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223: HIF prolyl hydroxylase PHD3 maintains hypoxic cell cycle through cyclin-dependent kinase inhibitor P27
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 flow cytometry. Cell fractionation was performed to investigate subcellular localization of MUC1-C. Tumor growth was analyzed in NOD/SCID xenograft model. Results and Discussion: Data from public microarrays and our data show that C1GALT1 is frequently up-regulated in breast cancer and increased C1GALT1 expression is correlated with higher histological grade and advanced tumor stage. C1GALT1 is differentially expressed in multiple breast cancer cell lines. C1GALT1 expression modified O-glycan structures on glycoproteins in breast cancer cells. Overexpression of C1GALT1 enhanced cell growth in MCF-7 cells, whereas knockdown of C1GALT1 suppressed cell growth in T47D cells. In vivo results revealed that C1GALT1 enhanced tumor growth in xenograft mice. Interestingly, our results showed that C1GALT1 modified Tn- and T-antigen expression on MUC1 oncoprotein and promoted MUC1-C translocation to nucleus. Conclusion: C1GALT1 is up-regulated in breast cancer tissues and overexpression of C1GALT1 enhances breast cancer cell growth probably through modification of MUC1 O-glycosylation and MUC1-C localization. No conflict of interest. 221 The T-box transcription factor, TBX3, promotes tumourigenesis in soft tissue and bone sarcomas: a possible therapeutic target T. Willmer1 , S. Prince2 . 1 Groote Schuur Hospital and University of Cape Town, Human Biology, Cape Town, South Africa, 2 University of Cape Town, Human Biology, Cape Town, South Africa Background: Sarcomas comprise a subset of malignant tumours derived from mesenchymal tissue and while they are rare, they represent some of the most aggressive cancers due to their highly metastatic nature and their resistance. Furthermore, they are resistant to conventional chemo- and radiation therapies which limits the options of their treatment. There has therefore been a great interest in developing single targeted therapies for sarcomas and indeed inhibitors to tyrosine kinases and c-KIT have proven promising. The transcription factor, TBX3, is overexpressed in several epithelial cancers and plays a direct role in their development which suggests that it may be a novel target for anti-cancer treatments. However whether this is the case for cancers of mesenchymal origin is not known and hence this study explores a possible tumour-promoting role for TBX3 in sarcomas. Material and Methods: TBX3 protein and mRNA expression were analysed in a panel of sarcoma cell lines by western blot analysis and quantitative real time PCR respectively. TBX3 protein expression was also determined in paraffin embedded sarcoma tissue sections by immunohistochemistry. TBX3 was silenced in chondrosarcoma cell lines using a shRNA approach, and the impact of this on the cancer phenotype was assessed using proliferation and cell cycle analyses, soft agar and cell motility assays and western blot analysis with antibodies to key cell cycle regulators. Results: We show for the first time that TBX3 is overexpressed in a panel of sarcoma cell lines and tissue. Importantly, we demonstrate that knocking down TBX3 levels in two chondrosarcoma cell lines is sufficient to reverse key features of the sarcoma phenotype. We show that TBX3 depletion triggers an S-phase arrest and inhibits substrate-dependent and -independent cell proliferation. This is shown to be accompanied by the activation of the p38-MAPK pathway and increased levels of p53 and p21 which may suggest a mechanism by which TBX3 promotes cell proliferation in sarcomas. Interestingly, knocking down TBX3 also inhibited chondrosarcoma cell migration which was reproducible in liposarcoma and rhabdomyosarcoma cell lines. Conclusions: Our data show for the first time that TBX3 is overexpressed in a diverse subset of sarcomas and that it is a key driver of the oncogenic process in these cancers. This work extends our current understanding of the role of TBX3 in cancer and provides additional support for its use in single targeted therapies to treat this highly aggressive disease. No conflict of interest. 222 Chronic intermittent hypoxia triggers adaptive changes that promote protection against cell death J. Matschke1 , H. Riffkin1 , R. Handrick2 , L. Klein-Hitpass1 , V. Jendrossek1 . 1 University Hospital Essen, Institute of Cell Biology, Essen, Germany, 2 Institute for Pharmaceutical Biotechnology Biberach, University of Applied Sciences, Biberach, Germany Introduction: Hypoxia is considered as one main biological factor driving malignant progression and promoting tumor cell resistance to chemotherapy and radiotherapy. We showed in previous work that chronic intermittent hypoxia drives the evolution of hypoxia-tolerant lung cancer cells that display increased resistance to stimuli of the intrinsic apoptosis pathway. Aim of the present study was to gain further insight into the molecular changes responsible for hypoxia tolerance and to understand the molecular mechanisms that promote apoptosis resistance in hypoxia-tolerant lung cancer cells. Methods: Human lung adenocarcinoma cells (NCI-H460) were subjected to 25 cycles of severe hypoxia (48 h <0.1% O2 ) and reoxygenation (120 h 21% O2 ).
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We analyzed tumor cell growth and sensitivity to treatments involving the generation of toxic reactive oxygen species (ROS), e.g. ionizing radiation. Cell function was determined by measuring apoptosis, cell death, mitochondrial membrane potential and ROS under control or starvation conditions. Finally, we compared gene expression profiles of hypoxia-selected and control cells by microarray analysis and validated genes of interest by qRTPCR. Results: The hypoxia-selected NCI-H460 cells were characterized by decreased formation of ROS and increased survival in response to ionizing radiation. Moreover, the hypoxia-tolerant cells differed from the control cells in their response to glucose, glutamine or serum starvation in normoxia and hypoxia. These changes were linked to complex alterations in gene expression hinting to adaptive changes in cell metabolism. Finally, the hypoxia-tolerant cells and the control cells showed distinct responses to drugs targeting cell metabolism. Conclusions: Our data suggest that improved ROS-defense of the hypoxia tolerant NCI H460 cells may contribute to their decreased sensitivity to ionizing radiation. We speculate that the identification of specific metabolic dependencies of hypoxia-tolerant cancer cells may offer novel opportunities for the treatment of chronically hypoxic tumors. No conflict of interest. 223 HIF prolyl hydroxylase PHD3 maintains hypoxic cell cycle through cyclin-dependent kinase inhibitor P27 H. Hogel ¨ 1 , P. Miikkulainen2 , L. Bino3 , P.M. Jaakkola4 . 1 Turku Centre for Biotechnology, Turku, Finland, 2 Turku Centre for Biotechnology, University of Turku, Turku, Finland, 3 Institute of Biophysics, The Academy of Sciences of the Czech Republic, Brno, Czech Republic, 4 Faculty of Medicine, University of Turku, Turku, Finland Introduction: Hypoxia is a common feature of solid tumours and has multiple effects on cancer progression. It has a major role in many of the events considered as hallmarks of cancer. Moreover, hypoxia causes a cell cycle arrest at G1/S interface which can be overcome by carcinoma cells. Growth control and cellular survival in hypoxic environment require changes in cellular signaling many of which are mediated by activity of master regulator of hypoxic response, hypoxia-inducible factor HIF-1. HIF prolyl hydroxylases (PHDs) are considered as cellular oxygen sensors as they regulate the activity of HIF-1. However, one of the three PHD isoforms, PHD3, has been shown having other substrates as well and it is upregulated under hypoxia. PHD3 is overexpressed in various cancer types including head and neck squamous cell carcinomas, renal cell carcinoma and pancreatic cancer. Recently we have shown that PHD3 is essential for the progression of cell cycle from G1 to S phase in hypoxia, and that the inhibition of PHD3 expression causes non-apoptotic cell death. Moreover, we found that PHD3 depletion causes induction of cyclindependent kinase inhibitor p27. Materials and Methods: Quantitative RT-PCR was used to study transcription and western blotting to monitor protein expression. siRNA and plasmid transfections were performed using manufacturers’ protocols. Cycloheximide chase was used to study stability of p27 at different phases of cell cycle. For cell cycle analysis with flow cytometry, cells were synchronized using serum starvation or aphidicolin treatment. Results and Discussion: We have previously shown that the cellular oxygen sensor PHD3 enhances hypoxic cell cycle entry at G1 to S transition. Here we show that PHD3 knockdown stabilizes the expression of cyclin-dependent kinase inhibitor p27. PHD3 inhibition led to increased p27 expression under hypoxia and p27 was required for hypoxic cell cycle block induced by PHD3 knockdown. PHD3 depletion increased the p27 half-life in G0 to G1 but did not affect p27 transcription. PHD3 inhibition led to an increase in p27 phosphorylation at Ser10 without affecting other p27 phosphorylation sites. Intact Ser10 was required for normal hypoxic degradation of p27. Conclusions: Our data shows that PHD3 enhances hypoxic cell cycle entry from G1 to S phase by decreasing the half-life of p27 through regulation of phosphorylation. No conflict of interest. 224 Effect of HuIFN-a and Royal jelly on the proliferation of human colon cancer (CaCo-2) cells in vitro K. Rihar1 , D. Gregoric Exel1 , S. Sladoljev2 , B. Filipic3 . 1 Ljubljana, Slovenia, 2 Institute of Immunology, Zagreb, Croatia, 3 Institute for Microbiology and Immunology, University of Ljubljana, Laboratory for Interferon Research, Slovenia Background: Royal jelly is a milky substance secreted by the pharyngeal glands of worker bees. In order to determine its unusual nature, different studies were conducted where biological properties and possible antitumor activity were examined. Interferon alpha (IFNa) was used in the treatment of different types of cancer, although its molecular mechanism remained fairly unknown. The main role in its antitumor activity is the antiproliferative (AP) effect. Experiments were performed to measure the effect of HuIFN-a and
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We analyzed tumor cell growth and sensitivity to treatments involving the generation of toxic reactive oxygen species (ROS), e.g. ionizing radiation. Cell function was determined by measuring apoptosis, cell death, mitochondrial membrane potential and ROS under control or starvation conditions. Finally, we compared gene expression profiles of hypoxia-selected and control cells by microarray analysis and validated genes of interest by qRTPCR. Results: The hypoxia-selected NCI-H460 cells were characterized by decreased formation of ROS and increased survival in response to ionizing radiation. Moreover, the hypoxia-tolerant cells differed from the control cells in their response to glucose, glutamine or serum starvation in normoxia and hypoxia. These changes were linked to complex alterations in gene expression hinting to adaptive changes in cell metabolism. Finally, the hypoxia-tolerant cells and the control cells showed distinct responses to drugs targeting cell metabolism. Conclusions: Our data suggest that improved ROS-defense of the hypoxia tolerant NCI H460 cells may contribute to their decreased sensitivity to ionizing radiation. We speculate that the identification of specific metabolic dependencies of hypoxia-tolerant cancer cells may offer novel opportunities for the treatment of chronically hypoxic tumors. No conflict of interest. 223 HIF prolyl hydroxylase PHD3 maintains hypoxic cell cycle through cyclin-dependent kinase inhibitor P27 H. Hogel ¨ 1 , P. Miikkulainen2 , L. Bino3 , P.M. Jaakkola4 . 1 Turku Centre for Biotechnology, Turku, Finland, 2 Turku Centre for Biotechnology, University of Turku, Turku, Finland, 3 Institute of Biophysics, The Academy of Sciences of the Czech Republic, Brno, Czech Republic, 4 Faculty of Medicine, University of Turku, Turku, Finland Introduction: Hypoxia is a common feature of solid tumours and has multiple effects on cancer progression. It has a major role in many of the events considered as hallmarks of cancer. Moreover, hypoxia causes a cell cycle arrest at G1/S interface which can be overcome by carcinoma cells. Growth control and cellular survival in hypoxic environment require changes in cellular signaling many of which are mediated by activity of master regulator of hypoxic response, hypoxia-inducible factor HIF-1. HIF prolyl hydroxylases (PHDs) are considered as cellular oxygen sensors as they regulate the activity of HIF-1. However, one of the three PHD isoforms, PHD3, has been shown having other substrates as well and it is upregulated under hypoxia. PHD3 is overexpressed in various cancer types including head and neck squamous cell carcinomas, renal cell carcinoma and pancreatic cancer. Recently we have shown that PHD3 is essential for the progression of cell cycle from G1 to S phase in hypoxia, and that the inhibition of PHD3 expression causes non-apoptotic cell death. Moreover, we found that PHD3 depletion causes induction of cyclindependent kinase inhibitor p27. Materials and Methods: Quantitative RT-PCR was used to study transcription and western blotting to monitor protein expression. siRNA and plasmid transfections were performed using manufacturers’ protocols. Cycloheximide chase was used to study stability of p27 at different phases of cell cycle. For cell cycle analysis with flow cytometry, cells were synchronized using serum starvation or aphidicolin treatment. Results and Discussion: We have previously shown that the cellular oxygen sensor PHD3 enhances hypoxic cell cycle entry at G1 to S transition. Here we show that PHD3 knockdown stabilizes the expression of cyclin-dependent kinase inhibitor p27. PHD3 inhibition led to increased p27 expression under hypoxia and p27 was required for hypoxic cell cycle block induced by PHD3 knockdown. PHD3 depletion increased the p27 half-life in G0 to G1 but did not affect p27 transcription. PHD3 inhibition led to an increase in p27 phosphorylation at Ser10 without affecting other p27 phosphorylation sites. Intact Ser10 was required for normal hypoxic degradation of p27. Conclusions: Our data shows that PHD3 enhances hypoxic cell cycle entry from G1 to S phase by decreasing the half-life of p27 through regulation of phosphorylation. No conflict of interest. 224 Effect of HuIFN-a and Royal jelly on the proliferation of human colon cancer (CaCo-2) cells in vitro K. Rihar1 , D. Gregoric Exel1 , S. Sladoljev2 , B. Filipic3 . 1 Ljubljana, Slovenia, 2 Institute of Immunology, Zagreb, Croatia, 3 Institute for Microbiology and Immunology, University of Ljubljana, Laboratory for Interferon Research, Slovenia Background: Royal jelly is a milky substance secreted by the pharyngeal glands of worker bees. In order to determine its unusual nature, different studies were conducted where biological properties and possible antitumor activity were examined. Interferon alpha (IFNa) was used in the treatment of different types of cancer, although its molecular mechanism remained fairly unknown. The main role in its antitumor activity is the antiproliferative (AP) effect. Experiments were performed to measure the effect of HuIFN-a and