- Email: [email protected]
220: Up-regulation of C1GALT1 promotes breast cancer cell growth through MUC1-C signaling pathway
S50
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
217 The identification of novel targets in b-catenin-driven acute myeloid leukemic stem cells H. Yi1 , J. Wang2 , M. Kavallaris1 , J.Y. Wang3 . 1 Children’s Cancer Institute Australia for Medical Research, Sydney NSW, Australia, 2 Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, USA, 3 School of Women’s and Children’s Health, Faculty of Medicine University of New South Wales, Sydney NSW, Australia Introduction: Leukemic stem cells (LSCs) are a small population of stem-like cancer cells that are believed to be responsible for the high rate of patient relapse in acute myeloid leukemia (AML). Although aberrant Wnt/b-catenin signaling has recently been shown to be required for the development of LSCs in AML, key components of the pathway remain elusive. Material and Methods: Hematopoietic stem cells were transduced with MLLAF9 or Hoxa9/Meis1a to generate pre-LSCs. Stable knockdown was achieved by lentiviral vector system to express short hairpin RNAs. Tumorigenic potential was assessed in vitro (by methylcellulose colony forming assays) and in vivo (by transplantations into syngeneic C57BL/6 mice). Differential gene expressions were assessed by microarray analysis, and target genes were confirmed by Western blot. Other techniques such as Wright Giemsa staining and apoptosis assay were used to examine changes in cell phenotypes. Results and Discussion: Gene expression analysis of MLL-rearranged AML patient samples revealed a 3-fold increase in expressions of Lgr4 and Ctnnb1 compared to normal hematopoietic stem cells. Here we demonstrated that depletion of Lgr4 impaired pre-LSC growth and significantly prolonged mouse survival through inhibition of b-catenin expression. Furthermore, overexpression of Lgr4 promoted cell growth and accelerated leukemia onset. Differential gene expressions by microarray analysis identified groups of Wnt target genes and genes that regulate G-protein signaling. We showed that inhibition of Gaq by a Gaq specific inhibitor caused marked reduction of colony forming capacity as well as increased differentiation and apoptosis of preLSCs. Conclusion: Our results suggest Lgr4 and Gaq as potential therapeutic targets for elimination of b-catenin-driven LSCs in AML. No conflict of interest. 218 The role of WWOX gene in EMT process in endometrial cancer E. Pluciennik1 , M. Nowakowska1 , K. Pospiech1 , K. Kosla1 , I. Baryla1 , K. Wojcik-Krowiranda2 , A. Bienkiewicz2 , M. Galdyszynska3 , M. Popeda3 , A.K. Bednarek1 . 1 Medical University of Lodz, Department of Molecular Cancerogenesis, Lodz, Poland, 2 Medical University of Lodz, Clinical Division of Gynecological Oncology, Lodz, Poland, 3 Medical University of Lodz, Faculty of of Biomedical Sciences and Postgraduate Education, Lodz, Poland Background: In Poland endometrial cancer is on the first place in morbidity statistics and the third in mortality within reproductive track-related cancers among women. WWOX is a newly described tumour suppressor gene which is affected in many types of tumor including steroid hormones regulated such as breast, ovary, prostate cancer. The EMT process is also of importance in this cancer type with unknown role of WWOX in its regulation. The aim of this study was to precise the role of WWOX in regulation of epithelial to mezenchymal transition in endometrial cancer. Material and Methods: This study was performed on series of 164 endometrial adenocarcinoma patients and well-differentiated steroid-responsive endometrial cell line − ECC1, which was transducted cDNA WWOX by retroviral system. We evaluated correlation between expression of WWOX gene and EMT markers (CDH1, VIM, ZEB1, SNAI1) on mRNA (real-time RT-PCR) and protein level (Western Blot). The EMT process were also analysed in in vitro model with biological assays such as adhesion of cells to six of extracellular matrix proteins, migration through basement membrane, anchorage-independent growth (soft agar assay), activity of MMPs (zymography test). Using DNA microarrays (HumanOneArray™;) we determined WWOX dependent pathways in ECC1 cell line. Results: In patients positive correlation between WWOX and ZEB1 (Rs = 0.26; p = 0.00037), and negative correlation with CDH1 and VIM (Rs = −0.5118, p = 0.0001; Rs = −0.2687, p = 0.0005, respectively) was observed. We also noticed that expression of WWOX inversely correlated with the risk of recurrence, i.e. tumors with low risk had higher WWOX expression, than those with medium and high risk, with the statistically significant difference between low and medium risk of disease recurrence. The negative correlation with VIM and positive with CDH1 was confirmed on protein/mRNA level in transfected cancer cell line. ECC1 cell line with overexpression of WWOX protein revealed increased migratory capacity, with increased expression of metalloproteinases MMP2/MMP9. On the other hand, those cells were not able to form colonies in suspension, which indicate that they loose the aggressive potential. Moreover, this transductants revealed tendency to decrease adhesion to fibronectin and statistically significant decrease in adhesion to fibrinogen. Microarray analysis demonstrated that WWOX has impact on variety of cellular pathway including cadherin and integrin signalling pathways.
Conclusion: Our results suggest the role of WWOX gene in the regulation of EMT process in endometrial cancer via regulation of expression of cell motility associated proteins and thus influencing tissue remodeling, with the important suppression of mezenchymal marker. Acknowledgements: This study was funded by the National Center of Sciences N N407 168940. No conflict of interest. 219 Inactivation of nucleoside-derived anticancer drugs by catabolic prokaryotic enzymes J. Vande Voorde1 , S. Sabuncuoglu1 , J. Balzarini1 , S. Liekens1 . 1 Rega Institute for Medical Research KU Leuven, Microbiology and Immunology, Leuven, Belgium Background: Nucleoside analogues (NAs) target tumor cell replication and represent ~20% of the approved anticancer drugs. NA treatment efficiency depends on the expression and activity of nucleo(s)(t)ide-metabolizing enzymes. Since prokaryotic enzymes are often endowed with a different activity and substrate specificity compared with their mammalian counterparts, bacterial presence in the tumor microenvironment may affect NA-based cancer treatment. Mycoplasmas are part of the normal flora of the human body and several mycoplasma species (e.g. Mycoplasma hyorhinis) are reported to preferentially colonize tumors in cancer patients. These prokaryotes interfere with host cell nucleoside metabolism and may influence the therapeutic efficacy of NAs. Material and Methods: The cytostatic activity of different NAs was evaluated in M. hyorhinis-infected and control tumor cell cultures. The stability and/or metabolism of radiolabeled cladribine and gemcitabine was monitored in mycoplasma-infected and control human MCF-7 breast carcinoma cells. The antitumor activity of gemcitabine and floxuridine was studied in mice bearing M. hyorhinis-infected or uninfected murine mammary FM3A tumors. Results: The cytostatic activity of several NAs (e.g. gemcitabine, cladribine and floxuridine) was severely compromised in various tumor cell cultures upon infection with M. hyorhinis. Accordingly, a significantly decreased antitumor effect of gemcitabine and floxuridine was observed in mice bearing M. hyorhinis-infected tumors. Mycoplasma-mediated drug inactivation of cladribine, floxuridine and gemcitabine was studied in detail. Cladribine and floxuridine were catabolised to their (inactive) free nucleobases by mycoplasma-encoded nucleoside phosphorylases. For gemcitabine, a tandem-mechanism of action in which two catabolic mycoplasma enzymes act in concert was identified: (i) mycoplasma cytidine deaminase (Cyd-DA) catalyzes deamination of gemcitabine to a poorly cytostatic metabolite and (ii) natural inhibitors of Cyd-DA are catabolised by a mycoplasma pyrimidine nucleoside phosphorylase. The cytostatic activity of these NAs was restored by co-administration of a mycoplasma-directed antibiotic or relevant inhibitor of the mycoplasma-encoded catabolic enzymes. These observations suggest that the presence of mycoplasmas in the tumor microenvironment may limit the anticancer efficiency of certain NAs. Conclusions: Recently, commensals were found to be essential for an optimal response to immunotherapy and cyclophosphamide- or platinumbased chemotherapy [Iida et al., Science (2013) 342: 967−70 & Viaud et al., Science (2013) 342: 971−76]. Such findings stress the potential risks of using antibiotics prior to, or during, cancer therapy. However, our results indicate that targeting the microbiome using antibiotics may improve the therapeutic outcome of NA-based cancer treatment. No conflict of interest. 220 Up-regulation of C1GALT1 promotes breast cancer cell growth through MUC1-C signaling pathway C. Chou1 , M.C. Huang1 . 1 National Taiwan University College of Medicine, Graduate Institute of Anatomy and Cell Biology, Taipei City, Taiwan Introduction: Breast cancer is the most frequently diagnosed malignancy in women and the fifth leading cause of cancer-related death worldwide. Core 1 b1,3-galactosyltransferase (C1GALT1) is the exclusive enzyme that catalyzes the formation of T antigens, a cancer associated structure, through the addition of galactose (Gal) to N-acetylgalactosamine (GalNAc)-Ser/Thr. C1GALT1 is pivotal in many biological functions in the developmental process. However, little is known of the function of C1GALT1 in association with cancer development, particularly, breast cancer. This study aims to correlate C1GALT1 expression with breast cancer clinicopathological characteristics and to investigate the role of C1GALT1 in breast cancer malignant behaviors and its underlying mechanisms. Materials and Methods: C1GALT1 expression in breast cancer tissue array (Pantomics) was analyzed by immunohistochemistry. C1GALT1 was knocked down with specific siRNA and C1GALT1/pcDNA3.1A plasmid was constructed to overexpress C1GALT1 in breast cancer cell lines. C1GALT1, MUC1 and MUC1-C expression in cells were analyzed by Western blotting. Vicia villosa (VVA) and peanut agglutinin (PNA) lectins were used to detect Tn- and T-antigens, respectively, in lectin pull-down, Western blotting, and
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 ).
S51
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
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
217 The identification of novel targets in b-catenin-driven acute myeloid leukemic stem cells H. Yi1 , J. Wang2 , M. Kavallaris1 , J.Y. Wang3 . 1 Children’s Cancer Institute Australia for Medical Research, Sydney NSW, Australia, 2 Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, USA, 3 School of Women’s and Children’s Health, Faculty of Medicine University of New South Wales, Sydney NSW, Australia Introduction: Leukemic stem cells (LSCs) are a small population of stem-like cancer cells that are believed to be responsible for the high rate of patient relapse in acute myeloid leukemia (AML). Although aberrant Wnt/b-catenin signaling has recently been shown to be required for the development of LSCs in AML, key components of the pathway remain elusive. Material and Methods: Hematopoietic stem cells were transduced with MLLAF9 or Hoxa9/Meis1a to generate pre-LSCs. Stable knockdown was achieved by lentiviral vector system to express short hairpin RNAs. Tumorigenic potential was assessed in vitro (by methylcellulose colony forming assays) and in vivo (by transplantations into syngeneic C57BL/6 mice). Differential gene expressions were assessed by microarray analysis, and target genes were confirmed by Western blot. Other techniques such as Wright Giemsa staining and apoptosis assay were used to examine changes in cell phenotypes. Results and Discussion: Gene expression analysis of MLL-rearranged AML patient samples revealed a 3-fold increase in expressions of Lgr4 and Ctnnb1 compared to normal hematopoietic stem cells. Here we demonstrated that depletion of Lgr4 impaired pre-LSC growth and significantly prolonged mouse survival through inhibition of b-catenin expression. Furthermore, overexpression of Lgr4 promoted cell growth and accelerated leukemia onset. Differential gene expressions by microarray analysis identified groups of Wnt target genes and genes that regulate G-protein signaling. We showed that inhibition of Gaq by a Gaq specific inhibitor caused marked reduction of colony forming capacity as well as increased differentiation and apoptosis of preLSCs. Conclusion: Our results suggest Lgr4 and Gaq as potential therapeutic targets for elimination of b-catenin-driven LSCs in AML. No conflict of interest. 218 The role of WWOX gene in EMT process in endometrial cancer E. Pluciennik1 , M. Nowakowska1 , K. Pospiech1 , K. Kosla1 , I. Baryla1 , K. Wojcik-Krowiranda2 , A. Bienkiewicz2 , M. Galdyszynska3 , M. Popeda3 , A.K. Bednarek1 . 1 Medical University of Lodz, Department of Molecular Cancerogenesis, Lodz, Poland, 2 Medical University of Lodz, Clinical Division of Gynecological Oncology, Lodz, Poland, 3 Medical University of Lodz, Faculty of of Biomedical Sciences and Postgraduate Education, Lodz, Poland Background: In Poland endometrial cancer is on the first place in morbidity statistics and the third in mortality within reproductive track-related cancers among women. WWOX is a newly described tumour suppressor gene which is affected in many types of tumor including steroid hormones regulated such as breast, ovary, prostate cancer. The EMT process is also of importance in this cancer type with unknown role of WWOX in its regulation. The aim of this study was to precise the role of WWOX in regulation of epithelial to mezenchymal transition in endometrial cancer. Material and Methods: This study was performed on series of 164 endometrial adenocarcinoma patients and well-differentiated steroid-responsive endometrial cell line − ECC1, which was transducted cDNA WWOX by retroviral system. We evaluated correlation between expression of WWOX gene and EMT markers (CDH1, VIM, ZEB1, SNAI1) on mRNA (real-time RT-PCR) and protein level (Western Blot). The EMT process were also analysed in in vitro model with biological assays such as adhesion of cells to six of extracellular matrix proteins, migration through basement membrane, anchorage-independent growth (soft agar assay), activity of MMPs (zymography test). Using DNA microarrays (HumanOneArray™;) we determined WWOX dependent pathways in ECC1 cell line. Results: In patients positive correlation between WWOX and ZEB1 (Rs = 0.26; p = 0.00037), and negative correlation with CDH1 and VIM (Rs = −0.5118, p = 0.0001; Rs = −0.2687, p = 0.0005, respectively) was observed. We also noticed that expression of WWOX inversely correlated with the risk of recurrence, i.e. tumors with low risk had higher WWOX expression, than those with medium and high risk, with the statistically significant difference between low and medium risk of disease recurrence. The negative correlation with VIM and positive with CDH1 was confirmed on protein/mRNA level in transfected cancer cell line. ECC1 cell line with overexpression of WWOX protein revealed increased migratory capacity, with increased expression of metalloproteinases MMP2/MMP9. On the other hand, those cells were not able to form colonies in suspension, which indicate that they loose the aggressive potential. Moreover, this transductants revealed tendency to decrease adhesion to fibronectin and statistically significant decrease in adhesion to fibrinogen. Microarray analysis demonstrated that WWOX has impact on variety of cellular pathway including cadherin and integrin signalling pathways.
Conclusion: Our results suggest the role of WWOX gene in the regulation of EMT process in endometrial cancer via regulation of expression of cell motility associated proteins and thus influencing tissue remodeling, with the important suppression of mezenchymal marker. Acknowledgements: This study was funded by the National Center of Sciences N N407 168940. No conflict of interest. 219 Inactivation of nucleoside-derived anticancer drugs by catabolic prokaryotic enzymes J. Vande Voorde1 , S. Sabuncuoglu1 , J. Balzarini1 , S. Liekens1 . 1 Rega Institute for Medical Research KU Leuven, Microbiology and Immunology, Leuven, Belgium Background: Nucleoside analogues (NAs) target tumor cell replication and represent ~20% of the approved anticancer drugs. NA treatment efficiency depends on the expression and activity of nucleo(s)(t)ide-metabolizing enzymes. Since prokaryotic enzymes are often endowed with a different activity and substrate specificity compared with their mammalian counterparts, bacterial presence in the tumor microenvironment may affect NA-based cancer treatment. Mycoplasmas are part of the normal flora of the human body and several mycoplasma species (e.g. Mycoplasma hyorhinis) are reported to preferentially colonize tumors in cancer patients. These prokaryotes interfere with host cell nucleoside metabolism and may influence the therapeutic efficacy of NAs. Material and Methods: The cytostatic activity of different NAs was evaluated in M. hyorhinis-infected and control tumor cell cultures. The stability and/or metabolism of radiolabeled cladribine and gemcitabine was monitored in mycoplasma-infected and control human MCF-7 breast carcinoma cells. The antitumor activity of gemcitabine and floxuridine was studied in mice bearing M. hyorhinis-infected or uninfected murine mammary FM3A tumors. Results: The cytostatic activity of several NAs (e.g. gemcitabine, cladribine and floxuridine) was severely compromised in various tumor cell cultures upon infection with M. hyorhinis. Accordingly, a significantly decreased antitumor effect of gemcitabine and floxuridine was observed in mice bearing M. hyorhinis-infected tumors. Mycoplasma-mediated drug inactivation of cladribine, floxuridine and gemcitabine was studied in detail. Cladribine and floxuridine were catabolised to their (inactive) free nucleobases by mycoplasma-encoded nucleoside phosphorylases. For gemcitabine, a tandem-mechanism of action in which two catabolic mycoplasma enzymes act in concert was identified: (i) mycoplasma cytidine deaminase (Cyd-DA) catalyzes deamination of gemcitabine to a poorly cytostatic metabolite and (ii) natural inhibitors of Cyd-DA are catabolised by a mycoplasma pyrimidine nucleoside phosphorylase. The cytostatic activity of these NAs was restored by co-administration of a mycoplasma-directed antibiotic or relevant inhibitor of the mycoplasma-encoded catabolic enzymes. These observations suggest that the presence of mycoplasmas in the tumor microenvironment may limit the anticancer efficiency of certain NAs. Conclusions: Recently, commensals were found to be essential for an optimal response to immunotherapy and cyclophosphamide- or platinumbased chemotherapy [Iida et al., Science (2013) 342: 967−70 & Viaud et al., Science (2013) 342: 971−76]. Such findings stress the potential risks of using antibiotics prior to, or during, cancer therapy. However, our results indicate that targeting the microbiome using antibiotics may improve the therapeutic outcome of NA-based cancer treatment. No conflict of interest. 220 Up-regulation of C1GALT1 promotes breast cancer cell growth through MUC1-C signaling pathway C. Chou1 , M.C. Huang1 . 1 National Taiwan University College of Medicine, Graduate Institute of Anatomy and Cell Biology, Taipei City, Taiwan Introduction: Breast cancer is the most frequently diagnosed malignancy in women and the fifth leading cause of cancer-related death worldwide. Core 1 b1,3-galactosyltransferase (C1GALT1) is the exclusive enzyme that catalyzes the formation of T antigens, a cancer associated structure, through the addition of galactose (Gal) to N-acetylgalactosamine (GalNAc)-Ser/Thr. C1GALT1 is pivotal in many biological functions in the developmental process. However, little is known of the function of C1GALT1 in association with cancer development, particularly, breast cancer. This study aims to correlate C1GALT1 expression with breast cancer clinicopathological characteristics and to investigate the role of C1GALT1 in breast cancer malignant behaviors and its underlying mechanisms. Materials and Methods: C1GALT1 expression in breast cancer tissue array (Pantomics) was analyzed by immunohistochemistry. C1GALT1 was knocked down with specific siRNA and C1GALT1/pcDNA3.1A plasmid was constructed to overexpress C1GALT1 in breast cancer cell lines. C1GALT1, MUC1 and MUC1-C expression in cells were analyzed by Western blotting. Vicia villosa (VVA) and peanut agglutinin (PNA) lectins were used to detect Tn- and T-antigens, respectively, in lectin pull-down, Western blotting, and
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 ).
S51
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