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Dual Inhibitor Sensitizes Pancreatic Cancer Cells to Chemotherapy Drugs
AGA Abstracts
p<0.01) and PC (-6.55, -2.11; p<0.01),upregulation in CP (3.64, 2.07; p<0.01) without much changes at the transcript level. Conclusion: Human transcriptome array and next generation sequencing identify decreased expression and abundant splice variants of GAS5 and MYH9 genes association with oncogenic transformation of chronic pancreatitis to pancreatic cancer.
Figure 2: Fold Changes in Free Fatty Acids (Unstimulated/ Stimulated)
Su2050 GASTRIC CANCER AND O-GLCNACYLATION Yosuke Inoue, Shoko Edogawa, Kazumasa Moriwaki, Shinpei Kawaguchi, Yoshiaki Takahashi, Yuichi Kojima, Toshihisa Takeuchi, Michio Asahi, Kazuhide Higuchi Background In diabetic patients, elevated glucose increases the O-GlcNAcylation in the various tissues through upregulation of the hexosamine biosynthesis pathway. Increased OGlcNAcylation could contribute to a part of diabetic complications such as retinopathy, nephropathy.On the other hand, increased O-GlcNAcylation is a general feature of cancer cells and contributes to various cancer phenotypes, including cell proliferation, survival, invasion, metastasis, energy metabolism, and epigenetics. Recently, it was reported that OGlcNAcylation, was upregulated in patients with both diabetes and cancer. Furthermore, it was shown that the expression level of FoxM1, which played a key role in carcinogenesis, was increased by O-GlcNAcylation in cancer cells such as prostate cancer cell line. There are many reports about diabetes as the risk factor for carcinogenesis. Aims we examined the molecular mechanism underlying the regulation of FoxM1 function by O-GlcNAcylation in gastric cancer cells. Methods We used human gastric cancer cell lines. We first examined the effect of glucose concentration on the levels of O-GlcNAcylated proteins and FoxM1 expression by Western blot analyses and Immunofluorescence staining. We next determined the levels of O-GlcNAcylated proteins and FoxM1 expression in the presence or absence of O-GlcNAc transferase (OGT) inhibitor, BADGP or O-GlcNAcase (OGA) inhibitor, Thiamet G, by Western blot analyses and IF staining.And we assessed cellular proliferation after Thiamet G or BADGP treatment using Cell Counting Kit(CCK8) to investigate the relationship between proliferation in gastric cancer cells and O-GlcNAcylation. Then We used co immunoprecipitation to detect the relationships between ubquitination of FoxM1 and O-GlcNAcylation. Results The levels of O-GlcNAcylation and FoxM1 expression were increased under high glucose concentration, whereas they were decreased under low glucose concentration in all cancer cell lines we used. The levels of O-GlcNAcylation and FoxM1 expression were upregulated by Thiamet G and downregulated by BADGP in all of the cells. And Elevated O-GlcNAcylation and Upregulation of FoxM1 expression increased cell proliferation in gastric cancer cells.Then ThiametG decreased FoxM1 ubiquitination in FoxM1 trancefected HEK293 Conclusions Our data indicate that O-GlcNAcylation increases FoxM1 protein level without affecting the mRNA level and blockade of proteasomal proteolysis also increases FoxM1 protein level in gastric cancer cells. Previous reports suggest that O-GlcNAcylation hampers proteasome system through modulating activities of ubiquitin ligases or blocking ubiquitination of target proteins. In diabetes patients, elevated O-GlcNAcylation might decrease FoxM1 degradation mediated by proteasome system resulting in upregulation of FoxM1 which promotes cancer development and progression .
Su2052 DUAL INHIBITOR SENSITIZES PANCREATIC CANCER CELLS TO CHEMOTHERAPY DRUGS Mouad Edderkaoui, Chintan Chheda, Badr Soufi, Fouzia Zayou, Aida Habtezion, Michael Lewis, Ramachandran Murali, Stephen J. Pandol Background: Pancreatic cancer cells are highly proliferative and metastatic; and pancreatic ductal adenocarcinoma (PDAC) has no effective treatment. We have developed a new agent, Metavert, for treatment of PDAC and have previously shown that it provides a survival benefit in experiment PDAC mouse models. Metavert targets simultaneously glycogen synthase kinase 3 beta (GSK-3β) and histone deacetylase (HDAC), important mediators of cancer progression. For the present study we determined the effects of Metavert in combination with chemotherapy drugs in vitro and in vivo. Methods: PDAC and normal cells were treated with different doses of Metavert with gemcitabine and paclitaxel. Cell survival, epithelial to mesenchymal transition (EMT) and cancer stemness markers were measured. In vivo, KrasLSLG12D/+ ;Trp53LSL-R172H/+;Pdxcre (KPC) mice were intra-peritoneally injected with Metavert with and without gemcitabine and paclitaxel from age 2 months until death. Survival and number of metastasis were determined. Pancreatic lesions and tumor grades as well as fibrosis and inflammation were measured by immunohistochemistry. Markers of cancer stemness in cells were measured by flow cytometry. Cytokine production was measured in blood and in tumor cells and in cells present in the tumor microenvironment. Results: Metavert significantly (IC50 =600nM) decreased cancer cell survival and increased apoptosis in several PDAC cells lines. The same doses of Metavert did not affect survival of normal cells. Metavert had an additive or synergistic effect on cancer cell survival when combined with gemcitabine or paclitaxel. Metavert decreased EMT markers when combined with chemotherapy drugs. Furthermore, Metavert decreased the expression of cancer stemness markers (sox2, CD24, CD44) and reversed the increase in the expression of these markers induced by paclitaxel in cancer cells. Treatment with Metavert significantly increased KPC mice survival by ~50%. Distal metastasis was decreased from 29% in control KPC mice to 0% in Metavert treated KPC mice. Metavert significantly decreased the level of pro-cancer cytokines in mice blood. Combination of gemcitabine or paclitaxel with Metavert further slowed down the progression of the disease and increased the survival of KPC mice. Conclusion: We have shown a significant anti-cancer effect in vitro and in vivo of Metavert combined with standard chemotherapy drugs in experimental PDAC. Metavert is a suitable drug to be used in combination with chemotherapy for treating PDAC patients.
Su2051 HUMAN TRANSCRIPTOME ARRAY REVEALS ASSOCIATION OF SPLICED TRANSCRIPTS OF GAS5 AND MYH9 GENES AND ABERRANTLY EXPRESSED SMALL NUCLEAR RNA IN ONCOGENIC TRANSFORMATION OF CHRONIC PANCREATITIS TO PANCREATIC CANCER Ravi Kanth V V, Mitnala Sasikala, Steffie Ua, Rupjyoti Talukdar, G. V. Rao, Pradeep Rebala, Ramji C, Anuradha S, Duvur N. Reddy Background: Early onset chronic pancreatitis (CP; eg. hereditary and tropical) patients have a higher risk of pancreatic cancer (PC). Although prolonged inflammation leading to neoplastic transformation is known, molecular mechanisms of oncogenic transformation of CP to PC are not clear. It is therefore necessary to identify genetic risks and delineate molecular pathways involved in malignant transformation of CP. The objective of the present study was to identify aberrant gene expression at the transcript level using whole transcriptome array in patients with CP who progressed to PC. Methods: Twenty one archived tissues (5CP, 9 PC on the background of CP [PCCP] Figure 1, 2PC, 5 control without any CP/PC) were subjected to RNA isolation using QiagenRNeasy FFPE kit for gene expression studies using transcriptome arrays and RNA sequencing. cRNA was synthesized and twelve good quality cRNA from 3CP,4PCCP,2PC,3 controls were hybridized to Affymetrix Human Transcriptome arrays 2.0. cRNA were also sequenced on Ion Proton (NGS) employing Lifetechnologies Ion Total RNA-Seq Kit v2.Transcriptome data was analyzed using Transciptome Analysis Console 3.0.0.466 and RNA seq data using Partek Genomics Suite v6.6. Gene and transcript level expression were analyzed (ANOVA) between control and CP (Group1), control and PCCP (Group2) and control and PC (Group3). Common genes between groups 1 and 2 were identified and were interpreted to be involved in progression from CP to PC. Results: Gene expression analysis revealed dysregulated expression of 83genes common between Group 1 and 2 among 306 in Group1, 937 in Group2. Majority of these were small nuclear RNA (snRNA) involved in methylation of rRNA and pre-mRNA processing. Alternative splicing analysis revealed significant down regulation of tumor suppressor genes; Growth arrest specific 5 (GAS5-long non coding RNA) and Myosin heavychain9-non muscle (MYH9).While GAS5 was downregulated by -596.3 fold in Group1, -326.64 in Group2 and -171.17 fold in Group3; p=0.008, MYH9 was downregulated by -4.51, -26.83 and -9.37 folds in Groups 1,2 and 3 respectively; p<0.01). Further, the number of splicing events was higher (21Vs 9 for GAS5 and 37 Vs 12 for MYH9;p <0.01; Figure 2) in PCCP Vs CP with predominantly intron retention in GAS5 and cassette exons in MYH9 genes. While these gene expressions were similar in CP and PCCP, another set of genes involved in tumor suppression &cell cycle arrest (BTG2, PLA2G12A) showed downregulation in PCCP (-2.55, -2.22;
AGA Abstracts
Su2053 USE OF A COMBINATION OF LENTIVIRUS PARTICLES AND A SPECIFIC PEPTIDE FOR ERADICATION OF CD24-EXPRESSING MALIGNANT CELLS: A NOVEL APPROACH FOR CANCER THERAPY Nadir Arber, Eynat Finkelshtein, Abraham Loyter, Shiran Shapira Background Lentiviral replication is driven by a molecular motor consisting of the three viral enzymes: the reverse transcriptase, protease and integrase (IN). The genomic RNA of the virus is used to produce a copy of viral DNA by reverse transcription, and the integrase
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p<0.01) and PC (-6.55, -2.11; p<0.01),upregulation in CP (3.64, 2.07; p<0.01) without much changes at the transcript level. Conclusion: Human transcriptome array and next generation sequencing identify decreased expression and abundant splice variants of GAS5 and MYH9 genes association with oncogenic transformation of chronic pancreatitis to pancreatic cancer.
Figure 2: Fold Changes in Free Fatty Acids (Unstimulated/ Stimulated)
Su2050 GASTRIC CANCER AND O-GLCNACYLATION Yosuke Inoue, Shoko Edogawa, Kazumasa Moriwaki, Shinpei Kawaguchi, Yoshiaki Takahashi, Yuichi Kojima, Toshihisa Takeuchi, Michio Asahi, Kazuhide Higuchi Background In diabetic patients, elevated glucose increases the O-GlcNAcylation in the various tissues through upregulation of the hexosamine biosynthesis pathway. Increased OGlcNAcylation could contribute to a part of diabetic complications such as retinopathy, nephropathy.On the other hand, increased O-GlcNAcylation is a general feature of cancer cells and contributes to various cancer phenotypes, including cell proliferation, survival, invasion, metastasis, energy metabolism, and epigenetics. Recently, it was reported that OGlcNAcylation, was upregulated in patients with both diabetes and cancer. Furthermore, it was shown that the expression level of FoxM1, which played a key role in carcinogenesis, was increased by O-GlcNAcylation in cancer cells such as prostate cancer cell line. There are many reports about diabetes as the risk factor for carcinogenesis. Aims we examined the molecular mechanism underlying the regulation of FoxM1 function by O-GlcNAcylation in gastric cancer cells. Methods We used human gastric cancer cell lines. We first examined the effect of glucose concentration on the levels of O-GlcNAcylated proteins and FoxM1 expression by Western blot analyses and Immunofluorescence staining. We next determined the levels of O-GlcNAcylated proteins and FoxM1 expression in the presence or absence of O-GlcNAc transferase (OGT) inhibitor, BADGP or O-GlcNAcase (OGA) inhibitor, Thiamet G, by Western blot analyses and IF staining.And we assessed cellular proliferation after Thiamet G or BADGP treatment using Cell Counting Kit(CCK8) to investigate the relationship between proliferation in gastric cancer cells and O-GlcNAcylation. Then We used co immunoprecipitation to detect the relationships between ubquitination of FoxM1 and O-GlcNAcylation. Results The levels of O-GlcNAcylation and FoxM1 expression were increased under high glucose concentration, whereas they were decreased under low glucose concentration in all cancer cell lines we used. The levels of O-GlcNAcylation and FoxM1 expression were upregulated by Thiamet G and downregulated by BADGP in all of the cells. And Elevated O-GlcNAcylation and Upregulation of FoxM1 expression increased cell proliferation in gastric cancer cells.Then ThiametG decreased FoxM1 ubiquitination in FoxM1 trancefected HEK293 Conclusions Our data indicate that O-GlcNAcylation increases FoxM1 protein level without affecting the mRNA level and blockade of proteasomal proteolysis also increases FoxM1 protein level in gastric cancer cells. Previous reports suggest that O-GlcNAcylation hampers proteasome system through modulating activities of ubiquitin ligases or blocking ubiquitination of target proteins. In diabetes patients, elevated O-GlcNAcylation might decrease FoxM1 degradation mediated by proteasome system resulting in upregulation of FoxM1 which promotes cancer development and progression .
Su2052 DUAL INHIBITOR SENSITIZES PANCREATIC CANCER CELLS TO CHEMOTHERAPY DRUGS Mouad Edderkaoui, Chintan Chheda, Badr Soufi, Fouzia Zayou, Aida Habtezion, Michael Lewis, Ramachandran Murali, Stephen J. Pandol Background: Pancreatic cancer cells are highly proliferative and metastatic; and pancreatic ductal adenocarcinoma (PDAC) has no effective treatment. We have developed a new agent, Metavert, for treatment of PDAC and have previously shown that it provides a survival benefit in experiment PDAC mouse models. Metavert targets simultaneously glycogen synthase kinase 3 beta (GSK-3β) and histone deacetylase (HDAC), important mediators of cancer progression. For the present study we determined the effects of Metavert in combination with chemotherapy drugs in vitro and in vivo. Methods: PDAC and normal cells were treated with different doses of Metavert with gemcitabine and paclitaxel. Cell survival, epithelial to mesenchymal transition (EMT) and cancer stemness markers were measured. In vivo, KrasLSLG12D/+ ;Trp53LSL-R172H/+;Pdxcre (KPC) mice were intra-peritoneally injected with Metavert with and without gemcitabine and paclitaxel from age 2 months until death. Survival and number of metastasis were determined. Pancreatic lesions and tumor grades as well as fibrosis and inflammation were measured by immunohistochemistry. Markers of cancer stemness in cells were measured by flow cytometry. Cytokine production was measured in blood and in tumor cells and in cells present in the tumor microenvironment. Results: Metavert significantly (IC50 =600nM) decreased cancer cell survival and increased apoptosis in several PDAC cells lines. The same doses of Metavert did not affect survival of normal cells. Metavert had an additive or synergistic effect on cancer cell survival when combined with gemcitabine or paclitaxel. Metavert decreased EMT markers when combined with chemotherapy drugs. Furthermore, Metavert decreased the expression of cancer stemness markers (sox2, CD24, CD44) and reversed the increase in the expression of these markers induced by paclitaxel in cancer cells. Treatment with Metavert significantly increased KPC mice survival by ~50%. Distal metastasis was decreased from 29% in control KPC mice to 0% in Metavert treated KPC mice. Metavert significantly decreased the level of pro-cancer cytokines in mice blood. Combination of gemcitabine or paclitaxel with Metavert further slowed down the progression of the disease and increased the survival of KPC mice. Conclusion: We have shown a significant anti-cancer effect in vitro and in vivo of Metavert combined with standard chemotherapy drugs in experimental PDAC. Metavert is a suitable drug to be used in combination with chemotherapy for treating PDAC patients.
Su2051 HUMAN TRANSCRIPTOME ARRAY REVEALS ASSOCIATION OF SPLICED TRANSCRIPTS OF GAS5 AND MYH9 GENES AND ABERRANTLY EXPRESSED SMALL NUCLEAR RNA IN ONCOGENIC TRANSFORMATION OF CHRONIC PANCREATITIS TO PANCREATIC CANCER Ravi Kanth V V, Mitnala Sasikala, Steffie Ua, Rupjyoti Talukdar, G. V. Rao, Pradeep Rebala, Ramji C, Anuradha S, Duvur N. Reddy Background: Early onset chronic pancreatitis (CP; eg. hereditary and tropical) patients have a higher risk of pancreatic cancer (PC). Although prolonged inflammation leading to neoplastic transformation is known, molecular mechanisms of oncogenic transformation of CP to PC are not clear. It is therefore necessary to identify genetic risks and delineate molecular pathways involved in malignant transformation of CP. The objective of the present study was to identify aberrant gene expression at the transcript level using whole transcriptome array in patients with CP who progressed to PC. Methods: Twenty one archived tissues (5CP, 9 PC on the background of CP [PCCP] Figure 1, 2PC, 5 control without any CP/PC) were subjected to RNA isolation using QiagenRNeasy FFPE kit for gene expression studies using transcriptome arrays and RNA sequencing. cRNA was synthesized and twelve good quality cRNA from 3CP,4PCCP,2PC,3 controls were hybridized to Affymetrix Human Transcriptome arrays 2.0. cRNA were also sequenced on Ion Proton (NGS) employing Lifetechnologies Ion Total RNA-Seq Kit v2.Transcriptome data was analyzed using Transciptome Analysis Console 3.0.0.466 and RNA seq data using Partek Genomics Suite v6.6. Gene and transcript level expression were analyzed (ANOVA) between control and CP (Group1), control and PCCP (Group2) and control and PC (Group3). Common genes between groups 1 and 2 were identified and were interpreted to be involved in progression from CP to PC. Results: Gene expression analysis revealed dysregulated expression of 83genes common between Group 1 and 2 among 306 in Group1, 937 in Group2. Majority of these were small nuclear RNA (snRNA) involved in methylation of rRNA and pre-mRNA processing. Alternative splicing analysis revealed significant down regulation of tumor suppressor genes; Growth arrest specific 5 (GAS5-long non coding RNA) and Myosin heavychain9-non muscle (MYH9).While GAS5 was downregulated by -596.3 fold in Group1, -326.64 in Group2 and -171.17 fold in Group3; p=0.008, MYH9 was downregulated by -4.51, -26.83 and -9.37 folds in Groups 1,2 and 3 respectively; p<0.01). Further, the number of splicing events was higher (21Vs 9 for GAS5 and 37 Vs 12 for MYH9;p <0.01; Figure 2) in PCCP Vs CP with predominantly intron retention in GAS5 and cassette exons in MYH9 genes. While these gene expressions were similar in CP and PCCP, another set of genes involved in tumor suppression &cell cycle arrest (BTG2, PLA2G12A) showed downregulation in PCCP (-2.55, -2.22;
AGA Abstracts
Su2053 USE OF A COMBINATION OF LENTIVIRUS PARTICLES AND A SPECIFIC PEPTIDE FOR ERADICATION OF CD24-EXPRESSING MALIGNANT CELLS: A NOVEL APPROACH FOR CANCER THERAPY Nadir Arber, Eynat Finkelshtein, Abraham Loyter, Shiran Shapira Background Lentiviral replication is driven by a molecular motor consisting of the three viral enzymes: the reverse transcriptase, protease and integrase (IN). The genomic RNA of the virus is used to produce a copy of viral DNA by reverse transcription, and the integrase
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