- Email: [email protected]
beta-catenin signaling and epithelial-mesenchymal transition in human prostate cancer cells
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
Annexin A5, a member of annexin protein family, is a calcium-dependent phospholipid binding protein. Previous studies showed that the biological function of annexin A5 is related to apoptotic features. But the role of annexin A5 in cell proliferation is not yet clear. In this study, we explored the role of annexin A5 in PC-3 human prostate cancer cells. To clarify whether annexin A5 regulates cell proliferation, we examined the cell viability with annexin A5-knockdowned cells and it showed that annexin A5 inhibits cell proliferation. To identify the detailed mechanism of annexin A5 further, we performed microarray with annexin A5-knockdowned PC-3 cells and selected a number of genes that significantly changed by alteration of annexin A5 expression. Of these, PAI-2, plasminogen activator inhibitor-2 increased about 1.6-fold in annexin A5knockdowned cells and decreased about 0.8-fold when cells were overexpressed with annexin A5. To investigate whether PAI-2 is involved in anti-proliferative mechanism induced by annexin A5, we suppressed PAI2 with specific siRNA and found that PAI-2 regulates cell proliferation. In conclusion, we suggest that PAI-2 is downregulated by annexin A5 and may play an important role in annexin A5-mediated cellular function. P111 CONSTITUTIVE OMEGA-3 FATTY ACID PRODUCTION IN TRANSGENIC MICE PROTECTS AGAINST UVB-INDUCED INFLAMMATION AND CARCINOGENESIS
FAT-1 SKIN
Hye-Won Yum 1, Joydeb Kumar Kundu 2, Yong-Yeon Cho 3, Young-Joon Surh 1. 1 Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea; 2 College of Pharmacy, Keimyung University, Daegu, South Korea; 3 College of Pharmacy, The Catholic University of Korea, Bucheon, South Korea Exposure to solar radiation, especially ultraviolet B (UVB), is the most prevalent cause of skin photocarcinogenesis. Omega-6 (u-6) and omega3 (u-3) polyunsaturated fatty acids (PUFAs) are known to have opposite effects on the inflammatory and carcinogenesis processes. In general, u6 PUFAs promote inflammation and tumor development, whereas u-3 PUFAs possess anti-inflammatory as well as antioxidative activity and enhance host immune response. In the present study, we investigated the protective effects of u-3 PUFAs on UVB-induced skin inflammation and carcinogenesis using fat-1 transgenic mice harboring u-3 desaturase gene capable of converting u-6 to u-3 PUFAs. The u-3/u-6 PUFA ratio was significantly higher in the dorsal skin of fat-1 mice than that in the wild-type (WT) C57BL/6 mice. fat-1 mice expressed heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1 and their mRNA transcripts to a greater extent than did WT mice, which was attributable to the elevated activation of nuclear factor erythroid-related factor-2 (Nrf2) responsible for upregulation of cytoprotective and stress-responsive proteins. Upon single exposure to UVB (500 mJ/cm2) irradiation, fat1 mice exhibited a significantly lower degree of skin inflammation and phototoxicity and the expression of the pro-inflammatory enzyme, cyclooxygenases-2 (COX-2) as compared to those observed in WT mouse skin. The protection of fat-1 mice from UVB-induced skin inflammation was associated with decreased phosphorylation of signal transducer and activator of transcription 3 (STAT3), one of the major proinflammatory transcription factors. To determine whether the elevated u-3 PUFA level also protects against UVB-induced skin papillomagenesis, hairless fat-1 / albino and fat-1+/ albino mice were generated by cross-breeding of male fat-1+/ transgenic mice with female SKH-1 hairless albino mice. After repeated UVB irradiation (180 mJ/cm2) thrice a week for 23 weeks, the incidence and the multiplicity of papillomas were markedly reduced in the fat-1+/ albino group as compared to the fat-1 / albino group. Moreover, the expression of COX-2 and activation of STAT3 in papillomas from fat-1+/ albino mice were significantly lower than those found in UVB-irradiated fat-1 / albino mice. Taken together, our results indicate that functional fat-1 potentiates cellular defense against UVB-induced skin inflammation and tumor development through elevated activation of Nrf2 and upregulation of cytoprotective gene expression.
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P112 ABSTRACT WITHDRAWN P113 HUMAN STEROID SULFATASE INDUCES AEROBIC GLYCOLYSIS THROUGH HIF1A INDUCTION IN HELA CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, YoungJin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Human steroid sulfatase (STS) is known as an important target enzyme for suppressing estrogen-mediated carcinogenesis. Although high level of STS expression is known to involve in carcinogenesis, the precise mechanism of STS-induced cancer progression is still not clear. STS converts estrone sulfate to estrone and dehydroepiandrosterone sulfate (DHEA-S) to dehydroepiandrosterone (DHEA). On the contrast to normal cells, cancer cells generate cellular energy by inefficient way, aerobic glycolysis. To elucidate whether STS is able to regulate energy production of cancer cells, the effects on aerobic glycolysis were determined. Oxygen consumption rate (OCR) which represents mitochondria respiration is significantly suppressed by STS overexpression, STS specific inhibitor STX064 enhanced STS-induced OCR repression. DHEA also suppressed OCR level, whereas inactive form DHEA-S did not affect mitochondria respiration of cells. Rapid production of lactic acid, which is a characteristic of tumorigenesis, was up-regulated by STS. Treatment of DHEA also enhanced lactic acid production level, while DHEA-S had no effect on production of lactic acid. To understand molecular mechanism of STSinduced cancer metabolism, we investigated glycolytic enzymes. Rate limit enzymes of glycolysis such as hexokinase 2 (HK-2) and pyruvate kinase M2 (PKM2) were highly increased by STS overexpression and DHEA treatment in both protein and mRNA levels. Inhibition of STS reduced increased glycolytic proteins such as HK-2 and PKM2. One of pivotal regulator of aerobic glycolysis, HIF1a was enhanced by STS and DHEA in protein, mRNA, and promoter activity level in HeLa cells. Inhibition of HIF1a with siRNA treatment, glycolytic enzymes STS-induced GLUT-1, HK-2, and PKM2 were suppressed. In conclusion, we suggested that STS and the major product DHEA regulate aerobic glycolysis through HIF1a induction. P114 HUMAN STEROID SULFATASE INDUCES THE WNT/BETA-CATENIN SIGNALING AND EPITHELIAL-MESENCHYMAL TRANSITION IN HUMAN PROSTATE CANCER CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Although steroid sulfatase (STS) is considered as a therapeutic target for estrogen-dependent diseases, the cellular functions of STS are still not clear. We found STS expression induced the Wnt/b-catenin signaling in PC-3 human prostate cancer cells and showed that STS overexpression increased levels of b-catenin, phospho-b-catenin, and phospho-GSK3b. Enhanced translocation of b-catenin to the nucleus by STS may activate transcription of target genes such as cyclin D1, c-myc, and MMP-7. STS knockdown by siRNA resulted in downregulation of the Wnt/b-catenin signaling. b-Catenin/TCF-mediated transcription was enhanced by STS expression. STS induced epithelial-mesenchymal transition (EMT) program because STS expression reduced the level of E-cadherin, whereas levels of mesenchymal markers such as N-cadherin and vimentin were enhanced. Moreover, EMT-inducing transcription factors such as Zeb1, Zeb2, Snai1, Twist1, and FOXC2 were significantly increased by STS expression. Treatment with a STS inhibitor prevented STS-mediated Wnt/b-catenin signaling and EMT program. However, STS metabolite DHEA was able to induce EMT program. Interestingly, cell migration and invasion were also induced when STS was expressed in PC-3 cells. Taken together, these data strongly suggest that STS expression induces the Wnt/b-catenin signaling and EMT program by upregulating EMT-inducing transcription factors. The ability of STS to induce the Wnt/b-catenin signaling and the EMT program has profound
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Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
implications on estrogen-mediated carcinogenesis in human prostate cancer cells. P115 NRF2 AND STAT3 INTERACTION: IMPLICATIONS FOR BREAST CANCER PROGRESSION Su-Jung Kim 1, 2, Soma Saeidi 1, 2, Young-Joon Surh 1, 3. 1 Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea; 2 Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, South Korea; 3 College of Pharmacy, Seoul National University, Seoul, South Korea Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a redox-sensitive transcription factor plays a key role in cellular cytoprotection against oxidative and electrophilic stress. Although Nrf2 protects normal cells against chemically induced tumor formation, it confers advantage for survival and growth of many different types of cancer cells. Another transcription factor STAT3 functions as an oncogene. Persistently activated STAT3 is associated with proliferation, survival, and invasiveness of tumor cells including those of breast, lung, pancreatic, and head and neck origin. Recent studies have suggested the possibility of the interplay or cross talk between Nrf2 and STAT3. Here, we present evidence for Nrf2 and STAT3 cooperation in breast cancer cells. Knockdown of Nrf2 expression with siRNA significantly decreased STAT3 tyrosine phosphorylation in H-Rastransformed human breast (MCF10A-ras) cells and MDA-MD-231 breast cancer cells. Conversely, we observed a significant decrease in Nrf2 protein levels in STAT3 siRNA transfected breast cancer cells. siRNA knockdown of Keap1, a cytosolic repressor of Nrf2, induced STAT3 tyrosine phosphorylation as well as elevated expression of Nrf2. Furthermore, silencing of Nrf2 significantly decreased STAT3 transcriptional activity. Several STAT3regulated genes, including Bcl-x, cyclin D1, LIF and survivin, were downregulated in MCF10A-ras cells when Nrf2 was depleted. In addition, knockdown of STAT3 expression reduced transcriptional activity of Nrf2 and its target gene expression. However, Nrf2 activation did not affect STAT3 tyrosine phosphorylation in normal MCF10A cells. Likewise, IL-6induced tyrosine phosphorylation of STAT3 did not affect the Nrf2 levels in normal MCF10A cells. Silencing STAT3 by siRNA had no effect on Keap1 protein expression and STAT3 did not interact with Keap1 in MCF10A-ras cells. Co-immunoprecipitation assays demonstrate that Nrf2 interacts with STAT3 in MCF10A-ras and MDA-MB-231 cells but not normal MCF10A cells. Taken together, these results suggest that Nrf2 and STAT3 may enhance survival and invasiveness of human breast cancer cells through direct interaction. P116 PKM2 MEDIATED EMT INDUCTION BY STEROID SULFATASE IN HUMAN PROSTATE CANCER CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Although STS is considered a therapeutic target for estrogen-dependent diseases, the cellular functions of STS are still not clear. Important metabolic pathway, glycolysis modulates and potentially alter cancer metabolism on requirement. Pyruvate kinase which is final rate-limiting step of glycolysis has four isoform, L, R, M1, and M2. PKM1 and PKM2 isozymes are made from single gene by alternative splicing. In previous study, we found that human steroid sulfatase (STS) enhances PKM2 expression. So we studied about PKM2 as mediator of carcinogenesis. Overexpression of STS regulated PKM alternative splicing, induced PKM2 and suppressed PKM1 in both mRNA and protein level. We confirm that STS induce PKM splicing through alternative splicing assay. STS increased lactic acid production level, PKM2 knock down repressed lactic acid production. Oxygen consumption also mediated by PKM2. PKM2 is highly associated STS-induced EMT. Cancer cell invasion and migration were induced by PKM2 overexpression. STS-induced cell invasion and migration also suppressed by PKM2 knockdown. EMT and MET markers, Twist1, Snail, Zeb2, E-cadherin, and uPAR. MMPs also regulated by PKM2 and STS overxepression. Taken together, these data suggest that
PKM2 regulates STS-induced glycolysis. STS-mediated cancer cell invasion and metastasis also highley assocaiated with PKM2 induction. Our study showed that role of PKM2 in STS-induced carcinogenesis. P117 RNA EDITING MODULATES HUMAN AHR AND ITS DOWNSTREAM P450 EXPRESSION Masataka Nakano 1, Tatsuki Fukami 1, Saki Gotoh 1, Masataka Takamiya 2, Yasuhiro Aoki 2, Miki Nakajima 1. 1 Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan; 2 Iwate Medical University School of Medicine, Morioka, Japan RNA editing refers to a post-transcriptional process that alters the nucleotide sequence of RNA transcripts. Adenosine-to-inosine (A-to-I) RNA editing is the most frequent type of RNA editing in mammals. Inosine forms base pair with cytidine as if it was guanosine, and the conversion possibly affects amino acid sequence, splicing, and microRNA (miRNA) targeting. The A-to-I RNA editing is catalyzed by adenosine deaminases acting on RNA (ADAR) enzymes, which act specifically on double strand RNAs. It became apparent that aberrant RNA editing is associated with several diseases including cancer, neurological disorders, metabolic diseases, viral infections and autoimmune disorders. However, the significance of the A-to-I RNA editing in human liver largely remains to be clarified. We sought to investigate the roles of RNA editing on human aryl hydrocarbon receptor (AhR), which has possible RNA editing sites in the 3’untranslated region (3’-UTR). To investigate whether AhR 3’-UTR in human liver is edited, we performed direct sequencing of AhR cDNA and genomic DNA. It was experimentally demonstrated that multiple RNA editing sites existed within the Alu elements in AhR 3’-UTR. In human hepatocellular carcinoma-derived Huh-7 cells, knockdown of ADAR1 decreased the RNA editing levels and increased AhR protein levels, but not mRNA expression levels. Knockdown of ADAR2 had no effects. These results indicated that ADAR1 negatively regulates AhR expression in the post-transcriptional manner. The 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated induction of CYP1A1, a downstream gene of AhR, was augmented by ADAR1 knockdown, suggesting that RNA editing affected the expression of P450 isoform. We surmised that A-to-I RNA editing created miRNA targeting sites in AhR mRNA. By in silico analysis, miR-378 was predicted as a candidate miRNA whose binding affinity to the edited AhR 3’-UTR would be higher than that to the unedited AhR 3’-UTR. Interestingly, overexpression of miR378 decreased AhR protein expression levels, and the miR-378-dependent downregulation of AhR was abolished by knockdown of ADAR1. The results indicated that the mechanism of the ADAR1-mediated downregulation of AhR would be attributed to the creation of the miR-378 recognition site in AhR 3’-UTR. The interindividual differences in the RNA editing levels within the AhR 3’-UTR in a panel of 32 human liver samples were relatively small, whereas the differences in ADAR1 expression were large (220-fold). In the human liver samples, a significant inverse association was observed between the miR-378 and AhR protein levels, suggesting that the RNA editing-dependent down-regulation of AhR by miR378 contributes to the variability in the constitutive hepatic expression of AhR. In conclusion, this study uncovered that A-to-I RNA editing modulates the potency of xenobiotic metabolism in the human liver. P118 THE PEPTIDYL PROLYL ISOMERASE PIN1 UPREGULATES NRF2 EXPRESSION IN H-RAS TRANSFORMED MAMMARY EPITHELIAL CELLS Soma Saeidi 1, 2, Su-Jung Kim 1, Hyeong-jun Han 1, Juan-Yu Piao 1, YoungJoon Surh 1, 3. 1 Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea; 2 Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, South Korea; 3 College of Pharmacy, Seoul National University, Seoul, South Korea The nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is a transcription factor that integrates cellular stress signals and rescues cells from a wide range of noxious stimuli. However, recent studies have revealed the constitutive overexpression of Nrf2 in many transformed or cancerous cell lines and human tumor tissues, which may confer an
Annexin A5, a member of annexin protein family, is a calcium-dependent phospholipid binding protein. Previous studies showed that the biological function of annexin A5 is related to apoptotic features. But the role of annexin A5 in cell proliferation is not yet clear. In this study, we explored the role of annexin A5 in PC-3 human prostate cancer cells. To clarify whether annexin A5 regulates cell proliferation, we examined the cell viability with annexin A5-knockdowned cells and it showed that annexin A5 inhibits cell proliferation. To identify the detailed mechanism of annexin A5 further, we performed microarray with annexin A5-knockdowned PC-3 cells and selected a number of genes that significantly changed by alteration of annexin A5 expression. Of these, PAI-2, plasminogen activator inhibitor-2 increased about 1.6-fold in annexin A5knockdowned cells and decreased about 0.8-fold when cells were overexpressed with annexin A5. To investigate whether PAI-2 is involved in anti-proliferative mechanism induced by annexin A5, we suppressed PAI2 with specific siRNA and found that PAI-2 regulates cell proliferation. In conclusion, we suggest that PAI-2 is downregulated by annexin A5 and may play an important role in annexin A5-mediated cellular function. P111 CONSTITUTIVE OMEGA-3 FATTY ACID PRODUCTION IN TRANSGENIC MICE PROTECTS AGAINST UVB-INDUCED INFLAMMATION AND CARCINOGENESIS
FAT-1 SKIN
Hye-Won Yum 1, Joydeb Kumar Kundu 2, Yong-Yeon Cho 3, Young-Joon Surh 1. 1 Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea; 2 College of Pharmacy, Keimyung University, Daegu, South Korea; 3 College of Pharmacy, The Catholic University of Korea, Bucheon, South Korea Exposure to solar radiation, especially ultraviolet B (UVB), is the most prevalent cause of skin photocarcinogenesis. Omega-6 (u-6) and omega3 (u-3) polyunsaturated fatty acids (PUFAs) are known to have opposite effects on the inflammatory and carcinogenesis processes. In general, u6 PUFAs promote inflammation and tumor development, whereas u-3 PUFAs possess anti-inflammatory as well as antioxidative activity and enhance host immune response. In the present study, we investigated the protective effects of u-3 PUFAs on UVB-induced skin inflammation and carcinogenesis using fat-1 transgenic mice harboring u-3 desaturase gene capable of converting u-6 to u-3 PUFAs. The u-3/u-6 PUFA ratio was significantly higher in the dorsal skin of fat-1 mice than that in the wild-type (WT) C57BL/6 mice. fat-1 mice expressed heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1 and their mRNA transcripts to a greater extent than did WT mice, which was attributable to the elevated activation of nuclear factor erythroid-related factor-2 (Nrf2) responsible for upregulation of cytoprotective and stress-responsive proteins. Upon single exposure to UVB (500 mJ/cm2) irradiation, fat1 mice exhibited a significantly lower degree of skin inflammation and phototoxicity and the expression of the pro-inflammatory enzyme, cyclooxygenases-2 (COX-2) as compared to those observed in WT mouse skin. The protection of fat-1 mice from UVB-induced skin inflammation was associated with decreased phosphorylation of signal transducer and activator of transcription 3 (STAT3), one of the major proinflammatory transcription factors. To determine whether the elevated u-3 PUFA level also protects against UVB-induced skin papillomagenesis, hairless fat-1 / albino and fat-1+/ albino mice were generated by cross-breeding of male fat-1+/ transgenic mice with female SKH-1 hairless albino mice. After repeated UVB irradiation (180 mJ/cm2) thrice a week for 23 weeks, the incidence and the multiplicity of papillomas were markedly reduced in the fat-1+/ albino group as compared to the fat-1 / albino group. Moreover, the expression of COX-2 and activation of STAT3 in papillomas from fat-1+/ albino mice were significantly lower than those found in UVB-irradiated fat-1 / albino mice. Taken together, our results indicate that functional fat-1 potentiates cellular defense against UVB-induced skin inflammation and tumor development through elevated activation of Nrf2 and upregulation of cytoprotective gene expression.
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P112 ABSTRACT WITHDRAWN P113 HUMAN STEROID SULFATASE INDUCES AEROBIC GLYCOLYSIS THROUGH HIF1A INDUCTION IN HELA CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, YoungJin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Human steroid sulfatase (STS) is known as an important target enzyme for suppressing estrogen-mediated carcinogenesis. Although high level of STS expression is known to involve in carcinogenesis, the precise mechanism of STS-induced cancer progression is still not clear. STS converts estrone sulfate to estrone and dehydroepiandrosterone sulfate (DHEA-S) to dehydroepiandrosterone (DHEA). On the contrast to normal cells, cancer cells generate cellular energy by inefficient way, aerobic glycolysis. To elucidate whether STS is able to regulate energy production of cancer cells, the effects on aerobic glycolysis were determined. Oxygen consumption rate (OCR) which represents mitochondria respiration is significantly suppressed by STS overexpression, STS specific inhibitor STX064 enhanced STS-induced OCR repression. DHEA also suppressed OCR level, whereas inactive form DHEA-S did not affect mitochondria respiration of cells. Rapid production of lactic acid, which is a characteristic of tumorigenesis, was up-regulated by STS. Treatment of DHEA also enhanced lactic acid production level, while DHEA-S had no effect on production of lactic acid. To understand molecular mechanism of STSinduced cancer metabolism, we investigated glycolytic enzymes. Rate limit enzymes of glycolysis such as hexokinase 2 (HK-2) and pyruvate kinase M2 (PKM2) were highly increased by STS overexpression and DHEA treatment in both protein and mRNA levels. Inhibition of STS reduced increased glycolytic proteins such as HK-2 and PKM2. One of pivotal regulator of aerobic glycolysis, HIF1a was enhanced by STS and DHEA in protein, mRNA, and promoter activity level in HeLa cells. Inhibition of HIF1a with siRNA treatment, glycolytic enzymes STS-induced GLUT-1, HK-2, and PKM2 were suppressed. In conclusion, we suggested that STS and the major product DHEA regulate aerobic glycolysis through HIF1a induction. P114 HUMAN STEROID SULFATASE INDUCES THE WNT/BETA-CATENIN SIGNALING AND EPITHELIAL-MESENCHYMAL TRANSITION IN HUMAN PROSTATE CANCER CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Although steroid sulfatase (STS) is considered as a therapeutic target for estrogen-dependent diseases, the cellular functions of STS are still not clear. We found STS expression induced the Wnt/b-catenin signaling in PC-3 human prostate cancer cells and showed that STS overexpression increased levels of b-catenin, phospho-b-catenin, and phospho-GSK3b. Enhanced translocation of b-catenin to the nucleus by STS may activate transcription of target genes such as cyclin D1, c-myc, and MMP-7. STS knockdown by siRNA resulted in downregulation of the Wnt/b-catenin signaling. b-Catenin/TCF-mediated transcription was enhanced by STS expression. STS induced epithelial-mesenchymal transition (EMT) program because STS expression reduced the level of E-cadherin, whereas levels of mesenchymal markers such as N-cadherin and vimentin were enhanced. Moreover, EMT-inducing transcription factors such as Zeb1, Zeb2, Snai1, Twist1, and FOXC2 were significantly increased by STS expression. Treatment with a STS inhibitor prevented STS-mediated Wnt/b-catenin signaling and EMT program. However, STS metabolite DHEA was able to induce EMT program. Interestingly, cell migration and invasion were also induced when STS was expressed in PC-3 cells. Taken together, these data strongly suggest that STS expression induces the Wnt/b-catenin signaling and EMT program by upregulating EMT-inducing transcription factors. The ability of STS to induce the Wnt/b-catenin signaling and the EMT program has profound
S60
Abstracts / Drug Metabolism and Pharmacokinetics 32 (2017) S27eS107
implications on estrogen-mediated carcinogenesis in human prostate cancer cells. P115 NRF2 AND STAT3 INTERACTION: IMPLICATIONS FOR BREAST CANCER PROGRESSION Su-Jung Kim 1, 2, Soma Saeidi 1, 2, Young-Joon Surh 1, 3. 1 Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea; 2 Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, South Korea; 3 College of Pharmacy, Seoul National University, Seoul, South Korea Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a redox-sensitive transcription factor plays a key role in cellular cytoprotection against oxidative and electrophilic stress. Although Nrf2 protects normal cells against chemically induced tumor formation, it confers advantage for survival and growth of many different types of cancer cells. Another transcription factor STAT3 functions as an oncogene. Persistently activated STAT3 is associated with proliferation, survival, and invasiveness of tumor cells including those of breast, lung, pancreatic, and head and neck origin. Recent studies have suggested the possibility of the interplay or cross talk between Nrf2 and STAT3. Here, we present evidence for Nrf2 and STAT3 cooperation in breast cancer cells. Knockdown of Nrf2 expression with siRNA significantly decreased STAT3 tyrosine phosphorylation in H-Rastransformed human breast (MCF10A-ras) cells and MDA-MD-231 breast cancer cells. Conversely, we observed a significant decrease in Nrf2 protein levels in STAT3 siRNA transfected breast cancer cells. siRNA knockdown of Keap1, a cytosolic repressor of Nrf2, induced STAT3 tyrosine phosphorylation as well as elevated expression of Nrf2. Furthermore, silencing of Nrf2 significantly decreased STAT3 transcriptional activity. Several STAT3regulated genes, including Bcl-x, cyclin D1, LIF and survivin, were downregulated in MCF10A-ras cells when Nrf2 was depleted. In addition, knockdown of STAT3 expression reduced transcriptional activity of Nrf2 and its target gene expression. However, Nrf2 activation did not affect STAT3 tyrosine phosphorylation in normal MCF10A cells. Likewise, IL-6induced tyrosine phosphorylation of STAT3 did not affect the Nrf2 levels in normal MCF10A cells. Silencing STAT3 by siRNA had no effect on Keap1 protein expression and STAT3 did not interact with Keap1 in MCF10A-ras cells. Co-immunoprecipitation assays demonstrate that Nrf2 interacts with STAT3 in MCF10A-ras and MDA-MB-231 cells but not normal MCF10A cells. Taken together, these results suggest that Nrf2 and STAT3 may enhance survival and invasiveness of human breast cancer cells through direct interaction. P116 PKM2 MEDIATED EMT INDUCTION BY STEROID SULFATASE IN HUMAN PROSTATE CANCER CELLS Sangyun Shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, Young-Jin Chun. College of Pharmacy, Chung-Ang University, Seoul, South Korea Although STS is considered a therapeutic target for estrogen-dependent diseases, the cellular functions of STS are still not clear. Important metabolic pathway, glycolysis modulates and potentially alter cancer metabolism on requirement. Pyruvate kinase which is final rate-limiting step of glycolysis has four isoform, L, R, M1, and M2. PKM1 and PKM2 isozymes are made from single gene by alternative splicing. In previous study, we found that human steroid sulfatase (STS) enhances PKM2 expression. So we studied about PKM2 as mediator of carcinogenesis. Overexpression of STS regulated PKM alternative splicing, induced PKM2 and suppressed PKM1 in both mRNA and protein level. We confirm that STS induce PKM splicing through alternative splicing assay. STS increased lactic acid production level, PKM2 knock down repressed lactic acid production. Oxygen consumption also mediated by PKM2. PKM2 is highly associated STS-induced EMT. Cancer cell invasion and migration were induced by PKM2 overexpression. STS-induced cell invasion and migration also suppressed by PKM2 knockdown. EMT and MET markers, Twist1, Snail, Zeb2, E-cadherin, and uPAR. MMPs also regulated by PKM2 and STS overxepression. Taken together, these data suggest that
PKM2 regulates STS-induced glycolysis. STS-mediated cancer cell invasion and metastasis also highley assocaiated with PKM2 induction. Our study showed that role of PKM2 in STS-induced carcinogenesis. P117 RNA EDITING MODULATES HUMAN AHR AND ITS DOWNSTREAM P450 EXPRESSION Masataka Nakano 1, Tatsuki Fukami 1, Saki Gotoh 1, Masataka Takamiya 2, Yasuhiro Aoki 2, Miki Nakajima 1. 1 Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan; 2 Iwate Medical University School of Medicine, Morioka, Japan RNA editing refers to a post-transcriptional process that alters the nucleotide sequence of RNA transcripts. Adenosine-to-inosine (A-to-I) RNA editing is the most frequent type of RNA editing in mammals. Inosine forms base pair with cytidine as if it was guanosine, and the conversion possibly affects amino acid sequence, splicing, and microRNA (miRNA) targeting. The A-to-I RNA editing is catalyzed by adenosine deaminases acting on RNA (ADAR) enzymes, which act specifically on double strand RNAs. It became apparent that aberrant RNA editing is associated with several diseases including cancer, neurological disorders, metabolic diseases, viral infections and autoimmune disorders. However, the significance of the A-to-I RNA editing in human liver largely remains to be clarified. We sought to investigate the roles of RNA editing on human aryl hydrocarbon receptor (AhR), which has possible RNA editing sites in the 3’untranslated region (3’-UTR). To investigate whether AhR 3’-UTR in human liver is edited, we performed direct sequencing of AhR cDNA and genomic DNA. It was experimentally demonstrated that multiple RNA editing sites existed within the Alu elements in AhR 3’-UTR. In human hepatocellular carcinoma-derived Huh-7 cells, knockdown of ADAR1 decreased the RNA editing levels and increased AhR protein levels, but not mRNA expression levels. Knockdown of ADAR2 had no effects. These results indicated that ADAR1 negatively regulates AhR expression in the post-transcriptional manner. The 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated induction of CYP1A1, a downstream gene of AhR, was augmented by ADAR1 knockdown, suggesting that RNA editing affected the expression of P450 isoform. We surmised that A-to-I RNA editing created miRNA targeting sites in AhR mRNA. By in silico analysis, miR-378 was predicted as a candidate miRNA whose binding affinity to the edited AhR 3’-UTR would be higher than that to the unedited AhR 3’-UTR. Interestingly, overexpression of miR378 decreased AhR protein expression levels, and the miR-378-dependent downregulation of AhR was abolished by knockdown of ADAR1. The results indicated that the mechanism of the ADAR1-mediated downregulation of AhR would be attributed to the creation of the miR-378 recognition site in AhR 3’-UTR. The interindividual differences in the RNA editing levels within the AhR 3’-UTR in a panel of 32 human liver samples were relatively small, whereas the differences in ADAR1 expression were large (220-fold). In the human liver samples, a significant inverse association was observed between the miR-378 and AhR protein levels, suggesting that the RNA editing-dependent down-regulation of AhR by miR378 contributes to the variability in the constitutive hepatic expression of AhR. In conclusion, this study uncovered that A-to-I RNA editing modulates the potency of xenobiotic metabolism in the human liver. P118 THE PEPTIDYL PROLYL ISOMERASE PIN1 UPREGULATES NRF2 EXPRESSION IN H-RAS TRANSFORMED MAMMARY EPITHELIAL CELLS Soma Saeidi 1, 2, Su-Jung Kim 1, Hyeong-jun Han 1, Juan-Yu Piao 1, YoungJoon Surh 1, 3. 1 Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea; 2 Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul National University, Seoul, South Korea; 3 College of Pharmacy, Seoul National University, Seoul, South Korea The nuclear factor erythroid 2 (NFE2)-related factor 2 (Nrf2) is a transcription factor that integrates cellular stress signals and rescues cells from a wide range of noxious stimuli. However, recent studies have revealed the constitutive overexpression of Nrf2 in many transformed or cancerous cell lines and human tumor tissues, which may confer an