292: Cytotoxic effect and apoptosis induction by phytohemagglutinin erythroagglutinating on lung cancer cells

EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 project is to open a gate for defining novel molecular diagnostic and thrapeutic methods by elucidating the functions of p33ING1 ING1 splicing variant in carcinogenesis and metastasis of head and neck cancer. Materials and Methods: We have used head and neck cancer cell lines as material derived from the primary tumor and their lymph node metastasis, which belong to our collaborator Prof. Grenman from Turku University, Finland. First expressions of the ING1 splicing variants and p53 mutation status will be examined in these cells. Then expression vector of the splicing variants will be prepared and overexpressed in the cells by transfecting each of them, followed by analysis of apoptosis, cell cycle and cell growth. Moreover their roles in metastasis will also be investigated with specific tests. Results: We have shown that splice variant of p33ING1 suppresses the proliferation of primary and metastatic cells and also it restrains the cancer cell movement via suppressing the migration of head and neck cells. Conclusion: Splice variant of p33ING1 has oncogenic function in head and neck cancer cells. It suppresses the proliferation and migration of head and neck cancer cells. No conflict of interest. 291 The 5 -untranslated region of p16INK4a acts as a cellular IRES, controls mRNA translation during hypoxic and energetic stresses, and is a target of YBX1 A. Bisio1 , E. Latorre2 , V. Andreotti3 , P. Ghiorzo3 , B. Bressac-de Paillerets4 , R.C. Spitale5 , A. Provenzani2 , A. Inga1 . 1 Laboratory of Transcriptional Networks Centre for Integrative Biology CIBIO, University of Trento, Trento, Italy, 2 Laboratory of Genomics ScreeningsCentre for Integrative Biology CIBIO, University of Trento, Trento, Italy, 3 Laboratory of Genetics of Rare Hereditary Cancers, DiMI University of Genoa, Genoa, Italy, 4 Institut Gustave Rousy, Villejuif, France, 5 Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford CA, USA Background: In mammalian cells, controlled progression through the cell cycle is essential for normal proliferation and its loss is a hallmark of malignancy. p16INK4a is a well known tumor suppressor gene acting as an inhibitor of cell cycle progression and its deregulation is often associated with many types of cancer, including melanoma. Here we report that p16INK4a belongs to the expanding group of proteins whose translation is influenced by sequence/structural features of the 5 UTR mRNA that are endowed of socalled cellular Internal Ribosome Entry Site (IRES) activity. Material and Methods: To study the potential for p16INK4a 5 UTR to drive capindependent translation we developed a dual-luciferase assay using bicistronic vectors, where wild type or deletion mutants of the p16INK4a 5 UTRs can be studied. Results: Quantification of reporters’ relative activities coupled to control analyses for actual bicistronic mRNA transcription, indicated that the wild type p16INK4a 5 UTR could stimulate cap-independent translation. Notably, hypoxic stress in particular, but also the treatment with mTOR inhibitors, enhanced the translation-stimulating property of the wild type p16INK4a 5 UTR. RNA immuno-precipitation (RIP) assays performed in the p16INK4a -positive melanoma-derived cell line SK-Mel-28, and in melanoma patients-derived lymphoblastoid cell lines, indicated that the RNA-binding protein YBX1, known to act in translation control, can target wild type p16INK4a mRNA and enhance its translation, particularly during hypoxic stress. Experiments where YBX1 was over-expressed or knocked-down confirmed its involvement in p16INK4a capindependent translational regulation. The p16INK4a c.−42T>A sequence-variant was instead no longer influenced by changes in YBX1 protein level, consistent with predictions of the binding site of this RBP to the p16INK4a 5 UTR and with results based on RNA SHAPE assays. Conclusions: Taken collectively, our results suggest that the p16INK4a 5 UTR region acts as cellular IRES that can modulate mRNA translation efficiency and can be positively regulated by YBX1. No conflict of interest. 292 Cytotoxic effect and apoptosis induction by phytohemagglutinin erythroagglutinating on lung cancer cells K.Y. Chen1 , W.T. Kuo2 , Y.J. Ho3 , C.H. Yao3 . 1 National Yunlin University of Science and Technology, Department of Chemical and Materials Engineering, Yunlin, Taiwan, 2 China Medical University, Graduate Institute of Clinical Medical Science, Taichung, Taiwan, 3 China Medical University, Department of Biomedical Imaging and Radiological Science, Taichung, Taiwan Introduction: Lung cancer is currently the leading cause of cancer deaths in the world. Therefore, it is critical to study new and effective drug treatments for lung cancer. Phytohemagglutinin, a lectin derived from red kidney beans, has been reported to inhibit the growth of cancer cells. In this study, the anticancer effects of phytohemagglutinin erythroagglutinating, one of isoforms of phytohemagglutinin, on lung cancer cell A549 were evaluated. Material and Method: Human A549 lung cancer cells were treated with various concentrations of phytohemagglutinin erythroagglutinating. After 2 days of culture, the cytotoxicity and apoptosis-inducing potential of phytohemagglutinin

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erythroagglutinating were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay, glucose-6-phosphate dehydrogenase) release assay and flow cytometry. Results and Discussion: Phytohemagglutinin erythroagglutinating caused a dose-dependent increase in cell growth inhibition and cell death of A549 cells. The percentage of apoptotic cells was increased with increasing phytohemagglutinin erythroagglutinating concentrations. Moreover, dead cells and live cells were a dose-dependent increase and decrease. These results suggest that phytohemagglutinin erythroagglutinating induced growth inhibition and cytotoxicity of A549 cells is mediated through an induction of apoptosis. Conclusion: Phytohemagglutinin erythroagglutinating effectively inhibited the growth of A549 cells and induced their apoptosis. Therefore, phytohemagglutinin erythroagglutinating could be developed into an effective anti-lung cancer drug. No conflict of interest. 293 MNT roles and expression in the absence of MAX M.C. Lafita1 , A. Quintanilla1 , J. Rodr´ıguez2 , I. Varela1 , A. Von Kriegsheim2 , J. Leon ´ 1 . 1 IBBTEC Universidad de Cantabria, Biolog´ıa Molecular, Santander, Spain, 2 Conway Institute, System Biology Ireland, Dublin, Ireland Introduction: MNT is a transcription factor of the MXD family. MXDs proteins take part in MYC/MAX/MXD network regulating genes involved in cell proliferation, differentiation, metabolism, cell growth and apoptosis. Alterations in MYC/MAX/MXD network have been found to be responsible of cancer development. We wanted to explore whether MNT has biological functions independent from MAX. Material and Methods: UR61 cells derivated from rat pheochromocytoma cell line PC12 that lacks MAX wt protein; UR61MAX cells, UR61 expressing a zinc-inducible human MAX gene; K562 human chronic myeloid leukemia cells and 293T human embryonic kidney cell line. siRNA against human MAX and rat MNT to downregulate their expression. RT-qPCR and WB to study gene expression at mRNA and protein levels. High-throughput sequencing techniques to analyse chromatin protein binding (ChIPseq). High-throughput proteomic techniques, IP-mass spectrometry, to look for new interacting partners. IP and ChIP to confirm the results obtained. Results and Discussion: To study the role of MNT in cells that lack MAX protein we silenced MNT protein in UR61 with siRNA and observed that it became lethal for the cells, suggesting a pro-survival role of MNT in these cells lacking MAX protein. Therefore, MNT down-regulation provokes cell growth inhibition in a MAX independent manner. We analysed MNT expression in UR61 and UR61MAX. In UR61, MAX expression leads to a downregulation of MNT at mRNA and protein levels. In K562 cells, silencing of MAX provoked MNT upregulation, confirming the results obtained in UR61. Bioinformatic analysis of the promoter of MNT gene showed 2 Eboxes within −1Kb from the transcription start site in rat and human MNT genes. ChIP assays for MNT and MAX proteins indicates that MNT and MAX are bound to MNT promoter in UR61MAX cells but not in UR61 cells. This suggests that MNT binds to its own promoter and regulates its own expression only when there is MAX in the cell. To elucidate whether MNT have different target genes when MAX is not present we did a ChIPseq assay in UR61 and UR61MAX and we found differences in MNT-DNA binding depending on MAX presence. To look for new possible partners of MNT, IP-mass spectrometry assay was performed in UR61 and UR61MAX. At least 70 protein interactions were detected. However, MNT interacting partners are different when MAX is present. Conclusion: We can conclude that MNT down-regulation impairs cell growth in a MAX-independent manner and that MAX induces a decrease in MNT expression. We have revealed new MNT interacting partners that might be involved in a pro-survival role of MNT in the cell. We think it is worthy to take into a count our work since understanding cancer development mechanisms can help design new treatments for cancer therapies. No conflict of interest. 294 In vitro modulation of CITED4 gene expression in a colorectal cancer cell line M.A. Rogers1 , V. Kalter1 , G. Marcias1 , M. Zapatka1 , S. Barbus1 , B. Radlwimmer1 , P. Lichter1 . 1 German Cancer Research Center, Division of Molecular Genetics (B060), Heidelberg, Germany Background: CITED4 is one member of the CITED family of transcriptional cofactors. Several of the CITED family members are deregulated in a variety of tumors. Analysis of data from the literature points to a possible role of CITED4 in colon carcinogenesis. In this study we deregulate CITED4 expression, in vitro, in a human colorectal cancer cell line, and analyze the phenotypic and gene expression changes induced by modulation of CITED4. Methods: CITED4 overexpressing- and shRNA-mediated knockdown cell lines, as well as control cell lines, were established in the colorectal cancer (CRC) cell line SW480. The cells were analyzed phenotypically, in vitro, for changes in proliferation, apoptosis/cell cycle, migration, invasion, colony formation and adhesion. Changes in mRNA expression were determined by