Mee-Hyun Lee

Magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway

BackgroundMagnolin is a natural compound abundantly found in Magnolia flos, which has been traditionally used in oriental medicine to treat headaches, nasal congestion and anti-inflammatory reactions. Our recent results have demonstrated that magnolin targets the active pockets of ERK1 and ERK2, which are important signaling molecules in cancer cell metastasis. The aim of this study is to evaluate the effects of magnolin on cell migration and to further explore the molecular mechanisms involved.MethodsMagnolin-mediated signaling inhibition was confirmed by Western blotting using RSK2+/+ and RSK2−/− MEFs, A549 and NCI-H1975 lung cancer cells, and by NF-κB and Cox-2 promoter luciferase reporter assays. Inhibition of cell migration by magnolin was examined by wound healing and/or Boyden Chamber assays using JB6 Cl41 and A549 human lung cancer cells. The molecular mechanisms involved in cell migration and epithelial-to-mesenchymal transition were determined by zymography, Western blotting, real-time PCR and immunocytofluorescence.ResultsMagnolin inhibited NF-κB transactivation activity by suppressing the ERKs/RSK2 signaling pathway. Moreover, magnolin abrogated the increase in EGF-induced COX-2 protein levels and wound healing. In human lung cancer cells such as A549 and NCI-H1975, which harbor constitutive active Ras and EGFR mutants, respectively, magnolin suppressed wound healing and cell invasion as seen by a Boyden chamber assay. In addition, it was observed that magnolin inhibited MMP-2 and −9 gene expression and activity. The knockdown or knockout of RSK2 in A549 lung cancer cells or MEFs revealed that magnolin targeting ERKs/RSK2 signaling suppressed epithelial-to-mesenchymal transition by modulating EMT marker proteins such as N-cadherin, E-cadherin, Snail, Vimentin and MMPs.ConclusionsThese results demonstrate that magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway.

RSK2-induced stress tolerance enhances cell survival signals mediated by inhibition of GSK3β activity

Our previous studies demonstrated that RSK2 plays a key role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor (EGF) in mouse and human skin cells. However, no direct evidence has been found regarding the relationship of RSK2 and cell survival. In this study, we found that RSK2 interacted and phosphorylated GSK3β at Ser9. Notably, GSK3β phosphorylation at Ser9 was suppressed in RSK2(-/-) MEFs compared with RSK2(+/+) MEFs by stimulation of EGF and calcium ionophore A23187, a cellular calcium stressor. In proliferation, we found that RSK2 deficiency suppressed cell proliferation compared with RSK2(+/+) MEFs. In contrast, GSK3β(-/-) MEFs induced the cell proliferation compared with GSK3β(+/+) MEFs. Importantly, RSK2(-/-) MEFs were induced severe cellular morphology change by A23187 and enhanced G1/G0 and sub-G1 accumulation of the cell cycle phase compared with RSK2(+/+) MEFs. The sub-G1 induction in RSK2(-/-) MEFs by A23187 was correlated with increase of cytochrome c release, caspase-3 cleavage and apoptotic DNA fragmentation compared with RSK2(+/+) MEFs. Notably, return back of RSK2 into RSK2(-/-) MEFs restored A23187-induced morphological change, and decreased apoptosis, apoptotic DNA fragmentation and caspase-3 induction compared with RSK2(-/-)/mock MEFs. Taken together, our results demonstrated that RSK2 plays an important role in stress-tolerance and cell survival, resulting in cell proliferation and cancer development.

Kaempferol targeting on the fibroblast growth factor receptor 3-ribosomal S6 kinase 2 signaling axis prevents the development of rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic inflammatory disease that mainly affects the synovial joints. Although involvement of the fibroblast growth factor (FGF) signaling pathway has been suggested as an important modulator in RA development, no clear evidence has been provided. In this study, we found that synovial fluid basic FGF (bFGF) concentration was significantly higher in RA than in osteoarthritis (OA) patients. bFGF stimulates proliferation and migration of human fibroblast-like synoviocytes (FLSs) by activation of the bFGF-FGF receptor 3 (FGFR3)-ribosomal S6 kinase 2 (RSK2) signaling axis. Moreover, a molecular docking study revealed that kaempferol inhibited FGFR3 activity by binding to the active pocket of the FGFR3 kinase domain. Kaempferol forms hydrogen bonds with the FGFR3 backbone oxygen of Glu555 and Ala558 and the side chain of Lys508. Notably, the inhibition of bFGF-FGFR3–RSK2 signaling by kaempferol suppresses the proliferation and migration of RA FLSs and the release of activated T-cell-mediated inflammatory cytokines, such as IL-17, IL-21, and TNF-α. We further found that activated phospho-FGFR3 and -RSK2 were more highly observed in RA than in OA synovium. The hyperplastic lining and sublining lymphoid aggregate layers of RA synovium showed p-RSK2-expressing CD68+ macrophages with high frequency, while pRSK2-expressing CD4+ T-cells was observed at a lower frequency. Notably, kaempferol administration in collagen-induced arthritis mice relieved the frequency and severity of arthritis. Kaempferol reduced osteoclast differentiation in vitro and in vivo relative to the controls and was associated with the inhibition of osteoclast markers, such as tartrate-resistant acid phosphatase, integrin β3, and MMP9. Conclusively, our data suggest that bFGF-induced FGFR3–RSK2 signaling may play a critical role during the initiation and progression of RA in terms of FLS proliferation and enhanced osteoclastogenesis, and that kaempferol may be effective as a new treatment for RA.

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

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

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

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 1917: Targeting wildtype and L858R/T790M mutant EGFR by isoliquiritigenin induces apoptosis and Inhibits tumor growth of NSCLC

Genetic alteration of epidermal growth factor receptor (EGFR) is a high risk factor of non-small-cell lung cancer (NSCLC). Isoliquiritigenin (ILQ), a chalcone derivative, reported anti-cancer activities. In this study, we investigated the effects of ILQ on the growth of tyrosine kinase inhibitor -sensitive and -resistant NSCLC cells and elucidated its molecular mechanisms. Treatment with ILQ inhibited growth and induced apoptosis in both tyrosine kinase inhibitor-sensitive (HCC827, H1650; delE746-A750 EGFR) and -resistant (H1975; L858R/T790M EGFR) NSCLC cells. However, ILQ didn9t showed the inhibitory effects of cell growth and induction of apoptosis in Met amplified-HCC827GR or K-ras mutated-A549 cells. ILQ induced apoptotic markers, the cleavage of caspase-3 and poly-(ADP ribose)-polymerase, increased expression of Bim and reduced expression of Bcl-2. ILQ inhibited the catalytic activity of both wildtype and double mutant (L858R/T790M) EGFR by kinase assay. Treatment with ILQ inhibited the anchorage-independent growth of NIH3T3 cells stably transfected with either wildtype or double-mutant EGFR with or without EGF stimulation. ILQ also reduced the phosphorylation of Akt and ERK1/2 in both TKI-sensitive and -resistant NSCLC cells in the western blotting analysis. ILQ directly bound both wildtype and double-mutant EGFR by ATP-competitive manner. Computational docking model expected that ILQ interacted with Glu762 and Met793 sites of wildtype EGFR and Lys745, Met793 and Asp855 of mutant EGFR. ILQ attenuated the tumor growth of H1975 cell xenografted athymic mice in vivo, which was caused by decreased expression of Ki-67 and diminished phosphorylation of Akt and ERK1/2. Taken together, ILQ suppresses NSCLC cell growth by directly targeting wildtype or mutant EGFR. Citation Format: Mee-Hyun Lee, Sung Keun Jung, Do Young Lim, Yong-Yeon Cho, Cheol-Jung Lee, Ji-Hong Song, Myoung Ok Kim, Sung-Hyun Kim, Ann M. Bode, Zigang Dong. Targeting wildtype and L858R/T790M mutant EGFR by isoliquiritigenin induces apoptosis and Inhibits tumor growth of NSCLC. [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 1917. doi:10.1158/1538-7445.AM2015-1917

Abstract 2471: Direct down-regulation of eEF1A2 by Tumor suppressor p16INK4a inhibits cancer cell growth

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnThe tumor suppressor protein p16INK4a is a member of the INK4 family of cyclin-dependent kinase (Cdk) inhibitors, which are involved in the regulation of the eukaryotic cell cycle. However, the mechanisms underlying the anti-proliferative effects of p16INK4a have not been fully elucidated. Using yeast two-hybrid screening, we identified the eukaryotic elongation factor (eEF)1A2 as a novel interacting partner of p16INK4a. eEF1A2 is thought to function as an oncogene in cancers. The p16INK4a protein interacted with all but the D2 (250-327 aa) domain of eEF1A2. Computational docking study predicted that D24/D131 residues of p16INK4a interacted with eEF1A2 and it was confirmed by pull-down assay with mutant p16INK4a (D24A/D131E). Ectopic expression of p16INK4a decreased the expression of eEF1A2 and inhibited cancer cell growth. Furthermore, suppression of protein synthesis by expression of p16INK4a ex vivo was verified by luciferase reporter activity. Microinjection of p16INK4a mRNA into the cytoplasm of Xenopus embryos suppressed the luciferase mRNA translation, whereas the combination of p16INK4a and morpholino-eEF1A2 resulted in a further reduction in translational activity. We conclude that the interaction of p16INK4a with eEF1A2, and subsequent downregulation of the expression and function of eEF1A2 is a novel mechanism explaining the anti-proliferative effects of p16INK4a.nnCitation Format: Mee-Hyun Lee, Bu Young Choi, Yong-Yeon Cho, Myoung Ok Kim, Sung-Hyun Kim, Cheol-Jung Lee, Ji Hong Song, Ann M. Bode, Zigang Dong, Yong-Joon Surh. Direct down-regulation of eEF1A2 by Tumor suppressor p16INK4a inhibits cancer cell growth. [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 2471. doi:10.1158/1538-7445.AM2014-2471

Abstract 5293: GSK3β phosphorylation at serine9 by RSK2 enhances calcium-induced stress tolerance and cell survival

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnOur previous studies demonstrated that RSK2 plays a key role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor in mouse and human skin cells. However, no direct evidence has been found regarding the relationship between RSK2 and cell survival. In this study, we found that RSK2 interacted and phosphorylated GSK3β at Ser9. Notably, GSK3β phosphorylation at Ser9 was suppressed in RSK2−/− MEFs compared with RSK2+/+ MEFs by stimulation of EGF and calcium ionophore A23187, a cellular calcium stressor. In proliferation, we found that RSK2 deficiency suppressed cell proliferation compared with RSK2+/+ MEFs. In contrast, GSK3β−/− MEFs induced the cell proliferation compared with GSK3β+/+ MEFs. Importantly, RSK2−/− MEFs were induced severe cellular morphology change by A23187 and enhanced G1/G0 and sub-G1 accumulation of the cell cycle phase compared with RSK2+/+ MEFs. The sub-G1 induction in RSK2−/− MEFs by A23187 was correlated with increase of cytochrome c release, caspase-3 cleavage and apoptotic DNA fragmentation compared with RSK2+/+ MEFs. Notably, return back of RSK2 into RSK2−/− MEFs restored A23187-induced morphological change, and decreased apoptosis, apoptotic DNA fragmentation and caspase-3 induction compared with RSK2−/− /mock MEFs. Taken together, our results demonstrated that RSK2 plays an important role in stress-tolerance and cell survival, resulting in cell proliferation and cancer development.nnCitation Format: Ji-Young Lee, Cheol-Jung Lee, Mee-Hyun Lee, Ji Hong Song, Yong-Yeon Cho. GSK3β phosphorylation at serine9 by RSK2 enhances calcium-induced stress tolerance and cell survival. [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 5293. doi:10.1158/1538-7445.AM2014-5293

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

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 IC 50 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 Ras G12V - harboring A549 human lung cancer cells and NIH3T3 cells stably expressing Ras G12V 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

Abstract A24: Targeting of ERKs with magnolin inhibits EGF-induced anchorage-independent cell transformation

An activated Ras mutation (RasG12V; constitutively active Ras) has exhibited in many human solid cancers with high occurrences including colon cancer (45%), pancreatic cancer (90%), non-small-cell lung cancer (35%) and melanomas (15%). Our previous results demonstrated that the ERKs signaling pathways including the ERKs/RSK2/histone H3 and ERKs/RSK2/cAMP-dependent transcription factor 1 (ATF1) signaling axes plays an important role in cell proliferation and transformation induced by growth factor stimulation such as epidermal growth factor (EGF). However, although ERKs are an important target molecule of anti-cancer drugs or chemopreventive agents, the identification or development of small molecules as ERKs inhibitors has been barely studied. Recently, several ERK specific inhibitors have been found by high-throughput phosphorylation assays with an in-house chemical library. For example, small molecule FR180204 is an inhibitor of ERK1 and ERK2 with IC50 values of 0.51 nM and 0.33 nM, respectively. More recently, FR148083 was identified as an ERK2 inhibitor and it suppressed ERK2 activity with an IC50 value of 80 nM on an enzyme assay screening. However, these compounds also showed inhibition of other enzymes such as TGF-β-induced AP-1-dependent luciferase expression with an IC50 value of 50 nM. The Magnolia species has been traditionally used as an oriental medicine to treat nasal congestion associated with headache, sinusitis, anti-inflammation, and allergic rhinitis. Magnolin is an ingredient found in the Magnolia species, and it has been widely used in oriental medicine to treat human diseases including empyema, nasal congestion, sinusitis and inflammation, and has been shown to inhibit the production of tumor necrosis factor-α (TNF-α). Recent studies have shown that magnolin influences nitric oxide (NO) and prostaglandin E2 (PGE2) production by inhibiting extracellular signal-regulated kinase (ERK). In this study, we found that magnolin 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. Notably, EGF-induced p90RSKs phosphorylation at Thr359/Ser363 was inhibited by magnolin treatment, resulted in inhibition of cell proliferation by suppression of the G1/S cell cycle transition. Importantly, inhibition of ERKs/RSK2 signaling axis by magnolin suppresses the EGF-induced anchorage-independent cell transformation by the abrogation of ATF1 and AP-1 transactivation activities. 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, Hye Suk Lee, Hyung Won Ryu, Mee-Hyun Lee, Ji-Young Lee, Hyoung-Kyu Lee, Sei-Ryang Oh, Yong-Yeon Cho. Targeting of ERKs with magnolin inhibits EGF-induced anchorage-independent cell transformation. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A24.