Activation Of Ribosomal S6 Kinase Research Articles

Arginase‐II Induces Vascular Smooth Muscle Cell Senescence and Apoptosis Through p66Shc and p53 Independently of Its l ‐Arginine Ureahydrolase Activity: Implications for Atherosclerotic Plaque Vulnerability

BackgroundVascular smooth muscle cell (VSMC) senescence and apoptosis are involved in atherosclerotic plaque vulnerability. Arginase‐II (Arg‐II) has been shown to promote vascular dysfunction and plaque vulnerability phenotypes in mice through uncoupling of endothelial nitric oxide synthase and activation of macrophage inflammation. The function of Arg‐II in VSMCs with respect to plaque vulnerability is unknown. This study investigated the functions of Arg‐II in VSMCs linking to plaque vulnerability.Methods and ResultsIn vitro studies were performed on VSMCs isolated from human umbilical veins, whereas in vivo studies were performed on atherosclerosis‐prone apolipoprotein E‐deficient (ApoE−/−) mice. In nonsenescent VSMCs, overexpressing wild‐type Arg‐II or an l‐arginine ureahydrolase inactive Arg‐II mutant (H160F) caused similar effects on mitochondrial dysfunction, cell apoptosis, and senescence, which were abrogated by silencing p66Shc or p53. The activation of p66Shc but not p53 by Arg‐II was dependent on extracellular signal‐regulated kinases (ERKs) and sequential activation of 40S ribosomal protein S6 kinase 1 (S6K1)—c‐Jun N‐terminal kinases (JNKs). In senescent VSMCs, Arg‐II and S6K1, ERK‐p66Shc, and p53 signaling levels were increased. Silencing Arg‐II reduced all these signalings and cell senescence/apoptosis. Conversely, silencing p66Shc reduced ERK and S6K1 signaling and Arg‐II levels and cell senescence/apoptosis. Furthermore, genetic ablation of Arg‐II in ApoE−/− mice reduced the aforementioned signaling and apoptotic VSMCs in the plaque of aortic roots.ConclusionsArg‐II, independently of its l‐arginine ureahydrolase activity, promotes mitochondrial dysfunction leading to VSMC senescence/apoptosis through complex positive crosstalk among S6K1‐JNK, ERK, p66Shc, and p53, contributing to atherosclerotic vulnerability phenotypes in mice.

Abstract 1296: Involvement of RSK2 in human skin cancer.

Abstract 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

Abstract 5243: The novel calcium-dependent direct binding of S100B to p90 ribosomal S6 kinase (RSK) decreases MAP kinase-mediated RSK activation in malignant melanoma.

Abstract S100B is an effective and extensively used prognostic marker for melanoma, with increasing serum S100B being predictive of disease stage, increased recurrence, and low overall patient survival. Establishing the mechanism by which S100B alters cell signaling provides insight into how it may facilitate the progression of melanoma and aid in developing new pharmacological drugs to inhibit cancer advancement. To evaluate specific roles for S100B in malignant melanoma, our S100B knock-down and over-expression studies established a correlation between S100B expression and cell viability. ERK phosphorylation was also found to correlate with S100B levels. We have discovered RSK, a downstream ERK target, as a novel binding partner of S100B. RSK is known to regulate MAPK signaling in melanoma but the mechanism is unclear. We observed an inverse relationship between activated and phosphorylated RSK and S100B levels. We used a Ca2+-binding mutant of S100B (E31A + E72A) to demonstrate that the S100B-RSK interaction was calcium-dependent. Pull-down experiments revealed the C-terminal domain of RSK to be necessary for S100B binding. In vitro, S100B was found to reduce ERK-dependent phosphorylation of RSK at Thr573. Our cellular fractionation and immuno-fluorescence studies showed that S100B protein prevented nuclear localization of phosphorylated RSK. These data are consistent with a mechanism in which elevated S100B binds directly to RSK in a calcium-dependent manner, preventing ERK-mediated phosphorylation of RSK and inhibiting RSK localization to the nucleus. We have identified the S100B-RSK interaction as a novel drug target in melanoma cells. We are currently screening compounds for inhibitors of the S100B-RSK interaction. Citation Format: Adam D. Pierce, Kira G. Hartman, David Weber. The novel calcium-dependent direct binding of S100B to p90 ribosomal S6 kinase (RSK) decreases MAP kinase-mediated RSK activation in malignant melanoma. [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 5243. doi:10.1158/1538-7445.AM2013-5243

Fisetin regulates obesity by targeting mTORC1 signaling

Fisetin, a flavonol present in vegetables and fruits, possesses antioxidative and anti-inflammatory properties. In this study, we have demonstrated that fisetin prevents diet-induced obesity through regulation of the signaling of mammalian target of rapamycin complex 1 (mTORC1), a central mediator of cellular growth, cellular proliferation and lipid biosynthesis. To evaluate whether fisetin regulates mTORC1 signaling, we investigated the phosphorylation and kinase activity of the 70-kDa ribosomal protein S6 kinase 1 (S6K1) and mTORC1 in 3T3-L1 preadipocytes. Fisetin treatment of preadipocytes reduced the phosphorylation of S6K1 and mTORC1 in a time- and concentration-dependent manner. To further our understanding of how fisetin negatively regulates mTORC1 signaling, we analyzed the phosphorylation of S6K1, mTOR and Akt in fisetin-treated TSC2-knockdown cells. The results suggested that fisetin treatment inhibits mTORC1 activity in an Akt-dependent manner. Recent studies have shown that adipocyte differentiation is dependent on mTORC1 activity. Fisetin treatment inhibited adipocyte differentiation, consistent with the negative effect of fisetin on mTOR. The inhibitory effect of fisetin on adipogenesis is dependent of mTOR activity, suggesting that fisetin inhibits adipogenesis and the accumulation of intracellular triglycerides during adipocyte differentiation by targeting mTORC1 signaling. Fisetin supplementation in mice fed a high-fat diet (HFD) significantly attenuated HFD-induced increases in body weight and white adipose tissue. We also observed that fisetin efficiently suppressed the phosphorylation of Akt, S6K1 and mTORC1 in adipose tissue. Collectively, these results suggest that inhibition of mTORC1 signaling by fisetin prevents adipocyte differentiation of 3T3-L1 preadipocytes and obesity in HFD-fed mice. Therefore, fisetin may be a useful phytochemical agent for attenuating diet-induced obesity.

Inhibition of S6 kinase suppresses the apoptotic effect of eIF4E ablation by inducing TGF-β-dependent G1 cell cycle arrest

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of cap-dependent translation through its direct activation of ribosomal protein p70 S6 kinase (S6 kinase) and indirect activation of eukaryotic initiation factor 4E (eIF4E). We recently reported that inhibition of eIF4E expression caused apoptosis in cancer cells in the absence of serum. This was indicated by treatment with the mTORC1 inhibitor rapamycin, which suppressed both S6 kinase and 4E-BP1 phosphorylation (dephosphorylated 4E-BP1 binds and inactivates eIF4E), or by knockdown of eIF4E. We report here that knockdown of eIF4E also causes apoptosis in the presence of serum. This was unexpected because rapamycin induces G1 cell cycle arrest in the presence of serum. Upon investigation, we have found that inactivated S6 kinase prevents the apoptotic effect observed by singular knockdown of eIF4E and results in G1 cell cycle arrest. This effect is dependent on TGF-β (transforming growth factor-β) signaling which contributes to G1 cell cycle arrest. Suppression of S6 kinase phosphorylation alone is insufficient to mediate cell cycle arrest, indicating that complete G1 cell cycle arrest is due to suppression of both S6 kinase and eIF4E. These data indicate that the cytostatic effect of rapamycin is suppression of both S6 kinase and eIF4E, while the cytotoxic effects are due suppression of eIF4E in the absence of S6 kinase-dependent activation of TGF-β signals. Our findings place an importance on the evaluating the activity/expression level of S6 kinase and eIF4E as readouts for rapamycin/rapalog efficacy.

Integrins promote cytokinesis through the RSK signaling axis.

Cytokinesis is the final stage in cell division. Although integrins can regulate cytokinesis, the mechanisms involved are not fully understood. In this study, we demonstrate that integrin-regulated ERK (extracellular signal-related kinase) and RSK (p90 ribosomal S6 kinase) signaling promotes successful cytokinesis. Inhibiting the activation of ERK and RSK in CHO cells by a mutation in the integrin β1 cytoplasmic tail or with pharmacological inhibitors results in the accumulation of cells with midbodies and the formation of binucleated cells. Activation of ERK and RSK signaling by the expression of constitutively active RAF1 suppresses the mutant phenotype in a RSK-dependent manner. Constitutively active RSK2 also restores cytokinesis inhibited by the mutant integrin. Importantly, the regulatory role of the RSK pathway is not specific to CHO cells. MCF-10A human mammary epithelial cells and HPNE human pancreatic ductal epithelial cells exhibit a similar dependence on RSK for successful cytokinesis. In addition, depriving mitotic MCF10A cells of integrin-mediated adhesion by incubating them in suspension suppressed ERK and RSK activation and resulted in a failure of cytokinesis. Furthermore, inhibition of RSK or integrins within the 3D context of a developing salivary gland organ explant also leads to an accumulation of epithelial cells with midbodies, suggesting a similar defect in cytokinesis. Interestingly, neither ERK nor RSK regulates cytokinesis in human fibroblasts, suggesting cell-type specificity. Taken together, our results identify the integrin-RSK signaling axis as an important regulator of cytokinesis in epithelial cells. We propose that the proper interaction of cells with their microenvironment through integrins contributes to the maintenance of genomic stability by promoting the successful completion of cytokinesis.

Abstract B55: Involvement of RSK2 in human skin cancer

Abstract 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.

Abstract B48: Anticancer effect of guava leaves (Psidium guajava L.) in prostate cancer cells

Abstract This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer. Citation Format: Woong Mo Yang, Mi Hye Kim, Sang-Yoon Park, Kwang Seok Ahn, Seok-Geun Lee. Anticancer effect of guava leaves (Psidium guajava L.) in prostate cancer cells. [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 B48.

Glucocorticoid mediates water avoidance stress-sensitized colon-bladder cross-talk via RSK2/PSD-95/NR2B in rats

Unexpected environmental and social stimuli could trigger stress. Although coping with stress is essential for survival, long-term stress impacts visceral functions, and therefore, it plays a role in the development and exacerbation of symptoms of gastrointestinal/urogenital disorders. The aim of this study is to characterize the role of corticosterone in stress-sensitized colon-bladder cross-talk, a phenomenon presumed to underlie the comorbidity of functional bowel and bladder disorders. Cystometry and protein/mRNA expression in the lumbosacral dorsal horn (L6-S1) in response to intracolonic mustard oil (MO) instillation were analyzed in female Wistar-Kyoto rats subjected to water avoidance stress (WAS; 1 h/day for 10 days) or sham stress (WAsham). Whereas it had no effect on baseline-voiding function, chronic stress upregulated plasma corticosterone concentration and dorsal horn spinal p90 ribosomal S6 kinase 2 (RSK2) protein/mRNA levels, and RSK2 immunoreactivity colocalized with NeuN-positive neurons. Intracolonic MO dose-dependently decreased intrercontraction intervals and threshold pressure, provoked spinal RSK2 and NR2B phosphorylation, and enhanced PSD-95-RSK2 and PSD-95-NR2B coupling. Intrathecal kaempferol (a RSK2 activation antagonist; 30 min before MO instillation), bilateral adrenalectomy (7 days prior the stress paradigm), and subcutaneous RU-38486 (a glucocorticoid receptor antagonist; 30 min daily before stress sessions), but not RU-28318 (a mineralocorticoid receptor antagonist), attenuated MO-induced bladder hyperactivity, protein phosphorylation, and protein-protein interactions in the WAS group. Our results suggest that stress-associated glucocorticoid release mediates WAS-dependent sensitization of colon-bladder cross-talk via the spinal RSK2/PSD-95/NR2B cascade and offer a possibility for developing pharmacological strategies for the treatment of stress-related pelvic pain.

DIFFERENTIAL SIGNALLING THROUGH P90 RIBOSOMAL S6 KINASES (RSKS) TO CARDIOMYOCYTE GENE EXPRESSION BY ENDOTHELIN-1 VERSUS α-ADRENERGIC AGONISTS

Endothelin or α1-adrenergic receptor agonists (eg, A61603) promote cardiomyocyte hypertrophy. Both signal through extracellular signal-regulated kinases1/2 (ERKs) to induce changes in gene expression. However, although there is overlap, the transcriptomic...

Anchored p90 Ribosomal S6 Kinase 3 Is Required for Cardiac Myocyte Hypertrophy

Cardiac myocyte hypertrophy is the main compensatory response to chronic stress on the heart. p90 ribosomal S6 kinase (RSK) family members are effectors for extracellular signal-regulated kinases that induce myocyte growth. Although increased RSK activity has been observed in stressed myocytes, the functions of individual RSK family members have remained poorly defined, despite being potential therapeutic targets for cardiac disease. To demonstrate that type 3 RSK (RSK3) is required for cardiac myocyte hypertrophy. RSK3 contains a unique N-terminal domain that is not conserved in other RSK family members. We show that this domain mediates the regulated binding of RSK3 to the muscle A-kinase anchoring protein scaffold, defining a novel kinase anchoring event. Disruption of both RSK3 expression using RNA interference and RSK3 anchoring using a competing muscle A-kinase anchoring protein peptide inhibited the hypertrophy of cultured myocytes. In vivo, RSK3 gene deletion in the mouse attenuated the concentric myocyte hypertrophy induced by pressure overload and catecholamine infusion. Taken together, these data demonstrate that anchored RSK3 transduces signals that modulate pathologic myocyte growth. Targeting of signaling complexes that contain select kinase isoforms should provide an approach for the specific inhibition of cardiac myocyte hypertrophy and for the development of novel strategies for the prevention and treatment of heart failure.

EGFR and PKC are involved in the activation of ERK1/2 and p90 RSK and the subsequent proliferation of SNU-407 colon cancer cells by muscarinic acetylcholine receptors

We have previously shown that muscarinic acetylcholine receptors (mAChRs) enhance SNU-407 colon cancer cell proliferation via the ERK1/2 pathway. Here, we examined the signaling pathways linking mAChR stimulation to ERK1/2 activation and the subsequent proliferation of SNU-407 cells. The inhibition of the epidermal growth factor receptor (EGFR) by AG1478 or protein kinase C (PKC) by GF109203X significantly reduced carbachol-stimulated ERK1/2 activation and cell proliferation. Cotreatment of the cells with AG1478 and GF109203X produced an additive effect on carbachol-stimulated ERK1/2 activation, suggesting that the EGFR and PKC pathways act in parallel. The p90 ribosomal S6 kinases (RSKs) are downstream effectors of ERK1/2 and are known to have important roles in cell proliferation. In SNU-407 cells, carbachol treatment induced RSK activation in an atropine-sensitive manner, and this RSK activation was decreased by the inhibition of either EGFR or PKC. Moreover, the RSK-specific inhibitor BRD7389 almost completely blocked carbachol-stimulated cell proliferation. Together, these data indicate that EGFR and PKC are involved in mAChR-mediated activation of ERK1/2 and RSK and the subsequent proliferation of SNU-407 colon cancer cells.

Epidermal Growth Factor-induced Vacuolar (H+)-ATPase Assembly: A ROLE IN SIGNALING VIA mTORC1 ACTIVATION

Using proteomics and immunofluorescence, we demonstrated epidermal growth factor (EGF) induced recruitment of extrinsic V(1) subunits of the vacuolar (H(+))-ATPase (V-ATPase) to rat liver endosomes. This was accompanied by reduced vacuolar pH. Bafilomycin, an inhibitor of V-ATPase, inhibited EGF-stimulated DNA synthesis and mammalian target of rapamycin complex 1 (mTORC1) activation as indicated by a decrease in eukaryotic initiation factor 4E-binding 1 (4E-BP1) phosphorylation and p70 ribosomal S6 protein kinase (p70S6K) phosphorylation and kinase activity. There was no corresponding inhibition of EGF-induced Akt and extracellular signal-regulated kinase (Erk) activation. Chloroquine, a neutralizer of vacuolar pH, mimicked bafilomycin effects. Bafilomycin did not inhibit the association of mTORC1 with Raptor nor did it affect AMP-activated protein kinase activity. Rather, the intracellular concentrations of essential but not non-essential amino acids were decreased by bafilomycin in EGF-treated primary rat hepatocytes. Cycloheximide, a translation elongation inhibitor known to augment intracellular amino acid levels, prevented the effect of bafilomycin on amino acids levels and completely reversed its inhibition of EGF-induced mTORC1 activation. In vivo administration of EGF stimulated the recruitment of Ras homologue enriched in brain (Rheb) but not mammalian target of rapamycin (mTOR) to endosomes and lysosomes. This was inhibited by chloroquine treatment. Our results suggest a role for vacuolar acidification in EGF signaling to mTORC1.

Tumor Necrosis Factor Receptor-associated Factor Family Protein 2 Is a Key Mediator of the Epidermal Growth Factor-induced Ribosomal S6 Kinase 2/cAMP-responsive Element-binding Protein/Fos Protein Signaling Pathway

TRAF2 has an important function in mediating the TNF-R signaling pathway toward activation of NF-κB and JNKs. Here we reveal a novel function of TRAF2 in the epidermal growth factor (EGF) signaling pathway. Knockdown of TRAF2 blocked EGF-induced AP-1 activity and anchorage- independent cell transformation. Notably, we showed that EGF induces ribosomal S6 kinase 2 (RSK2) ubiquitination, and knocking down TRAF2 suppresses ubiquitination of RSK2 induced by EGF. We also found that TRAF2 affects RSK2 activity through RSK2 ubiquitination. RSK2 plays a critical role in AP-1 activity mediated through CREB and c-Fos, which regulates anchorage-independent cell transformation. In addition, TRAF2 is overexpressed in colon cancer and required for colon cancer development, suggesting that TRAF2 might be a potential molecular target for cancer prevention and treatment.

Pristimerin, a Triterpenoid, Inhibits Tumor Angiogenesis by Targeting VEGFR2 Activation

Pristimerin is a triterpenoid isolated from Celastrus and Maytenus spp. thathas been shown to possess a variety of biological activities, including anti-cancer activity. However, little is known about pristimerin’s effects on tumor angiogenesis. In this study, we examined the function and the mechanism of this compound in tumor angiogenesisusing multiple angiogenesis assays. We found that pristimerin significantly reduced both the volume and weight of solid tumors and decreased angiogenesis in a xenograft mouse tumor model in vivo. Pristimerin significantly inhibited the neovascularization of chicken chorioallantoic membrane (CAM) in vivo and abrogated vascular endothelial growth factor (VEGF)-induced microvessel sprouting in an ex vivo rat aortic ring assay. Furthermore, pristimerin inhibited the VEGF-induced proliferation, migration and capillary-like structure formation of human umbilical vascular endothelial cells (HUVECs) in a concentration-dependent manner. Mechanistic studies revealed that pristimerin suppressed the VEGF-induced phosphorylation of VEGF receptor 2 kinase (KDR/Flk-1) and the activity of AKT, ERK1/2, mTOR, and ribosomal protein S6 kinase. Taken together, our results provide evidence for the first time that pristimerin potently suppresses angiogenesis by targeting VEGFR2 activation. These results provide a novel mechanism of action for pristimerin which may be important in the treatment of cancer.

A Hexane Fraction of Guava Leaves (Psidium guajavaL.) Induces Anticancer Activity by Suppressing AKT/Mammalian Target of Rapamycin/Ribosomal p70 S6 Kinase in Human Prostate Cancer Cells

This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer.

Hypoxia-induced invadopodia formation involves activation of NHE-1 by the p90 ribosomal S6 kinase (p90RSK).

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na+/H+ exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na+/H+ exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors.

Mammalian Target of Rapamycin Inhibition in Macrophages of Asymptomatic HIV+ Persons Reverses the Decrease in TLR-4–Mediated TNF-α Release through Prolongation of MAPK Pathway Activation

TLR-4-mediated signaling is significantly impaired in macrophages from HIV(+) persons, predominantly owing to altered MyD88-dependent pathway signaling caused in part by constitutive activation of PI3K. In this study we assessed in these macrophages if the blunted increase in TLR-4-mediated TNF-α release induced by lipid A (LA) is associated with PI3K-induced upregulation of mammalian target of rapamycin (mTOR) activity. mTOR inhibition with rapamycin enhanced TLR-4-mediated TNF-α release, but suppressed anti-inflammatory IL-10 release. Targeted gene silencing of mTOR in macrophages resulted in LA-induced TNF-α and IL-10 release patterns similar to those induced by rapamycin. Rapamycin restored MyD88/IL-1R-associated kinase interaction in a dose-dependent manner. Targeted gene silencing of MyD88 (short hairpin RNA) and mTOR (RNA interference) inhibition resulted in TLR-4-mediated 70-kDa ribosomal protein S6 kinase activation and enhanced TNF-α release, whereas IL-10 release was inhibited in both silenced and nonsilenced HIV(+) macrophages. Furthermore, mTOR inhibition augmented LA-induced TNF-α release through enhanced and prolonged phosphorylation of ERK1/2 and JNK1/2 MAPK, which was associated with time-dependent MKP-1 destabilization. Taken together, impaired TLR-4-mediated TNF-α release in HIV(+) macrophages is attributable in part to mTOR activation by constitutive PI3K expression in a MyD88-dependent signaling pathway. These changes result in MAPK phosphatase 1 stabilization, which shortens and blunts MAPK activation. mTOR inhibition may serve as a potential therapeutic target to upregulate macrophage innate immune host defense responsiveness in HIV(+) persons.

Increased 90-kDa ribosomal S6 kinase (Rsk) activity is protective against mutant huntingtin toxicity

BackgroundThe 90-kDa ribosomal S6 kinase (Rsk) family is involved in cell survival. Rsk activation is regulated by sequential phosphorylations controlled by extracellular signal-regulated kinase (ERK) 1/2 and 3-phosphoinositide-dependent protein kinase 1 (PDK1). Altered ERK1/2 and PDK1 phosphorylation have been described in Huntington's disease (HD), characterized by the expression of mutant huntingtin (mhtt) and striatal degeneration. However, the role of Rsk in this neurodegenerative disease remains unknown. Here, we analyzed the protein levels, activity and role of Rsk in in vivo and in vitro HD models.ResultsWe observed increased protein levels of Rsk1 and Rsk2 in the striatum of HdhQ111/Q111 and R6/1 mice, STHdhQ111/Q111 cells and striatal cells transfected with full-length mhtt. Analysis of the phosphorylation of Rsk in Hdh mice and STHdh cells showed reduced levels of phospho Ser-380 (dependent on ERK1/2), whereas phosphorylation at Ser-221 (dependent on PDK1) was increased. Moreover, we found that elevated Rsk activity in STHdhQ111/Q111 cells was mainly due to PDK1 activity, as assessed by transfection with Rsk mutant constructs. The increase of Rsk in STHdhQ111/Q111 cells occurred in the cytosol and in the nucleus, which results in enhanced phosphorylation of both cytosolic and nuclear Rsk targets. Finally, pharmacological inhibition of Rsk, knock-down and overexpression experiments indicated that Rsk activity exerts a protective effect against mhtt-induced cell death in STHdhQ7/Q7 cells transfected with mhtt.ConclusionThe increase of Rsk levels and activity would act as a compensatory mechanism with capacity to prevent mhtt-mediated cell death. We propose Rsk as a good target for neuroprotective therapies in HD.

Phosphorylation of Eukaryotic Translation Initiation Factor 4B (EIF4B) by Open Reading Frame 45/p90 Ribosomal S6 Kinase (ORF45/RSK) Signaling Axis Facilitates Protein Translation during Kaposi Sarcoma-associated Herpesvirus (KSHV) Lytic Replication

Open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus (KSHV) causes sustained activation of p90 ribosomal S6 kinase (RSK), which is crucial for KSHV lytic replication, but the exact functional roles remain to be determined. To characterize the biological consequence of persistent RSK activation by ORF45, we screened known cellular substrates of RSK. We found that ORF45 induced phosphorylation of eukaryotic translation initiation factor 4B (eIF4B), increased its assembly into translation initiation complex, and subsequently facilitated protein translation. The ORF45/RSK-mediated eIF4B phosphorylation was distinguishable from that caused by the canonical AKT/mammalian target of rapamycin/ribosomal S6 kinase and MEK/ERK/RSK pathways because it was resistant to both rapamycin (an mammalian target of rapamycin inhibitor) and U1026 (an MEK inhibitor). The rapamycin and U1026 doubly insensitive eIF4B phosphorylation was induced during KSHV reactivation but was abolished if either ORF45 or RSK1/2 were ablated by siRNA, a pattern that is correlated with reduced lytic gene expression as we observed previously. Ectopic expression of eIF4B but not its phosphorylation-deficient mutant form increased KSHV lytic gene expression and production of progeny viruses. Together, these results indicated that ORF45/RSK axis-induced eIF4B phosphorylation is involved in translational regulation and is required for optimal KSHV lytic replication.