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109 Tyrosine Kinase Receptors as Functional Markers and Therapeutical Targets of Glioblastoma Stem Cells
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
of Usp9x through the use of transposon mutagenesis reaffirms the utility of in vivo mouse cancer screens to complement the direct investigation of human cancer. 109 Tyrosine Kinase Receptors as Functional Markers and Therapeutical Targets of Glioblastoma Stem Cells F. De Bacco1 , E. Casanova1 , E. Medico1 , S. Pellegatta2 , F. Orzan1 , R. Albano1 , A. D’Ambrosio1 , B. Pollo3 , G. Finocchiaro2 , C. Boccaccio1 . 1 IRCC − Institute for Cancer Research @ Candiolo, Department of Molecular Oncology, Torino, Italy, 2 Fondazione I.R.C.C.S. Istituto Neurologico C. Besta, Unit of Molecular Neuro-Oncology, Milano, Italy, 3 Fondazione I.R.C.C.S. Istituto Neurologico C. Besta, Unit of Neuropathology, Torino, Italy Introduction: Glioblastoma (GB) is the most common and aggressive primary brain tumor, with estimated median survival of 15 months after diagnosis. However, thanks to the recent identification of genetic lesions and transcriptional profiles, GB can be classified into subtypes (‘proneural’, ‘classical’ or ‘mesenchymal’), each endowed with distinct biological and prognostic features, and associated with new potential therapeutical targets. The proneural profile is characterized by mutations of IDH1/2 gene and alterations of the PDGF signaling pathway. Classical and mesenchymal subtypes are characterized by activating lesions of the EGFR gene, or by mutations/deletions of PTEN and NF1 genes, respectively. GB tumors are also known to be organized into a cellular hierarchy, led by a stem-like cell subpopulation that retains the tumorigenic potential, and gives rise to a heterogeneous, pseudodifferentiated bulk population. GB cell subpopulations display different sensitivity to therapies, and the stem-like subpopulation has been shown to possess high inherent radio- and chemoresistance: thus, GB stem cells are likely responsible for treatment failure and tumor recurrence. We undertook systematic characterization of tyrosine kinase receptor expression and function in GB stem cells, in order to find out new therapeutical targets. Material and Methods: Neurospheres, i.e. cultures enriched in stem and progenitor cells, were isolated and propagated in vitro. Neurospheres underwent mutational and transcriptional profiling, immunophenotyping, analysis of proliferative and invasive abilities in vitro and tumorigenic potential in vivo. Results: We found that neurospheres displayed the same mutational profile, and mirrored the same gene expression profile as the original tumors, and could thus be classified as ‘classical’, ‘mesenchymal’ or ‘proneural’. We then specifically associated the expression of EGFR to the ‘classical’, and MET and FGFR to the ‘mesenchymal’ or ‘proneural’ subtypes, and showed that these cells rely on these receptors for their biological activities. Conclusions: We have shown that tyrosine kinase receptors are subtypespecific functional markers in glioblastoma, and can be considered as potential therapeutical targets. 110 EMT Followed in Three Dimensions in Vitro P.A. Cygan1 , A.M. Grabowska2 , S. Stolnik-Trenkic1 , P. Elvin3 , S.R. Wedge3 , S. Watson2 . 1 The University of Nottingham, School of Pharmacy, Nottingham, United Kingdom, 2 The University of Nottingham, School of Clinical Sciences, Nottingham, United Kingdom, 3 AstraZeneca, Cancer Bioscience, Macclesfield, United Kingdom Introduction: The clinically important process of metastasis is difficult to model in vitro because it is dependent on the tumour microenvironment. In this study two 3-dimensional (3D) in vitro models were created and used to study epithelial-mesenchymal transition (EMT), a programme hypothesised to drive the metastatic process. Materials and Methods: HCT116 colon cancer cells were encapsulated in alginate-chitosan-hyaluronan (Alg-Cs-HA) or alginate-fibronectin (Alg-Fn) matrices and co-cultured with separately encapsulated neuroblastoma (model A) or normal embryonic lung fibroblasts (model B), respectively. The internal structure of both capsule types was analysed. Cell morphology, metabolic activity, EMT-associated gene expression changes and protein expression of encapsulated cancer cells were examined. Results and Discussion: Distinctive differences between Alg-Cs-HA and AlgFn capsule internal structure, different morphologies of encapsulated epithelial cells and different patterns of metabolic activity were observed. Following coculture with fibroblasts, a more EMT-like phenotype was observed in both models (E-cadherin down-regulation and up-regulation of the mesenchymal marker, vimentin) but only in model A was the effect significant (p < 0.01, N = 2 and p < 0.05, N = 2) and simultaneous up-regulation of E-cadherin transcriptional repressors, Slug and Zeb1, was observed. Analysis of matrix metalloproteinases (MMPs), involved in extracellular matrix (ECM) remodelling and cell migration, revealed significant MMP2 up-regulation (day 6, p < 0.01; day 15, p < 0.05, N = 2) in model A and modest up-regulation of MMP9 on day 20 in both models. Upon co-culture, both models showed somewhat reduced expression of integrin a5, a receptor mediating cellECM interactions. Altered morphology of adherens junctions and strong
S27
MMP2 protein expression was revealed by immunostaining of cryo-sections of encapsulated cancer cells. Conclusion: Two tumour microenvironment models used in this study, rich in different ECM components and containing two types of stromal fibroblasts, induced different changes in the epithelial phenotype of cancer cells. This 3D in vitro approach may help to understand how cancer cells interact with both adjacent and distant tissues and can potentially be used to provide in vitro models for therapeutic target validation, in particular for approaches targeting EMT or the metastatic phenotype, prior to in vivo studies. 111 Autophagic and Apoptotic Mechanisms of Death Induced by Sesbania Grandiflora Flower in Human Leukemic Cells R. Roy1 , D. Kumar1 , C. Chowdhury2 , P. Das1 . 1 CSIR-Indian Institute of Chemical Biology, Cancer and Cell Biology Division, Kolkata, India, 2 CSIR-Indian Institute of Chemical Biology, Chemistry Division, Kolkata, India Background: Ethnobotanical knowledge coupled with rationale-driven scientific research has formed an important facet of anti-cancer drug discovery because medicinal plants have a very long history of safe consumption, and bioactive compounds obtained from them are normally non-toxic or less toxic to humans. Sesbania grandiflora, a small tree, is widely used in Indian traditional medicine for the treatment of a broad spectrum of diseases. The present study provides evidence for the antiproliferative effect of S. grandiflora flowers (SG) extract in leukemic cell lines and delineates the underlying involvement of various apoptotic and autophagic pathways. Material and Methods: Cytotoxicity of SG was checked in U937 cells by MTT assay along with flow cytometric measurement of reactive oxygen species using CMH2 DCFDA, phosphatidylserine exposure by Annexin V-FITC, mitochondrial membrane potential using JC-1 and western blot analysis of alteration in levels of apoptotic and autophagic proteins. Transmission electron microscope and fluorescence microscope were used to identify the formation of acidic vesicular organelles (AVO) during autophagy. Rate of oxygen consumption was polarographically measured with a Clark-type oxygen electrode. Caspase activity was measured spectrophotometrically. DNA degradation and changes in nuclear morphology were determined by confocal microscopy and cell cycle analysis by flow cytometry. Results: SG exhibited an IC50 of 18.6 mg/ml in U937 cells. Inhibition of growth is associated with increased ROS generation which enhanced Annexin-V positivity through stimulation of pro-apoptotic proteins and concomitant inhibition of anti-apoptotic protein expressions producing mitochondrial disruption and the release of cytochrome-c which initiate caspase activation. Additionally, autophagic vacuoles formation clearly observed after treatment with SG and accompanied by LC3 processing and increased Atg protein expression levels. These phenomena culminated in DNA fragmentation, cell cycle arrest and eventually cell death. Conclusions: Taken together, the findings suggest that both apoptosis and autophagy contribute to the antileukemic activity of SG. Thus, SG merits consideration and further investigation as a therapeutic option for the treatment of leukemia. 112 Inhibition of Heat Shock Protein (Hsp) 27 Potentiates the Suppressive Effect of Hsp90 Inhibitors in Targeting Breast Cancer Stem-like Cells W.W. Chang1 , C.H. Lee2 , H.M. Hong1 , Y.Y. Chang1 . 1 Chung Shan Medical University, School of Biomedical Sciences, Taichung City, Taiwan, 2 China Medical University, Department of Microbiology, Taichung City, Taiwan Background: Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44+ cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. Materials and Methods: The activity of Hsp90 was inhibited by specific inhibitors, geldanamycin (GA) or 17-desmethoxy-17-N,N-dimethylaminoethylaminogeldanamycin (17-DMAG). The activity of Hsp27 or heat shock factors were inhibited by quercetin or KNK437, respectively. The changes of the population of BCSCs in human breast cancer cells were determined by fluorescence-based ALDH activity assay and flow cytometric analysis. The cell proliferation or migration was determined by mitochondria activity assay or wound-healing assay, respectively. The specific knockdown of Hsp27 expression was performed by RNA interference. The protein expression was determined by western blot analysis and quantified with ImageJ software. Results: We first discovered that the expression of Hsp90a was increased in ALDH+ human breast cancer cells. GA or 17-DMAG could suppress ALDH+ breast cancer cells in a dose dependent manner. We are interesting in the
european journal of cancer 48, suppl. 5 (2012) S25–S288
of Usp9x through the use of transposon mutagenesis reaffirms the utility of in vivo mouse cancer screens to complement the direct investigation of human cancer. 109 Tyrosine Kinase Receptors as Functional Markers and Therapeutical Targets of Glioblastoma Stem Cells F. De Bacco1 , E. Casanova1 , E. Medico1 , S. Pellegatta2 , F. Orzan1 , R. Albano1 , A. D’Ambrosio1 , B. Pollo3 , G. Finocchiaro2 , C. Boccaccio1 . 1 IRCC − Institute for Cancer Research @ Candiolo, Department of Molecular Oncology, Torino, Italy, 2 Fondazione I.R.C.C.S. Istituto Neurologico C. Besta, Unit of Molecular Neuro-Oncology, Milano, Italy, 3 Fondazione I.R.C.C.S. Istituto Neurologico C. Besta, Unit of Neuropathology, Torino, Italy Introduction: Glioblastoma (GB) is the most common and aggressive primary brain tumor, with estimated median survival of 15 months after diagnosis. However, thanks to the recent identification of genetic lesions and transcriptional profiles, GB can be classified into subtypes (‘proneural’, ‘classical’ or ‘mesenchymal’), each endowed with distinct biological and prognostic features, and associated with new potential therapeutical targets. The proneural profile is characterized by mutations of IDH1/2 gene and alterations of the PDGF signaling pathway. Classical and mesenchymal subtypes are characterized by activating lesions of the EGFR gene, or by mutations/deletions of PTEN and NF1 genes, respectively. GB tumors are also known to be organized into a cellular hierarchy, led by a stem-like cell subpopulation that retains the tumorigenic potential, and gives rise to a heterogeneous, pseudodifferentiated bulk population. GB cell subpopulations display different sensitivity to therapies, and the stem-like subpopulation has been shown to possess high inherent radio- and chemoresistance: thus, GB stem cells are likely responsible for treatment failure and tumor recurrence. We undertook systematic characterization of tyrosine kinase receptor expression and function in GB stem cells, in order to find out new therapeutical targets. Material and Methods: Neurospheres, i.e. cultures enriched in stem and progenitor cells, were isolated and propagated in vitro. Neurospheres underwent mutational and transcriptional profiling, immunophenotyping, analysis of proliferative and invasive abilities in vitro and tumorigenic potential in vivo. Results: We found that neurospheres displayed the same mutational profile, and mirrored the same gene expression profile as the original tumors, and could thus be classified as ‘classical’, ‘mesenchymal’ or ‘proneural’. We then specifically associated the expression of EGFR to the ‘classical’, and MET and FGFR to the ‘mesenchymal’ or ‘proneural’ subtypes, and showed that these cells rely on these receptors for their biological activities. Conclusions: We have shown that tyrosine kinase receptors are subtypespecific functional markers in glioblastoma, and can be considered as potential therapeutical targets. 110 EMT Followed in Three Dimensions in Vitro P.A. Cygan1 , A.M. Grabowska2 , S. Stolnik-Trenkic1 , P. Elvin3 , S.R. Wedge3 , S. Watson2 . 1 The University of Nottingham, School of Pharmacy, Nottingham, United Kingdom, 2 The University of Nottingham, School of Clinical Sciences, Nottingham, United Kingdom, 3 AstraZeneca, Cancer Bioscience, Macclesfield, United Kingdom Introduction: The clinically important process of metastasis is difficult to model in vitro because it is dependent on the tumour microenvironment. In this study two 3-dimensional (3D) in vitro models were created and used to study epithelial-mesenchymal transition (EMT), a programme hypothesised to drive the metastatic process. Materials and Methods: HCT116 colon cancer cells were encapsulated in alginate-chitosan-hyaluronan (Alg-Cs-HA) or alginate-fibronectin (Alg-Fn) matrices and co-cultured with separately encapsulated neuroblastoma (model A) or normal embryonic lung fibroblasts (model B), respectively. The internal structure of both capsule types was analysed. Cell morphology, metabolic activity, EMT-associated gene expression changes and protein expression of encapsulated cancer cells were examined. Results and Discussion: Distinctive differences between Alg-Cs-HA and AlgFn capsule internal structure, different morphologies of encapsulated epithelial cells and different patterns of metabolic activity were observed. Following coculture with fibroblasts, a more EMT-like phenotype was observed in both models (E-cadherin down-regulation and up-regulation of the mesenchymal marker, vimentin) but only in model A was the effect significant (p < 0.01, N = 2 and p < 0.05, N = 2) and simultaneous up-regulation of E-cadherin transcriptional repressors, Slug and Zeb1, was observed. Analysis of matrix metalloproteinases (MMPs), involved in extracellular matrix (ECM) remodelling and cell migration, revealed significant MMP2 up-regulation (day 6, p < 0.01; day 15, p < 0.05, N = 2) in model A and modest up-regulation of MMP9 on day 20 in both models. Upon co-culture, both models showed somewhat reduced expression of integrin a5, a receptor mediating cellECM interactions. Altered morphology of adherens junctions and strong
S27
MMP2 protein expression was revealed by immunostaining of cryo-sections of encapsulated cancer cells. Conclusion: Two tumour microenvironment models used in this study, rich in different ECM components and containing two types of stromal fibroblasts, induced different changes in the epithelial phenotype of cancer cells. This 3D in vitro approach may help to understand how cancer cells interact with both adjacent and distant tissues and can potentially be used to provide in vitro models for therapeutic target validation, in particular for approaches targeting EMT or the metastatic phenotype, prior to in vivo studies. 111 Autophagic and Apoptotic Mechanisms of Death Induced by Sesbania Grandiflora Flower in Human Leukemic Cells R. Roy1 , D. Kumar1 , C. Chowdhury2 , P. Das1 . 1 CSIR-Indian Institute of Chemical Biology, Cancer and Cell Biology Division, Kolkata, India, 2 CSIR-Indian Institute of Chemical Biology, Chemistry Division, Kolkata, India Background: Ethnobotanical knowledge coupled with rationale-driven scientific research has formed an important facet of anti-cancer drug discovery because medicinal plants have a very long history of safe consumption, and bioactive compounds obtained from them are normally non-toxic or less toxic to humans. Sesbania grandiflora, a small tree, is widely used in Indian traditional medicine for the treatment of a broad spectrum of diseases. The present study provides evidence for the antiproliferative effect of S. grandiflora flowers (SG) extract in leukemic cell lines and delineates the underlying involvement of various apoptotic and autophagic pathways. Material and Methods: Cytotoxicity of SG was checked in U937 cells by MTT assay along with flow cytometric measurement of reactive oxygen species using CMH2 DCFDA, phosphatidylserine exposure by Annexin V-FITC, mitochondrial membrane potential using JC-1 and western blot analysis of alteration in levels of apoptotic and autophagic proteins. Transmission electron microscope and fluorescence microscope were used to identify the formation of acidic vesicular organelles (AVO) during autophagy. Rate of oxygen consumption was polarographically measured with a Clark-type oxygen electrode. Caspase activity was measured spectrophotometrically. DNA degradation and changes in nuclear morphology were determined by confocal microscopy and cell cycle analysis by flow cytometry. Results: SG exhibited an IC50 of 18.6 mg/ml in U937 cells. Inhibition of growth is associated with increased ROS generation which enhanced Annexin-V positivity through stimulation of pro-apoptotic proteins and concomitant inhibition of anti-apoptotic protein expressions producing mitochondrial disruption and the release of cytochrome-c which initiate caspase activation. Additionally, autophagic vacuoles formation clearly observed after treatment with SG and accompanied by LC3 processing and increased Atg protein expression levels. These phenomena culminated in DNA fragmentation, cell cycle arrest and eventually cell death. Conclusions: Taken together, the findings suggest that both apoptosis and autophagy contribute to the antileukemic activity of SG. Thus, SG merits consideration and further investigation as a therapeutic option for the treatment of leukemia. 112 Inhibition of Heat Shock Protein (Hsp) 27 Potentiates the Suppressive Effect of Hsp90 Inhibitors in Targeting Breast Cancer Stem-like Cells W.W. Chang1 , C.H. Lee2 , H.M. Hong1 , Y.Y. Chang1 . 1 Chung Shan Medical University, School of Biomedical Sciences, Taichung City, Taiwan, 2 China Medical University, Department of Microbiology, Taichung City, Taiwan Background: Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and its expression has been reported to be associated with poor prognosis of breast cancer. Cancer stem cells (CSCs) are particular subtypes of cells in cancer which have been demonstrated to be important to tumor initiation, drug resistance and metastasis. In breast cancer, breast CSCs (BCSCs) are identified as CD24-CD44+ cells or cells with high intracellular aldehyde dehydrogenase activity (ALDH+). Although the clinical trials of Hsp90 inhibitors in breast cancer therapy are ongoing, the BCSC targeting effect of them remains unclear. Materials and Methods: The activity of Hsp90 was inhibited by specific inhibitors, geldanamycin (GA) or 17-desmethoxy-17-N,N-dimethylaminoethylaminogeldanamycin (17-DMAG). The activity of Hsp27 or heat shock factors were inhibited by quercetin or KNK437, respectively. The changes of the population of BCSCs in human breast cancer cells were determined by fluorescence-based ALDH activity assay and flow cytometric analysis. The cell proliferation or migration was determined by mitochondria activity assay or wound-healing assay, respectively. The specific knockdown of Hsp27 expression was performed by RNA interference. The protein expression was determined by western blot analysis and quantified with ImageJ software. Results: We first discovered that the expression of Hsp90a was increased in ALDH+ human breast cancer cells. GA or 17-DMAG could suppress ALDH+ breast cancer cells in a dose dependent manner. We are interesting in the