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Frequent and functional derepression of the Iroquois homeobox gene IRX3 in human acute leukemia
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218 with somatostatin analogues. According to the literature, the expression of somatostatin receptors, especially SSTR1, is positively correlated with the patient survival rate which justifies the use of these receptor genes as prognostic markers in NETs. Conclusions: Identification of gene profile may represent a sensitive tool for diagnosis and therapy in NETs. The virtual device will allow the pathologist, instead of using a broad panel of antibodies, to detect and quickly quantify the degree of differentiation, specific hormone production expression of somatostatin receptor, the potential of proliferation and angiogenesis. Acknowledgement: The study was supported by Project RENET − PN-II-PTPCCA-2011-3.2.0623 91/20012. No conflict of interest.
Sunday 10 July 2016 Poster Session
Cell and Tumour Biology I 215 Frequent and functional derepression of the Iroquois homeobox gene IRX3 in human acute leukemia T. Somervaille1 , T. Somerville1 . 1 Cancer Research UK Manchester Institute, Leukaemia Biology, Manchester, United Kingdom Background: Transcription factors are master regulators of cell fate and their tissue-inappropriate mis-expression is an important and emerging mechanism of oncogenic transformation. Methods and Results: Using bioinformatics methods and quantitative PCR, we identified the Iroquois homeobox transcription factor gene IRX3 as highly expressed in ~30% of cases of primary human acute myeloid leukemia (AML), but minimally expressed in prospectively purified normal human hematopoietic populations (stem, progenitors and mature). High IRX3 expression in AML is strongly associated with mutated NPM1, FLT3-ITD and high concomitant HOXA gene expression. High IRX3 expression is also observed in both T-acute lymphoblastic leukemia (T-ALL) (~50% of cases) and B-ALL (~20% of cases), also in association with high HOX gene expression. Iroquois members have essential developmental roles in mesodermal tissues such as heart, fat and bone, but to date there is no reported functional role for IRX3 in leukemic hematopoiesis. Compared with control cells, forced expression of IRX3 in murine CD117+ bone marrow hematopoietic stem and progenitor cells (BM HSPCs) resulted in enhanced serial replating in semisolid culture and significantly impaired morphologic differentiation. In transplantation experiments, murine recipients of IRX3-expressing CD117+ BM HSPCs exhibited a significant expansion of LY6C+ T-cells and later developed donor-derived T-cell leukemias with incomplete penetrance. In coexpression experiments, Hoxa9/IRX3 double transduced CD117+ BM HSPCs exhibited significantly greater clonogenic cell frequencies and morphologic differentiation block by comparison with control Hoxa9/empty vector (MTV) cells. In vivo, in the post-transplant period Hoxa9/IRX3 BM HSPCs exhibited multilineage differentiation but with skewing away from the myeloid lineage and towards the B-lineage by comparison with control Hoxa9/MTV cells. Nevertheless, donor-derived AML subsequently developed in all mice in both cohorts. Unexpectedly, recipients of control Hoxa9/MTV cells developed AML significantly more rapidly than recipients of Hoxa9/IRX3 cells (median 125 days versus 270 days). However, mice receiving Hoxa9/IRX3 cells developed leukemias with a much more pronounced differentiation block by comparison with Hoxa9/MTV recipients, as evidenced by immunophenotype, morphology and transcriptome. IRX3 knockdown in both human and murine AML cells induced differentiation and loss of clonogenic potential, whereas similar experiments in primary human CD34+ cells did not affect clonogenic potential. Conclusions: De-repression of the mesodermal transcription factor gene IRX3 in human acute leukemia is both frequent and functional, contributing to leukemic transformation through skewing differentiation, enhancing selfrenewal and modulating the cellular consequences of HOX gene expression. No conflict of interest. 216 Tumour suppressor genes of common fragile sites: Active players in DNA damage response?! R. Aqeilan1 . 1 Hebrew University-Hadasah Medical School, Immunology & Cancer Research, Jerusalem, Israel The role of common fragile sites (CFSs) in cancer is controversial. Two main views dominate the discussion: one view suggests that CFSs are inert structures associated with genomic instability leading to inactivation of genes associated within them. The opposing view suggests that CFSs are functional units harboring genes and other elements that when altered lead to selective pressure contributing to cancer development and/or other diseases. This view is supported by emerging evidence showing that inactivation of these residing tumour suppressor genes leads to dysregulation of the DNA damage response
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(DDR) and increased genomic instability. Emerging evidence from our lab and others directly links CFS gene products with the DDR, genomic instability, carcinogenesis and other disease states. These findings underscore the critical roles of these genes and how their inactivation represents selective advantage in pathogenesis. No conflict of interest. 218 Study of the biomarker potential of circulating non-coding PRNCR1 & CCAT2 RNAs in plasma of breast cancer patients E. Babaei1 , V. Montazeri2 . 1 Genetics, School of Natural Sciences, Tabriz, Iran, 2 Noor Najat Hospital, Torax, Tabirz, Iran Introduction: Despite recent breakthroughs in the treatment of breast cancer, early non-invasive detection and prognosis remain poor. Emerging evidence shows that long non-coding RNAs (lncRNAs) play an important role in cancer initiation and progression. Here, we evaluated the expression of PRNCR1 & CCAT8 as novel lncRNAs in plasma of breast cancer patients. Materials and Methods: The expression levels of PRNCR1 & CCAT8 in 50 pairs of breast cancer patients and normal women were detected by quantitative real-time PCR (qRT-PCR). Results and Discussion: Our data showed that both lncRNAs are upregulated in plasma of breast cancer patients in comparison with normal ones. Furthermore, statistical data revealed that lncPRNCR1 & lncCCAT2 are significantly expressed in advanced stages rather than lower stages. Also, ROC analysis demonstrated the biomarker potential of circulating CCAT2. Conclusion: Our data provides the first evidence detecting lncRNAs; PRNCR1 & CCAT2 in blood of cancer patients. Meanwhile, the positive correlation between their expression levels and carcinomas as well as their TNM stage highlights PRNCR1 & CCAT2 as a biomarker for detection and prognosis of breast malignancies. No conflict of interest. 219 Determination of pyruvate metabolic fate regulates head and neck tumourigenesis W.C. Li1,2,3 , T.Y. Chen1 , J.M. Huang1 , C.W. Chang1 , C.Y. Chen1 , W.J. Chang1 , C.J. Liu1,2,4,5 , H.M. Chen6 , J.F. Lo1,2,3,7 . 1 Institute of Oral Biology, School of Dentistry- National Yang-Ming University, Taipei, Taiwan, 2 Department of Dentistry, School of Dentistry- National Yang-Ming University, Taipei, Taiwan, 3 Genome Research Center, National Yang-Ming University, Taipei, Taiwan, 4 Department of Medical Research, MacKay Memorial Hospital, Taipei, Taiwan, 5 Department of Oral and Maxillofacial Surgery, MacKay Memorial Hospital, Taipei, Taiwan, 6 Department of Dentistry, National Taiwan University Hospital- College of Medicine, Taipei, Taiwan, 7 Department of Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan Introduction: Most cancer cells preferentially obtain cellular energy and biomacromolecules via aerobic glycolysis over mirochondrial oxidative phosphorylation. Indeed, previous studies have shown that suppression of glycolytic enzymes could modulate cell malignancy in various cancers. Pyruvate is the main glycolytic metabolite that could be sequentially converted into either lactate or acetyl-CoA by Lactate Dehydrogenase A (LDHA) and Pyruvate Dehydrogenase Complex (PDC), repectively, under various physiological conditions. In tumour cells, lactate is largely procuded and exported out of cells resulting in acidic environment whereas the conversion of pyruvate to acetly-CoA, a key reactant for TriCarboxylic Acid (TCA) cycle in mitochondrion, is often less active. Owing to this variance, the determination of pyruvate metabolism might be of great potential to regulate tumorous phenotype in cancer cells. Material and Method: The cellular and molecular regulations of LDHA and Pyruvate dehydrogenase E1 component a subunit (PDHA1), a catalytic subunit of mitochondrial PDC, for cell malignancy in Head and Neck Squamous Cell Carcinoma (HNSCC) were examined. The multifaceted cancer phenotypes were identified in vitro in shRNA-mediated LDHA and PDHA1 deficient HNSCC cells while in vivo growth of LDHA and PDHA1 deficient HNSCC derived xenografic tumours were analysed. Results and Discussion: In vitro analysis showed that decreased cell growth and cell migration/invasion capacity as well as up-regulated cell differentiation and drug sensitivity to chemotherapeutic agents in LDHA deficient HNSCC cells were observed. Loss of PDHA1, on the contrary, enhanced cell growth, cell motility, drug resistance but inhibited cell differentiation. The xenografic tumours deficient for LDHA grew slower than control tumours whereas PDHA1 knockdown resulted in larger tumour mass in vivo. In molecular level, the protein array analysis demonstrated that LDHA/PDHA1 mediated regulation for HNSCC tumourigenesis could possibly be through the modulation of ERK/JNK/p38-c-jun pathway. Conclusion: Taken together, the choices of pyruvate metabolic fates regulated cell malignancy in HNSCC cells revealing that the balance between lactate production and oxidative phosphorylation activity plays a significant role for HNSCC development. No conflict of interest.
Sunday 10 July 2016 Poster Session
Cell and Tumour Biology I 215 Frequent and functional derepression of the Iroquois homeobox gene IRX3 in human acute leukemia T. Somervaille1 , T. Somerville1 . 1 Cancer Research UK Manchester Institute, Leukaemia Biology, Manchester, United Kingdom Background: Transcription factors are master regulators of cell fate and their tissue-inappropriate mis-expression is an important and emerging mechanism of oncogenic transformation. Methods and Results: Using bioinformatics methods and quantitative PCR, we identified the Iroquois homeobox transcription factor gene IRX3 as highly expressed in ~30% of cases of primary human acute myeloid leukemia (AML), but minimally expressed in prospectively purified normal human hematopoietic populations (stem, progenitors and mature). High IRX3 expression in AML is strongly associated with mutated NPM1, FLT3-ITD and high concomitant HOXA gene expression. High IRX3 expression is also observed in both T-acute lymphoblastic leukemia (T-ALL) (~50% of cases) and B-ALL (~20% of cases), also in association with high HOX gene expression. Iroquois members have essential developmental roles in mesodermal tissues such as heart, fat and bone, but to date there is no reported functional role for IRX3 in leukemic hematopoiesis. Compared with control cells, forced expression of IRX3 in murine CD117+ bone marrow hematopoietic stem and progenitor cells (BM HSPCs) resulted in enhanced serial replating in semisolid culture and significantly impaired morphologic differentiation. In transplantation experiments, murine recipients of IRX3-expressing CD117+ BM HSPCs exhibited a significant expansion of LY6C+ T-cells and later developed donor-derived T-cell leukemias with incomplete penetrance. In coexpression experiments, Hoxa9/IRX3 double transduced CD117+ BM HSPCs exhibited significantly greater clonogenic cell frequencies and morphologic differentiation block by comparison with control Hoxa9/empty vector (MTV) cells. In vivo, in the post-transplant period Hoxa9/IRX3 BM HSPCs exhibited multilineage differentiation but with skewing away from the myeloid lineage and towards the B-lineage by comparison with control Hoxa9/MTV cells. Nevertheless, donor-derived AML subsequently developed in all mice in both cohorts. Unexpectedly, recipients of control Hoxa9/MTV cells developed AML significantly more rapidly than recipients of Hoxa9/IRX3 cells (median 125 days versus 270 days). However, mice receiving Hoxa9/IRX3 cells developed leukemias with a much more pronounced differentiation block by comparison with Hoxa9/MTV recipients, as evidenced by immunophenotype, morphology and transcriptome. IRX3 knockdown in both human and murine AML cells induced differentiation and loss of clonogenic potential, whereas similar experiments in primary human CD34+ cells did not affect clonogenic potential. Conclusions: De-repression of the mesodermal transcription factor gene IRX3 in human acute leukemia is both frequent and functional, contributing to leukemic transformation through skewing differentiation, enhancing selfrenewal and modulating the cellular consequences of HOX gene expression. No conflict of interest. 216 Tumour suppressor genes of common fragile sites: Active players in DNA damage response?! R. Aqeilan1 . 1 Hebrew University-Hadasah Medical School, Immunology & Cancer Research, Jerusalem, Israel The role of common fragile sites (CFSs) in cancer is controversial. Two main views dominate the discussion: one view suggests that CFSs are inert structures associated with genomic instability leading to inactivation of genes associated within them. The opposing view suggests that CFSs are functional units harboring genes and other elements that when altered lead to selective pressure contributing to cancer development and/or other diseases. This view is supported by emerging evidence showing that inactivation of these residing tumour suppressor genes leads to dysregulation of the DNA damage response
S39
(DDR) and increased genomic instability. Emerging evidence from our lab and others directly links CFS gene products with the DDR, genomic instability, carcinogenesis and other disease states. These findings underscore the critical roles of these genes and how their inactivation represents selective advantage in pathogenesis. No conflict of interest. 218 Study of the biomarker potential of circulating non-coding PRNCR1 & CCAT2 RNAs in plasma of breast cancer patients E. Babaei1 , V. Montazeri2 . 1 Genetics, School of Natural Sciences, Tabriz, Iran, 2 Noor Najat Hospital, Torax, Tabirz, Iran Introduction: Despite recent breakthroughs in the treatment of breast cancer, early non-invasive detection and prognosis remain poor. Emerging evidence shows that long non-coding RNAs (lncRNAs) play an important role in cancer initiation and progression. Here, we evaluated the expression of PRNCR1 & CCAT8 as novel lncRNAs in plasma of breast cancer patients. Materials and Methods: The expression levels of PRNCR1 & CCAT8 in 50 pairs of breast cancer patients and normal women were detected by quantitative real-time PCR (qRT-PCR). Results and Discussion: Our data showed that both lncRNAs are upregulated in plasma of breast cancer patients in comparison with normal ones. Furthermore, statistical data revealed that lncPRNCR1 & lncCCAT2 are significantly expressed in advanced stages rather than lower stages. Also, ROC analysis demonstrated the biomarker potential of circulating CCAT2. Conclusion: Our data provides the first evidence detecting lncRNAs; PRNCR1 & CCAT2 in blood of cancer patients. Meanwhile, the positive correlation between their expression levels and carcinomas as well as their TNM stage highlights PRNCR1 & CCAT2 as a biomarker for detection and prognosis of breast malignancies. No conflict of interest. 219 Determination of pyruvate metabolic fate regulates head and neck tumourigenesis W.C. Li1,2,3 , T.Y. Chen1 , J.M. Huang1 , C.W. Chang1 , C.Y. Chen1 , W.J. Chang1 , C.J. Liu1,2,4,5 , H.M. Chen6 , J.F. Lo1,2,3,7 . 1 Institute of Oral Biology, School of Dentistry- National Yang-Ming University, Taipei, Taiwan, 2 Department of Dentistry, School of Dentistry- National Yang-Ming University, Taipei, Taiwan, 3 Genome Research Center, National Yang-Ming University, Taipei, Taiwan, 4 Department of Medical Research, MacKay Memorial Hospital, Taipei, Taiwan, 5 Department of Oral and Maxillofacial Surgery, MacKay Memorial Hospital, Taipei, Taiwan, 6 Department of Dentistry, National Taiwan University Hospital- College of Medicine, Taipei, Taiwan, 7 Department of Stomatology, Taipei Veterans General Hospital, Taipei, Taiwan Introduction: Most cancer cells preferentially obtain cellular energy and biomacromolecules via aerobic glycolysis over mirochondrial oxidative phosphorylation. Indeed, previous studies have shown that suppression of glycolytic enzymes could modulate cell malignancy in various cancers. Pyruvate is the main glycolytic metabolite that could be sequentially converted into either lactate or acetyl-CoA by Lactate Dehydrogenase A (LDHA) and Pyruvate Dehydrogenase Complex (PDC), repectively, under various physiological conditions. In tumour cells, lactate is largely procuded and exported out of cells resulting in acidic environment whereas the conversion of pyruvate to acetly-CoA, a key reactant for TriCarboxylic Acid (TCA) cycle in mitochondrion, is often less active. Owing to this variance, the determination of pyruvate metabolism might be of great potential to regulate tumorous phenotype in cancer cells. Material and Method: The cellular and molecular regulations of LDHA and Pyruvate dehydrogenase E1 component a subunit (PDHA1), a catalytic subunit of mitochondrial PDC, for cell malignancy in Head and Neck Squamous Cell Carcinoma (HNSCC) were examined. The multifaceted cancer phenotypes were identified in vitro in shRNA-mediated LDHA and PDHA1 deficient HNSCC cells while in vivo growth of LDHA and PDHA1 deficient HNSCC derived xenografic tumours were analysed. Results and Discussion: In vitro analysis showed that decreased cell growth and cell migration/invasion capacity as well as up-regulated cell differentiation and drug sensitivity to chemotherapeutic agents in LDHA deficient HNSCC cells were observed. Loss of PDHA1, on the contrary, enhanced cell growth, cell motility, drug resistance but inhibited cell differentiation. The xenografic tumours deficient for LDHA grew slower than control tumours whereas PDHA1 knockdown resulted in larger tumour mass in vivo. In molecular level, the protein array analysis demonstrated that LDHA/PDHA1 mediated regulation for HNSCC tumourigenesis could possibly be through the modulation of ERK/JNK/p38-c-jun pathway. Conclusion: Taken together, the choices of pyruvate metabolic fates regulated cell malignancy in HNSCC cells revealing that the balance between lactate production and oxidative phosphorylation activity plays a significant role for HNSCC development. No conflict of interest.