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Hypermethylation and downregulation of glutathione peroxidase 3 are related to pathogenesis of melanoma
S190
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
can give rise to CDX models in immune-compromised mice. Here we compare the pathobiology of 16 newly generated CDX models generated from blood samples of extensive stage SCLC patients. Methods: Histopathology and immunohistochemistry (IHC) analyses of 16 CDX was performed and compared to available matched diagnostic clinical specimens. CDX were also molecularly characterised using Whole Exome Sequencing (WES) and RNA-sequencing (RNASeq). Results: All CDX models had common as well as distinct pathological features. 15/16 CDX expressed (NE) markers and had 85% Ki67 positive cells consistent with the rapid proliferation of SCLC cells. Patient specimens and CDXs showed densely packed small cells, scant cytoplasm, finely granular chromatin organised in nests, trabeculae and rosette patterns. The NE progenitor marker ASCL1 is predominantly expressed in SCLC patients and the Genetically Engineered Mouse Model of SCLC developed by A. Berns demonstrated the neuroendocrine cell of origin for SCLC. However, 2/16 CDX models expressed an alternative NE progenitor marker NeuroD1 in the absence of ASCL1. Profound differences between CDX models were observed regarding the degree of angiogenesis (murine specific anti-CD31) and prevalence of mesenchymal phenotypes (anti-vimentin). Human SCLC noted for a relatively minimal stromal component and this held true in 13/16 CDX models; however 3/16 CDX had significantly higher stromal composition. WES data confirmed prevalent SCLC abnormalities (including CNA loss and mutations in remaining alleles of p53 and RB1) and considerable heterogeneity amongst CDX models also observed in the RNA seq data sets. Conclusion: We show here that CDX models, derived from a minimally invasive ‘liquid biopsy’ recapitulate donor patient histopathology. CDX models cannot be considered true ‘avatars’ because the time-frame for their generation and characterisation extends the lifespan of the donor patients. Nevertheless, they do provide a unique opportunity to increase our understanding of this devastating disease and are being used test novel therapies. The biology associated with the differential expression of ASCL1 and NeuroD1 amongst the CDX models is currently under investigation alongside the impact of stromaltumour cell interactions. No conflict of interest. 827 NUB1 protein regulates cullins and E3 ligases in estrogen receptor negative breast cancers K.L. Tan1 , S. Haider2 , C. Zois2 , H. Turley1 , R. Leek1 , A. Harris2 , F. Buffa2 , O. Acuto3 , F. Pezzella1 . 1 University of Oxford, Radcliffe Department of Medicine, Oxford, United Kingdom, 2 University of Oxford, Department of Oncology, Oxford, United Kingdom, 3 University of Oxford, Sir William Dunn School of Pathology, Oxford, United Kingdom Background: The NEDD8 ultimate buster (NUB1) protein is a tumour suppressor that causes degradation of neddylated and fat10ylated proteins through the ubiquitin proteasome system. Aryl Hydrocarbon Receptor Interacting Protein-Like 1 (AIPL1) interacts with NUB1 and restricts the nucleus entry of NUB1 protein. Upregulated NUB1 expression inhibits proliferation of IFNa-resistant cancer cells. The mechanistic roles of NUB1 protein remain unexplored. Materials and Methods: NUB1 proteins expression and global protein neddylation and fat10ylation were examined by immunohistochemistry and immunoblotting. Cell counts and cell cycle profiles were determined upon neddylation inhibition by MLN4924, a NEDD8-activating enzyme inhibitor. Survival was analysed with Kaplan–Meier methods and compared by the logrank test. All statistical tests were two sided. Results: The microarray study and meta-analysis of cancer databases revealed that expression transcripts of chaperones, including AIPL1, were downregulated in lung and pancreatic cancers in comparison to the adjacent normal tissues. Bioinformatics using METABRIC dataset showed that low NUB1 transcripts had poor survival in ER negative subgroup of breast cancer patients: hazard ratio (HR) = 0.66, 95% confidence interval (CI) 0.5–0.87, p = 0.003 and triple negative subgroup of breast cancer patients: HR = 0.67, 95% CI 0.47–0.96, p = 0.028. NUB1 silencing promotes in vitro cell growth in MDA-MB-231, MCF7 and BT474. NUB1 proteins move into nucleus upon chronic severe hypoxia, in timely manner. NUB1-silent HCC1806 cells expressed higher p21 and p27 proteins. Upon the treatment of neddylation inhibition, HCC1806 cells with siNUB1 transfection showed decreased CUL1 which led to accumulation of tumour suppressive cullin-RING E3 ligase substrates, p21 (marker of senescence) & p27 (marker of cell cycle arrest). In the same condition, more observed dead cells were thought to be caused by senescent cells that drive cellular aging and depletion. It suggest that the NUB1 induced-cell death of cancer cells upon hypoxia happen through neddylationdependent CUL1-p27-p21 axis. We demonstrated that HIF1a protein could be both neddylated and FAT10ylated upon reoxygenation. In comparison to RCC4plusVHL cells, NUB1-silent null-VHL RCC4 cells expressed higher HIF1a protein. It suggests the upregulated HIF1a protein level is CUL2VHL dependent. In tissue microarray (TMA) study, compared with high cytoplasmic expression, those with low cytoplasmic expression had worse overall: HR = 0.432, 95% CI 0.201–0.663, p = 0.002.
Conclusions and Further study: Our study highlights the cell cycle regulation mechanism of NUB1 proteins in breast cancers. NUB1 could be a promising therapeutic target that prevents cancer progression. We plan to utilise multiplexed ion beam imaging technique to study ER-negative subgroup primary tumour against its metastatic nodes. No conflict of interest. 828 Hypermethylation and downregulation of glutathione peroxidase 3 are related to pathogenesis of melanoma J.Y. Bae1 , X. Zhang2 . 1 Center for RNA Research- Institute for Basic Science, School of Biological Sciences- College of Natural Sciences- Seoul National University, Seoul, Korea, 2 Oral Cancer Research Institute, Department of Oral Pathology- Yonsei University College of Dentistry, Seoul, Korea Introduction: As well known potential carcinogens, reactive oxygen species (ROS) are involved in both carcinogenesis and tumor progression via ROSinduced DNA damage. During normal metabolism, cells avoid oxidative damage by means of antioxidant defense mechanisms that control the balance between the generation and removal of oxygen radicals. The antioxidant system includes various enzymes, such as superoxide dismutase and catalase, as well as glutathione peroxidase (GPX). GPX3, a member of the GPX family, has been found to be frequently repressed in many cancers due to promoter hypermethylation and is known as a possible tumor suppressor gene. Materials and Methods: mRNA and protein expression of GPX3 was comparatively investigated in human epidermal melanocyte (HEMs) and 2 melanoma cell lines using RT-PCR analysis and immunocytochemistry. The influence of GPX3 expression on biological behavior of melanoma cells were investigated in GPX3 depleted melanoma cell line in vitro. The clinicopathological significance of GPX3 was investigated 105 melanoma patients by immunohistochemistry. Methylation of GPX3 was detected by methylation specific PCR in HEMs, 2 melanoma cell lines, 3 normal skin samples and 105 melanoma tissues. Results and Discussion: GPX3 expression was downregulated and methylated in melanoma cell lines and tissue samples. In melanoma cell lines, GPX3 expression was restored by treatment with 5-aza-2 -deoxycytidine. Depletion of GPX3 was found to increase the proliferative ability, motility, and invasiveness of melanoma cells. Moreover, negative expression of GPX3 was related to poor prognosis in melanoma patients. These results suggest that methylation-mediated GPX3 repression may have critical implications for melanoma pathogenesis. Conclusion: The epigenetic inactivation of GPX3 may be a crucial mechanism in the pathogenesis of melanoma, and GPX3 may serve as a possible predictive or prognostic biomarker and therapeutic target for melanoma patients. No conflict of interest. 829 Bisphosphonates potentiate the activity of pitavastatin against ovarian cancer cells M. Abdullah1 , A. Richardson2 . 1 Keele university, Institute for Science and Technology in Medicine-Guy Hilton Research Centre, Stoke on Trent, United Kingdom, 2 Keele university, Institute for Science and Technology in MedicineGuy Hilton Research Centre, Stoke on Trent, United Kingdom Background: Although many ovarian cancer patients respond to chemotherapy, the majority relapse and drug resistance usually prevents further effective treatment. As a result, the five year survival rate is only 40%. Novel therapeutic approaches are required. We have shown that statins, which inhibit the synthesis of mevalonate by HMG-CoA reductase, have the potential to treat ovarian cancer. Statins inhibit the production of isoprenoids which are necessary for the membrane localization of several small G-protein oncogenes. However, a major limitation for the clinical application of statin as anti-tumor therapy is the requirement for a high dose which may cause adverse effects such as myopathy. Bisphosphonates (BP), drugs used to treat osteoporosis, inhibits farnesyl diphosphate synthase and consequently the synthesis of isoprenoids. We hypothesized that bisphosphonates would potentiate the activity of the statins in ovarian cancer cells. This may allow a reduction in the dose of statin necessary to treat ovarian cancer and minimize the risk of adverse effects. Consequently, we evaluated the activity of the BPs, zoledronic acid and risedronate, in combination with pitavastatin against a panel of ovarian cancer cell lines. Methods: The effect of combinations of pitavastatin and zoledronic acid or risedronate on ovarian cancer cell lines was assessed in several different assays including cell growth, trypan blue exclusion, intracellular ATP, Caspase -3/7, -8, and -9 activity as well as PARP cleavage. Results: When tested as single agents, pitavastatin (IC50 = 0.6−14 mM) was the most potent of the drugs in cell growth assays, whereas zoledronic acid (IC50 = 21−57 mM) and risedronate (IC50 >100mM) showed only modest activity. Combinations of pitavastatin with zoledronic acid, and to lesser extend risedronate, displayed synergistic activity in A2780, CisA2780, Ovcar-4, Igrov-1 and Skov-3 cells or additivity in Cov-318, Cov-362, Ovcar-5 and Ovcar-8 cells.
EACR24 Poster Sessions / European Journal of Cancer 61, Suppl. 1 (2016) S9–S218
can give rise to CDX models in immune-compromised mice. Here we compare the pathobiology of 16 newly generated CDX models generated from blood samples of extensive stage SCLC patients. Methods: Histopathology and immunohistochemistry (IHC) analyses of 16 CDX was performed and compared to available matched diagnostic clinical specimens. CDX were also molecularly characterised using Whole Exome Sequencing (WES) and RNA-sequencing (RNASeq). Results: All CDX models had common as well as distinct pathological features. 15/16 CDX expressed (NE) markers and had 85% Ki67 positive cells consistent with the rapid proliferation of SCLC cells. Patient specimens and CDXs showed densely packed small cells, scant cytoplasm, finely granular chromatin organised in nests, trabeculae and rosette patterns. The NE progenitor marker ASCL1 is predominantly expressed in SCLC patients and the Genetically Engineered Mouse Model of SCLC developed by A. Berns demonstrated the neuroendocrine cell of origin for SCLC. However, 2/16 CDX models expressed an alternative NE progenitor marker NeuroD1 in the absence of ASCL1. Profound differences between CDX models were observed regarding the degree of angiogenesis (murine specific anti-CD31) and prevalence of mesenchymal phenotypes (anti-vimentin). Human SCLC noted for a relatively minimal stromal component and this held true in 13/16 CDX models; however 3/16 CDX had significantly higher stromal composition. WES data confirmed prevalent SCLC abnormalities (including CNA loss and mutations in remaining alleles of p53 and RB1) and considerable heterogeneity amongst CDX models also observed in the RNA seq data sets. Conclusion: We show here that CDX models, derived from a minimally invasive ‘liquid biopsy’ recapitulate donor patient histopathology. CDX models cannot be considered true ‘avatars’ because the time-frame for their generation and characterisation extends the lifespan of the donor patients. Nevertheless, they do provide a unique opportunity to increase our understanding of this devastating disease and are being used test novel therapies. The biology associated with the differential expression of ASCL1 and NeuroD1 amongst the CDX models is currently under investigation alongside the impact of stromaltumour cell interactions. No conflict of interest. 827 NUB1 protein regulates cullins and E3 ligases in estrogen receptor negative breast cancers K.L. Tan1 , S. Haider2 , C. Zois2 , H. Turley1 , R. Leek1 , A. Harris2 , F. Buffa2 , O. Acuto3 , F. Pezzella1 . 1 University of Oxford, Radcliffe Department of Medicine, Oxford, United Kingdom, 2 University of Oxford, Department of Oncology, Oxford, United Kingdom, 3 University of Oxford, Sir William Dunn School of Pathology, Oxford, United Kingdom Background: The NEDD8 ultimate buster (NUB1) protein is a tumour suppressor that causes degradation of neddylated and fat10ylated proteins through the ubiquitin proteasome system. Aryl Hydrocarbon Receptor Interacting Protein-Like 1 (AIPL1) interacts with NUB1 and restricts the nucleus entry of NUB1 protein. Upregulated NUB1 expression inhibits proliferation of IFNa-resistant cancer cells. The mechanistic roles of NUB1 protein remain unexplored. Materials and Methods: NUB1 proteins expression and global protein neddylation and fat10ylation were examined by immunohistochemistry and immunoblotting. Cell counts and cell cycle profiles were determined upon neddylation inhibition by MLN4924, a NEDD8-activating enzyme inhibitor. Survival was analysed with Kaplan–Meier methods and compared by the logrank test. All statistical tests were two sided. Results: The microarray study and meta-analysis of cancer databases revealed that expression transcripts of chaperones, including AIPL1, were downregulated in lung and pancreatic cancers in comparison to the adjacent normal tissues. Bioinformatics using METABRIC dataset showed that low NUB1 transcripts had poor survival in ER negative subgroup of breast cancer patients: hazard ratio (HR) = 0.66, 95% confidence interval (CI) 0.5–0.87, p = 0.003 and triple negative subgroup of breast cancer patients: HR = 0.67, 95% CI 0.47–0.96, p = 0.028. NUB1 silencing promotes in vitro cell growth in MDA-MB-231, MCF7 and BT474. NUB1 proteins move into nucleus upon chronic severe hypoxia, in timely manner. NUB1-silent HCC1806 cells expressed higher p21 and p27 proteins. Upon the treatment of neddylation inhibition, HCC1806 cells with siNUB1 transfection showed decreased CUL1 which led to accumulation of tumour suppressive cullin-RING E3 ligase substrates, p21 (marker of senescence) & p27 (marker of cell cycle arrest). In the same condition, more observed dead cells were thought to be caused by senescent cells that drive cellular aging and depletion. It suggest that the NUB1 induced-cell death of cancer cells upon hypoxia happen through neddylationdependent CUL1-p27-p21 axis. We demonstrated that HIF1a protein could be both neddylated and FAT10ylated upon reoxygenation. In comparison to RCC4plusVHL cells, NUB1-silent null-VHL RCC4 cells expressed higher HIF1a protein. It suggests the upregulated HIF1a protein level is CUL2VHL dependent. In tissue microarray (TMA) study, compared with high cytoplasmic expression, those with low cytoplasmic expression had worse overall: HR = 0.432, 95% CI 0.201–0.663, p = 0.002.
Conclusions and Further study: Our study highlights the cell cycle regulation mechanism of NUB1 proteins in breast cancers. NUB1 could be a promising therapeutic target that prevents cancer progression. We plan to utilise multiplexed ion beam imaging technique to study ER-negative subgroup primary tumour against its metastatic nodes. No conflict of interest. 828 Hypermethylation and downregulation of glutathione peroxidase 3 are related to pathogenesis of melanoma J.Y. Bae1 , X. Zhang2 . 1 Center for RNA Research- Institute for Basic Science, School of Biological Sciences- College of Natural Sciences- Seoul National University, Seoul, Korea, 2 Oral Cancer Research Institute, Department of Oral Pathology- Yonsei University College of Dentistry, Seoul, Korea Introduction: As well known potential carcinogens, reactive oxygen species (ROS) are involved in both carcinogenesis and tumor progression via ROSinduced DNA damage. During normal metabolism, cells avoid oxidative damage by means of antioxidant defense mechanisms that control the balance between the generation and removal of oxygen radicals. The antioxidant system includes various enzymes, such as superoxide dismutase and catalase, as well as glutathione peroxidase (GPX). GPX3, a member of the GPX family, has been found to be frequently repressed in many cancers due to promoter hypermethylation and is known as a possible tumor suppressor gene. Materials and Methods: mRNA and protein expression of GPX3 was comparatively investigated in human epidermal melanocyte (HEMs) and 2 melanoma cell lines using RT-PCR analysis and immunocytochemistry. The influence of GPX3 expression on biological behavior of melanoma cells were investigated in GPX3 depleted melanoma cell line in vitro. The clinicopathological significance of GPX3 was investigated 105 melanoma patients by immunohistochemistry. Methylation of GPX3 was detected by methylation specific PCR in HEMs, 2 melanoma cell lines, 3 normal skin samples and 105 melanoma tissues. Results and Discussion: GPX3 expression was downregulated and methylated in melanoma cell lines and tissue samples. In melanoma cell lines, GPX3 expression was restored by treatment with 5-aza-2 -deoxycytidine. Depletion of GPX3 was found to increase the proliferative ability, motility, and invasiveness of melanoma cells. Moreover, negative expression of GPX3 was related to poor prognosis in melanoma patients. These results suggest that methylation-mediated GPX3 repression may have critical implications for melanoma pathogenesis. Conclusion: The epigenetic inactivation of GPX3 may be a crucial mechanism in the pathogenesis of melanoma, and GPX3 may serve as a possible predictive or prognostic biomarker and therapeutic target for melanoma patients. No conflict of interest. 829 Bisphosphonates potentiate the activity of pitavastatin against ovarian cancer cells M. Abdullah1 , A. Richardson2 . 1 Keele university, Institute for Science and Technology in Medicine-Guy Hilton Research Centre, Stoke on Trent, United Kingdom, 2 Keele university, Institute for Science and Technology in MedicineGuy Hilton Research Centre, Stoke on Trent, United Kingdom Background: Although many ovarian cancer patients respond to chemotherapy, the majority relapse and drug resistance usually prevents further effective treatment. As a result, the five year survival rate is only 40%. Novel therapeutic approaches are required. We have shown that statins, which inhibit the synthesis of mevalonate by HMG-CoA reductase, have the potential to treat ovarian cancer. Statins inhibit the production of isoprenoids which are necessary for the membrane localization of several small G-protein oncogenes. However, a major limitation for the clinical application of statin as anti-tumor therapy is the requirement for a high dose which may cause adverse effects such as myopathy. Bisphosphonates (BP), drugs used to treat osteoporosis, inhibits farnesyl diphosphate synthase and consequently the synthesis of isoprenoids. We hypothesized that bisphosphonates would potentiate the activity of the statins in ovarian cancer cells. This may allow a reduction in the dose of statin necessary to treat ovarian cancer and minimize the risk of adverse effects. Consequently, we evaluated the activity of the BPs, zoledronic acid and risedronate, in combination with pitavastatin against a panel of ovarian cancer cell lines. Methods: The effect of combinations of pitavastatin and zoledronic acid or risedronate on ovarian cancer cell lines was assessed in several different assays including cell growth, trypan blue exclusion, intracellular ATP, Caspase -3/7, -8, and -9 activity as well as PARP cleavage. Results: When tested as single agents, pitavastatin (IC50 = 0.6−14 mM) was the most potent of the drugs in cell growth assays, whereas zoledronic acid (IC50 = 21−57 mM) and risedronate (IC50 >100mM) showed only modest activity. Combinations of pitavastatin with zoledronic acid, and to lesser extend risedronate, displayed synergistic activity in A2780, CisA2780, Ovcar-4, Igrov-1 and Skov-3 cells or additivity in Cov-318, Cov-362, Ovcar-5 and Ovcar-8 cells.