- Email: [email protected]
370 Suppression of Breast Cancer Metastasis by BMP4
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
Although advanced prostate cancer is typically regarded as not expressing detectable p63 levels, we have unexpectedly found low expression levels of the DNp63a variant in the metastatic PC3 cell line. Surprisingly, expression of DNp63a increases when cells are grown in stem cell-favoring conditions. Furthermore, FACS purification and analysis of the CD44/CD133-positive cancer stem cell population reveals that expression of DNp63a is highest in these cells. Conclusion: Together, this work describes how DNp63a is a critical mediator of normal prostate stem cell function. Surprisingly, we have also uncovered expression of DNp63a in a subset of cancer stem cells in prostate cancer that suggests that tumor cells may utilize the stem cell-promoting function of p63 to facilitate tumor maintenance and metastatic colonization. 366 Phosphatidylcholine-specific Phospholipase C as a Target to Manipulate CXCR4-CXCL12 Signaling Pathway in Human Lymphoblastoid Cells S. Cecchetti1 , A. Ricci1 , E. Iorio1 , L. Paris1 , M.E. Pisanu1 , L. Portella2 , ` Department S. Scala2 , G. Carpinelli1 , F. Podo1 . 1 Istituto Superiore di Sanita, of Cell Biology and Neurosciences, Rome, Italy, 2 Istituto Nazionale Tumori Fondazione G. Pascale, Immunological Oncology, Naples, Italy Background: Chemokines, a family of small pro-inflammatory cytokines, and their receptors regulate a variety of immune responses. Cancer cells are known to exploit signaling through chemokine receptors for key steps of tumor initiation and progression. In particular, CXCR4 receptor overexpression is detected in cancers of different origins. The functional consequence is a sustained CXCR4 activation by the CXCL12 chemokine which regulates tumor proliferation, survival, chemotaxis and migration. We showed that phosphatidylcholine (PC)-specific phospholipase C (PCPLC) is able to modulate the membrane expression, and hence the signal transduction, of specific cell receptors such as CD16 in NK cells and HER2 in breast cancer cells, interfering with cell-signaling dependent functions. In this study we investigated the alterations of CXCR4 expression and PC metabolism induced by the PC-PLC inhibition in a human T-lymphoblastoid cell line (CEM). Material and Method: Membrane localization and direct interaction of PC-PLC with CXCR4 were investigated by confocal laser scanning microscopy (CLSM), flow cytometry and immunoprecipitation techniques. The effects induced by a selective PLC inhibitor (D609) on CXCR4 membrane expression and its overall contents were evaluated in comparison with those of a well-known CXCR4 antagonist, AMD3100. MRS experiments were performed on ethanolic extracts of CEM cells, previously exposed to the D609 until 24h, using a Bruker Avance 400 spectrometer equipped with a 1H-X multinuclear inverse probehead. Results and Discussion: PC-PLC was preferentially localized on the membrane surface of CEM cells. Fine analysis of CXCR4 and PC-PLC subcellular distribution demonstrated that the two proteins are physically associated and partially accumulated in lipid rafts.D609 induced a strong downmodulation of the CXCR4 receptor from the plasma membrane within 5 h. At this time point most of CXCR4 molecules internalized into early and late endosomes and a substantial amount of the receptor co-localized with lysosomes, indicating that PC-PLC deactivation leads to CXCR4 degradation, as also confirmed by Western blot experiments. Under these conditions, MRS experiments provided direct evidence of PC-PLC inhibition showing an about 42% reduction of phosphocholine, direct product of PC-PLC activity (in agreement with enzyme assays) in cells exposed for 24h to D609. Moreover, PC-PLC appeared to negatively regulate the CXCR4-CXCL12 signaling pathway. Indeed, inhibition of this phospholipase induced inactivation of both ERK1/2 and AKT, even in the presence of the CXCL12 stimulus, this effect being higher than that observed for AMD3100 only. Conclusion: Altogether, these data indicate that PC-PLC could play an important role in regulating the CXCR4-CXCL12 signaling pathway and suggest that, by manipulating this axis, PC-PLC may represent a potential therapeutic target for cancer treatment. 367 N-glycan Biosynthesis Inhibitors Induce in Vitro Anticancer Activity in Colorectal Cancer Cells J. de Freitas1 , L.G. Bastos2 , C.A. Freire-Neto2 , B.D.R. D’Aguiar-Silva3 , ˆ E.S. Abdelhay3 , J.A. Morgado-D´ıaz2 . 1 Instituto Nacional de Cancer / UERJ, ˜ de Biologia Celular / PPGB, Rio de Janeiro, Brazil, 2 Instituto Nacional Divisao ˆ ˜ de Biologia Celular, Rio de Janeiro, Brazil, 3 Instituto de Cancer, Divisao ´ ˆ Nacional de Cancer, Centro de Transplante de Medula Ossea, Rio de Janeiro, Brazil Background: During malignant transformation, changes in the expression profile of glycans may be involved in a variety of events, including the loss of cell-cell and cell-matrix adhesion, migration, invasion, and evasion of apoptosis. Therefore, modulation of glycan expression with drugs has promising therapeutic potential for various cancer types. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors (swainsonine and tunicamycin) in cells derived from colorectal
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cancer. We also examined whether these inhibitors are able to induce radiosensitization and toxicity when used in combination with cisplatin or irinotecan, two current anticancer drugs. Material and Method: Three colorectal cancer cell lines were used: Caco-2, HCT-116, and HT-29. Cells were treated with drug concentrations ranging from 1 to 8 mg/mL for swainsonine and 0.25 to 2 mg/mL for tunicamycin. We examined the effects of swainsonine and tunicamycin treatment on cytotoxicity, migration, invasion, anchorage-dependent and anchorage-independent colony formation, and radiosensitivity, after 24, 48, 72, and 96 h. Results and Discussion: Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in undifferentiated HCT-116 colon cancer cells, whereas swainsonine only inhibits cell migration. We also observed that tunicamycin, but not swainsonine, caused radiosensitivity in HCT-116 cells. Moreover, the combination of swainsonine with cisplatin or irinotecan enhanced their toxicity in HCT-116 cells, while the combination of tunicamycin with these drugs had no effect. Conclusion: Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for colorectal cancer treatment because inhibition of this process induced anticancer activity in vitro. 369 Anticancer Immune Responses Induced by Chemotherapeutic Agents Depends on Autophagy M. Michaud1 , A.Q. Sukkurwala1 , I. Martins1 , S. Adjemian1 , G. Kroemer1 . 1 Institut Gustave Roussy, U848, Villejuif, France Immunogenic cell death can be obtained by using antineoplastic chemotherapies and provokes an anticancer immune response, thus increasing the efficiency of the treatment. Here, we demonstrate that autophagy, often disabled in cancer, is required for immunogenicity of chemotherapy-induced cell death. We observed that autophagy-competent, but not autophagydeficient cancers attracted dendritic cells and T lymphocytes into the tumor bed in response to chemotherapy. Moreover, autophagy inhibition decreased the amount of adenosine triphosphate (ATP) released from dying tumor cells. This effect could be reversed by inhibiting extracellular ATP-degrading enzymes, which led to increased pericellular ATP in autophagy-deficient tumors, and thus, to the re-establishment of the recruitment of immune cells and restoration of the chemotherapeutic responses in immunocompetent hosts. All together, this study shows that autophagy is essential for the release of ATP from dying cells and the triggering of the immune system, and that increased extracellular ATP concentrations can enhance the efficiency of antineoplastic chemotherapies in the case of autophagy-deficient cancers. 370 Suppression of Breast Cancer Metastasis by BMP4 R. Anderson1 , B.L. Eckhardt2 , S. Loi3 , Y. Cao1 . 1 Peter MacCallum Cancer Centre, Research Division, East Melbourne Victoria, Australia, 2 MD Anderson Cancer Center, Genitourinary Medical Oncology, Houston Texas, USA, 3 L’Universite´ Libre de Bruxelles, Institut Jules Bordet, Brussels, Belgium Background: The majority of deaths due to breast cancer result from the formation of metastases that arise in distant organs such as lymph nodes, lung, liver and bone. To date, there is no effective cure and current treatments are largely palliative. Methods and Results: We have utilised a mammary tumour model comprising a series of isogenic tumours with varying metastatic capacities to reveal genetic regulators of metastasis, one of which is BMP4, a ligand in the TGF-b superfamily. The extent of metastasis in mice bearing highly metastatic 4T1.2 mammary tumours engineered to express exogenous BMP4 is markedly reduced compared to those bearing the parental 4T1.2 tumours. The inhibition of metastasis is attributable both to decreased tumour cell escape from the primary site and a marked reduction in growth of macrometastases in distant organs. BMP4 induces a number of genes in the tumour cells, including SMAD7. Enforced expression of SMAD7 can reproduce the metastasis suppression seen in BMP4 expressing tumours whilst stable knockdown of SMAD7 in BMP4 expressing 4T1.2 tumours reverses the inhibition of metastasis driven by BMP4. BMP4 also has paracrine activity leading to suppression of metastasis. Expression of BMP4 in 4T1.2 tumours reduces the mobilisation of CD11b+ Gr1+ myeloid derived suppressor cells (MDSC) that have been implicated in many studies as suppressors of T cell activity and promoters of metastasis. MDSC are mobilised from bone marrow by G-CSF, which is secreted by the tumour cells. BMP4 inhibits the secretion of G-CSF, thereby leading to reduced mobilisation and enhanced anti-tumour immunity. Forced expression of G-CSF in 4T1.2-BMP4 cells partially restores their metastatic capacity. The clinical potential of BMP4 as an anti-metastatic therapy is revealed by administration of recombinant BMP4 to mice bearing 4T1.2 tumours, resulting in a significant increase in overall survival. Further, high expression of BMP4
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in primary breast tumours correlates with improved disease-free survival and overall survival in a large cohort of patients. Conclusion: Activation of the BMP4 signalling pathway is a potential therapeutic approach for patients with advanced breast cancer. 371 Ageing and Cancer-related Gene Expression of the Human Cell Lines Transfected With K-RAS12V, BMI-1 and BCL-2 Or/and TERT M. Kamada1 , T. Kumazaki1 , Y. Kawahara1 , T. Matsuo1 , Y. Mitsui1 , T. Takahashi1 . 1 Tokushima Bunri University, Kagawa Prefecture, Japan Introduction: Human normal cells are limited in cell sources for drug screening because of the short life span in vitro, and that they hardly transform to cancer cells in vitro with defined factors, which make us difficult to analyze mechanisms of human tumor formation and screen the drugs for cancer therapy in vitro. Thus, we aimed to establish useful human cell lines for the study of aging and cancer attaching great importance to generation of their model cells. Materials and Methods: K-RAS12V, BMI-1, BCL-2 and/or TERT cDNA were transfected into human lung fibroblast, TIG-1. Hygromycin B, Phleomycin D1, Puromycin or G418 were used for drug selection of their transformants. Population Doubling Level (PDL) during subcultivation was recorded. Senescence was confirmed by the decline in growth rate and appearance of senescent associated b-Galactosidase (Sa-b-Gal) staining cells. Immortality was defined as division potential with more than 200 PDL. Expressions of various genes of cell cycle, cancer specific characters, and cell death were examined by RT-PCR. Colony formation in soft agar gel was performed as a marker of malignant growth. Results and Discussion: Of 74 individual clones of TERT transfected cells, we obtained 55 cultures with normal range of life span (<75 PDL), 16 cultures with extended life span (75–140 PDL). In addition, three immortal cell strains, and unexpectedly one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and extremely low level of p16INK4A. They showed vigorous growth even at 450 PDL. Transformants of mutated K-RAS, BMI-1 and BCL-2 yielded higher growth rate and higher expression of BCL-2 without forming colony in soft agar gel. ULT-1, however, exhibited shortening of telomere length, high expression of p16INK4A, moderate level of Sa-b-Gal and finally reached senescence at 166 PDL with normal senescent morphology and karyotypes. Expression system of GFP and luciferase under the control of p16 promoter was developed to utilize this ULT-1 for the screening of senescence modulating agents. Conclusion: ULT-1 is useful for screening senescence-modulating factors with extremely long life span, and IMT-1, or -2 from the same parent cells are useful for the study of human cancer formation from normal cells. 372 The Subset of CD133+/CXCR4+/EpCAM− Cancer Initiating Cells is Responsible for Lung Tumor Metastatic Spreading G. Bertolini1 , M. Moro1 , M. Tortoreto1 , R. Caserini1 , U. Pastorino2 , L. Roz1 , G. Sozzi1 . 1 Fondazione IRCCS Istituto Nazionale Tumori, Department of Experimental Oncology and Molecular Medicine, Milan, Italy, 2 Fondazione IRCCS Istituto Nazionale Tumori, Unit of Thoracic Surgery, Milan, Italy Background: Late diagnosis at metastatic phase is the main reason for treatment failure of lung cancer. We previously demonstrated that the subset of CD133+ cells represents the fraction of lung cancer initiating cells (CICs) able to initiate and sustain tumor formation. Furthermore we showed that cisplatin treatment enriches for a subpopulation of CD133+ lung cancer cells co-expressing CXCR4, a chemokine receptor involved in invasion and metastasis. We suggest that chemoresistant CD133+ /CXCR4+ subset may represent the migrating fraction of CICs involved in metastatic process. Results: We analyzed by FACS a series of primary lung tumors (n = 30) and found that the subpopulation of CD133+ /CXCR4+ cells represents 0.3% of the total population. To test the metastatic potential, CD133+ /CXCR4+ and CD133− /CXCR4− cells were sorted from patient derived xenografts (PDXs), established by direct implant of primary lung tumor tissue in immunocompromised mice, and injected intravenously in SCID mice. FACS analysis of lungs for human HLA antigen showed that CD133+ /CXCR4+ cells had a much higher ability to colonize mice lung than double negative cells. Phenotypic study of spontaneous lung metastases derived from subcutaneous injection of H460 lung cancer cell line revealed an increase of CD133+ /CXCR4+ population in metastases compared to parental tumors. Treatment of a primary lung cancer cell line with TGF-b verified a direct link between epithelial to mesenchymal transition (EMT) activation and acquisition of stem like properties (i.e. increase of CD133+ cells fraction, increased expression of stemness genes and enhanced tumorigenicty in vivo). Moreover we observed that EMT process could generate a subset of migrating CD133/CXCR4 lung CICs that do not express the epithelial marker EpCAM.
Sunday 8 − Tuesday 10 July 2012
Using PDX models we observed that tumor cells colonizing mice lungs were enriched for CD133+ /CXCR4+ /EpCAM− subset and expressed high levels of stemness genes and EMT-related genes compared to bulk tumor cells, suggesting that disseminating lung tumor cells are endowed with stem like features and mesenchymal traits. In vitro invasion assays performed on PDXs tumor cells confirmed that invading cells are enriched for CD133+ /CXCR4+ / EpCAM− subset, as evaluated by FACS. The assessment of our findings in the clinical setting proved that the fraction of CD133+ /EpCAM− cells, expressing high level of CXCR4, was particularly enriched in patients’ lymph node metastases compared to primary tumors. Conclusions: Altogether these results suggest that the subset of CD133+ /CXCR4+ / EpCAM− lung CICs endowed with mesenchymal traits could be responsible for tumor dissemination and could drive metastases formation. Our findings provide new insights into metastatic process and may lead to the identification of novel targets for both lung cancer treatment and prevention of metastases formation. 373 Loss of BRCA1-interacting Helicase BRIP1 Leads to Abnormal Mammary Acinar Morphogenesis − Implications for Breast Tumorigenesis K. Daino1 , T. Imaoka1 , T. Morioka1 , M. Nishimura1 , Y. Shimada1 . 1 National Institute of Radiological Sciences, Research Center for Radiation Protection Radiobiology for Children’s Health Program, Chiba, Japan Background: BRIP1 is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast susceptibility protein BRCA1 and plays an important role in BRCA1-dependent DNA repair and DNA damageinduced checkpoint control. Recent studies implicate BRIP1 as moderate/low penetrance breast cancer susceptibility gene. However, the phenotypic effects and their molecular basis involved in the tumorigenesis of breast cancer due to the dysfunction of BRIP1 remain unclear. Material and Methods: In this study, we generated BRIP1-knockdown MCF10A cells by shRNA-mediated RNA interference, and examined its effect in a three-dimensional culture model. In addition, microarray analysis was performed to identify the differentially expressed genes in the knockdown cells. Results: Knockdown of BRIP1 in MCF-10A cells by RNA interference induced neoplastic-like changes such as abnormal cell adhesion, increased acinar size, irregular-shaped acinar structure, luminal filling, and invasive growth. Dysregulation of multiple genes and signaling pathways, involving LPA receptor signaling, oxygen homeostasis, telomerase regulation, c-Myc, and Wnt signaling, in the knockdown cells was revealed by microarray analysis. Conclusions: These results suggest that BRIP1 is involved in the development of mammary glands through multiple genes and signaling pathways. Our results also provide, in part, the molecular basis involved in the tumorigenesis of breast cancer due to the dysfunction of BRIP1. 374 Determination of Synthetic Lethal Interactions in KRAS Oncogene Dependent Cancer Cells Reveals Novel Therapeutic Targeting Strategies M. Molina Arcas1 , D. Hancock1 , M. Steckel1 , J. Downward1 . 1 Cancer Research UK − London Research Institute, Signal Transduction Laboratory, London, United Kingdom Introduction: Oncogenic mutations in RAS genes are very common in many human cancers, including those affecting the lung and the gastrointestinal tract. RAS mutant cancers tend to be resistant to most targeted therapies, emphasising a clear need for the development of more effective treatments. Oncogenic KRAS deregulates numerous downstream pathways, resulting in cells with well-characterised selective advantages, but also less well understood vulnerabilities. Materials and Methods: In order to affect tumour-specific vulnerabilities associated with the oncogenic state, we used a panel of twenty-six non-small cell lung cancer (NSCLC) cell lines, half of which carry an activating KRAS mutation, to examine the activity of small molecule inhibitors targeting pathways directly controlled by RAS, such as RAS/RAF/MEK or PI3K/AKT/mTOR, as well as drugs directed against other targets (such as HSP90, topoisomerases or the proteasome). Results: Knock-down of KRAS using siRNA indicates that KRAS mutant cells require KRAS expression to maintain viability. Moreover, KRAS depletion produces a clear inhibition of ERK and AKT signaling. Viability assays showed that cells harbouring an activated KRAS oncogene were more sensitive to MEK, RAF and IGF-1R inhibitors than cells carrying only wild-type KRAS alleles. In contrast, no differential loss of cell viability was observed when cells were treated with either PI3K or AKT inhibitors. Combinations of IGF1R inhibitors with MEK or RAF inhibitors resulted in a synergistic antiproliferative effect in KRAS mutant cell lines. Similar effects were observed upon simultaneous treatment with PI3K and MEK inhibitors. However, in this case, wild-type KRAS cells were also affected by PI3K inhibition,
european journal of cancer 48, suppl. 5 (2012) S25–S288
Although advanced prostate cancer is typically regarded as not expressing detectable p63 levels, we have unexpectedly found low expression levels of the DNp63a variant in the metastatic PC3 cell line. Surprisingly, expression of DNp63a increases when cells are grown in stem cell-favoring conditions. Furthermore, FACS purification and analysis of the CD44/CD133-positive cancer stem cell population reveals that expression of DNp63a is highest in these cells. Conclusion: Together, this work describes how DNp63a is a critical mediator of normal prostate stem cell function. Surprisingly, we have also uncovered expression of DNp63a in a subset of cancer stem cells in prostate cancer that suggests that tumor cells may utilize the stem cell-promoting function of p63 to facilitate tumor maintenance and metastatic colonization. 366 Phosphatidylcholine-specific Phospholipase C as a Target to Manipulate CXCR4-CXCL12 Signaling Pathway in Human Lymphoblastoid Cells S. Cecchetti1 , A. Ricci1 , E. Iorio1 , L. Paris1 , M.E. Pisanu1 , L. Portella2 , ` Department S. Scala2 , G. Carpinelli1 , F. Podo1 . 1 Istituto Superiore di Sanita, of Cell Biology and Neurosciences, Rome, Italy, 2 Istituto Nazionale Tumori Fondazione G. Pascale, Immunological Oncology, Naples, Italy Background: Chemokines, a family of small pro-inflammatory cytokines, and their receptors regulate a variety of immune responses. Cancer cells are known to exploit signaling through chemokine receptors for key steps of tumor initiation and progression. In particular, CXCR4 receptor overexpression is detected in cancers of different origins. The functional consequence is a sustained CXCR4 activation by the CXCL12 chemokine which regulates tumor proliferation, survival, chemotaxis and migration. We showed that phosphatidylcholine (PC)-specific phospholipase C (PCPLC) is able to modulate the membrane expression, and hence the signal transduction, of specific cell receptors such as CD16 in NK cells and HER2 in breast cancer cells, interfering with cell-signaling dependent functions. In this study we investigated the alterations of CXCR4 expression and PC metabolism induced by the PC-PLC inhibition in a human T-lymphoblastoid cell line (CEM). Material and Method: Membrane localization and direct interaction of PC-PLC with CXCR4 were investigated by confocal laser scanning microscopy (CLSM), flow cytometry and immunoprecipitation techniques. The effects induced by a selective PLC inhibitor (D609) on CXCR4 membrane expression and its overall contents were evaluated in comparison with those of a well-known CXCR4 antagonist, AMD3100. MRS experiments were performed on ethanolic extracts of CEM cells, previously exposed to the D609 until 24h, using a Bruker Avance 400 spectrometer equipped with a 1H-X multinuclear inverse probehead. Results and Discussion: PC-PLC was preferentially localized on the membrane surface of CEM cells. Fine analysis of CXCR4 and PC-PLC subcellular distribution demonstrated that the two proteins are physically associated and partially accumulated in lipid rafts.D609 induced a strong downmodulation of the CXCR4 receptor from the plasma membrane within 5 h. At this time point most of CXCR4 molecules internalized into early and late endosomes and a substantial amount of the receptor co-localized with lysosomes, indicating that PC-PLC deactivation leads to CXCR4 degradation, as also confirmed by Western blot experiments. Under these conditions, MRS experiments provided direct evidence of PC-PLC inhibition showing an about 42% reduction of phosphocholine, direct product of PC-PLC activity (in agreement with enzyme assays) in cells exposed for 24h to D609. Moreover, PC-PLC appeared to negatively regulate the CXCR4-CXCL12 signaling pathway. Indeed, inhibition of this phospholipase induced inactivation of both ERK1/2 and AKT, even in the presence of the CXCL12 stimulus, this effect being higher than that observed for AMD3100 only. Conclusion: Altogether, these data indicate that PC-PLC could play an important role in regulating the CXCR4-CXCL12 signaling pathway and suggest that, by manipulating this axis, PC-PLC may represent a potential therapeutic target for cancer treatment. 367 N-glycan Biosynthesis Inhibitors Induce in Vitro Anticancer Activity in Colorectal Cancer Cells J. de Freitas1 , L.G. Bastos2 , C.A. Freire-Neto2 , B.D.R. D’Aguiar-Silva3 , ˆ E.S. Abdelhay3 , J.A. Morgado-D´ıaz2 . 1 Instituto Nacional de Cancer / UERJ, ˜ de Biologia Celular / PPGB, Rio de Janeiro, Brazil, 2 Instituto Nacional Divisao ˆ ˜ de Biologia Celular, Rio de Janeiro, Brazil, 3 Instituto de Cancer, Divisao ´ ˆ Nacional de Cancer, Centro de Transplante de Medula Ossea, Rio de Janeiro, Brazil Background: During malignant transformation, changes in the expression profile of glycans may be involved in a variety of events, including the loss of cell-cell and cell-matrix adhesion, migration, invasion, and evasion of apoptosis. Therefore, modulation of glycan expression with drugs has promising therapeutic potential for various cancer types. In this study, we investigated the in vitro anticancer activity of the N-glycan biosynthesis inhibitors (swainsonine and tunicamycin) in cells derived from colorectal
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cancer. We also examined whether these inhibitors are able to induce radiosensitization and toxicity when used in combination with cisplatin or irinotecan, two current anticancer drugs. Material and Method: Three colorectal cancer cell lines were used: Caco-2, HCT-116, and HT-29. Cells were treated with drug concentrations ranging from 1 to 8 mg/mL for swainsonine and 0.25 to 2 mg/mL for tunicamycin. We examined the effects of swainsonine and tunicamycin treatment on cytotoxicity, migration, invasion, anchorage-dependent and anchorage-independent colony formation, and radiosensitivity, after 24, 48, 72, and 96 h. Results and Discussion: Our results show that treatment with tunicamycin inhibits cellular mechanisms related to the malignant phenotype, such as anchorage-dependent and anchorage-independent colony formation, migration and invasion, in undifferentiated HCT-116 colon cancer cells, whereas swainsonine only inhibits cell migration. We also observed that tunicamycin, but not swainsonine, caused radiosensitivity in HCT-116 cells. Moreover, the combination of swainsonine with cisplatin or irinotecan enhanced their toxicity in HCT-116 cells, while the combination of tunicamycin with these drugs had no effect. Conclusion: Given these results, we suggest that the modulation of N-glycan biosynthesis appears to be a potential therapeutic tool for colorectal cancer treatment because inhibition of this process induced anticancer activity in vitro. 369 Anticancer Immune Responses Induced by Chemotherapeutic Agents Depends on Autophagy M. Michaud1 , A.Q. Sukkurwala1 , I. Martins1 , S. Adjemian1 , G. Kroemer1 . 1 Institut Gustave Roussy, U848, Villejuif, France Immunogenic cell death can be obtained by using antineoplastic chemotherapies and provokes an anticancer immune response, thus increasing the efficiency of the treatment. Here, we demonstrate that autophagy, often disabled in cancer, is required for immunogenicity of chemotherapy-induced cell death. We observed that autophagy-competent, but not autophagydeficient cancers attracted dendritic cells and T lymphocytes into the tumor bed in response to chemotherapy. Moreover, autophagy inhibition decreased the amount of adenosine triphosphate (ATP) released from dying tumor cells. This effect could be reversed by inhibiting extracellular ATP-degrading enzymes, which led to increased pericellular ATP in autophagy-deficient tumors, and thus, to the re-establishment of the recruitment of immune cells and restoration of the chemotherapeutic responses in immunocompetent hosts. All together, this study shows that autophagy is essential for the release of ATP from dying cells and the triggering of the immune system, and that increased extracellular ATP concentrations can enhance the efficiency of antineoplastic chemotherapies in the case of autophagy-deficient cancers. 370 Suppression of Breast Cancer Metastasis by BMP4 R. Anderson1 , B.L. Eckhardt2 , S. Loi3 , Y. Cao1 . 1 Peter MacCallum Cancer Centre, Research Division, East Melbourne Victoria, Australia, 2 MD Anderson Cancer Center, Genitourinary Medical Oncology, Houston Texas, USA, 3 L’Universite´ Libre de Bruxelles, Institut Jules Bordet, Brussels, Belgium Background: The majority of deaths due to breast cancer result from the formation of metastases that arise in distant organs such as lymph nodes, lung, liver and bone. To date, there is no effective cure and current treatments are largely palliative. Methods and Results: We have utilised a mammary tumour model comprising a series of isogenic tumours with varying metastatic capacities to reveal genetic regulators of metastasis, one of which is BMP4, a ligand in the TGF-b superfamily. The extent of metastasis in mice bearing highly metastatic 4T1.2 mammary tumours engineered to express exogenous BMP4 is markedly reduced compared to those bearing the parental 4T1.2 tumours. The inhibition of metastasis is attributable both to decreased tumour cell escape from the primary site and a marked reduction in growth of macrometastases in distant organs. BMP4 induces a number of genes in the tumour cells, including SMAD7. Enforced expression of SMAD7 can reproduce the metastasis suppression seen in BMP4 expressing tumours whilst stable knockdown of SMAD7 in BMP4 expressing 4T1.2 tumours reverses the inhibition of metastasis driven by BMP4. BMP4 also has paracrine activity leading to suppression of metastasis. Expression of BMP4 in 4T1.2 tumours reduces the mobilisation of CD11b+ Gr1+ myeloid derived suppressor cells (MDSC) that have been implicated in many studies as suppressors of T cell activity and promoters of metastasis. MDSC are mobilised from bone marrow by G-CSF, which is secreted by the tumour cells. BMP4 inhibits the secretion of G-CSF, thereby leading to reduced mobilisation and enhanced anti-tumour immunity. Forced expression of G-CSF in 4T1.2-BMP4 cells partially restores their metastatic capacity. The clinical potential of BMP4 as an anti-metastatic therapy is revealed by administration of recombinant BMP4 to mice bearing 4T1.2 tumours, resulting in a significant increase in overall survival. Further, high expression of BMP4
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in primary breast tumours correlates with improved disease-free survival and overall survival in a large cohort of patients. Conclusion: Activation of the BMP4 signalling pathway is a potential therapeutic approach for patients with advanced breast cancer. 371 Ageing and Cancer-related Gene Expression of the Human Cell Lines Transfected With K-RAS12V, BMI-1 and BCL-2 Or/and TERT M. Kamada1 , T. Kumazaki1 , Y. Kawahara1 , T. Matsuo1 , Y. Mitsui1 , T. Takahashi1 . 1 Tokushima Bunri University, Kagawa Prefecture, Japan Introduction: Human normal cells are limited in cell sources for drug screening because of the short life span in vitro, and that they hardly transform to cancer cells in vitro with defined factors, which make us difficult to analyze mechanisms of human tumor formation and screen the drugs for cancer therapy in vitro. Thus, we aimed to establish useful human cell lines for the study of aging and cancer attaching great importance to generation of their model cells. Materials and Methods: K-RAS12V, BMI-1, BCL-2 and/or TERT cDNA were transfected into human lung fibroblast, TIG-1. Hygromycin B, Phleomycin D1, Puromycin or G418 were used for drug selection of their transformants. Population Doubling Level (PDL) during subcultivation was recorded. Senescence was confirmed by the decline in growth rate and appearance of senescent associated b-Galactosidase (Sa-b-Gal) staining cells. Immortality was defined as division potential with more than 200 PDL. Expressions of various genes of cell cycle, cancer specific characters, and cell death were examined by RT-PCR. Colony formation in soft agar gel was performed as a marker of malignant growth. Results and Discussion: Of 74 individual clones of TERT transfected cells, we obtained 55 cultures with normal range of life span (<75 PDL), 16 cultures with extended life span (75–140 PDL). In addition, three immortal cell strains, and unexpectedly one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and extremely low level of p16INK4A. They showed vigorous growth even at 450 PDL. Transformants of mutated K-RAS, BMI-1 and BCL-2 yielded higher growth rate and higher expression of BCL-2 without forming colony in soft agar gel. ULT-1, however, exhibited shortening of telomere length, high expression of p16INK4A, moderate level of Sa-b-Gal and finally reached senescence at 166 PDL with normal senescent morphology and karyotypes. Expression system of GFP and luciferase under the control of p16 promoter was developed to utilize this ULT-1 for the screening of senescence modulating agents. Conclusion: ULT-1 is useful for screening senescence-modulating factors with extremely long life span, and IMT-1, or -2 from the same parent cells are useful for the study of human cancer formation from normal cells. 372 The Subset of CD133+/CXCR4+/EpCAM− Cancer Initiating Cells is Responsible for Lung Tumor Metastatic Spreading G. Bertolini1 , M. Moro1 , M. Tortoreto1 , R. Caserini1 , U. Pastorino2 , L. Roz1 , G. Sozzi1 . 1 Fondazione IRCCS Istituto Nazionale Tumori, Department of Experimental Oncology and Molecular Medicine, Milan, Italy, 2 Fondazione IRCCS Istituto Nazionale Tumori, Unit of Thoracic Surgery, Milan, Italy Background: Late diagnosis at metastatic phase is the main reason for treatment failure of lung cancer. We previously demonstrated that the subset of CD133+ cells represents the fraction of lung cancer initiating cells (CICs) able to initiate and sustain tumor formation. Furthermore we showed that cisplatin treatment enriches for a subpopulation of CD133+ lung cancer cells co-expressing CXCR4, a chemokine receptor involved in invasion and metastasis. We suggest that chemoresistant CD133+ /CXCR4+ subset may represent the migrating fraction of CICs involved in metastatic process. Results: We analyzed by FACS a series of primary lung tumors (n = 30) and found that the subpopulation of CD133+ /CXCR4+ cells represents 0.3% of the total population. To test the metastatic potential, CD133+ /CXCR4+ and CD133− /CXCR4− cells were sorted from patient derived xenografts (PDXs), established by direct implant of primary lung tumor tissue in immunocompromised mice, and injected intravenously in SCID mice. FACS analysis of lungs for human HLA antigen showed that CD133+ /CXCR4+ cells had a much higher ability to colonize mice lung than double negative cells. Phenotypic study of spontaneous lung metastases derived from subcutaneous injection of H460 lung cancer cell line revealed an increase of CD133+ /CXCR4+ population in metastases compared to parental tumors. Treatment of a primary lung cancer cell line with TGF-b verified a direct link between epithelial to mesenchymal transition (EMT) activation and acquisition of stem like properties (i.e. increase of CD133+ cells fraction, increased expression of stemness genes and enhanced tumorigenicty in vivo). Moreover we observed that EMT process could generate a subset of migrating CD133/CXCR4 lung CICs that do not express the epithelial marker EpCAM.
Sunday 8 − Tuesday 10 July 2012
Using PDX models we observed that tumor cells colonizing mice lungs were enriched for CD133+ /CXCR4+ /EpCAM− subset and expressed high levels of stemness genes and EMT-related genes compared to bulk tumor cells, suggesting that disseminating lung tumor cells are endowed with stem like features and mesenchymal traits. In vitro invasion assays performed on PDXs tumor cells confirmed that invading cells are enriched for CD133+ /CXCR4+ / EpCAM− subset, as evaluated by FACS. The assessment of our findings in the clinical setting proved that the fraction of CD133+ /EpCAM− cells, expressing high level of CXCR4, was particularly enriched in patients’ lymph node metastases compared to primary tumors. Conclusions: Altogether these results suggest that the subset of CD133+ /CXCR4+ / EpCAM− lung CICs endowed with mesenchymal traits could be responsible for tumor dissemination and could drive metastases formation. Our findings provide new insights into metastatic process and may lead to the identification of novel targets for both lung cancer treatment and prevention of metastases formation. 373 Loss of BRCA1-interacting Helicase BRIP1 Leads to Abnormal Mammary Acinar Morphogenesis − Implications for Breast Tumorigenesis K. Daino1 , T. Imaoka1 , T. Morioka1 , M. Nishimura1 , Y. Shimada1 . 1 National Institute of Radiological Sciences, Research Center for Radiation Protection Radiobiology for Children’s Health Program, Chiba, Japan Background: BRIP1 is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast susceptibility protein BRCA1 and plays an important role in BRCA1-dependent DNA repair and DNA damageinduced checkpoint control. Recent studies implicate BRIP1 as moderate/low penetrance breast cancer susceptibility gene. However, the phenotypic effects and their molecular basis involved in the tumorigenesis of breast cancer due to the dysfunction of BRIP1 remain unclear. Material and Methods: In this study, we generated BRIP1-knockdown MCF10A cells by shRNA-mediated RNA interference, and examined its effect in a three-dimensional culture model. In addition, microarray analysis was performed to identify the differentially expressed genes in the knockdown cells. Results: Knockdown of BRIP1 in MCF-10A cells by RNA interference induced neoplastic-like changes such as abnormal cell adhesion, increased acinar size, irregular-shaped acinar structure, luminal filling, and invasive growth. Dysregulation of multiple genes and signaling pathways, involving LPA receptor signaling, oxygen homeostasis, telomerase regulation, c-Myc, and Wnt signaling, in the knockdown cells was revealed by microarray analysis. Conclusions: These results suggest that BRIP1 is involved in the development of mammary glands through multiple genes and signaling pathways. Our results also provide, in part, the molecular basis involved in the tumorigenesis of breast cancer due to the dysfunction of BRIP1. 374 Determination of Synthetic Lethal Interactions in KRAS Oncogene Dependent Cancer Cells Reveals Novel Therapeutic Targeting Strategies M. Molina Arcas1 , D. Hancock1 , M. Steckel1 , J. Downward1 . 1 Cancer Research UK − London Research Institute, Signal Transduction Laboratory, London, United Kingdom Introduction: Oncogenic mutations in RAS genes are very common in many human cancers, including those affecting the lung and the gastrointestinal tract. RAS mutant cancers tend to be resistant to most targeted therapies, emphasising a clear need for the development of more effective treatments. Oncogenic KRAS deregulates numerous downstream pathways, resulting in cells with well-characterised selective advantages, but also less well understood vulnerabilities. Materials and Methods: In order to affect tumour-specific vulnerabilities associated with the oncogenic state, we used a panel of twenty-six non-small cell lung cancer (NSCLC) cell lines, half of which carry an activating KRAS mutation, to examine the activity of small molecule inhibitors targeting pathways directly controlled by RAS, such as RAS/RAF/MEK or PI3K/AKT/mTOR, as well as drugs directed against other targets (such as HSP90, topoisomerases or the proteasome). Results: Knock-down of KRAS using siRNA indicates that KRAS mutant cells require KRAS expression to maintain viability. Moreover, KRAS depletion produces a clear inhibition of ERK and AKT signaling. Viability assays showed that cells harbouring an activated KRAS oncogene were more sensitive to MEK, RAF and IGF-1R inhibitors than cells carrying only wild-type KRAS alleles. In contrast, no differential loss of cell viability was observed when cells were treated with either PI3K or AKT inhibitors. Combinations of IGF1R inhibitors with MEK or RAF inhibitors resulted in a synergistic antiproliferative effect in KRAS mutant cell lines. Similar effects were observed upon simultaneous treatment with PI3K and MEK inhibitors. However, in this case, wild-type KRAS cells were also affected by PI3K inhibition,