosis gene family, has been shown to prevent cell death and control mitosis. It complexes with caspases and aurora kinase B during mitosis. In this study, we examined whether irradiation affected survivin and Aurora kinase expression in a mesothelioma cell line, and how inhibition of these molecules may result in radiosensitization. Radiation increased the levels of survivin and aurora B kinase as well as its activity. Cell survival as measured by clonogenic assay showed that dual inhibition of survivin by its antisense oligonucleotides (ASOs) and Aurora kinase B, by a small molecule inhibitor, ZM447439 (ZM), respectively, resulted in the greatest radiosensitization in mesothelioma cells, although survivin ASOs alone also showed strong radiosensitizing ability. Combined inhibition of survivin and Aurora B increased the formation of multinucleated cells following irradiation, but did not increase levels of cleaved caspase 3. Specifically, these results suggest that inhibition of survivin and Aurora B induces mitotic cell arrest in mesothelioma cells following irradiation. Therefore, we propose that survivin and Aurora kinase B may be therapeutic targets for radiation sensitization of mesothelioma.
Friday, October 20, 2006 / Poster discussion session: Tumor biology
intervals at single cell resolution. BCL-2, MCL-1 and BCL-XL expression was not significantly altered at this timepoint. Conclusions: Bortezomib potentiates direct activation of BAX and BAK in MMP mesothelioma cells suggesting priming of these proapoptotic proteins.
97
Cisplatin-induced apoptosis in malignant pleural mesothelioma cells involves p53 but not p14ARF
S. Hopkins-Donaldson 1 , T.M. Marti 1 , L.L. Belyanskaya 1 , A.P. Simões-Wüst 1 , B. Sigrist 1 , S. Kurtz 1 , U. Zangemeister-Wittke 2 , R. Stahel 1 . 1 University Hospital Zurich, Zurich, Switzerland; 2 University of Bern, Bern, Switzerland Background: Malignant pleural mesothelioma (MPM) tumours often contain homozygous deletions in the INK4A locus that encodes p14ARF, a tumour suppressor protein that promotes p53 stability by blocking the p53 inhibitor MDM2. It has been suggested that p53 is inactive in MPM due to the absence of p14ARF expression. Methods and Results: We investigated p53 activation in p14ARF negative MPM cells or primary cultures after cisplatin treatment and found increased levels of phospho-p53 (serine-15), total p53 protein and p53 specific DNAbinding activity in the majority of cell lines. In addition, the expression levels of p53-positively controlled genes p21WAF, Bax, pig3 and MDM2 increased on incubation with cisplatin, whereas levels of the negatively regulated p53 target gene survivin decreased. Cisplatin-induced apoptosis was detected in p14ARF-deficient cells expressing wild type p53 but not in a p53 mutant cell line. Knockdown of wild type p53 with siRNA rendered MPM cells more resistant to cisplatin-induced apoptosis. Over-expression of p14ARF in p53 wild type MPM cells by transient transfection did not increase their sensitivity to cisplatininduced apoptosis, however, pre-incubation with antisense oligonucleotides targeting the inhibitor of apoptosis protein survivin lead to p53 activation and increased apoptosis sensitivity to cisplatin. Conclusions: These results suggest that p53 is functional in MPM in the absence of p14ARF, and that therapies that target the inhibitor of apoptosis protein survivin are worthy of investigation in MPM.
98
Anoikis resistance in malignant pleural mesothelioma cells
J. Daubriac, J. Fleury-Feith, J.M.C. Jaurand. INSERM U674, Paris, France Background: Pleural fluid (PF) accumulation is a frequent clinical observation in diffuse malignant pleural mesothelioma (MM). The cytological analysis of PF often revealed the presence of spherical clusters of malignant cells, addressing the question of how non-adherent tumor cells survive and are able to develop new tumor foci in the pleural cavity. As anoikis is the mechanism whereby cells should die following loss of matricial anchorage, the aim of the present work was to determine whether MM cells are resistant to anoïkis. Methods: To study anoikis resistance of mesothelioma cells, 4 MM cell lines were cultured on poly-2-hydroxyethyl methacrylate (polyHEMA)-coated plates. The effect of the specific PI3K/Akt pathway inhibitor, LY294002, on cell death was determined by assessing DNA fragmentation in situ by flow cytometry. Apoptosis mechanism was also investigated by the measurement of caspase-3 activation, and the percentage of cycling cells was evaluated by studying Ki-67 antigen expression by flow cytometry. Activation of Akt, PAK and Rac proteins was estimated by western-immunoblot using phospho-specific antibodies. Results: MM cells are regularly cultured in adherent conditions, but loss of anchorage results in the formation of spherical clusters, able to survive more than one week in a cycling state. The percentage of Ki-67 positive cells decreases as the size and the compactness of the clusters increase, but cells remain able to reattach and grow on regular tissue culture plates. While LY294002 can reduce apoptosis resistance of MM cells growing on a substratum, the drug has a poor effect on three cell lines cultured in anchorage-independent conditions. Besides, phospho-Akt, phospho-PAK and phospho-Rac proteins were found to be downregulated following the lost of anchorage. Conclusion: Human malignant pleural MM cells are resistant to anoikis, in a uncommon PI3K/Akt independent pathway. Loss of anchorage can reduce the sensitivity of the MM cells to inhibitors of survival. Hence, it is of importance to better investigate signaling pathways in both non-adherent and adherent MM cells.
99
Role of notch signaling in human malignant mesothelioma
I. Graziani 1 , M.A. De Marco 1 , Y. Chen 1 , R.M. DeMay 2 , H.I. Pass 3 , M. Bocchetta 1 . 1 Loyola University Chicago, USA; 2 University of Chicago, Chicago, USA; 3 New York University, New York, USA Notch signaling regulates key cell fate decisions during development and postnatal life. Increasingly, Notch receptors, their ligands and downstream effectors have been implicated in carcinogenesis. The biological effects of Notch signaling appear highly context-dependent. Therefore, both tumor suppressor
and oncogenic activities of Notch have been reported in different tumor models. Here we analyzed a panel of human malignant mesothelioma cell lines, SV40-transformed human mesothelial cell lines and primary human mesothelial cell cultures for Notch expression, both at the protein and at the mRNA level. Additionally, we analyzed a panel of 21 matched biopsies of frozen lung and malignant mesothelioma. We found that Notch receptors and downstream effectors are deregulated in mesotheliomas and their derived cell lines compared to normal tissues. SV40-transformed mesothelial cell lines displayed a specific pattern of Notch receptors contrasting that of SV40 Large T antigen negative mesothelioma cell lines. One common feature of all malignant cells was a marked downregulation of Notch-2. Reintroduction of constitutively active Notch-2 caused apoptosis in both SV40-transformed mesothelial cells and mesothelioma cell lines. The putative mechanisms of Notch-2 functions in mesothelial cells are discussed. In conclusion, it appears that Notch-2 may have tumor suppressor activities in the human mesothelium.
100
Bax and Bak immunoreactivity in malignant pleural mesothelioma (MPM)
A. Klabatsa 1 , S. Bax 1 , J.P.C. Steele 1 , R.M. Rudd 1 , M.T. Sheaff 1 , D.A. Fennell 2 . 1 St Bartholomew’s Hospital, London, UK; 2 Belfast City Hospital, Belfast, Northern Ireland Background: Two proteins of the Bcl-2 family, Bax and Bak, are known to have a key role in mediating apoptosis via the mitochondrial pathway. Recent double knockout studies have shown that BAX and BAK knockout confers resistance to chemotherapy. In this study we therefore examined the co-expression of BAX and BAK and its relationship to survival. Methods: A tissue microarray (TMA) of 100 cases of MPM was used (epithelioid 58%, sarcomatoid 8%, biphasic 29%, reactive mesothelium 3%, normal mesothelium 2%). Immunohistochemistry was semi-quantitatively scored (0, +, ++, +++) according to the number of cells stained and intensity of staining. Results: Seventy-two cases were suitable for analysis; Bax and Bak positivity was seen in 65.3% and 63.4% of cases respectively. BAX/BAK double immunonegativity was seen in 31% of cases. BAX/BAK expression was not influenced by histology. Overall median survival (MS) for the population was 8 months (mo). MS for BAX- was 3 mo versus 11 for BAX+ (Log Rank statistic= 10.34, p= 0.0013), and 3 mo for BAK- versus 9 mo for BAK+ (Log Rank statistic= 8.12, p= 0.0044). Conclusion: BAX/BAK double immunonegativity is a potential prognostic factor in mesothelioma and suggests an important role for these proteins as tumour suppressors.
101
Inhibition of survivin and aurora B kinase radiosensitizes mesothelioma cells by enhancing mitotic arrests
K.W. Kim, B. Lu. Vanderbilt University, Nashville, USA Survivin, a member of the inhibitor of apoptosis gene family, has been shown to prevent cell death and control mitosis. It complexes with caspases and aurora kinase B during mitosis. In this study, we examined whether irradiation affected survivin and Aurora kinase expression in a mesothelioma cell line, and how inhibition of these molecules may result in radiosensitization. Radiation increased the levels of survivin and aurora B kinase as well as its activity. Cell survival as measured by clonogenic assay showed that dual inhibition of survivin by its antisense oligonucleotides (ASOs) and Aurora kinase B, by a small molecule inhibitor, ZM447439 (ZM), respectively, resulted in the greatest radiosensitization in mesothelioma cells, although survivin ASOs alone also showed strong radiosensitizing ability. Combined inhibition of survivin and Aurora B increased the formation of multinucleated cells following irradiation, but did not increase levels of cleaved caspase 3. Specifically, these results suggest that inhibition of survivin and Aurora B induces mitotic cell arrest in mesothelioma cells following irradiation. Therefore, we propose that survivin and Aurora kinase B may be therapeutic targets for radiation sensitization of mesothelioma.