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Previously, no member of the mixed-lineage kinase (MLK) protein family was known to function as an oncogene. Here, we demonstrate that MLK-like mitogen-activated protein triple kinase (MLTK)-α, a member of the MLK family, induced neoplastic cell transformation and tumorigenesis in athymic nude mice. Introduction of small interference RNA (siRNA)-MLTK-α into MLTK-α-overexpressing cells dramatically suppressed cell transformation. Nuclear accumulation of the pHisG-MLTK-α fusion protein was observed after epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate treatment. Phosphorylation of downstream mitogen-activated protein kinase-targeted transcription factors including c-Myc, Elk-1, c-Jun, and activating transcription factor (ATF) 2 was also differentially enhanced in MLTK-α-overexpressing cells exposed to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate stimulation compared with cells expressing mock vector or siRNA-MLTK-α. Very importantly, MLTK-α-overexpressing cells formed fibrosarcomas when injected s.c. into athymic nude mice, whereas almost no tumor formation was observed in mice that received injections of mock or siRNA-MLTK-α stably transfected cells. These results are the first to indicate that MLTK-α plays a key role in neoplastic cell transformation and cancer development.

Phosphorylation at Ser727 in signal transducer and activator of transcription 1 (STAT1) is essential for its activation and signal transduction. However, the upstream kinases responsible for phosphorylating Ser727 are still elusive. Here, we provide evidence showing that UVA-induced mitogen-activated protein kinase (MAPK) signaling pathways lead to STAT1 Ser727 phosphorylation. Our experimental results show that UVA-induced Ser727 phosphorylation of STAT1 was, to different degrees, diminished by PD98059 and U0126, two specific inhibitors of MEKs, and SB202190 and PD169316, inhibitors of p38 kinase and c-Jun N-terminal kinases (JNKs), respectively. STAT1 phosphorylation was also blocked by a dominant negative mutant of p38beta kinase or JNK1, JNK1- or JNK2-deficiency, or an N-terminal or C-terminal kinase-dead mutant of mitogen- and stress-activated protein kinase 1 (MSK1), a downstream kinase closer to p38 kinase and extracellular signal-regulated kinases (ERKs). In vitro kinase assays using the combined STAT1 proteins as substrates from immunoprecipitation and glutathione S-transferase pull down show that active ERK1, JNK1, p38 kinase, MEK1 and MSK1 stimulated phosphorylation of STAT1 (Ser727) indirectly through an unidentified factor or a downstream kinase. Overall, our data indicate that phosphorylation of STAT1 at Ser727 occurs through diverse MAPK cascades including MEK1, ERKs, p38 kinase, JNKs and MSK1 in the cellular response to UVA.

The relationship between cell transformation and p38 MAP kinase, a major mitogen-activated protein (MAP) kinase pathway converting signals of various extracellular stimuli into expression of specific target genes through activation of transcription factors, still remains unclear. The aim of the present study was to investigate the role of the p38 MAP kinase pathway in epidermal growth factor (EGF)-induced cell transformation in JB6 cells. Our data show that a dominant negative mutant of p38 MAP (DN-p38) kinase inhibits EGF-promoted JB6 Cl41 cell transformation and that SB202190, an inhibitor of p38 MAP kinase, also inhibits JB6 Cl41 cell transformation in a dose-dependent manner. Moreover, our results show that DN-p38 MAP kinase inhibits the phosphorylation of EGF-stimulated activating transcription factor-2 (ATF-2) and signal transducer and activator of transcription 1 (STAT1). Additionally, DN-p38 MAP kinase inhibits EGF-induced phosphorylation of c-Myc (Thr58/Ser62). Gel shift assays indicate that DN-p38 MAP kinase inhibits EGF-induced activator protein-1 (AP-1) DNA binding in a dose-dependent manner. These results show that p38 MAP kinase plays a key role in the regulation of EGF-induced cell transformation in JB6 cells through regulation of phosphorylation of p38 MAP kinase and activation of its target genes in phosphorylation, c-Myc cell transformation-related genes, and AP-1 binding ability.

Phosphorylation at Ser(727) is known to be required for complete activation of STAT3 by diverse stimuli including UV irradiation, but the kinase(s) responsible for phosphorylating STAT3 (Ser(727)) is still not well discerned. In the present study, we observed that activation of ATM is required for a UVA-stimulated increase in Ser(727) phosphorylation of STAT3 as well as in activation and phosphorylation of p90 ribosomal protein S6 kinases (RSKs). Moreover, UVA-stimulated activation of upstream kinases, such as c-Jun N-terminal kinases (JNKs) and ERKs, involved in mediating phosphorylation of RSKs and STAT3 was defective or delayed in ATM-deficient cells. Furthermore, we provide evidence that RSK2-deficient cells were defective for UV-induced Ser(727) phosphorylation of STAT3, and the defect was restored after ectopic expression of transfected full-length RSK2. In vitro experiments showed that active RSK2 and JNK1 induce the phosphorylation of STAT3 precipitates from immunoprecipitation but not from glutathione S-transferase (GST) pull-down. Interestingly, the GST fusion STAT3 proteins mixed together with STAT3 immunoprecipitates can be phosphorylated by JNK. However, the in vitro phosphorylation of STAT3 was reduced by the GST-STAT3 beta protein, a dominant negative form of STAT3. Taken together, our results demonstrate that the STAT3 phosphorylation at Ser(727) is triggered by active RSK2 or JNK1 in the presence of a downstream kinase or a cofactor, and thereby the intracellular phosphorylation process is stimulated through a signaling pathway involving ATM, MAPKs, RSK2, and an as yet unidentified kinase or cofactor. Additionally, RSK2-mediated phosphorylation of STAT3 (Ser(727)) was further determined to be required for basal and UVA-stimulated STAT3 transcriptional activities.

Yong-Yeon Cho,1 Hyeon-Uk Jeong,1 Jeong-Han Kim,2 Hye Suk Lee1 1College of Pharmacy, The Catholic University of Korea, Bucheon, Korea; 2Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea Abstract: Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 µM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5–50 µM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans. Keywords: honokiol, human hepatocytes, drug interactions, cytochrome P450, UDP-glucuronosyltransferases

The 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. Citation 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

The c-Jun transcription factor is a highly unstable oncoprotein. Several ubiquitin ligases mediate c-Jun degradation. However, c-Jun can be stabilized once it is phosphorylated at the N-terminus by c-Jun N-terminal kinases (JNKs) or other protein kinases. This phosphorylation decreases c-Jun ubiquitination and degradation. The underlying mechanism for this phenomenon is still unknown. We show that receptor for activated C-kinase 1 (Rack1) can bind with c-Jun and ubiquitin ligase Fbw7 to form a complex. When c-Jun is phosphorylated at the N-terminus, c-Jun is released from the complex and cannot be ubiquitinated by Fbw7, which leads to increased stabilization and accumulation of c-Jun. These results reveal that Rack1 plays a very important role in tumorigenesis by maintaining the stability of c-Jun that has been phosphorylated at its N- terminus by JNKs or other kinases. Citation Format: Jinhui Zhang, Feng Zhu, Xiang Li, Yanming Xu, Cong Peng, Shengqing Li, Yong-Yeon Cho, Ke Yao, Tatyana A. Zykova, Ann M. Bode, Zigang Dong. Rack1 protects N-terminal phosphorylated c-Jun from Fbw7-mediated degradation. [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 3178. doi:10.1158/1538-7445.AM2014-3178

Our 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. Citation 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

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

Arachidonate released by various stimuli is rapidly reesterified into membrane phospholipids initiated by acyl-CoA synthetase (ACS) and subsequent acyl-transfer reactions. ACS4 is an arachidonate-preferring enzyme abundant in steroidogenic tissues and postulated to modulate eicosanoid production. Female mice heterozygous for ACS4 deficiency become pregnant less frequently and produce small litters with extremely low transmission of the disrupted alleles. Striking morphological changes, including extremely enlarged uteri and lumina filled with numerous proliferative cysts of various sizes, were detected in ACS4+/- females. Furthermore, marked accumulation of prostaglandins was seen in the uterus of the heterozygous females. These results indicate that ACS4 modulates female fertility and uterine prostaglandin production.

Acyl-CoA synthetase 4 (ACS4) is an arachidonate-preferring enzyme abundant in steroidogenic tissues. We demonstrate that ACS4 expression in steroidogenic tissues in vivo is induced by adrenocorticotropic hormone (ACTH) and suppressed by glucocorticoid. ACTH also induced ACS4 protein but not its mRNA in Y1 adrenocortical tumor cells, whereas both ACS4 mRNA and protein were increased by dibutyryl cAMP (db-cAMP) and forskolin. Furthermore, the levels of ACS4 mRNA and protein in Y1 cells were induced by arachidonate. These data suggest that ACS4 expression in steroidogenic cells is regulated in coordination with induced steroidogenesis and arachidonate released by cholesterol ester hydrolase.

In arachidonate metabolism, acyl-CoA synthetase(ACS) plays a key role in esterification of free arachidonate into membrane phospholipids. ACS4 is an arachidonate preferring ACS abundant in steroidogenic tissues. The human gene for ACS4 (FACL4) is mapped chromosome band Xq22-23, a region close to the a5 chain of the type IV collagen gene that is related to X-linked Alport Syndrome. Recent studies have revealed that FACL4 is deleted in patients with Alport Syndrome, elliptocytosis and mental retardation, suggesting that the enzyme may play a role in intellectual growth in humans.

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.

Our previous study demonstrated that the total p90 ribosomal S6 kinase 2 (RSK2) protein level was significantly higher in human skin cancer tissues and cancer cells compared with normal skin tissues and premalignant cell lines. However, the role of RSK2 activation and signaling in human skin cancer has not yet been fully elucidated. In this study, we provided evidences that RSK2 plays a key role in human skin cancer. We examined the effect of knocking down RSK2 expression on EGF-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma (MM) cells. We found that UVB stimulation induced increased in not only the total and phosphorylated protein levels of ERKs and RSK2, but also the nuclear localization and gene expression of RSK2. Moreover, RSK2−/− mouse embryonic fibroblasts (MEFs) showed enhanced sub-G1 accumulation induced by UVB stimulation compared with RSK2+/+ MEFs, indicating that RSK2 might play an important role in tolerance against stress associated with UV. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of MM cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Taken together, our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth. Citation Format: Cheol-Jung Lee, Hye Suk Lee, Ji-Young Lee, Yong Yeon Cho. Involvement of RSK2 in human skin cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1296. doi:10.1158/1538-7445.AM2013-1296

Our previous study demonstrated that the total p90 ribosomal S6 kinase 2 (RSK2) protein level was significantly higher in human skin cancer tissues and cancer cells compared with normal skin tissues and premalignant cell lines. We also demonstrated that the N-terminal kinase domain of RSK2 plays an important role in transducing the RSK2 activation signal to its substrates. RSK2 requires activation of its C-terminal domain by its upstream kinases, ERKs, in order to phosphorylate and activate its substrates. For example, a recombinant RSK2 protein purified from E. Coli cannot phosphorylate the RSK2 substrate, NFAT3, but phosphorylation of RSK2 by ERKs at the linker region of RSK2 gives RSK2 the ability to phosphorylate its substrates through its N-terminal kinase domain. This suggests that ERKs/RSK2 signaling plays an important role in RSK2-mediated cell proliferation and cell transformation. Importantly, our study demonstrated that co-expression of RasG12V and RSK2 in NIH3T3 cells increased foci formation compared with RasG12V alone and si-RNA knockdown of RSK2 in RasG12V- or RasG12V/RSK2-co-expressing NIH3T3 cells completely blocked foci formation. Furthermore, we demonstrated that kaempferol, a natural compound abundantly found in dietary foods, inhibited RSK2 N-terminal kinase activity and EGF-induced neoplastic cell transformation. These indicated that the Ras/ERKs/RSK2 signaling pathway plays a key role in cell transformation. However, the role of RSK2 activation and signaling in human skin cancer has not yet been fully elucidated. To elucidate the relationship of RSK2 activity and human skin cancer, we examined the effect of knocking down RSK2 expression on EGF-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma (MM) cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. We found that UVB stimulation induced increased in not only the total and phosphorylated protein levels of ERKs and RSK2, but also the nuclear localization and gene expression of RSK2. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of MM cells. Moreover, RSK2−/− mouse embryonic fibroblasts (MEFs) showed enhanced sub-G1 accumulation induced by UVB stimulation compared with RSK2+/+ MEFs, indicating that RSK2 might play an important role in tolerance against stress associated with UV. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Taken together, our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth. Citation Format: Cheol-Jung Lee, Mee Hyun Lee, Ke Yao, Hye Suk Lee, Ann M. Bode, Zigang Dong, Yong Yeon Cho. Involvement of RSK2 in human skin cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B55.

The ribosomal S6 kinase 2 (RSK2) is a member of the p90 ribosomal S6 kinase (p90RSK) family of proteins and plays a critical role in proliferation, cell cycle and transformation. Here, we report that RSK2 phosphorylates caspase-8 and Thr263 was identified as a novel caspase-8 phosphorylation site. Phosphorylation of Thr263 is associated with the ubiquitination and degradation of caspase through the proteasome pathway. Results suggested that RSK2 regulates caspase-8 stability through its phosphorylation of this caspase. These data provide a direct link between RSK2 and caspase-8 and identify a novel molecular mechanism for caspase-8 modulation by RSK2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4968. doi:1538-7445.AM2012-4968

The c-Jun N-terminal kinases (JNKs) pathway plays a important role in many physiological processes including cell proliferation, survival, death, DNA repair and metabolism induced by a variety of stress signals and pro-inflammatory stimuli. The JNK proteins are encoded by three genes, jnk1, jnk2, jnk3. The protein products of jnk1 and jnk2 are found in all cells and tissues, whereas the JNK3 protein is found primarily in the brain, but is also found in the heart and the testes. The unequal expression of the different isoforms of JNK is possibly determined by some unique characteristics. Each JNK appears to have a distinct function in cancer, asthma, diabetes, and Parkinson's disease. Recent studies demonstrate that JNK1 is a key contributor for the serine phosphorylation of the IRS-1. Several other studies also demonstrated the importance of JNK1, but not JNK2, in lung fibrosis and gastric cancer. We have identified a novel flavonoid isolated from natural sources, herein referred to Compound-A, that suppressed JNK1 activity, with no effect on JNK2 in vitro. At 10 μM, Compound-A specifically inhibited JNK1 by about 50%. We also found that compound-A inhibits UVB-induced c-Jun phosphorylation in a cell culture system. In contrast to SP600125, a non-selective JNKs inhibitor that binds to the ATP active site, compound-A appears to target the protein-protein interaction between JNK1 and its binding proteins, including the JNK-interacting protein-1(JIP1) rather than the ATP binding site. In a computer simulation model, compound-A complexed with JNK1, but not JNK2, and formed strong hydrogen bonding at Glu329. Glu329 is a key residue for the JNK complex with JIP1. This interaction induces a hinge motion between the JNK N- and C-terminal domains and decreases the affinity of the kinase and ATP. These results demonstrate that compound-A is a selective JNK1 inhibitor. In humans, JNK1 play a key role in obesity-induced type 2 diabetes mellitus and the absence of JNK1 results in decreased adiposity, significantly improved insulin sensitivity and an enhanced insulin receptor signaling capacity in mouse obesity models. Therefore, we suggest that because of JNK1's critical role in diabetes, compound-A might have therapeutic potential against this devastating disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1034. doi:10.1158/1538-7445.AM2011-1034

The cancer micro-environment 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 micro-environmental model, we provide evidence showing that the ES micro-environment inhibits proliferation and anchorage-independent growth of SK-MEL-5 and SK-MEL-28 human melanoma cells, which is accompanied by the down-regulation of c-jun and c-fos gene expression. We also found that the ES micro-environment suppresses telomerase activity and thereby induces senescence in SK-MEL-5 and SK-MEL-28 cells. Furthermore, we observed that gremlin, an antagonist of bone morphogenic protein 4 (BMP4), is secreted from ES cells and plays an important role in cellular senescence. Taken together, these results demonstrated that gremlin is a crucial factor responsible for abrogating cancer properties in the ES-preconditioned 3-D micro-environment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1055. doi:10.1158/1538-7445.AM2011-1055

Histone H2AX is a histone H2A variant that is ubiquitously expressed throughout the genome. It plays a key role in the cellular response to DNA damage and has been designated as the histone guardian of the genome. H2AX deficiency decreases genomic stability and increases tumor susceptibility of normal cells and tissues. However, the role of H2AX phosphorylation in malignant transformation and cancer development is not totally clear. We found that ribosomal S6 kinase 2 (RSK2) directly phosphorylates H2AX at Ser139 and also at a newly discovered site, Ser16. EGF-induced phosphorylation of H2AX at both sites was decreased in RSK2 knockout cells. Phosphorylated RSK2 and H2AX co-localized in the nucleus following EGF treatment and the phosphorylation of H2AX by RSK2 enhanced the stability of H2AX and prevented cell transformation induced by EGF. RSK2 and DNA-PK, but not ATM or ATR, are required for EGF-induced phosphorylation of H2AX at Ser139. However, only RSK2 is required for phosphorylation of H2AX at Ser16. Phosphorylation of histone H3 was suppressed in cells expressing wildtype H2AX compared with H2AX knockout (H2AX-/-) cells. EGF-associated AP-1 transactivation activity was dramatically lower in H2AX-/- cells overexpressing wildtype H2AX compared with H2AX-/- cells expressing mutant H2AX-AA. Thus, the RSK2/H2AX signaling pathway negatively regulates the RSK2/histone H3 pathway and therefore maintains normal cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1017. doi:10.1158/1538-7445.AM2011-1017

The ribosomal S6 kinase 2 (RSK2), a member of the p90RSK (RSK) family of proteins, is a widely expressed serine/threonine kinase and its activation enhances cell proliferation. Here we report that activating transcription factor 1(ATF1) is a novel substrate of RSK2 and the RSK2-ATF1 signaling axis plays an important role in neoplastic cell transformation. RSK2 phosphorylates ATF1 at Ser63 and enhances the transactivation and transcriptional activities of ATF1. Computational modeling, high-through put screening and in vitro pull down assays demonstrated that eriodictiol, a flavanone found in fruits, binds with the N-terminal kinase domain and linker region of RSK2 and inhibits RSK2 N-terminal kinase activity. In a cell culture system, eriodictyol treatment suppressed phosphorylation of ATF1, but did not affect phosphorylation of RSK, MEK1/2, ERK1/2, p38 or JNKs, indicating that eriodictyol specifically inhibits RSK2 signaling. Furthermore, eriodictyol inhibited RSK2-mediated ATF1 transactivation activity and cell transformation induced by tumor promoters in JB6 Cl41 mouse skin epidermal cells. In a foci formation assay, knockdown of RSK2 or ATF1 suppressed foci formation compared with RasG12V, RasG12V/RSK2, RasG12V/ATF1 and RasG12V/RSK2/ATF1 expressing cells. In addition, eriodictyol treatment showed the same effect as RSK2 knockdown in foci formation. Taken together, these results indicated that the RSK2-ATF1 signaling axis plays an important role in neoplastic cell transformation and eriodictyol is a new natural compound for selectively inhibiting RSK2 kinase activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1227.

The ribosomal S6 kinase 2 (RSK2) mediates proliferation, survival, and malignant transformation. Activation of nuclear factor kappa B (NF-κB) results in the transcription of anti-apoptotic genes and is mediated by I-kappaB kinases (IKKs). IKK(α/β) phosphorylates IκBα at Ser32/Ser36 in the presence of TNF-α to induce IκBα degradation and NF-κB activation. The purpose of this study was to determine the potential role of RSK2 in mediating TNF-α-induced signaling. Notably, we found that RSK2 is activated by TNF-α and directly phosphorylates IκBα at Ser32 leading to IκBα degradation to promote the activation and translocation of the NF-κB subunits, p65 and p50, to the nucleus. This resulted in increased NF-κB activity, which served as a mechanism for RSK2 blockage of TNF-α induction of apoptosis and enhanced cell survival. Thus a new kinase that directly phosphorylates IκBα to promote its degradation is identified in TNF-R1 pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 309.

Bladder cancer is a common genitourinary malignant disease in the USA. EGFR and/or HER2 are overexpressed in these cancers, and EGFR/HER2 inhibitors are suggested as treatment. Gefitinib (i.e., Iressa) is a selective inhibitor of the epidermal growth factor receptor (EGFR) and Lapatinib is a dual inhibitor of both the EGFR and HER2/neu (Human EGFR type 2) receptor. Both compounds compete with the binding of ATP to the tyrosine kinase domain of the respective receptors to inhibit autophosphorylation causing suppression of signal transduction. Unfortunately, resistance to these inhibitors is a problem. The purpose of this study was to compare the signaling pathway(s) induced by Gefitinib and Lapatinib, in UM-UC-5 (Gefitinib-sensitive) and UM-UC-14 (Gefitinib-resistant) bladder cancer cells and to identify molecular markers that might be useful predictors of efficacy. Each cell line was cultured to 90% confluence and then incubated 24 h in serum-free media. They were treated, respectively, with 2.5 μM Gefitinib or Lapatinib for 24 h and harvested. Cell lysates were subjected to Proteome Profiler™ Arrays (R&D, Minneapolis, MN) including RTK, phospho-MAP kinase, phospho-kinase, and apoptosis arrays. Results indicate that Gefitinib and Lapatinib had differential effects on proteins known to play a role in apoptosis. Iressa appeared to decrease anti-apoptotic Bcl2, whereas Lapatinib increased pro-apoptotic Bax expression. Lapatinib, but not Iressa, also suppressed anti-apoptotic clusterin. Both agents decreased anti-apoptotic claspin, survivin, and XIAP expression but the effect of Lapatinib was 2- to 8-fold greater than Iressa. Notably, in the drug-resistant UM-UC-14 cells, Iressa had little affect whereas Lapatinib induced pro-apoptotic BAD and inhibited expression of anti-apoptotic Bcl2, Bcl-XL, and clusterin. Perhaps the most notable difference between these two drugs, was the effectiveness of Lapatinib in suppressing EGFR/HER2 expression in both cell lines. In the sensitive line, Lapatinib suppressed EGFR/HER2 expression 2-3 fold more than did Iressa and was almost as effective in the resistant line, whereas Iressa had no effect on UM-UC-14 cells. In contrast, Iressa was at least 8-fold more effective than Lapatinib in suppressing ERK1/2 expression in drug-sensitive cell lines but neither compound affected ERKs in resistant cells. Overall, the effect of these compounds on MAP kinase signaling appeared to be similar in sensitive cells; however, Lapatinib was able to suppress some MAP kinase signaling in the resistant line. Other notable differences included increased phosphorylation of p53, p27 and p70S6 kinase only by Lapatinib and decreased phosphorylation of eNOS only by Iressa in sensitive cells. Overall results suggest that human bladder cancer cells display some similarity but also marked heterogeneity in response to “specific” EGFR inhibitors ex vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1190.

During embryonic development, the extracellular matrix (ECM) plays a critical role in the regulation of stem cells to different lineages, cell migration and proliferation. A recently developed 3-dimensional (3D) ES-conditioned matrigel matrix culture of metastatic melanoma cells induces reprogramming and differentiation. However, although the ES-cell conditioned microenvironment induces melanoma cell reprogramming to suppress invasive behavior, the mechanisms have not yet been clearly understood. To investigate the effect of the cancer cell microenvironment, we hypothesized that 1) the microenvironment might induce nuclear reprogramming; 2) microenvironment-induced reprogramming might affect cell proliferation; 3) microenvironment-induced reprogramming might induce differentiation-mediated cellular senescence; and 4) microenvironment-induced reprogramming causes a loss of anchorage-independent cell growth, which is a hallmark of cancer cell properties. Here, we found that the ES-conditioned 3D-microenvironment induced a dramatic change in the morphology of A431 human skin epidermoid carcinoma cells and SK-MEL-28 human skin malignant melanoma cells. In the anchorage-independent cell growth assay, we found that A431 and SK-MEL-28 cells grown in the 3D ES-conditioned microenvironment exhibited dramatically decreased colony growth in soft agar. Real time PCR analysis indicated that ES-conditioned microenvironment suppressed endogenous Oct4 and Nanog gene expression in A431 and SK-MEL-28 cells and inhibited c-jun and c-fos promoter activities, which are components of the AP-1 transcriptional factor complex. Very importantly, we found that the ES cell-conditioned microenvironment inhibited telomerase activity, resulting in the induction of cellular senescence in human skin melanoma cells. We found that ES-conditioned medium contained a greater amount of Gremlin, a natural inhibitor of TGF-β signaling. Gremlin treatment of A431 and SK-MEL-28 cells induced morphological changes as observed in the 3D ES-conditioned matrigel matrix. Our results demonstrated that the ES-cell conditioned microenvironment inhibited proliferation and induced cellular senescence of A431 and SK-Mel-28 cells through the inhibition of TGF-β signaling by Gremlin. Furthermore, our data strongly suggests that modulation of TGF-β signaling in the cancer microenvironment is a potential strategy to modulate cancer cell behaviors and properties. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2337.

Background: Activation of tyrosine kinase receptors, including EGFR, HER2, HER3 and HER4, plays a key role in the prognosis of mammary cancer. EGFR and HER2 are validated therapeutic targets; but unfortunately, only a small percentage of patients with EGFR or HER2-overexpressing tumors respond to therapy, and resistance develops even in responsive patients. Lapatinib is a small molecule dual tyrosine kinase inhibitor that suppresses the activation of EGFR and HER2. Completely understanding the molecular mechanisms and protein targets involved in the effects of Lapatinib and other RTK inhibitors can help determine why efficacy varies. This requires the simultaneous identification of specific molecular markers in the complex network of signaling pathways that are modulated by Lapatinib in mammary cancer.Methods and Materials: In this study, female Sprague-Dawley rats (50 days old) were given methylnitrosourea (MNU) by IV injection through the jugular vein (75 mg/kg BW). The rats were palpated for mammary cancers 2 times per week. When a palpable mammary cancer of approximately 200-250 mm2 was present, the rat was administered Lapatinib (150 mg/kg BW/day) by gavage for 7 days. This dose of lapatinib was effective in a prevention study (causing a greater than 80% inhibition of mammary cancers). At sacrifice, the mammary cancer was rapidly removed and fixed for immunohistochemistry or frozen for protein array analysis. Fixed samples were analyzed for phosphorylated EGFR (Tyr1177) and HER2 expression by incubating with specific primary antibodies to detect pEGFR (Tyr1177) or HER2 and secondary antibodies conjugated to a fluorescence dye. Samples were observed by confocal microscopy. Frozen tissues were subjected to Proteome Profiler™ Arrays (R&D, Minneapolis, MN) including phospho-MAP kinase, phospho-kinase, and apoptosis arrays.Results: The results indicated that Lapatinib very effectively suppressed the abundance of both HER2 and phosphorylated EGFR (Tyr1177) compared to tumors from untreated control rats. The array analyses allowed parallel determination of the effect of Lapatinib on the relative levels of protein phosphorylation and apoptosis. Interestingly, unlike Gefitinib (Iressa), which appears to target EGFR and EGFR signaling mediated primarily through ERK MAP kinases, Lapatinib seems to suppress EGFR/HER2 signaling through Src-related proteins, Lyn and Lck, leading to increased cytochrome c release. The complete signaling cascade involves a number of additional signaling molecules, including inhibition of p38δ and γ, JNKs, GSK3α and β, and increased phosphorylation and activation of p27 and p53 potentially leading to apoptosis or cell cycle arrest. A second pathway that might also have a role in the action of Lapatinib involves the suppression of RTK activation of STAT5α and STAT3 potentially leading to decreased cell proliferation.Conclusion: These results suggest that RTK inhibitors have a wide range of potential targets. More work is in progress to continue the dissection of the action of Lapatinib and other RTK inhibitors. Supported by NCI Contract Number HHSN-261200433009C - NO1-CN-55006-72. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1130.

An age-dependent cellular change of DNA contents in the testis of Sprague-Dawley rats was investigated by flow-cytometric method. Testicular cell suspensions at the age of 4, 5, 6, 7, 8, 10, 12, 16 and 26 weeks were prepared and stained with propidium iodide. The relative proportions in the number of mature and immature haploid (1n), diploid (2n), S-phase and tetraploid (4n) cells were calculated. The proportion in the number of mature haploid cells was sharply increased to the age of 10 weeks (about 38%), thereafter increased slightly to the level of 42% at the age of 26 weeks. The proportion of immature haploid cells was dramatically increased to the age of 6 weeks, then maintained at the level of 20 to 30% thereafter. The proportion of diploid cells was 64% at the age of 4 weeks, then decreased gradually through the age of 26 weeks. The proportion of S-phase cells was increased to the age of 4 weeks, then maintained at a plateau level to the age of 26 weeks. The proportion of tetraploid cells were about 26% at the age of 4 weeks, then decreased gradually to the age of 26 weeks. These results suggest that the proportions of testicular cells may depend on the age of the rat and that the flow cytometric method may be useful in the evaluation of the spermatogenic status with regard to accuracy and sensitivity.

The effects of ethylene glycol monoethyl ether (EGEE) on testicular cell populations in pubertal (5 weeks old) and adult (9 weeks old) male rats were investigated by a flow cytometric method. A total of 50 rats (in number, 25 pubertal and 25 adult rats) was divided into 5 experimental groups including 0 (control), 50, 100, 200, and 400 mg EGEE/kg of body weight. The animals were administered by gavage for 4 weeks. In adult rats, the treatment of EGEE at the dose of 400 mg/kg of body weight decreased significantly the populations of haploid, while it increased those of diploid and tetraploid cells. In pubertal rats, the treatment of EGEE at the dose of 400 mg/kg of body weight caused only minimal changes in the relative percent of testicular cell types. These results suggest that the effects of EGEE on testicular function in pubertal rats appear to be less pronounced than in adult rats.

Background Magnolin 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. Methods Magnolin-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. Results Magnolin 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. Conclusions These results demonstrate that magnolin inhibits cell migration and invasion by targeting the ERKs/RSK2 signaling pathway. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1580-7) contains supplementary material, which is available to authorized users.