Dong Hoon Yu

ERK1 and ERK2 regulate embryonic stem cell self-renewal through phosphorylation of Klf4

Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism regulating this role of Klf4 is unclear. We found that ERK1 or ERK2 binds the activation domain of Klf4 and directly phosphorylates Klf4 at Ser123. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins βTrCP1 or βTrCP2 (components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. Overall, our data provide a molecular basis for the role of ERK1 and ERK2 in regulating Klf4-mediated mouse embryonic stem cell self-renewal.

ERK1 phosphorylates Nanog to regulate protein stability and stem cell self-renewal

Nanog regulates human and mouse embryonic stem (ES) cell self-renewal activity. Activation of ERKs signaling negatively regulates ES cell self-renewal and induces differentiation, but the mechanisms are not understood. We found that ERK1 binds and phosphorylates Nanog. Activation of MEK/ERKs signaling and phosphorylation of Nanog inhibit Nanog transactivation, inducing ES cell differentiation. Conversely, suppression of MEK/ERKs signaling enhances Nanog transactivation to inhibit ES cell differentiation. We observed that phosphorylation of Nanog by ERK1 decreases Nanog stability through ubiquitination-mediated protein degradation. Further, we found that this phosphorylation induces binding of FBXW8 with Nanog to reduce Nanog protein stability. Overall, our results demonstrated that ERKs-mediated Nanog phosphorylation plays an important role in self-renewal of ES cells through FBXW8-mediated Nanog protein stability.

Kaempferol Targets RSK2 and MSK1 to Suppress UV Radiation-Induced Skin Cancer

Solar UV (SUV) irradiation is a major factor in skin carcinogenesis, the most common form of cancer in the United States. The MAPK cascades are activated by SUV irradiation. The 90 kDa ribosomal S6 kinase (RSK) and mitogen and stress-activated protein kinase (MSK) proteins constitute a family of protein kinases that mediate signal transduction downstream of the MAPK cascades. In this study, phosphorylation of RSK and MSK1 was upregulated in human squamous cell carcinoma (SCC) and SUV-treated mouse skin. Kaempferol, a natural flavonol, found in tea, broccoli, grapes, apples, and other plant sources, is known to have anticancer activity, but its mechanisms and direct target(s) in cancer chemoprevention are unclear. Kinase array results revealed that kaempferol inhibited RSK2 and MSK1. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that kaempferol interacts with RSK2 and MSK1 at the ATP-binding pocket and inhibits their respective kinase activities. Mechanistic investigations showed that kaempferol suppresses RSK2 and MSK1 kinase activities to attenuate SUV-induced phosphorylation of cAMP-responsive element binding protein (CREB) and histone H3 in mouse skin cells. Kaempferol was a potent inhibitor of SUV-induced mouse skin carcinogenesis. Further analysis showed that skin from the kaempferol-treated group exhibited a substantial reduction in SUV-induced phosphorylation of CREB, c-Fos, and histone H3. Overall, our results identify kaempferol as a safe and novel chemopreventive agent against SUV-induced skin carcinogenesis that acts by targeting RSK2 and MSK1. Cancer Prev Res; 7(9); 958–67. ©2014 AACR.

Embryonic stem‐cell‐preconditioned microenvironment induces loss of cancer cell properties in human melanoma cells

The cancer microenvironment affects cancer cell proliferation and growth. Embryonic stem (ES)‐preconditioned 3‐dimensional (3‐D) culture of cancer cells induces cancer cell reprogramming and results in a change in cancer cell properties such as differentiation and migration in skin melanoma. However, the mechanism has not yet been clarified. Using the ES‐preconditioned 3‐D microenvironment model, we provide evidence showing that the ES microenvironment inhibits proliferation and anchorage‐independent growth of SK‐MEL‐28 melanoma cells. We also found that the ES microenvironment suppresses telomerase activity and thereby induces senescence in SK‐MEL‐28 cells. Furthermore, we observed that gremlin, an antagonist of BMP4, is secreted from ES cells and plays an important role in cellular senescence. Knocking down gremlin in the ES microenvironment increases proliferation and anchorage‐independent growth of SK‐MEL‐28 melanoma cells. Taken together, these results demonstrated that gremlin is a crucial factor responsible for abrogating melanoma properties in the ES‐preconditioned 3‐D microenvironment.

JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.

Embryonic stem (ES) cells are pluripotent cells with the capacity for unlimited self-renewal or differentiation. Inhibition of MAPK pathways enhances mouse ES cell pluripotency characteristics. Compared to wildtype ES cells, jnk2(-/-) ES cells displayed a much higher growth rate. To determine whether JNKs are required for stem cell self-renewal or differentiation, we performed a phosphorylation kinase array assay to compare mouse ES cells under LIF+ or LIF- culture conditions. The data showed that activation of JNKs was induced by LIF withdrawal. We also found that JNK1 or 2 phosphorylated Klf4 at threonines 224 and 225. Activation of JNK signaling and phosphorylation of Klf4 inhibited Klf4 transcription and transactivation activity. Importantly, jnk1(-/-) and jnk2(-/-) murine embryonic fibroblasts (MEFs) exhibited a significantly greater potency in the ability to increase the number of iPS colonies compared with jnk wildtype MEFs. Overall, our results demonstrated that JNK1 and 2 play a negative role in reprogramming to pluripotent stem cells by suppressing Klf4 activity.

Fyn is a redox sensor involved in solar ultraviolet light-induced signal transduction in skin carcinogenesis

Solar ultraviolet (UV) light is a major etiological factor in skin carcinogenesis, with solar UV-stimulated signal transduction inducing pathological changes and skin damage. The primary sensor of solar UV-induced cellular signaling has not been identified. We use an experimental system of solar simulated light (SSL) to mimic solar UV and we demonstrate that Fyn is a primary redox sensor involved in SSL-induced signal transduction. Reactive oxygen species (ROS) generated by SSL exposure directly oxidize Cys488 of Fyn, resulting in increased Fyn kinase activity. Fyn oxidation was increased in mouse skin after SSL exposure and Fyn-knockout mice formed larger and more tumors compared with Fyn wild-type mice when exposed to SSL for an extended period of time. Murine embryonic fibroblasts (MEFs) lacking Fyn and cells in which Fyn expression was knocked down were resistant to SSL-induced apoptosis. Furthermore, cells expressing mutant Fyn (C448A) were resistant to SSL-induced apoptosis. These findings suggest that Fyn acts as a regulatory nexus between solar UV, ROS and signal transduction during skin carcinogenesis.

Abstract 1241: Kaempferol suppresses solar ultraviolet radiation-induced skin cancers by targeting RSK2 and MSK1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, which is the most common form of cancer in the United States. Discovering novel chemopreventive agents against this disease is extremely important. Kaempferol, a natural flavonol isolated from tea, broccoli, grapes, apples and other plant sources, is known to have anticancer activity, but its molecular mechanisms and direct target(s) in cancer chemoprevention are still unclear. In this study, our pull-down assay results showed that RSK2 and MSK1 directly interact with kaempferol in both ex vivo and in vitro systems. ATP competition and in vitro kinase assay data revealed that kaempferol interacts with RSK2 and MSK1 at the ATP-binding pocket and inhibits their respective kinase activities. Mechanistic investigations determined that kaempferol acts as an inhibitor of RSK2 and MSK1 kinase activities to attenuate solar UV-induced phosphorylation in mitogen-activated protein kinase signaling cascades in JB6 P+ mouse skin epidermal cells. In a mouse skin tumorigenesis study, kaempferol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis showed that the kaempferol-treated group had a substantial reduction in solar UV-induced phosphorylation of CREB and c-Fos in mouse skin. Taken together, our results identify kaempferol as a safe and novel chemopreventive agent against solar UV-induced skin carcinogenesis that acts by targeting RSK2 and MSK1. Citation Format: Ke Yao, Hanyong Chen, Mee-Hyun Lee, Alyssa Langfald, Myoung Ok Kim, Dong Hoon Yu, Kangdong Liu, Wei-Ya Ma, Ann M. Bode, Ziming Dong, Zigang Dong. Kaempferol suppresses solar ultraviolet radiation-induced skin cancers by targeting RSK2 and MSK1. [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 1241. doi:10.1158/1538-7445.AM2014-1241

Serine 347 Phosphorylation by JNKs Negatively Regulates OCT4 Protein Stability in Mouse Embryonic Stem Cells

Summary The POU transcription factor OCT4 is critical for maintaining the undifferentiated state of embryonic stem cells (ESCs) and generating induced pluripotent stem cells (iPSCs), but its precise mechanisms of action remain poorly understood. Here, we investigated the role of OCT4 phosphorylation in the biological functions of ESCs. We observed that c-Jun N-terminal kinases (JNKs) directly interacted with and phosphorylated OCT4 at serine 347, which inhibited the transcriptional activity of OCT4. Moreover, phosphorylation of OCT4 induced binding of FBXW8, which reduced OCT4 protein stability and enhanced its proteasomal degradation. We also found that the mutant OCT4 (S347A) might delay the differentiation process of mouse ESCs and enhance the efficiency of generating iPSCs. These results demonstrated that OCT4 phosphorylation on serine 347 by JNKs plays an important role in its stability, transcriptional activities, and self-renewal of mouse ESCs.

Abstract 5746: TRAF1 is required for solar UV-induced skin carcinogenesis

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a member of the TRAF protein family, which regulates the canonical and non-canonical NF-κB signaling cascades. Although aberrant TRAF1 expression in tumors is reported, the role of TRAF1 remains elusive. Here, we report that TRAF1 is required for skin carcinogenesis induced by chronic solar UV radiation. In vivo studies with solar UV exposure indicate that the deletion of TRAF1 results in the inhibition of AP-1 activity by down-regulating the induction of c-Fos and c-Jun by regulating ERK5 activity. Furthermore, we show that TRAF1 is required for solar UV-induced ERK5 activation. Mechanistic studies revealed that TRAF1 expression enhances the ubiquitination of ERK5 on K184, which is necessary for AP-1 activation. Altogether, our results suggest that TRAF1 mediates ERK5 activity by regulating the upstream effectors of ERK5 and also by modulating its ubiquitination status. Targeting TRAF1 function might lead to strategies for preventing and treating skin cancer. Citation Format: Hiroyuki Yamamoto, Joohyun Ryu, Eli Min, Naomi Oi, Ruihua Bai, Tatyana A. Zykova, Dong Hoon Yu, Margarita Malakhova, Kenji Moriyama, Ann M. Bode, Zigang Dong. TRAF1 is required for solar UV-induced skin carcinogenesis [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 5746. doi:10.1158/1538-7445.AM2017-5746