Sangyun Shin

CYP1B1 Enhances Cell Proliferation and Metastasis through Induction of EMT and Activation of Wnt/β-Catenin Signaling via Sp1 Upregulation

Cytochrome P450 1B1 (CYP1B1) is a major E2 hydroxylase involved in the metabolism of potential carcinogens. CYP1B1 expression has been reported to be higher in tumors compared to normal tissues, especially in hormone-related cancers including breast, ovary, and prostate tumors. To explore the role of CYP1B1 in cancer progression, we investigated the action of CYP1B1 in cells with increased CYP1B1 via the inducer 7,12-dimethylbenz[α]anthracene (DMBA) or an overexpression vector, in addition to decreased CYP1B1 via the inhibitor tetramethoxystilbene (TMS) or siRNA knockdown. We observed that CYP1B1 promoted cell proliferation, migration, and invasion in MCF-7 and MCF-10A cells. To understand its molecular mechanism, we measured key oncogenic proteins including β-catenin, c-Myc, ZEB2, and matrix metalloproteinases following CYP1B1 modulation. CYP1B1 induced epithelial-mesenchymal transition (EMT) and activated Wnt/β-catenin signaling via upregulation of CTNNB1, ZEB2, SNAI1, and TWIST1. Sp1, a transcription factor involved in cell growth and metastasis, was positively regulated by CYP1B1, and suppression of Sp1 expression by siRNA or DNA binding activity using mithramycin A blocked oncogenic transformation by CYP1B1. Therefore, we suggest that Sp1 acts as a key mediator for CYP1B1 action. Treatment with 4-hydroxyestradiol (4-OHE2), a major metabolite generated by CYP1B1, showed similar effects as CYP1B1 overexpression, indicating that CYP1B1 activity mediated various oncogenic events in cells. In conclusion, our data suggests that CYP1B1 promotes cell proliferation and metastasis by inducing EMT and Wnt/β-catenin signaling via Sp1 induction.

Bacterial Lipopolysaccharides Induce Steroid Sulfatase Expression and Cell Migration through IL-6 Pathway in Human Prostate Cancer Cells

Steroid sulfatase (STS) is responsiblefor the conversion of estrone sulfate to estrone that can stimulate growth in endocrine-dependent tumors such as prostate cancer. Although STS is considered as a therapeutic target for the estrogen-dependent diseases, cellular function of STS are still not clear. Previously, we found that tumor necrosis factor (TNF)-α significantly enhances steroid sulfatase expression in PC-3 human prostate cancer cells through PI3K/Akt-dependent pathways. Here, we studied whether bacterial lipopolysaccharides (LPS) which are known to induce TNF-α may increase STS expression. Treatment with LPS in PC-3 cells induced STS mRNA and protein in concentration- and time-dependent manners. Using luciferase reporter assay, we found that LPS enhanced STS promoter activity. Moreover, STS expression induced by LPS increased PC-3 tumor cell migration determined by wound healing assay. We investigated that LPS induced IL-6 expression and IL-6 increased STS expression. Taken together, these data strongly suggest that LPS induces STS expression through IL-6 pathway in human prostate cancer cells.

Induction of steroid sulfatase expression in PC-3 human prostate cancer cells by insulin-like growth factor II.

Human steroid sulfatase (STS) plays an important role in regulating the formation of biologically active estrogens and may be a promising target for treating estrogen-mediated carcinogenesis. The molecular mechanism of STS gene expression, however, is still not clear. Growth factors are known to increase STS activity but the changes in STS expression have not been completely understood. To determine whether insulin-like growth factor (IGF)-II can induce STS gene expression, the effects of IGF-II on STS expression were studied in PC-3 human prostate cancer cells. RT-PCR and Western blot analysis showed that IGF-II treatment significantly increased the expression of STS mRNA and protein in concentration- and time-dependent manners. To understand the signaling pathway by which IGF-II induces STS gene expression, the effects of specific PI3-kinase/Akt and NF-κB inhibitors were determined. When the cells were treated with IGF-II and PI3-kinase/Akt inhibitors, such as LY294002, wortmannin, or Akt inhibitor IV, STS expression induced by IGF-II was significantly blocked. Moreover, we found that NF-κB inhibitors, such as MG-132, bortezomib, Bay 11-7082 or Nemo binding domain (NBD) binding peptide, also strongly prevented IGF-II from inducing STS gene expression. We assessed whether IGF-II activates STS promoter activity using transient transfection with a luciferase reporter. IGF-II significantly stimulated STS reporter activity. Furthermore, IGF-II induced expression of 17β-hydroxysteroid dehydrogenase (HSD) 1 and 3, whereas it reduced estrone sulfotransferase (EST) gene expression, causing enhanced estrone and β-estradiol production. Taken together, these results strongly suggest that IGF-II induces STS expression via a PI3-kinase/Akt-NF-κB signaling pathway in PC-3 cells and may induce estrogen production and estrogen-mediated carcinogenesis.

Human steroid sulfatase induces Wnt/β-catenin signaling and epithelial-mesenchymal transition by upregulating Twist1 and HIF-1α in human prostate and cervical cancer cells

Steroid sulfatase (STS) catalyzes the hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate (DHEAS) to their unconjugated biologically active forms. Although STS is considered a therapeutic target for estrogen-dependent diseases, the cellular functions of STS remain unclear. We found that STS induces Wnt/β-catenin s Delete ignaling in PC-3 and HeLa cells. STS increases levels of β-catenin, phospho-β-catenin, and phospho-GSK3β. Enhanced translocation of β-catenin to the nucleus by STS might activate transcription of target genes such as cyclin D1, c-myc, and MMP-7. STS knockdown by siRNA resulted in downregulation of Wnt/β-catenin signaling. β-Catenin/TCF-mediated transcription was also enhanced by STS. STS induced an epithelial-mesenchymal transition (EMT) as it reduced the levels of E-cadherin, whereas levels of mesenchymal markers such as N-cadherin and vimentin were enhanced. We found that STS induced Twist1 expression through HIFα activation as HIF-1α knockdown significantly blocks the ability of STS to induce Twist1 transcription. Furthermore, DHEA, but not DHEAS is capable of inducing Twist1. Treatment with a STS inhibitor prevented STS-mediated Wnt/β-catenin signaling and Twist1 expression. Interestingly, cancer cell migration, invasion, and MMPs expression induced by STS were also inhibited by a STS inhibitor. Taken together, these results suggest that STS induces Wnt/β-catenin signaling and EMT by upregulating Twist1 and HIF-1α. The ability of STS to induce the Wnt/β-catenin signaling and EMT has profound implications on estrogen-mediated carcinogenesis in human cancer cells.Steroid sulfatase (STS) catalyzes the hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate (DHEAS) to their unconjugated biologically active forms. Although STS is considered a therapeutic target for estrogen-dependent diseases, the cellular functions of STS remain unclear. We found that STS induces Wnt/β-catenin s Delete ignaling in PC-3 and HeLa cells. STS increases levels of β-catenin, phospho-β-catenin, and phospho-GSK3β. Enhanced translocation of β-catenin to the nucleus by STS might activate transcription of target genes such as cyclin D1, c-myc, and MMP-7. STS knockdown by siRNA resulted in downregulation of Wnt/β-catenin signaling. β-Catenin/TCF-mediated transcription was also enhanced by STS. STS induced an epithelial-mesenchymal transition (EMT) as it reduced the levels of E-cadherin, whereas levels of mesenchymal markers such as N-cadherin and vimentin were enhanced. We found that STS induced Twist1 expression through HIFα activation as HIF-1α knockdown significantly blocks the ability of STS to induce Twist1 transcription. Furthermore, DHEA, but not DHEAS is capable of inducing Twist1. Treatment with a STS inhibitor prevented STS-mediated Wnt/β-catenin signaling and Twist1 expression. Interestingly, cancer cell migration, invasion, and MMPs expression induced by STS were also inhibited by a STS inhibitor. Taken together, these results suggest that STS induces Wnt/β-catenin signaling and EMT by upregulating Twist1 and HIF-1α. The ability of STS to induce the Wnt/β-catenin signaling and EMT has profound implications on estrogen-mediated carcinogenesis in human cancer cells.

Annexin A5 suppresses cyclooxygenase-2 expression by downregulating the protein kinase C-ζ–nuclear factor-κB signaling pathway in prostate cancer cells

Annexin A5 (ANXA5) is a member of the annexin protein family. Previous studies have shown that ANXA5 is involved in anti-inflammation and cell death. However, the detailed mechanism of the role of ANXA5 in cancer cells is not well understood. In this study, we investigated the inhibitory effect of ANXA5 on cyclooxygenase-2 (COX-2) in prostate cancer cells. Expression of COX-2 induced by TNF-α was inhibited by overexpression of ANXA5 and inhibition of COX-2 expression by auranofin, which could induce ANXA5 expression, was restored by ANXA5 knockdown. In addition, ANXA5 knockdown induces phosphorylation of NF-κB p65 in prostate cancer cells, indicating that ANXA5 causes COX-2 downregulation through inhibition of p65 activation. We also found that protein kinase C (PKC)-ζ protein levels were upregulated by the inhibition of ANXA5, although the mRNA levels were unaffected. We have shown that upregulated COX-2 expression by inhibition of ANXA5 is attenuated by PKC-ζ siRNA. In summary, this study demonstrates that downregulation of PKC-ζ-NF-κB signaling by ANXA5 may inhibit COX-2 expression in prostate cancer.Annexin A5 (ANXA5) is a member of the annexin protein family. Previous studies have shown that ANXA5 is involved in anti-inflammation and cell death. However, the detailed mechanism of the role of ANXA5 in cancer cells is not well understood. In this study, we investigated the inhibitory effect of ANXA5 on cyclooxygenase-2 (COX-2) in prostate cancer cells. Expression of COX-2 induced by TNF-α was inhibited by overexpression of ANXA5 and inhibition of COX-2 expression by auranofin, which could induce ANXA5 expression, was restored by ANXA5 knockdown. In addition, ANXA5 knockdown induces phosphorylation of NF-κB p65 in prostate cancer cells, indicating that ANXA5 causes COX-2 downregulation through inhibition of p65 activation. We also found that protein kinase C (PKC)-ζ protein levels were upregulated by the inhibition of ANXA5, although the mRNA levels were unaffected. We have shown that upregulated COX-2 expression by inhibition of ANXA5 is attenuated by PKC-ζ siRNA. In summary, this study demonstrates that downregulation of PKC-ζ-NF-κB signaling by ANXA5 may inhibit COX-2 expression in prostate cancer.

Induction of Integrin Signaling by Steroid Sulfatase in Human Cervical Cancer Cells.

Steroid sulfatase (STS) is an enzyme responsible for the hydrolysis of aryl and alkyl sulfates. STS plays a pivotal role in the regulation of estrogens and androgens that promote the growth of hormone-dependent tumors, such as those of breast or prostate cancer. However, the molecular function of STS in tumor growth is still not clear. To elucidate the role of STS in cancer cell proliferation, we investigated whether STS is able to regulate the integrin signaling pathway. We found that overexpression of STS in HeLa cells increases the protein and mRNA levels of integrin β1 and fibronectin, a ligand of integrin α5β1. Dehydroepiandrosterone (DHEA), one of the main metabolites of STS, also increases mRNA and protein expression of integrin β1 and fibronectin. Further, STS expression and DHEA treatment enhanced phosphorylation of focal adhesion kinase (FAK) at the Tyr 925 residue. Moreover, increased phosphorylation of ERK at Thr 202 and Tyr 204 residues by STS indicates that STS activates the MAPK/ERK pathway. In conclusion, these results suggest that STS expression and DHEA treatment may enhance MAPK/ERK signaling through up-regulation of integrin β1 and activation of FAK.

Discovery of Ezrin Expression as a Potential Biomarker for Chemically Induced Ocular Irritation Using Human Corneal Epithelium Cell Line and a Reconstructed Human Cornea-like Epithelium Model

Numerous studies have attempted to develop a new in vitro eye irritation test (EIT). To obtain more reliable results from EIT, potential new biomarkers that reflect eye irritation by chemicals must be identified. We investigated candidate biomarkers for eye irritation, using a proteomics approach. Sodium lauryl sulfate (SLS) or benzalkonium chloride (BAC) was applied on a reconstructed human cornea-like epithelium model, MCTT HCE, and corneal protein expression was examined by two-dimensional gel electrophoresis. We found that ezrin (EZR) was significantly upregulated by SLS or BAC. In addition, upregulation of EZR in immortalized human corneal cells treated with SLS or BAC was confirmed by quantitative reverse transcription-PCR and western blot analysis. Furthermore, other well-known eye irritants such as cetylpyridinium bromide, Triton X-100, cyclohexanol, ethanol, 2-methyl-1-pentanol, and sodium hydroxide significantly increased EZR expression in immortalized human corneal cells. Induction of EZR promoter activity in irritant-treated human corneal cells was confirmed by a luciferase gene reporter assay. In conclusion, EZR expression may be a potential biomarker for detecting eye irritation, which may substantially improve the performance of in vitro EIT.

Abstract LB-A10: The carcinogenic role of CYP1B1 in cancer cells related to Wnt/β-catenin signaling and uPAR pathway

The carcinogenic role of CYP1B1 in cancer cells related to Wnt/β-catenin signaling and uPAR pathway Yeo-Jung Kwon*, Sangyun Shin, Dong-Jin Ye, Hyoung-Seok Baek, and Young-Jin Chun Department of Pharmaceutical Biochemistry, College of Pharmacy, Chung-Ang University, Seoul, Korea Cytochrome P450 1B1 (CYP1B1) belongs to the CYP1 family and shares the feature as an enzyme for oxidative metabolisms. It has been reported that CYP1B1 expression is higher in the tumor tissues than the normal ones, especially in hormone-related cancers such as breast, ovarian, and prostate cancer cells. To explore the role of CYP1B1 on cancer cell proliferation, we studied the effects of CYP1B1 inhibition and activation in MCF-7 and MCF-10A cells. Here, we confirmed that expression of CYP1B1 affects the level of cell proliferation using CCK assay. To elucidate whether the cell proliferation induced by CYP1B1 is related to Wnt/β-catenin signaling, we examined the alteration of β-catenin and expression using western blot, RT-PCR, immunostaining, and qPCR followed by over-expression of CYP1B1 or treatment with DMBA (7,12-dimethylbenz[α]anthracene), a well known CYP1B1 inducer and these studies also performed when CYP1B1 gene was knockdown or cells were treated with TMS (tetramethoxystilbene), a specific CYP1B1 inhibitor. These investigations all showed that CYP1B1 promotes β-catenin expression in mRNA and protein levels and induces β-catenin nuclear localization. To identify the effect of CYP1B1 on Wnt/β-catenin signaling further, the promoter activity of β-catenin/TCF/LEF or expression level of Wnt/β-catenin signaling target genes such as c-myc, cyclin D1 were examined and all factors also showed to be up-regulated by CYP1B1. To elucidate whether CYP1B1 induces cell metastasis, cell migration and invasion assay were conducted and it has been identified that CYP1B1 enhances cell motility by induction of mesenchymal-like cell morphological changes. Moreover, the uPAR-uPA pathway has been identified to be related to the CYP1B1-induced cell metastasis by detection of uPAR-uPA pathway factors using western blot and qPCR in MCF-7 and HeLa cells. Taken together, these data suggest that CYP1B1 promotes cell proliferation and metastasis via inducing Wnt/β-catenin signaling and uPAR pathway. Citation Format: Yeo-Jung Kwon, Sangyun Shin, Dong-Jin Ye, Hyoung-Seok Baek, Young-Jin Chun. The carcinogenic role of CYP1B1 in cancer cells related to Wnt/β-catenin signaling and uPAR pathway. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A10.

Abstract LB-A11: Expression of steroid sulfatase induces the integrin signaling pathway in HeLa cells

Steroid sulfatase (STS) is responsible for the hydrolysis of aryl and alkyl steroid sulfates. STS has a pivotal role in regulating the level of estrogen and androgen responsible for growth of hormone-dependent tumors, such as breast or prostate cancer. However, the molecular function for tumor growth of STS is still not clear. To elucidate possible role of STS on cancer cell proliferation, we investigated whether STS is able to regulate integrin signaling pathway. In this study, we observed that overexpression of STS in HeLa cells induces the expression of integrin β1 and fibronectin, a ligand of integrin α5β1 at protein and mRNA levels. Dehydroepiandrosterone (DHEA), one of the main metabolite of STS, also induces mRNA and protein level of integrin β1 and fibronectin. We found that STS expression and DHEA enhance phosphorylation of focal adhesion kinase (FAK) at tyrosine 925 residue. Moreover, phosphorylation of ERK at threonine 202 and tyrosine 204 residues was also induced, indicating that STS may activate Ras/Raf/MEK signaling pathway. In conclusion, these results suggest that STS expression and DHEA may enhance Ras/Raf/MEK signaling through upregulation of integrin β1 and activation of FAK. Citation Format: Dong-Jin Ye, Yeo-Jung Kwon, Sangyun Shin, Mihye Hong, Hyoungseok Baek, Seung-Ki Ahn, Dongwon Shin, Young-Jin Chun. Expression of steroid sulfatase induces the integrin signaling pathway in HeLa cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A11.

Abstract LB-A12: New target for cancer metabolism : Steroid Sulfatase

Steroid sulfatase (STS) expression has been considered to play a pivotal role in estrogen-dependent cancers. STS is known as a target enzyme for suppressing estrogen-mediated carcinogenesis. STS is able to convert estrogen sulfate to estrone form, like dehydroepiandrosterone sulfate (DHEA-S) to its9 active form dehydroepiandrosterone (DHEA). Aerobic glycolysis is a hallmark of cancer metabolism. Lactic acid production was also increased by STS and DHEA.STS and its9 major product DEHA reduced Oxygen consumption rate. STX064, a STS specific inhibitor inhibited DHEA formation and lactic acid production, recovered mitochondria resperation in HeLa cells. Hypoxia inducing factor 1 subunit HIF1α is recognized as an important regulator of aerobic glycolysis. To elucidate whether STS is able to regulate cancer metabolism, the effects on aerobic glycolysis were determined. STS overexpression significantly converted DHEA-S to DHEA. STS and the major product DHEA enhanced HIF1α protein, mRNA, promoter activity in HeLa cells. Glycolytic enzymes such as hexokinase 2 (HK-2) and glucose transporter (GLUT) were increased by STS overexpression and DHEA treatment in protein and mRNA levels. When cells were treated with HIF1α siRNA, all glycolytic enzymes induced by STS and DHEA were down-regulated. STS also regulated pyruvate kinase M (PKM)splicing. STS up-regulated PKM2 level and inhibited PKM1 level through transcription factor c-Myc. Phosphorylation of PKM2 was also induced by STS. In conclusion, STS and the major product DHEA may regulate aerobic glycolysis via HIF1α induction and c-Myc related PKM2 alternative splicing. Citation Format: sangyun shin, Yeo-Jung Kwon, Dong-Jin Ye, Hyoung-Seok Baek, Young-Jin Chun. New target for cancer metabolism : Steroid Sulfatase. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A12.