EGF-induced Cell Transformation Research Articles

Structure and chemopreventive activity of fucoidans from the brown alga Alaria angusta

Six fucoidan fractions were isolated from the brown alga Alaria angusta. Structures of enzymatic hydrolysis products of the fraction 1AaF2 (Fuc:Gal ~ 1:1; 33 % of sulfates) by fucanase from Wenyingzhuangia fucanilytica were studied by chemical and instrumental (NMR spectroscopy and mass-spectrometry) methods. It was shown that 1AaF2 consisted of two structurally different fucoidans: a sulfated 1,3;1,4-α-L-fucan and an enzyme-resistant sulfated and acetylated complex fucogalactan (Fuc:Gal ~ 1:2; 19 % of sulfates) 1AaF2_HMP containing extended 1,3-linked fucose and 1,3/1,4-linked galactose fragments (up to 5 residues). The fractions 1AaF2 and 1AaF2_HMP were a non-cytotoxic, possessed dose-dependent chemopreventive effect on EGF-induced neoplastic cell transformation of mouse normal epidermal JB6 Cl41 cells and inhibited the colony formation of human melanoma SK-MEL-28 cells.

Induction of p27 contributes to inhibitory effect of isorhapontigenin (ISO) on malignant transformation of human urothelial cells

ABSTRACT Bladder cancer (BC) is the most expensive cancer to manage on a per-patient basis, costing about $4 billion in total healthcare expenditure per annum in America alone. Therefore, identifying a natural compound for prevention of BC is of tremendous importance for managing this disease. Previous studies have identified isorhapontigenin (ISO) as having an 85% preventive effect against invasive BC formation induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The results showed here that ISO treatment inhibited EGF-induced cell transformation of human urothelial cells through induction of tumor suppressor p27 transcription secondary to activation of an E2F1-dependentpathway.ISOtreatmentrenderedcellsresistanttoEGF-induced anchorage-independent growth concurrent with p27 protein induction in both UROtsa and SV-HUC-1 cells. ISO inhibition of EGF-induced cell transformation could be completely reversed by knockdown of p27, indicating that this protein was essential for the noted ISO inhibitory action. Mechanistic studies revealed that ISO treatment resulted in increased expression of E2F1, which in turn bound to its binding site in p27 promoter and initiated p27 transcription. The E2F1 induction was due to the elevation of its translation caused by ISO-induced miR-205 downregulation. Consistently, miR-205 was found to be overexpressed in human BCs, and ectopic expression of miR-205 mitigated ISO inhibitory effects against EGF-induced outcomes. Collectively, the results here demonstrate that ISO exhibits its preventive effect on EGF-induced human urothelial cell transformation by induction of p27 through a miR-205/E2F1 axis. This is distinct from what has been described for the therapeutic effects of ISO on human BC cells.

Fargesin Inhibits EGF-Induced Cell Transformation and Colon Cancer Cell Growth by Suppression of CDK2/Cyclin E Signaling Pathway.

Although the lignan compound fargesin is a major ingredient in Shin-Yi, the roles of fargesin in carcinogenesis and cancer cell growth have not been elucidated. In this study, we observed that fargesin inhibited cell proliferation and transformation by suppression of epidermal growth factor (EGF)-stimulated G1/S-phase cell cycle transition in premalignant JB6 Cl41 and HaCaT cells. Unexpectedly, we found that signaling pathway analyses showed different regulation patterns in which fargesin inhibited phosphatidylinositol 3-kinase/AKT signaling without an alteration of or increase in mitogen activated protein kinase (MAPK) in JB6 Cl41 and HaCaT cells, while both signaling pathways were abrogated by fargesin treatment in colon cancer cells. We further found that fargesin-induced colony growth inhibition of colon cancer cells was mediated by suppression of the cyclin dependent kinase 2 (CDK2)/cyclin E signaling axis by upregulation of p21WAF1/Cip1, resulting in G1-phase cell cycle accumulation in a dose-dependent manner. Simultaneously, the suppression of CDK2/cyclin E and induction of p21WAF1/Cip1 were correlated with Rb phosphorylation and c-Myc suppression. Taken together, we conclude that fargesin-mediated c-Myc suppression inhibits EGF-induced cell transformation and colon cancer cell colony growth by the suppression of retinoblastoma (Rb)-E2F and CDK/cyclin signaling pathways, which are mainly regulated by MAPK and PKB signaling pathways.

Structure, antiproliferative and cancer preventive properties of sulfated α-d-fucan from the marine bacterium Vadicella arenosi

Sulfated fucose-containing glycopolymers are currently of great interest because of their wide spectrum of bioactivity, including anti-tumor properties. In this study, the structure of O-polysaccharide (OPS) of the marine bacterium Vadicella arenosi KMM 9024T, its effect on the proliferation of human breast cancer MCF-7 cells and cancer preventive properties were investigated. Two OPS fractions with different molecular weights were isolated and purified from the lipopolysaccharide by mild acid hydrolysis followed by anion-exchange chromatography. The OPS was found to consist of α-(1→3)-linked 2-O-sulfate-d-fucopyranosyl residues, whose structure was deduced by sugar analysis along with 2D NMR spectroscopy. The biological assay indicated that polysaccharide significantly reduced the proliferation and inhibited colony formation of MCF-7 cells in a dose-dependent manner. Besides, the experiment indicated the inhibitory role of polysaccharide on EGF-induced neoplastic cell transformation in mouse epidermal cells. The investigated polysaccharide is the first sulfated fucan isolated from the bacteria.

Abstract 265: Epi-magnolin suppresses EGF-induced neoplastic cell transformation by inhibition of Akt/mTOR signaling pathway in JBC Cl41 cells

Abstract The phosphatidylinositol-3-kinase (PI3K)/Akt/the mammalian target of rapamycin (mTOR) signaling pathway is frequently activated in human cancer and plays a pivotal role in cell proliferation and transformation. In this study, we reveal epi-magnolin, a phytochemical found in Magnolia flos, as an inhibitor of Akt/mTOR signaling pathway. Epi-magnolin inhibits EGF-induced Akt phosphorylation at Thr308 and Ser473, but not extracellular signal-regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway. Moreover, we demonstrate that epi-magnolin suppresses JB6 cell proliferation by impairing EGF-induced G1/S cell cycle transition in a dose-dependent manner. Notably, epi-magnolin abrogates phosphorylation of c-Jun at Ser63 and 73 as well as total c-Jun protein level, which leads to downregulation of AP-1 transactivation activity. The inhibition effect of Akt/mTOR signaling pathway and AP-1 transactivation activity by epi-magnolin suppressed EGF-induced anchorage-independent cell transformation. Based on these results, epi-magnolin might be a chemopreventive agent by targeting Akt/mTOR signaling pathway. Key words: chemoprevention, cell transformation, Akt/mTOR signaling pathway Citation Format: Cheol-Jung Lee, Sun-Mi Yoo, Seung-Min Kim, Seon-Yeon Cho, Juhee Park, Yu-Mi Shin, Yong-Yeon Cho, Hye Suk Lee. Epi-magnolin suppresses EGF-induced neoplastic cell transformation by inhibition of Akt/mTOR signaling pathway in JBC Cl41 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 265.

TRAF6 regulates EGF-induced cell transformation and cSCC malignant phenotype through CD147/EGFR

TRAF6, a well-known adapter molecule, plays pivotal role in TLR/IL-1R associated signaling pathway. Although TRAF6 has been shown to have oncogenic activity in various malignant tumors, the details remain unclear. In this study, we demonstrated that TRAF6 facilitates Ras (G12V) and EGF-induced cellular transformation through EGFR. Silencing of TRAF6 expression significantly downregulated AP-1 activity, as well as MMP-2,9 expression after EGF stimulation. Furthermore, we found that TRAF6 plays an essential role in cutaneous squamous cell carcinoma (cSCC) malignant phenotypes, affecting cell growth and migration. CD147/Basigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, is over-expressed in tumors and induces tumorigenesis. Our results showed that CD147 formed complex with EGFR and TRAF6. Knockdown of TRAF6 disrupted the CD147-EGFR complex, thereby inducing EGFR endocytosis. Therefore, TRAF6 might be a novel molecular target for cSCC prevention or therapy.

Topically Applied Carvedilol Attenuates Solar Ultraviolet Radiation Induced Skin Carcinogenesis.

In previous studies, the β-blocker carvedilol inhibited EGF-induced epidermal cell transformation and chemical carcinogen-induced mouse skin hyperplasia. As exposure to ultraviolet (UV) radiation leads to skin cancer, the present study examined whether carvedilol can prevent UV-induced carcinogenesis. Carvedilol absorbs UV like a sunscreen; thus, to separate pharmacological from sunscreen effects, 4-hydroxycarbazole (4-OHC), which absorbs UV to the same degree as carvedilol, served as control. JB6 P+ cells, an established epidermal model for studying tumor promotion, were used for evaluating the effect of carvedilol on UV-induced neoplastic transformation. Both carvedilol and 4-OHC (1 μmol/L) blocked transformation induced by chronic UV (15 mJ/cm2) exposure for 8 weeks. However, EGF-mediated transformation was inhibited by only carvedilol but not by 4-OHC. Carvedilol (1 and 5 μmol/L), but not 4-OHC, attenuated UV-induced AP-1 and NF-κB luciferase reporter activity, suggesting a potential anti-inflammatory activity. In a single-dose UV (200 mJ/cm2)-induced skin inflammation mouse model, carvedilol (10 μmol/L), applied topically after UV exposure, reduced skin hyperplasia and the levels of cyclobutane pyrimidine dimers, IL1β, IL6, and COX-2 in skin. In SKH-1 mice exposed to gradually increasing levels of UV (50-150 mJ/cm2) three times a week for 25 weeks, topical administration of carvedilol (10 μmol/L) after UV exposure increased tumor latency compared with control (week 18 vs. 15), decreased incidence and multiplicity of squamous cell carcinomas, while 4-OHC had no effect. These data suggest that carvedilol has a novel chemopreventive activity and topical carvedilol following UV exposure may be repurposed for preventing skin inflammation and cancer. Cancer Prev Res; 10(10); 598-606. ©2017 AACR.

Abstract 3127: Roles of ERKs-RSK2 signaling in human cancers

Abstract RSK2 is a member of p90RSK superfamily consisted with RSK1-4 and MSK1-2, and has suggested involving in many cellular processes including cell proliferation, differentiation, transformation and cancer development. Accumulating data obtained by our 16-year researches strongly demonstrated that RSK2 is a key player in cell transformation and cancer metastasis. We found that ectopic expression of RSK2 induced anchorage-independent cell transformation. Notably, knockdown of RSK2 strongly suppressed constitutive active-Ras (CA-Ras)-induced foci-formation, EGF-induced cell transformation and cancer cell growth in anchorage-independent condition. The results obtained from human skin cancer tissue array demonstrated that total protein levels and RSK2 activity were higher in cancer tissues than that of normal tissues. Molecular targeting of RSK2 or ERK1 and 2 with natural compounds demonstrated that ERKs-RSK2 signaling pathway plays a key role in cell transformation and cancer development. Moreover, inhibition of ERKs-RSK2 signaling suppressed NF-κB activity in JB6Cl41 cells. The EGF-induced NF-κB activity was abrogated up-regulation of COX-2 mRNA expression and COX-2 protein levels by inhibition of ERKs-RSK2 signaling pathway. Interestingly, magnolin, a specific inhibitor of ERK1 and ERK2, suppressed ERK-mediated MMP-2 and NF-κB-mediated MMP-9 expression. In addition, we found that the protein expression of N-cadherin involved in metastasis was markedly attenuated by inhibition of ERKs-RSK2 signaling pathway. Notably, magnolin suppressed the migration and invasion of lung cancer cells in a dose-dependent manner. Taken together, these results demonstrated that targeting of ERKs-RSK2 signaling is beneficial for the chemoprevention, and anti-invasion and -migration in cancer metastasis. Key words: MAPK pathway, Transformation, Metastasis, Molecular targets Citation Format: Yong Yeon Cho. Roles of ERKs-RSK2 signaling in human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3127. doi:10.1158/1538-7445.AM2017-3127

Eupatilin inhibits EGF-induced JB6 cell transformation by targeting PI3K.

Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that play fundamental roles in regulation of multiple signaling pathways, including cell proliferation, survival and cell cycle. Increasing evidence has shown that abnormal activation of PI3K pathway contributes to tumorigenesis and progression of various malignant tumors. Therefore, it is an attractive target of chemoprevention and chemotherapy. Eupatilin, a natural flavone compound extracted from Artemisia vulgaris, has antitumor and anti-inflammation efficacy. However, the direct target(s) of eupatilin in cancer chemoprevention are still elusive. In the present study, we reported eupatilin suppressed JB6 cell proliferation and its EGF-induced colony formation. Eupatilin attenuated phosphorylation of PI3K downstream signaling molecules. Downregulation ofcyclin D1 expression and arresting in G1 phase were induced through eupatilin treatment. Furthermore, we found it could bind to the p110α, a catalytic subunit of PI3K, by computational docking methods. Pull down assay outcomes also verified the binding of eupatilin with PI3K. Taken together, our results suggest that epatilin is a potential chemopreventive agent in inhibition of skin cell transformation by targeting PI3K.

PDZ-binding kinase/T-LAK cell-originated protein kinase is a target of the fucoidan from brown alga Fucus evanescens in the prevention of EGF-induced neoplastic cell transformation and colon cancer growth.

The fucoidan with high anticancer activity was isolated from brown alga Fucus evanescens. The compound effectively prevented EGF-induced neoplastic cell transformation through inhibition of TOPK/ERK1/2/MSK 1 signaling axis. In vitro studies showed that the fucoidan attenuated mitogen-activated protein kinases downstream signaling in a colon cancer cells with different expression level of TOPK, resulting in growth inhibition. The fucoidan exerts its effects by directly interacting with TOPK kinase in vitro and ex vivo and inhibits its kinase activity. In xenograft animal model, oral administration of the fucoidan suppressed HCT 116 colon tumor growth. The phosphorylation of TOPK downstream signaling molecules in tumor tissues was also inhibited by the fucoidan. Taken together, our findings support the cancer preventive efficacy of the fucoidan through its targeting of TOPK for the prevention of neoplastic cell transformation and progression of colon carcinomas in vitro and ex vivo.

Dipeptidyl peptidase 4 promotes epithelial cell transformation and breast tumourigenesis via induction of PIN1 gene expression.

Dipeptidyl peptidase 4 (DPP4) is an aminopeptidase that is widely expressed in different cell types. Recent studies suggested that DPP4 plays an important role in tumour progression in several human malignancies. Here we have examined the mechanisms by which up-regulation of DPP4 expression causes epithelial transformation and mammary tumourigenesis. Expression of DPP4 and the peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1), and the cytotoxic effects of combined treatment with sitagliptin and juglone were investigated by immunohistochemistry, immunoblotting, real-time PCR, TUNEL and soft agar assays, using MCF7 cells. The effects of sitagliptin on tumour development in vivo were studied in the syngeneic 4T1 metastatic breast cancer model. Activity of the transcription factor E2F1 induced by EGF was enhanced by DPP4, thus increasing PIN1 expression. Furthermore, DPP4 enhanced MEK/ERK and JNK/c-Jun signalling induced by EGF, inducing AP-1 activity and epithelial cell transformation. In contrast, DPP4 silencing or DPP4 inhibition in MCF7 cells inhibited PIN1 expression via E2F1 activity induced by EGF, decreasing colony formation and inducing DNA fragmentation. In the syngeneic 4T1 metastatic breast cancer model, DPP4 overexpression increased tumour development, whereas treatment with sitagliptin and/or juglone suppressed it. Consistent with these observations, DPP4 levels were positively correlated with PIN1 expression in human breast cancer. DPP4 promoted EGF-induced epithelial cell transformation and mammary tumourigenesis via induction of PIN1 expression, suggesting that sitagliptin targeting of DPP4 could be a treatment strategy in patients with breast cancer.

Aschantin targeting on the kinase domain of mammalian target of rapamycin suppresses epidermal growth factor-induced neoplastic cell transformation.

Mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, forms two different complexes, complex 1 and 2, and plays a key role in the regulation of Akt signaling-mediated cell proliferation and transformation. This study reveals aschantin, a natural compound abundantly found in Magnolia flos, as a novel mTOR kinase inhibitor. Aschantin directly targeted the active pocket of mTOR kinase domain by competing with adenosine triphosphate (ATP), but not PI3K and PDK1. Aschantin inhibited epidermal growth factor (EGF)-induced full activation of Akt by phosphorylation at Ser473/Thr308, resulting in inhibition of the mTORC2/Akt and Akt/mTORC1/p70S6K signaling pathways and activation of GSK3β by abrogation of Akt-mediated GSK3β phosphorylation at Ser9. The activated GSK3β inhibited cell proliferation by c-Jun phosphorylation at Ser243, which facilitated destabilization and degradation of c-Jun through the ubiquitination-mediated proteasomal degradation pathway. Notably, aschantin treatment decreased c-Jun stability through inhibition of the mTORC2-Akt signaling pathway, which suppressed EGF-induced anchorage-independent cell transformation in non-malignant JB6 Cl41 and HaCaT cells and colony growth of LNCaP and MIAPaCa-2 cancer cells in soft agar. Altogether, the results show that aschantin targets mTOR kinase and destabilizes c-Jun, which implicate aschantin as a potential chemopreventive or therapeutic agent.

Abstract 4641: Crucial role of c-Jun phosphorylation at Ser63/73 mediated by PHLPP protein degradation in the Cheliensisin A (Chel A) inhibition of cell transformation

Abstract Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its underlying molecular mechanisms have not been fully characterized yet. In the present study, we found that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP down-regulation by promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction by Chel A and further reversed the Chel A inhibition of EGF-induced cell transformation. With the findings, we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to c-Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel A. Citation Format: Chuanshu Huang, Junlan Zhu, Jingxia Li. Crucial role of c-Jun phosphorylation at Ser63/73 mediated by PHLPP protein degradation in the Cheliensisin A (Chel A) inhibition of cell transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4641. doi:10.1158/1538-7445.AM2015-4641

Abstract 39: Fibroblast growth factor induces neoplastic cell transformation through a non-canonical signaling pathway

Abstract Induction of cell proliferation is closely related with the cellular signaling pathway activation by stimulation of diverse growth factors such as epidermal growth factor (EGF) and fibroblast growth factor. The stimulation of growth factors induces activation of extracellular signal-regulated kinases (ERKs)/p90 ribosomal S6 kinases (p90RSK), resulting in induction of cell proliferation and cell transformation. Although fibroblast growth factor (FGF) is a well-known growth factor and acts as a ligand of FGF receptor (FGFR), a receptor tyrosine kinase, in cytoplasmic membrane, the tumor promoter potential has not been clearly understood. Here, we provided the evidences that FGF acted as a tumor promoter. We found that FGF-induced cell proliferation and anchorage-independent cell transformation were correlated with the induction of G1/S cell cycle transition. Importantly, we found that kaempferol targeted and inhibited FGFR phosphorylation by in vitro and ex vivo. Interestingly, FGF stimulation utilized a non-canonical signaling pathway to activate RSK2 and ATF-1, which was not transduced by EGF stimulation. We confirmed that kaempferol inhibited tyrosine phosphorylation of FGFR, resulted in nuclear accumulation of phospho-ATF-1 at Ser63. Importantly, kaempferol, PKC412, PD98059 and U0126 inhibited EGF-induced anchorage-independent cell transformation in JB6 Cl41 cells. In contrast, FGF-induced cell transformation in soft agar was not inhibited by PD98059 and U0126. Taken together, these results demonstrate that FGF acts as a tumor promoter and dual inhibition of kaempferol on the kinase activities of FGFR and RSK2 suppresses the FGF-induced neoplastic cell transformation through a non-canonical signaling pathway which is not utilized by EGF stimulation. Citation Format: Sun-Mi Yoo, Cheol-Jung Lee, Mee-Hyun Lee, Yong-Yeon Cho. Fibroblast growth factor induces neoplastic cell transformation through a non-canonical signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 39. doi:10.1158/1538-7445.AM2015-39

Abstract 5202: Magnolin suppresses cell migration by abrogation of ERK-mediated RSK2/NF-κB signaling pathway

Abstract Our previous study demonstrated that RSK2 directly phosphorylated IκBα at ser 32, resulted in its degradation which leads to promote NF-κB activation and might be an important role in metastasis. Therefore, it is reasonable the discovery of chemotherapeutic and/or chemopreventive agent. Magnolin, an ingredient of magnolia species, suppressed EGF-induced neoplastic cell transformation by targeting of ERK 1 and 2. In this study, we found that magnolin inhibited cell migration and invasion of normal cell and human lung cancer cells. Further, we demonstrated that magnolin suppressed epidermal growth factor-induced ERK/RSK2 signaling pathway, resulting in suppression of NF-κB activity in JB6Cl41 cells. The EGF-induced NF-κB activity was abrogated up-regulation of COX-2 mRNA expression and COX-2 protein levels by co-treatment of EGF and magnolin. Interestingly, magnolin suppressed ERK-mediated MMP-2 and NF-κB-mediated MMP-9 expression. In addition, we found that the protein expression of N-cadherin involved in metastasis was markedly attenuated upon treatment with magnolin. Notably, magnolin suppressed the migration and invasion of lung cancer cells in a dose-dependent manner. Taken together, these results demonstrated that magnolin is beneficial for the anti-invasion and -migration in cancer metastasis. Citation Format: Cheol-Jung Lee, Mee-Hyun Lee, Ji-Hong Song, Sun-Mi Yoo, Yong-Yeon Cho. Magnolin suppresses cell migration by abrogation of ERK-mediated RSK2/NF-κB signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5202. doi:10.1158/1538-7445.AM2015-5202

Abstract 2810: Identification of a natural compound as mammalian target of rapamycin kinase inhibitor

Abstract The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase, plays a critical role in the regulation of Akt signaling-mediated cell proliferation and transformation. Although several synthetic chemical compounds have developed to inhibit mTOR kinase activity, only few publications have been reported on the identification of natural compounds to target the mTOR kinase active pocket. In this study, we found that AME, a natural compound, inhibited cell proliferation by impairment of G1/S cell cycle transition when cells were co-treated with tumor promoters, such as epidermal growth factor. AME directly targeted the active pocket of mTOR kinase domain by competing with adenosine triphosphate (ATP), but not with PI3K and PDK1. We further confirmed that AME inhibited phosphorylation of Akt at Ser473, which is a target amino acid of mTOR complex 2 (mTORC2), and Akt-mediated GSK3β phosphorylation at Ser9, which resulted in activation of GSK3β. The activated GSK3β inhibited cell proliferation by c-Jun phosphorylation at Ser243, which facilitated destabilization and degradation of c-Jun through an ubiquitination-mediated proteasomal degradation pathway. Notably, decreased c-Jun stability by AME treatment suppressed EGF-induced neoplastic cell transformation in JB6 Cl41 mouse skin epidermal cells and HaCaT human skin keratinocytes in soft agar assay. Taken together, these results demonstrated that AME might be a natural chemopreventive agent targeting mTOR kinase active pocket. Key words: Natural compound, ATP-competitive mTOR kinase inhibitor, mTORC2/Akt/GSK3 signaling, chemoprevention Citation Format: Jeong-Hoon Jang, Cheol-Jung Lee, Mee-Hyun Lee, Young-Joon Surh, Yong-Yeon Cho. Identification of a natural compound as mammalian target of rapamycin kinase inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2015-2810

TNFR1 Regulates Ovarian Cancer Cell Tumorigenicity Through PIK3CB-p110Beta.

Studies have shown that TNFR1 is a key factor in the tumor microenvironment that is dependent on the TNF-α-initiated cascade for tumorigenesis. In this present study, we found that TNFR1 is over-expressed in ovarian cancer, which is relevant to both clinical survival and disease free status. Knockdown of TNFR1 dramatically attenuates malignant phenotypes, including proliferation and colony growth in soft agar, as well as glycolysis in ovarian cancer cells. Unexpectedly, knocking down TNFR1 blocks EGF-induced p-AKT and p-p70S6K expression and EGF-induced cell transformation through PIK3-p110beta rather than p110alpha expression. Taken together, our data provide evidence that TNFR1 plays a critical role in ovarian cancer and show that the EGF induced signaling pathway is independent of the TNF-α triggering cascade signal. Therefore, TNFR1 may serve as a prognostic molecule in ovarian cancer.

Cyclin-dependent kinase 2 (CDK2) is a key mediator for EGF-induced cell transformation mediated through the ELK4/c-Fos signaling pathway.

Cyclin dependent kinase 2 (CDK2) is a known regulator in the cell cycle control of the G1/S and S/G2 transitions. However, the role of CDK2 in tumorigenesis is controversial. Evidence from knockout mice as well as colon cancer cell lines indicated that CDK2 is dispensable for cell proliferation. In this study, we found that ectopic CDK2 enhances Ras (G12V)-induced foci formation and knocking down CDK2 expression dramatically decreases EGF-induced cell transformation mediated through the down-regulation of c-fos expression. Interestingly, CDK2 directly phosphorylates ELK4 at Thr194 and Ser387 and regulates ELK4 transcriptional activity, which serves as a mechanism to regulate c-fos expression. In addition, ELK4 is over-expressed in melanoma and knocking down ELK4 or CDK2 expression significantly attenuated the malignant phenotype of melanoma cells. Taken together, our study reveals a novel function of CDK2 in EGF-induced cell transformation and the associated signal transduction pathways. This indicates that CDK2 is a useful molecular target for chemoprevention and therapy against skin cancer.

Crucial role of c-Jun phosphorylation at Ser63/73 mediated by PHLPP protein degradation in the cheliensisin a inhibition of cell transformation.

Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its underlying molecular mechanisms have not been fully characterized yet. In the current study, we found that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP downregulation by promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction by Chel A, and further reversed the Chel A inhibition of EGF-induced cell transformation. With the findings, we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to c-Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel A.

Abstract 1249: ERK1/2 targeting with magnolin as a chemopreventive agent

Abstract Mitogen activated protein kinases (MAPKs) play a key role in cell proliferation, cell cycle progression and cell transformation, and activated Ras/ERKs/RSK2 signaling pathways have been widely identified in many solid tumors. In this study, we found that magnolin, a compound found in Magnolia species, directly targeted and inhibited ERK1 and ERK2 kinase activities with IC50 values of 87 nM and 16.5 nM by competing with ATP in an active pocket, respectively. Further, we demonstrated that magnolin inhibited EGF-induced p90RSKs phopshorylation at Thr359/Ser363, but not ERKs phosphorylation at Thr202/Tyr204, resulted in inhibition of cell proliferation by suppression of G1/S cell cycle transition. Additionally, p38 kinases, Jun N-terminal kinases, and Akts did not involved magnolin-mediated inhibitory signaling. Magnolin targeting on ERK1 and 2 activities suppressed the phosphorylation of RSK2 and downstream target proteins including ATF1 and c-Jun and AP-1, a dimer of Jun/Fos, and the transactivation activities of ATF1 and AP-1. Notably, ERKs inhibition by magnolin suppressed EGF-induced anchorage-independent cell transformation and colony growth of RasG12V- harboring A549 human lung cancer cells and NIH3T3 cells stably expressing RasG12V in soft agar. Taken together, these results demonstrated that magnolin might be a naturally occurring chemoprevention and therapeutic agent capable of inhibiting cell proliferation and transformation by targeting ERK1 and ERK2. Citation Format: Cheol-Jung Lee, Mee-Hyun Lee, Ji-Young Lee, Ji Hong Song, Yong-Yeon Cho. ERK1/2 targeting with magnolin as a chemopreventive agent. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1249. doi:10.1158/1538-7445.AM2014-1249