- Email: [email protected]
334 Changes in Bcl-2 Protein Expression Regulate Sensitivity to Chemotherapy Drugs in Multi-cellular Tumour Spheroids
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
of cellular growth both ’in vitro’ and ’in vivo’ models of cancer. Furthermore, GRK2 levels are increased in a very significant proportion of infiltrating ductal carcinoma samples from patients, strongly suggesting that GRK2 is a relevant modulator of tumor survival and progression 331 The Microenvironment Regulates Responses to Hepsin Overexpression in Prostate Cancer Cells R. Wittig1 , S.M. Wittig-Blaich2 , L.A. Kacprzyk2 , M. Bewerunge-Hudler2 , 2 T. Eismann3 , M. Schrader3 , W.S.L. Strauss1 , D. Mertens4 , H. Sultmann ¨ . 1 Institute for Laser Technologies in Medicine and Metrology (ILM) at Ulm 2 University, Biology, Ulm, Germany, German Cancer Research Centre (DKFZ) and National Centre for Tumour Diseases (NCT), Cancer Genome Research Unit, Heidelberg, Germany, 3 University of Ulm, Urology, Ulm, Germany, 4 University of Ulm, Internal Medicine III, Ulm, Germany Introduction: The serine protease hepsin was found to be overexpressed in approximately 90% of prostate cancer cases. Conversely, ectopic overexpression of the enzyme was described to suppress viability and invasive growth in cell lines of prostate, ovarian, and endometrial cancer origin. Although hepsin was demonstrated to contribute to matrix degradation and invasive growth of prostate cancer in vivo, this apparent paradox raised questions about the role of the enzyme in cancer cells. Material and Methods: We employed doxycycline (dox)-inducible overexpression of hepsin in PC3 prostate cancer cells to analyze cellular effects of the enzyme in a quantitative manner. Subsequent to graded induction of the gene in different microenvironments, cells were analyzed for viability, adhesion and the expression / phosphorylation state of signalling proteins using flow cytometry, cell-based assays, and Westernblot analysis. Results and Discussion: We observed a gradual loss of cell adhesion and viability with increased expression levels of hepsin during growth in conventional cell culture dishes and on extracellular matrix (ECM) secreted by PC3 cells. Adhesion and viability was partially restored during growth on ECM secreted by non-tumourigenic RWPE1 cells. During anchorageindependent growth, cell viability did not correlate with expression levels of hepsin. Dephosphorylation of AKT at serine473 correlated tightly to hepsin overexpression in PC3 cells, but could be specifically reversed by growth on RWPE1-derived ECM. Thus, ECM secreted by non-transformed prostate epithelial cells seems to provide a protective microenvironment for hepsinoverexpressing prostate cancer cells. Conclusions: Our findings suggest that expression levels of hepsin must be spatially and temporally restricted for the efficient development of tumours and metastases. Modulation of the microenvironment may therefore be a promising therapeutic option for hepsin-positive prostate tumours. 332 A New CXCR4 Receptor Antagonist − Effects on Cell Growth and Tumor Microenvironment in a Glioma Model S. Cecchetti1 , M.A. Ajmone-Cat1 , L. Mercurio1 , A. Ricci1 , L. Portella2 , P. Amodeo3 , R.M. Vitale4 , S. Scala2 , L. Minghetti1 , G. Carpinelli1 . 1 Istituto ` Department of Cell Biology and Neurosciences, Rome, Superiore di Sanita, Italy, 2 Istituto Nazionale Tumori Fondazione G. Pascale, Immunological Oncology, Naples, Italy, 3 Istituto di Biostrutture e Bioimmagini − Consiglio Nazionale delle Ricerche, Naples, Italy, 4 Istituto di Chimica Biomolecolare − Consiglio Nazionale delle Ricerche, Naples, Italy Background: The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor. Although initially linked with leukocyte trafficking, CXCR4 is expressed in various tumors, where it is strictly related to cell motility, proliferation and survival. In gliomas, CXCR4 activation by the agonist CXCL12 has been demonstrated to regulate the recruitment of the surrounding microglia/macrophages. Therefore, the CXCL12/CXCR4 signaling pathway is emerging as a potential therapeutic target. In this study we evaluated the effects mediated by a new CXCR4 receptor antagonist, the cyclic peptide Phe-7, compared with AMD3100, a well-known CXCR4 inhibitor, on a human glioma cell line (U87MG), and the influence of these drugs on microglial reactivity, by using intracranial xenografts. Material and Method: U87MG cell line was obtained from ATCC and for in vivo experiments cells were stereotaxically implanted into the caudate nucleus of CD1 nude mice then separated in: control group (PBS-treated), AMD3100and Phe-7-treated groups. Treatments were administered twice daily (for 15−20 days) by i.p. injection since U87MG cells implantation. To monitor tumor growth, tumor volume was assessed by MRI analyses, performed at 4.7 T on VARIAN Agilent system (Agilent, Palo Alto, US). Mice were then perfused and brains were fixed and frozen following standard protocols. Sections were stained for different tumor and microglial markers and analyzed by confocal laser scanning microscopy (CLSM). The effects of Phe-7 and AMD3100 on U87MG cells were investigated by using CLSM, flow cytometry, MTT assays and western blot analyses. Results and Discussion: The expression of CXCR4 was strongly downmodulated after treatment of U87MG cells with Phe-7 within 48−72h. Cell viability (MTT) and cell count assays showed a Phe-7-mediated decrease
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(about 40%) in the amount of living/proliferating cells after 72h of incubation, compared to control cells. Moreover, in these experimental conditions neither apoptosis nor necrosis were induced, thus suggesting a possible block of cell proliferation. Immunohistochemistry performed on brain sections of the Phe-7-treated group, showed a strong reduction of CD11b+ cells (microglial/macrophages marker) at the tumor edge, together with an increase of CD11b+ /iNOS+ cells (inducible nitric oxide synthase as a marker for the proinflammatory phenotype of microglia) in the tumor core. While, in the untreated gliomas a strong expression of arginase-1 at the level of endothelial cells was observed, together with a pool of CD11b− /iNOS+ cells in the peritumoral areas. These features were not observed in the Phe-7-treated tumors. Conclusion: These data indicate that the new CXCR4 receptor antagonist, the peptide Phe-7, could modulate glioma-microglia interactions mediated by the CXCR4/CXCL12 signaling axis, thus interfering with microglial recruitment and activation. 333 Myc/p27 Balance in Chronic Lymphocytic Leukemia J.M. Caraballo Otero1 , J.C. Acosta1 , M.T. Gomez-Casares2 , M.A. Cortes3 , A. Batlle4 , M.A. Cuadrado4 , D. Colomer5 , J. Leon1 . 1 University of Cantabria, Molecular Biology, Santander, Spain, 2 Hospital Doctor Negr´ın, Hematology Department, Las Palmas, Spain, 3 Hospital de Laredo, Analysis Service, Laredo, Spain, 4 Hospital Universitario Marques de Valdecilla, Hematology Department, Santander, Spain, 5 Hospital Clinic, Hematophatology Department, Barcelona, Spain Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. CLL is characterized by the accumulation of CD5+ B lymphocytes. However, clinical outcome of CLL may be different. Some patients have an indolent leukemia with long survival while others experience an aggressive disease. Contrary to other human malignancies, the cyclin-dependent kinase inhibitor p27kip1 (p27) has been described to be overexpressed in CLL cells. On the other hand, Myc is an oncogenic transcription factor overexpressed in many human tumours. In recent years, work in several cellular models have demonstrated that Myc, through different mechanisms, antagonize p27 expression and its anti-proliferatice activity. Thus we set out to study the interaction functional between p27 and Myc in B-CLL and whether changes in p27/Myc ratio correlated with the clinic features of the disease. Material and Methods: Protein and mRNA levels of Myc and p27 in CLL patients and Mec1 cells were analyzed by Western Blot and real time RTPCR. We studied p27 and Myc localization by immunofluorescence. Annexin assay was performed to study the fludarabine resistance. SPSS and GraphPad Prism were used for statistical studies. Results: We studied expression levels of p27 and Myc in more than 100 CLL patients using peripheral blood, tonsil and CD19+ lymphocytes as control. p27 and Myc levels were inversely correlated. Thus, the ratio between p27 (overexpressed) and Myc (downregulated) appears inverted in CLL with respect to the other tumours so far known. Moreover, low p27 and high Myc expression correlated with the expression of Skp2, a subunit of ubiquitin protein ligase complex SCF which is the main responsible for p27 degradation. Skp2 is a Myc target gene and promotes the degradation of p27, suggesting a pathway Myc–Skp2–p27 in CLL. To investigate why p27 is expressed in CLL we overexpressed in a CLL-derived cell line (Mec1) and observed that high levels of p27 provide resistance to fludarabine treatment. In fact, p27 expression was strongly correlated with Bcl2 levels, an antiapoptotic protein, in CLL samples. Conclusions: Our data provide new insights into the p27/Myc balance in CLL cells. The high percentage of p27 overexpressed patients suggests an important function in CLL, causing low levels of Myc and increasing resistance to apoptosis, likely through collaboration with Bcl2. These results suggest a functional explanation so as why p27 is overexpressed in CLL. 334 Changes in Bcl-2 Protein Expression Regulate Sensitivity to Chemotherapy Drugs in Multi-cellular Tumour Spheroids I. Barnawi1 . 1 University of Reading, Biological Science, Reading, United Kingdom Most chemotherapy drugs work through the induction of apoptosis in tumour cells. Mutations in apoptotic pathways, notably p53 and Bcl-2, are common in cancer and associated with increased resistance to apoptosis and therefore to chemotherapy. We have compared the sensitivity of two cell types, A172 human glioblastoma cells and MDA-MB-231 human breast cancer cells, for their sensitivity to the chemotherapy drugs cisplatin, doxorubicin, gemcitabine, vincristine and temozolomide. We have then determined the effect of growing these cells as two-dimensional monolayers or as three-dimensional multicellular spheroids on their sensitivity to these drugs and correlated changes in apoptotic sensitivity with changes in the expression profile of members of the Bcl-2 family of proteins. We found in both cell lines that growing the cells as multi-cellular spheroids significantly enhanced their resistance to apoptosis induced by each of the chemotherapy drugs when compared to
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european journal of cancer 48, suppl. 5 (2012) S25–S288
growing the cells as monolayers. In many cases cells in the tumour spheroids became completely resistant to apoptosis. We also found that growing cells in 3D culture by embedding them in Matrigel also increased their apoptosis resistance to chemotherapy drugs, although not to the same extent as when the cells were grown in multi-cellular spheroids. We found that growing cells in spheroids led to significant changes in the expression of the Bcl-2 family of proteins, including increases in expression of Bcl-2 and Bik and decreases in expression of Bid, Bim and Bok. In conclusion tumour cells grown in spheroids have altered expression of Bcl-2 molecules which may account for their increased resistance to apoptosis.
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335 Inhibition of P90RSK Sensitizes Ovarian Cancer Cells to Apoptosis
IHC staining for snail 1 significantly (p < 0.001) than patients with preserved histological grades (grades 2 and 3) and also patients with a diagnosis of BPH (p < 0.0001). On the other hand, cells with increased metastatic potential (PC3) had significantly higher means for DOI (p = 0.017) than those with less ability tumorigenic (LNCaP). Similar results were obtained by performing western blot with protein extracts from the same cell lines. Conclusions: Snail 1 is overexpressed in PCa and proves to be a good marker of aggressiveness, presenting a good index of correlation with histologic grade of Gleason. This quality makes it a promising tool to differentiate those more aggressive cancers. However, it is still necessary to correlate their expression with clinical variables such as survival and biochemical recurrence in patients with PCa. Grant: Nº 1110269 FONDECYT.
E. Torchiaro1 , M. Olivero1 , S. Pavan1 , M.F. Di renzo1 . 1 University of Torino School Medicine, Oncological Sciences, Candiolo (Torino), Italy
337 Scribble Deregulation of MAPK Impairs Normal Mammary Development and Promotes Tumourigenesis
Ovarian cancer is the leading cause of death among gynecologic cancers. In ovarian cancer, apoptosis is impaired by the anti-apoptotic action of several oncogenes, including growth factor receptors and other kinases. The MET oncogene is overexpressed in a consistent fraction of ovarian cancers. Its ligand HGF has been found in ovarian cancer ascitic fluids and in fluid of benign and malignant ovarian cysts. However, it was previously shown that HGF might sensitize ovarian carcinoma cells to the chemotherapeutics agents. We have carried out the search for signal transducers of the proapoptotic signalling triggered by the MET receptor. We found that long-term MET activation switched off the activation of the p90RSK triggered by apoptotic stimuli. A crucial role of this protein family has been reported in oncogenesis and tumour progression, so that p90RSK is considered an attractive therapeutic target for cancer treatment. Material and Methods: Western blot analysis was carried out to evaluate the phosphorylation of ERK1/2 and p90RSK in cells committed to death by the treament with HGF for 48 hours with or without cisplatin. Cell survival after exposure to the RSK specific and potent inhbitor BI-D1870 was evaluated by staining cells with Annexin V/DAPI in a panel of ovarian cancer cell lines (SKOV-3, IGROV-1, OV-90, TOV21G, OAW42, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8) in HUVEC primary cell line, immortalized human cell lines (MCF10A, 293T), and other tumor cell lines (GTL16, HELA). Results: Treatment of ovarian cancer cells with HGF for 48 hours abrogated the phophorylation of p90RSK at the Ser380 elicited by cisplatin. The simultaneous loss of the phosphorylation of its substrate BAD showed that the kinase activity and thus likely the anti-apoptotic activity was impaired. The switch off of the p90RSK in ovarian cancer cells committed to death also suggested that in these cells p90RSK might be indispensable for survival. BI-D1870 did not affect the viability of primary and immortalized nontransformed cell lines. On the contrary, the panel of ovarian cancer cell lines could be subdivided in three different groups according to their BI-D1870 susceptibility as resistant, intermediate and sensitive cell lines. Moreover, a further decreased viability was observed when sensitive and intermediate resistant cells were pretreated with BI-D1870 for 2 hours and then treated with cisplatin for 48 hours. Conclusion: In conclusion, p90RSK could be regarded as a suitable target for therapy in either mono-therapy or in combination with chemotherapeutic drugs. 336 Expression of Snail 1 in the Progression of Prostate Cancer J. Fulla1 , C. Poblete1 , E.A. Castellon ´ 1 , H.R. Contreras1 . 1 Instituto de Ciencias Biomedicas. Universidad de Chile, Programa de Fisiologia, Santiago, Chile Background: During the progression of the tumor cells lose their accessions intercellular and suffer from profound changes in its phenotype, which are known under the concept of epithelial-mesenchymal transition (TEM). In this process, snail 1 plays a central role in the acquisition of a phenotype mesenquimatico carrying the cells to acquire a invasive capability. The present work has as objective correlate the expression of snail 1 with the degree of malignancy of prostate cancer (PCa) evaluated according Gleason score. Materials and Methods: We obtained biopsies of patients with PCa and benign prostatic hyperplasia (BPH) as a control non-tumoral, which were revised by a pathologist. Then they built Microarrays of tissue (MTA) in which immunohistochemistry was performed for the transcription factor Snail 1. Photographs were taken of the samples in 400× magnification and quantitative immunohistochemistry was carried out by calculating the density integrated optics (DOI) through the use of the Image program J (v1.37c). In addition, we studied the expression of snail1 in cell lines from prostate cancer with low and high metastatic potential (LNCaP and PC3 respectively). Data were tabulated and analyzed using SPSS v17.0. Were considered significant at p < 0.05. Results: We studied a total of 36 patients with a diagnosis of PCa and eight with BPH. DOI values for immunohistochemical staining of snail 1, showed a normal distribution (KS test p = 0.689) with a correlation index between the DOI and the Gleason histological grade of 0.734 (Pearson test). Patients with a Gleason score of more undifferentiated (grades 4 and 5) had average DOI
N. Godde1 , J.M. Sheridan2 , L.K. Smith1 , H.B. Pearson1 , J.E. Visvader2 , P.O. Humbert1 . 1 Peter MacCallum Cancer Centre, Cell Cycle & Cancer Genetics Laboratory, Melbourne, Australia, 2 The Walter and Eliza Hall Institute of Medical Research, Stem Cells and Cancer, Melbourne, Australia Background: Loss of polarity is a defining feature of breast cancer progression however how polarity loss contributes to this process is unknown. Normal mammary epithelial cells are highly polarized, exhibit specialized cell-cell contacts and are attached to the basement membrane, whereas premalignant lesions are characterised by abnormal cell layers and luminal filling. Scribble is a key regulator of polarity. In Drosophila, Scribble acts as a neoplastic tumour suppressor where its loss induces tumourigenic growth and can cooperate with oncogenes such as activated Ras to promote invasion and metastatic-like spread. We have characterised a role for Scribble as a tumour suppressor in MCF10A mammary epithelial cells where Scribble regulates the MAPK pathway and restricts Ras-mediated invasion [1]. Materials and Methods: Since Scribble knock out mice are neonatal lethal, we have employed Cre-LoxP technology to specifically deplete Scribble within the mammary gland by crossing Scribble floxed mice with MMTV-Cre transgenic mice. Several developmental timepoints were then analysed to assess ductal morphogenesis, alveolae differentiation and mammary tumour formation. Results: These mutant mice display disorganized multilayered mammary ducts where the luminal space is absent due to an excess of luminal epithelial cells with increased ERK activation. Here we present our characterisation of this mouse model which reveals that Scribble loss in the mammary gland subverts normal tissue homeostasis to drive hyperplasia and mimic early phases of breast cancer. In addition, Scribble loss increases incidence and pathological grade of mammary tumours. Conclusions: This study provides important insights into the role of polarity in cancer and provides analysis of Scribble loss from morphogenesis to multiple steps of tumourigenesis. Reference(s) [1] Dow LE, et al, Loss of human Scribble cooperates with H-Ras to promote cell invasion through deregulation of MAPK signalling. Oncogene 2008. 27: 5988–6001. 338 The HER2 Amplicon Includes Several Genes Required for the Growth and Survival of HER2 Positive Breast Cancer Cells V. Hongisto1 , K. Kleivi Sahlberg2 , H. Edgren3 , R. Makel ¨ a¨ 1 , E. Due2 , H.K. Moen Vollan2 , N. Sahlberg1 , A.L. Børresen-Dale2 , M. Peral ¨ a¨ 1 , O. Kallioniemi3 . 1 VTT Technical Research Centre of Finland, Medical Biotechnology, Turku, Finland, 2 Oslo University Hospital, Department of Genetics, Oslo, Norway, 3 University of Helsinki, Institute for Molecular Medicine Finland, Helsinki, Finland Background: About 20% of breast cancers are characterized by amplification and overexpression of the HER2 oncogene. Although significant progress has been achieved for treating such patients with HER2 inhibitor Trastuzumab, more than half of the patients respond poorly or become resistant to the treatment. Since the HER2 amplicon at 17q12 contains multiple genes, we have systematically explored the role of the HER2 co-amplified genes in breast cancer cell growth and their relation to Trastuzumab resistance. Materials and Methods: We integrated aCGH data of the HER2 amplicon from 71 HER2 positive breast tumors and 10 cell lines with systematic functional RNA interference analysis of 23 core amplicon genes of Trastuzumab responding and non-responding HER2 positive breast cancer cells. Several key signaling components were used as screening readouts by utilizing protein lysate microarray technology. Results: Silencing of HER2 caused a greater growth arrest and apoptosis in the responding compared to the non-responding cell lines, indicating that the resistant cells are inherently less dependent on the HER2 pathway. Several other genes in the amplicon also showed clear effects on survival when silenced; indicating that expression of HER2 co-amplified genes may be needed to sustain the growth of breast cancer cells. Importantly, co-silencing
european journal of cancer 48, suppl. 5 (2012) S25–S288
of cellular growth both ’in vitro’ and ’in vivo’ models of cancer. Furthermore, GRK2 levels are increased in a very significant proportion of infiltrating ductal carcinoma samples from patients, strongly suggesting that GRK2 is a relevant modulator of tumor survival and progression 331 The Microenvironment Regulates Responses to Hepsin Overexpression in Prostate Cancer Cells R. Wittig1 , S.M. Wittig-Blaich2 , L.A. Kacprzyk2 , M. Bewerunge-Hudler2 , 2 T. Eismann3 , M. Schrader3 , W.S.L. Strauss1 , D. Mertens4 , H. Sultmann ¨ . 1 Institute for Laser Technologies in Medicine and Metrology (ILM) at Ulm 2 University, Biology, Ulm, Germany, German Cancer Research Centre (DKFZ) and National Centre for Tumour Diseases (NCT), Cancer Genome Research Unit, Heidelberg, Germany, 3 University of Ulm, Urology, Ulm, Germany, 4 University of Ulm, Internal Medicine III, Ulm, Germany Introduction: The serine protease hepsin was found to be overexpressed in approximately 90% of prostate cancer cases. Conversely, ectopic overexpression of the enzyme was described to suppress viability and invasive growth in cell lines of prostate, ovarian, and endometrial cancer origin. Although hepsin was demonstrated to contribute to matrix degradation and invasive growth of prostate cancer in vivo, this apparent paradox raised questions about the role of the enzyme in cancer cells. Material and Methods: We employed doxycycline (dox)-inducible overexpression of hepsin in PC3 prostate cancer cells to analyze cellular effects of the enzyme in a quantitative manner. Subsequent to graded induction of the gene in different microenvironments, cells were analyzed for viability, adhesion and the expression / phosphorylation state of signalling proteins using flow cytometry, cell-based assays, and Westernblot analysis. Results and Discussion: We observed a gradual loss of cell adhesion and viability with increased expression levels of hepsin during growth in conventional cell culture dishes and on extracellular matrix (ECM) secreted by PC3 cells. Adhesion and viability was partially restored during growth on ECM secreted by non-tumourigenic RWPE1 cells. During anchorageindependent growth, cell viability did not correlate with expression levels of hepsin. Dephosphorylation of AKT at serine473 correlated tightly to hepsin overexpression in PC3 cells, but could be specifically reversed by growth on RWPE1-derived ECM. Thus, ECM secreted by non-transformed prostate epithelial cells seems to provide a protective microenvironment for hepsinoverexpressing prostate cancer cells. Conclusions: Our findings suggest that expression levels of hepsin must be spatially and temporally restricted for the efficient development of tumours and metastases. Modulation of the microenvironment may therefore be a promising therapeutic option for hepsin-positive prostate tumours. 332 A New CXCR4 Receptor Antagonist − Effects on Cell Growth and Tumor Microenvironment in a Glioma Model S. Cecchetti1 , M.A. Ajmone-Cat1 , L. Mercurio1 , A. Ricci1 , L. Portella2 , P. Amodeo3 , R.M. Vitale4 , S. Scala2 , L. Minghetti1 , G. Carpinelli1 . 1 Istituto ` Department of Cell Biology and Neurosciences, Rome, Superiore di Sanita, Italy, 2 Istituto Nazionale Tumori Fondazione G. Pascale, Immunological Oncology, Naples, Italy, 3 Istituto di Biostrutture e Bioimmagini − Consiglio Nazionale delle Ricerche, Naples, Italy, 4 Istituto di Chimica Biomolecolare − Consiglio Nazionale delle Ricerche, Naples, Italy Background: The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor. Although initially linked with leukocyte trafficking, CXCR4 is expressed in various tumors, where it is strictly related to cell motility, proliferation and survival. In gliomas, CXCR4 activation by the agonist CXCL12 has been demonstrated to regulate the recruitment of the surrounding microglia/macrophages. Therefore, the CXCL12/CXCR4 signaling pathway is emerging as a potential therapeutic target. In this study we evaluated the effects mediated by a new CXCR4 receptor antagonist, the cyclic peptide Phe-7, compared with AMD3100, a well-known CXCR4 inhibitor, on a human glioma cell line (U87MG), and the influence of these drugs on microglial reactivity, by using intracranial xenografts. Material and Method: U87MG cell line was obtained from ATCC and for in vivo experiments cells were stereotaxically implanted into the caudate nucleus of CD1 nude mice then separated in: control group (PBS-treated), AMD3100and Phe-7-treated groups. Treatments were administered twice daily (for 15−20 days) by i.p. injection since U87MG cells implantation. To monitor tumor growth, tumor volume was assessed by MRI analyses, performed at 4.7 T on VARIAN Agilent system (Agilent, Palo Alto, US). Mice were then perfused and brains were fixed and frozen following standard protocols. Sections were stained for different tumor and microglial markers and analyzed by confocal laser scanning microscopy (CLSM). The effects of Phe-7 and AMD3100 on U87MG cells were investigated by using CLSM, flow cytometry, MTT assays and western blot analyses. Results and Discussion: The expression of CXCR4 was strongly downmodulated after treatment of U87MG cells with Phe-7 within 48−72h. Cell viability (MTT) and cell count assays showed a Phe-7-mediated decrease
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(about 40%) in the amount of living/proliferating cells after 72h of incubation, compared to control cells. Moreover, in these experimental conditions neither apoptosis nor necrosis were induced, thus suggesting a possible block of cell proliferation. Immunohistochemistry performed on brain sections of the Phe-7-treated group, showed a strong reduction of CD11b+ cells (microglial/macrophages marker) at the tumor edge, together with an increase of CD11b+ /iNOS+ cells (inducible nitric oxide synthase as a marker for the proinflammatory phenotype of microglia) in the tumor core. While, in the untreated gliomas a strong expression of arginase-1 at the level of endothelial cells was observed, together with a pool of CD11b− /iNOS+ cells in the peritumoral areas. These features were not observed in the Phe-7-treated tumors. Conclusion: These data indicate that the new CXCR4 receptor antagonist, the peptide Phe-7, could modulate glioma-microglia interactions mediated by the CXCR4/CXCL12 signaling axis, thus interfering with microglial recruitment and activation. 333 Myc/p27 Balance in Chronic Lymphocytic Leukemia J.M. Caraballo Otero1 , J.C. Acosta1 , M.T. Gomez-Casares2 , M.A. Cortes3 , A. Batlle4 , M.A. Cuadrado4 , D. Colomer5 , J. Leon1 . 1 University of Cantabria, Molecular Biology, Santander, Spain, 2 Hospital Doctor Negr´ın, Hematology Department, Las Palmas, Spain, 3 Hospital de Laredo, Analysis Service, Laredo, Spain, 4 Hospital Universitario Marques de Valdecilla, Hematology Department, Santander, Spain, 5 Hospital Clinic, Hematophatology Department, Barcelona, Spain Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in the western world. CLL is characterized by the accumulation of CD5+ B lymphocytes. However, clinical outcome of CLL may be different. Some patients have an indolent leukemia with long survival while others experience an aggressive disease. Contrary to other human malignancies, the cyclin-dependent kinase inhibitor p27kip1 (p27) has been described to be overexpressed in CLL cells. On the other hand, Myc is an oncogenic transcription factor overexpressed in many human tumours. In recent years, work in several cellular models have demonstrated that Myc, through different mechanisms, antagonize p27 expression and its anti-proliferatice activity. Thus we set out to study the interaction functional between p27 and Myc in B-CLL and whether changes in p27/Myc ratio correlated with the clinic features of the disease. Material and Methods: Protein and mRNA levels of Myc and p27 in CLL patients and Mec1 cells were analyzed by Western Blot and real time RTPCR. We studied p27 and Myc localization by immunofluorescence. Annexin assay was performed to study the fludarabine resistance. SPSS and GraphPad Prism were used for statistical studies. Results: We studied expression levels of p27 and Myc in more than 100 CLL patients using peripheral blood, tonsil and CD19+ lymphocytes as control. p27 and Myc levels were inversely correlated. Thus, the ratio between p27 (overexpressed) and Myc (downregulated) appears inverted in CLL with respect to the other tumours so far known. Moreover, low p27 and high Myc expression correlated with the expression of Skp2, a subunit of ubiquitin protein ligase complex SCF which is the main responsible for p27 degradation. Skp2 is a Myc target gene and promotes the degradation of p27, suggesting a pathway Myc–Skp2–p27 in CLL. To investigate why p27 is expressed in CLL we overexpressed in a CLL-derived cell line (Mec1) and observed that high levels of p27 provide resistance to fludarabine treatment. In fact, p27 expression was strongly correlated with Bcl2 levels, an antiapoptotic protein, in CLL samples. Conclusions: Our data provide new insights into the p27/Myc balance in CLL cells. The high percentage of p27 overexpressed patients suggests an important function in CLL, causing low levels of Myc and increasing resistance to apoptosis, likely through collaboration with Bcl2. These results suggest a functional explanation so as why p27 is overexpressed in CLL. 334 Changes in Bcl-2 Protein Expression Regulate Sensitivity to Chemotherapy Drugs in Multi-cellular Tumour Spheroids I. Barnawi1 . 1 University of Reading, Biological Science, Reading, United Kingdom Most chemotherapy drugs work through the induction of apoptosis in tumour cells. Mutations in apoptotic pathways, notably p53 and Bcl-2, are common in cancer and associated with increased resistance to apoptosis and therefore to chemotherapy. We have compared the sensitivity of two cell types, A172 human glioblastoma cells and MDA-MB-231 human breast cancer cells, for their sensitivity to the chemotherapy drugs cisplatin, doxorubicin, gemcitabine, vincristine and temozolomide. We have then determined the effect of growing these cells as two-dimensional monolayers or as three-dimensional multicellular spheroids on their sensitivity to these drugs and correlated changes in apoptotic sensitivity with changes in the expression profile of members of the Bcl-2 family of proteins. We found in both cell lines that growing the cells as multi-cellular spheroids significantly enhanced their resistance to apoptosis induced by each of the chemotherapy drugs when compared to
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european journal of cancer 48, suppl. 5 (2012) S25–S288
growing the cells as monolayers. In many cases cells in the tumour spheroids became completely resistant to apoptosis. We also found that growing cells in 3D culture by embedding them in Matrigel also increased their apoptosis resistance to chemotherapy drugs, although not to the same extent as when the cells were grown in multi-cellular spheroids. We found that growing cells in spheroids led to significant changes in the expression of the Bcl-2 family of proteins, including increases in expression of Bcl-2 and Bik and decreases in expression of Bid, Bim and Bok. In conclusion tumour cells grown in spheroids have altered expression of Bcl-2 molecules which may account for their increased resistance to apoptosis.
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335 Inhibition of P90RSK Sensitizes Ovarian Cancer Cells to Apoptosis
IHC staining for snail 1 significantly (p < 0.001) than patients with preserved histological grades (grades 2 and 3) and also patients with a diagnosis of BPH (p < 0.0001). On the other hand, cells with increased metastatic potential (PC3) had significantly higher means for DOI (p = 0.017) than those with less ability tumorigenic (LNCaP). Similar results were obtained by performing western blot with protein extracts from the same cell lines. Conclusions: Snail 1 is overexpressed in PCa and proves to be a good marker of aggressiveness, presenting a good index of correlation with histologic grade of Gleason. This quality makes it a promising tool to differentiate those more aggressive cancers. However, it is still necessary to correlate their expression with clinical variables such as survival and biochemical recurrence in patients with PCa. Grant: Nº 1110269 FONDECYT.
E. Torchiaro1 , M. Olivero1 , S. Pavan1 , M.F. Di renzo1 . 1 University of Torino School Medicine, Oncological Sciences, Candiolo (Torino), Italy
337 Scribble Deregulation of MAPK Impairs Normal Mammary Development and Promotes Tumourigenesis
Ovarian cancer is the leading cause of death among gynecologic cancers. In ovarian cancer, apoptosis is impaired by the anti-apoptotic action of several oncogenes, including growth factor receptors and other kinases. The MET oncogene is overexpressed in a consistent fraction of ovarian cancers. Its ligand HGF has been found in ovarian cancer ascitic fluids and in fluid of benign and malignant ovarian cysts. However, it was previously shown that HGF might sensitize ovarian carcinoma cells to the chemotherapeutics agents. We have carried out the search for signal transducers of the proapoptotic signalling triggered by the MET receptor. We found that long-term MET activation switched off the activation of the p90RSK triggered by apoptotic stimuli. A crucial role of this protein family has been reported in oncogenesis and tumour progression, so that p90RSK is considered an attractive therapeutic target for cancer treatment. Material and Methods: Western blot analysis was carried out to evaluate the phosphorylation of ERK1/2 and p90RSK in cells committed to death by the treament with HGF for 48 hours with or without cisplatin. Cell survival after exposure to the RSK specific and potent inhbitor BI-D1870 was evaluated by staining cells with Annexin V/DAPI in a panel of ovarian cancer cell lines (SKOV-3, IGROV-1, OV-90, TOV21G, OAW42, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8) in HUVEC primary cell line, immortalized human cell lines (MCF10A, 293T), and other tumor cell lines (GTL16, HELA). Results: Treatment of ovarian cancer cells with HGF for 48 hours abrogated the phophorylation of p90RSK at the Ser380 elicited by cisplatin. The simultaneous loss of the phosphorylation of its substrate BAD showed that the kinase activity and thus likely the anti-apoptotic activity was impaired. The switch off of the p90RSK in ovarian cancer cells committed to death also suggested that in these cells p90RSK might be indispensable for survival. BI-D1870 did not affect the viability of primary and immortalized nontransformed cell lines. On the contrary, the panel of ovarian cancer cell lines could be subdivided in three different groups according to their BI-D1870 susceptibility as resistant, intermediate and sensitive cell lines. Moreover, a further decreased viability was observed when sensitive and intermediate resistant cells were pretreated with BI-D1870 for 2 hours and then treated with cisplatin for 48 hours. Conclusion: In conclusion, p90RSK could be regarded as a suitable target for therapy in either mono-therapy or in combination with chemotherapeutic drugs. 336 Expression of Snail 1 in the Progression of Prostate Cancer J. Fulla1 , C. Poblete1 , E.A. Castellon ´ 1 , H.R. Contreras1 . 1 Instituto de Ciencias Biomedicas. Universidad de Chile, Programa de Fisiologia, Santiago, Chile Background: During the progression of the tumor cells lose their accessions intercellular and suffer from profound changes in its phenotype, which are known under the concept of epithelial-mesenchymal transition (TEM). In this process, snail 1 plays a central role in the acquisition of a phenotype mesenquimatico carrying the cells to acquire a invasive capability. The present work has as objective correlate the expression of snail 1 with the degree of malignancy of prostate cancer (PCa) evaluated according Gleason score. Materials and Methods: We obtained biopsies of patients with PCa and benign prostatic hyperplasia (BPH) as a control non-tumoral, which were revised by a pathologist. Then they built Microarrays of tissue (MTA) in which immunohistochemistry was performed for the transcription factor Snail 1. Photographs were taken of the samples in 400× magnification and quantitative immunohistochemistry was carried out by calculating the density integrated optics (DOI) through the use of the Image program J (v1.37c). In addition, we studied the expression of snail1 in cell lines from prostate cancer with low and high metastatic potential (LNCaP and PC3 respectively). Data were tabulated and analyzed using SPSS v17.0. Were considered significant at p < 0.05. Results: We studied a total of 36 patients with a diagnosis of PCa and eight with BPH. DOI values for immunohistochemical staining of snail 1, showed a normal distribution (KS test p = 0.689) with a correlation index between the DOI and the Gleason histological grade of 0.734 (Pearson test). Patients with a Gleason score of more undifferentiated (grades 4 and 5) had average DOI
N. Godde1 , J.M. Sheridan2 , L.K. Smith1 , H.B. Pearson1 , J.E. Visvader2 , P.O. Humbert1 . 1 Peter MacCallum Cancer Centre, Cell Cycle & Cancer Genetics Laboratory, Melbourne, Australia, 2 The Walter and Eliza Hall Institute of Medical Research, Stem Cells and Cancer, Melbourne, Australia Background: Loss of polarity is a defining feature of breast cancer progression however how polarity loss contributes to this process is unknown. Normal mammary epithelial cells are highly polarized, exhibit specialized cell-cell contacts and are attached to the basement membrane, whereas premalignant lesions are characterised by abnormal cell layers and luminal filling. Scribble is a key regulator of polarity. In Drosophila, Scribble acts as a neoplastic tumour suppressor where its loss induces tumourigenic growth and can cooperate with oncogenes such as activated Ras to promote invasion and metastatic-like spread. We have characterised a role for Scribble as a tumour suppressor in MCF10A mammary epithelial cells where Scribble regulates the MAPK pathway and restricts Ras-mediated invasion [1]. Materials and Methods: Since Scribble knock out mice are neonatal lethal, we have employed Cre-LoxP technology to specifically deplete Scribble within the mammary gland by crossing Scribble floxed mice with MMTV-Cre transgenic mice. Several developmental timepoints were then analysed to assess ductal morphogenesis, alveolae differentiation and mammary tumour formation. Results: These mutant mice display disorganized multilayered mammary ducts where the luminal space is absent due to an excess of luminal epithelial cells with increased ERK activation. Here we present our characterisation of this mouse model which reveals that Scribble loss in the mammary gland subverts normal tissue homeostasis to drive hyperplasia and mimic early phases of breast cancer. In addition, Scribble loss increases incidence and pathological grade of mammary tumours. Conclusions: This study provides important insights into the role of polarity in cancer and provides analysis of Scribble loss from morphogenesis to multiple steps of tumourigenesis. Reference(s) [1] Dow LE, et al, Loss of human Scribble cooperates with H-Ras to promote cell invasion through deregulation of MAPK signalling. Oncogene 2008. 27: 5988–6001. 338 The HER2 Amplicon Includes Several Genes Required for the Growth and Survival of HER2 Positive Breast Cancer Cells V. Hongisto1 , K. Kleivi Sahlberg2 , H. Edgren3 , R. Makel ¨ a¨ 1 , E. Due2 , H.K. Moen Vollan2 , N. Sahlberg1 , A.L. Børresen-Dale2 , M. Peral ¨ a¨ 1 , O. Kallioniemi3 . 1 VTT Technical Research Centre of Finland, Medical Biotechnology, Turku, Finland, 2 Oslo University Hospital, Department of Genetics, Oslo, Norway, 3 University of Helsinki, Institute for Molecular Medicine Finland, Helsinki, Finland Background: About 20% of breast cancers are characterized by amplification and overexpression of the HER2 oncogene. Although significant progress has been achieved for treating such patients with HER2 inhibitor Trastuzumab, more than half of the patients respond poorly or become resistant to the treatment. Since the HER2 amplicon at 17q12 contains multiple genes, we have systematically explored the role of the HER2 co-amplified genes in breast cancer cell growth and their relation to Trastuzumab resistance. Materials and Methods: We integrated aCGH data of the HER2 amplicon from 71 HER2 positive breast tumors and 10 cell lines with systematic functional RNA interference analysis of 23 core amplicon genes of Trastuzumab responding and non-responding HER2 positive breast cancer cells. Several key signaling components were used as screening readouts by utilizing protein lysate microarray technology. Results: Silencing of HER2 caused a greater growth arrest and apoptosis in the responding compared to the non-responding cell lines, indicating that the resistant cells are inherently less dependent on the HER2 pathway. Several other genes in the amplicon also showed clear effects on survival when silenced; indicating that expression of HER2 co-amplified genes may be needed to sustain the growth of breast cancer cells. Importantly, co-silencing