Dephosphorylation Of P53 Research Articles

RNA demethylase FTO participates in malignant progression of gastric cancer by regulating SP1-AURKB-ATM pathway

Gastric cancer (GC) is the 5th most prevalent cancer and the 4th primary cancer-associated mortality globally. As the first identified m6A demethylase for removing RNA methylation modification, fat mass and obesity-associated protein (FTO) plays instrumental roles in cancer development. Therefore, we study the biological functions and oncogenic mechanisms of FTO in GC tumorigenesis and progression. In our study, FTO expression is obviously upregulated in GC tissues and cells. The upregulation of FTO is associated with advanced nerve invasion, tumor size, and LNM, as well as the poor prognosis in GC patients, and promoted GC cell viability, colony formation, migration and invasion. Mechanistically, FTO targeted specificity protein 1 and Aurora Kinase B, resulting in the phosphorylation of ataxia telangiectasia mutated and P38 and dephosphorylation of P53. In conclusion, the m6A demethylase FTO promotes GC tumorigenesis and progression by regulating the SP1-AURKB-ATM pathway, which may highlight the potential of FTO as a diagnostic biomarker for GC patients’ therapy response and prognosis.

The repression of oncoprotein SET by the tumor suppressor p53 reveals a p53-SET-PP2A feedback loop for cancer therapy.

The oncoprotein SET is frequently overexpressed in many types of tumors and contributes to malignant initiation and progression through multiple mechanisms, including the hijacking of the tumor suppressors p53 and PP2A. Targeting aberrant SET represents a promising strategy for cancer intervention. However, the mechanism by which endogenous SET is regulated in cancer cells remains largely unknown. Here, we identified the tumor suppressor p53 as a key regulator that transcriptionally repressed the expression of SET in both normal and cancer cells. In addition, p53 stimulated PP2A phosphatase activity via p53-mediated transcriptional repression of SET, whereby SET-mediated inhibition of PP2A was alleviated. Moreover, targeting the interaction between SET and PP2A catalytic subunit (PP2Ac) with FTY720 enhanced stress-induced p53 activation via PP2A-mediated dephosphorylation of p53 on threonine 55 (Thr55). Therefore, our findings uncovered a previously unknown p53-SET-PP2A regulatory feedback loop. To functionally potentiate this feedback loop, we designed a combined therapeutic strategy by simultaneously administrating a p53 activator and SET antagonist in cancer cells and observed a dramatic synergistic effect on tumor suppression. Our study reveals mechanistic insight into the regulation of the oncoprotein SET and raises a potential strategy for cancer therapy by stimulating the p53-SET-PP2A feedback loop.

Melatonin rescues swim stress induced gastric ulceration by inhibiting matrix metalloproteinase-3 via down-regulation of inflammatory signaling cascade

AimThis study investigated the link between forced swim induced acute gastric ulceration, inflammation and MMP-3 along with the possible mechanism of protective efficacy of melatonin. Main methodsWe distributed Balb/c mice into four different groups. Group 1 and 2 were given PBS gavage. Group 3 and 4 were given melatonin (60 mg/kg b.wt.) and omeprazole (25 mg/kg b.wt.), respectively, an hour prior to forced swim. Ulcer index, tissue histology, immunohistochemistry, protein carbonylation, lipid peroxidation, Myeloperoxidase, Zymography, Western blotting, reactive oxygen species (ROS), mitochondrial dehydrogenase, mitochondrial transmembrane potential and bioinformatical analysis were performed. Key findingsOur data revealed that gastric ulceration due to forced swim stress is responsible for overproduction of ROS, which may be a prime reason for mitochondrial dysfunction and induction of apoptosis via activation of Caspase-3. ROS is also responsible for p38 phosphorylation which in turn increases the activity of MMP-3 in ulcerated milieu, along with the oxidation of proteins, peroxidation of lipids and altered expression patterns of heat shock protein (HSP)-70. Melatonin is shown to reduce the inflammatory burden in gastric milieu and offers gastroprotection by binding to the active site of MMP-3; thereby inhibiting its activity, as suggested by in silico studies. Melatonin also inhibits the downregulation of HSP-70 and activates p38 dephosphorylation and thereby, it rescues gastric mucosal cells from stress-induced ulceration. SignificanceOur findings suggest that, melatonin imparts its gastroprotective effect by down-regulating the activation of MAPK-ERK pathway along with binding to the active site of MMP-3.

MKP-1 modulates ubiquitination/phosphorylation of TLR signaling.

Ubiquitination and phosphorylation are reversible posttranslational protein modifications regulating physiological and pathological processes. MAPK phosphatase (MKP)-1 regulates innate and adaptive immunity. The multifaceted roles of MKP-1 were attributed to dephosphorylation of p38 and JNK MAPKs. We show that the lack of MKP-1 modulates the landscape of ubiquitin ligases and deubiquitinase enzymes (DUBs). MKP-1-/- showed an aberrant regulation of several DUBs and increased expression of proteins and genes involved in IL-1/TLR signaling upstream of MAPK, including IL-1R1, IRAK1, TRAF6, phosphorylated TAK1, and an increased K63 polyubiquitination on TRAF6. Increased K63 polyubiquitination on TRAF6 was associated with an enhanced phosphorylated form of A20. Among abundant DUBs, ubiquitin-specific protease-13 (USP13), which cleaves polyubiquitin-chains on client proteins, was substantially enhanced in murine MKP-1-deficient BMDMs. An inhibitor of USP13 decreased the K63 polyubiquitination on TRAF6, TAK1 phosphorylation, IL-1β, and TNF-α induction in response to LPS in BMDMs. Our data show for the first time that MKP-1 modulates the ligase activity of TRAF6 through modulation of specific DUBs.

Steroid Receptor RNA Activator, a Long Noncoding RNA, Activates p38, Facilitates Epithelial-Mesenchymal Transformation, and Mediates Experimental Melanoma Metastasis

Melanoma metastasis signals dismal prognosis even with current checkpoint inhibitors. Long noncoding RNAs (lncRNAs) regulate dynamic metastasis in several cancers, including melanoma. We became interested in a lncRNA, steroid receptor RNA activator (SRA), because it is the first lncRNA also encoding a conserved protein, SRAP, and it regulates progression of prostate and breast cancers. We investigated how SRA mediates melanoma proliferation, migration, invasion, epithelial-mesenchymal transformation (EMT), and metastasis by RNA interference. The expression of SRAP was measured in melanoma tissue and in human and mouse B16 melanoma cells by immunofluorescence and PCR. The results showed that SRA knockdown decreased B16 cell and A375 cell proliferation and inhibited B16 cell migration significantly. Transwell analysis revealed that CCL21-mediated invasion was abolished in SRA-deficient B16 cells. In parallel, p38 dephosphorylation and reciprocal phosphorylation of B-Raf and mitogen-activated protein kinase kinase 1/2 were present in B16-SRA inhibited cells. The induction of EMT markers, β-catenin and N-cadherin, by CCL21 was reduced in B16-SRA inhibited cells, suggesting that SRA promotes the EMT process. Invivo experimental metastasis showed that B16-SRA inhibited cells formed significantly fewer tumor nodules in the lungs grossly and microscopically. In summary, our results showed that SRA expression is increased in melanoma tissue and that SRA mediates p38 activation, cell invasion, and proliferation and regulates EMT and distant metastasis.

Flexible Tethering of ASPP Proteins Facilitates PP-1c Catalysis

ASPP (apoptosis-stimulating proteins of p53) proteins bind PP-1c (protein phosphatase 1) and regulate p53 impacting cancer cell growth and apoptosis. Here we determine the crystal structure of the oncogenic ASPP protein, iASPP, bound to PP-1c. The structure reveals a 1:1 complex that relies on interactions of the iASPP SILK and RVxF motifs with PP-1c, plus interactions of the PP-1c PxxPxR motif with the iASPP SH3 domain. Small-angle X-ray scattering analyses suggest that the crystal structure undergoes slow interconversion with more extended conformations in solution. We show that iASPP, and the tumor suppressor ASPP2, enhance the catalytic activity of PP-1c against the small-molecule substrate, pNPP as well as p53. The combined results suggest that PxxPxR binding to iASPP SH3 domain is critical for complex formation, and that the modular ASPP-PP-1c interface provides dynamic flexibility that enables functional binding and dephosphorylation of p53 and other diverse protein substrates.

Titanium dioxide nanoparticles induce endothelial cell apoptosis via cell membrane oxidative damage and p38, PI3K/Akt, NF-κB signaling pathways modulation

BackgroundTitanium dioxide nanoparticles (TiO2 NPs) are widely used nanoparticles. Despite, several studies investigated the toxic effects of TiO2 NPs on HUVECs, the results are contradictory and the possible underlying mechanisms remain unclear. MethodsIn the present study, we conducted an in vitro study to re-evaluate the possible toxic effects of TiO2 NPs on HUVECs including cell viability, lipids peroxidation, intracellular signaling pathways and nitric oxide syntheses enzymes. ResultsOur results demonstrated that, TiO2 NPs were internalized to HUVECs and induce intracellular reactive oxygen species production and cell membrane oxidative damage at the higher concentration. TiO2 NPs induce IKKα/β and Akt phosphorylation and p38 dephosphorylation. After 24 h treatment, pro-inflammatory cytokines, adhesion molecules and chemokine upregulated significantly. TiO2 NPs have no significant effects on eNOS enzymatic activation and iNOS gene expression. At cellular level, apoptosis is the main process that occur in response to TiO2 NPs treatment. HUVECs pretreatment with N-acetyl-l-cysteine (NAC) ameliorate the toxic effects of TiO2 NPs that indicate the oxidative stress is essential in TiO2 NPs -induced toxicity. Total antioxidant capacity show a trend to increase in response to TiO2 NPs exposure. ConclusionsTaken together, this study confirmed the effects of TiO2 NPs on endothelial cells and proposed multiple underlying mechanisms including cell membrane oxidative damage and intracellular processes.

Lipoteichoic Acid Isolated from Lactobacillus plantarum Maintains Inflammatory Homeostasis through Regulation of Th1- and Th2-Induced Cytokines.

Lipoteichoic acid isolated from Lactobacillus plantarum K8 (pLTA) alleviates lipopolysaccharide (LPS)-induced excessive inflammation through inhibition of TNF-α and interleukin (IL)-6. In addition, pLTA increases the survival rate of mice in a septic shock model. In the current study, we have found that pLTA contributes to homeostasis through regulation of pro- and anti-inflammatory cytokine production. In detail, pLTA decreased the production of IL-10 by phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells stimulated with prostaglandin E2 (PGE-2) and LPS. However, TNF-α production which was inhibited by PGE-2+LPS increased by pLTA treatment. The regulatory effects of IL-10 and TNF-α induced by PGE-2 and LPS in PMA-differentiated THP-1 cells were mediated by pLTA, but not by other LTAs isolated from either Staphylococcus aureus (aLTA) or L. sakei (sLTA). Further studies revealed that pLTA-mediated IL-10 inhibition and TNF-α induction in PGE-2+LPS-stimulated PMA-differentiated THP-1 cells were mediated by dephosphorylation of p38 and phosphorylation of c-Jun N-terminal kinase (JNK), respectively. Reduction of pLTA-mediated IL-10 inhibited the metastasis of breast cancer cells (MDA-MB-231), which was induced by IL-10 or conditioned media prepared from PGE-2+LPS-stimulated PMA-differentiated THP-1 cells. Taken together, our data suggest that pLTA contributes to inflammatory homeostasis through induction of repressed pro-inflammatory cytokines as well as inhibition of excessive anti-inflammatory cytokines.

The Selective Acetamidine-Based iNOS Inhibitor CM544 Reduces Glioma Cell Proliferation by Enhancing PARP-1 Cleavage In Vitro.

Gliomas are the most aggressive adult primary brain tumors. Expression of inducible Nitric Oxide Synthase has been reported as a hallmark of chemoresistance in gliomas and several studies have reported that inhibition of inducible Nitric Oxide Synthase could be related to a decreased proliferation of glioma cells. The present work was to analyze the molecular effects of the acetamidine derivative compound 39 (formally CM544, N-(3-{[(1-iminioethyl)amino]methyl}benzyl) prolinamide dihydrochloride), a newly synthetized iNOS inhibitor, in a C6 rat glioma cell model. There is evidence of CM544 selective binding to the iNOS, an event that triggers the accumulation of ROS/RNS, the expression of Nrf-2 and the phosphorylation of MAPKs after 3 h of treatment. In the long run, CM544 leads to the dephosphorylation of p38 and to a massive cleavage of PARP-1, confirming the block of C6 rat glioma cell proliferation in the G1/S checkpoint and the occurrence of necrotic cell death.

Post-treatment de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma

BackgroundDasatinib (Sprycel) was developed as a tyrosine kinase inhibitor targeting Bcr-Abl and the family of Src kinases. Dasatinib is commonly used for the treatment of acute lymphoblastic and chronic myelogenous leukemia. Previous clinical studies in melanoma returned inconclusive results and suggested that patients respond highly heterogeneously to dasatinib as single agent or in combination with standard-of-care chemotherapeutic dacarbazine. Reliable biomarkers to predict dasatinib responsiveness in melanoma have not yet been developed.ResultsHere, we collected comprehensive in vitro data from experimentally well-controlled conditions to study the effect of dasatinib, alone and in combination with dacarbazine, on cell proliferation and cell survival. Sixteen treatment conditions, covering therapeutically relevant concentrations ranges of both drugs, were tested in 12 melanoma cell lines with diverse mutational backgrounds. Melanoma cell lines responded heterogeneously and, importantly, dasatinib and dacarbazine did not synergize in suppressing proliferation or inducing cell death. Since dasatinib is a promiscuous kinase inhibitor, possibly affecting multiple disease-relevant pathways, we also determined if basal phospho-protein amounts and treatment-induced changes in phospho-protein levels are indicative of dasatinib responsiveness. We found that treatment-induced de-phosphorylation of p53 correlates with dasatinib responsiveness in malignant melanoma.ConclusionsLoss of p53 phosphorylation might be an interesting candidate for a kinetic marker of dasatinib responsiveness in melanoma, pending more comprehensive validation in future studies.

Anthraquinone and naphthopyrone glycosides from Cassia obtusifolia seeds mediate hepatoprotection via Nrf2-mediated HO-1 activation and MAPK modulation.

Cassia obtusifolia L. seed is one of the most popular traditional Chinese medicine for mutagenicity, genotoxicity, hepatotoxicity, and acute inflammatory diseases. We evaluated the hepatoprotective activity of anthraquinone and naphthopyrone glycosides isolated from the butanol fraction of C. obtusifolia seeds and explored their effects on cell signaling pathways. Continuous chromatographic separation led to the isolation of 1-desmethylaurantio-obtusin 2-O-β-D-glucopyranoside (1), rubrofusarin 6-O-β-D-apiofuranosyl-(1 → 6)-O-β-D-glucopyranoside (2) and rubrofusarin 6-O-β-gentiobioside (3). All glycosides were non-toxic at concentrations up to 80µM. The increased intracellular reactive oxygen species (ROS) and decreased glutathione levels observed after tert-butylhydroperoxide (t-BHP) intoxication were ameliorated by all three glycosides, with compound 3 being the most active. Pretreatment with the three glycosides increased nuclear factor erythroid-2-related factor 2 (Nrf2)-mediated heme oxidase-1 (HO-1) expression. All the glycosides enhanced the phosphorylation of c-JunN-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK), and the dephosphorylation of p38. The protective effects of the anthraquinone and naphthopyrone glycosides against t-BHP-induced oxidative damage in human liver-derived HepG2 cells were due to the prevention of ROS generation and up-regulated activity of HO-1 via Nrf2 activation and modulation of the JNK/ERK/MAPK signaling pathway. The data indicate the potential of these compounds as hepatoprotective agents in pharmaceuticals and/or nutraceuticals.

Abstract 2152: Protein phosphatase Mg2+ and Mn2+ dependent 1F promotes smoking-induced breast cancer by inactivating phosphorylated p53-induced signals

Abstract Previously we demonstrated that the activation of α9-nicotinic acetylcholine receptor (α9-nAchR) signaling by smoking promotes breast cancer formation. To investigate the downstream signaling molecules involved in α9-nAChR-induced breast tumorigenesis, we used real-time polymerase chain reactions and Western blotting to assess expression of protein phosphatase Mg2+/Mn2+ dependent 1F (PPM1F), a Ser/Thr protein phosphatase, in human breast cancer samples (n=167). Additionally, stable PPM1F-knockdown and overexpressing cell lines were established to evaluate the function of PPM1F. The phosphatase activity of PPM1F in nicotine-treated cells was assessed through Western blotting, confocal microscopy, and fluorescence resonance energy transfer. Higher levels of PPM1F were detected in the breast cancer tissues of heavy smokers (n=7, 12.8-fold) greater than of non-smokers (n= 28, 6.3-fold) (**p=0.01). In vitro, nicotine induced PPM1F expression, whereas α9-nAChR knockdown reduced the protein expression of PPM1F. A series of biochemical experiments using nicotine-treated cells suggested that the dephosphorylation of p53 (Ser-20) and BAX (Ser-184) by PPM1F is a critical posttranslational modification, as observed in breast cancer patients who were heavy smokers. These observations indicate that PPM1F may be a mediator downstream of α9-nAChR that activates smoking-induced carcinogenic signals. Thus, PPM1F expression could be used for prognostic diagnosis or inhibited for cancer prevention and therapy. Citation Format: Li-Ching Chen. Protein phosphatase Mg2+ and Mn2+ dependent 1F promotes smoking-induced breast cancer by inactivating phosphorylated p53-induced signals [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 2152. doi:10.1158/1538-7445.AM2017-2152

MAP kinase p38 is a novel target of CacyBP/SIP phosphatase

Mitogen-activated protein (MAP) kinases are important players in cellular signaling pathways. Recently, it has been shown that CacyBP/SIP serves as a phosphatase for one of the MAP kinases, ERK1/2. Through dephosphorylation of this kinase CacyBP/SIP modulates the transcriptional activity of Elk-1 and the activity of the CREB-BDNF pathway. In this work, using NB2a cell lysate and recombinant proteins, we show that CacyBP/SIP binds and dephosphorylates another member of the MAP kinase family, p38. Analysis of recombinant full-length CacyBP/SIP and its three major domains, N-terminal, middle CS and C-terminal SGS, indicates that the middle CS domain is responsible for p38 dephosphorylation. Moreover, we show that CacyBP/SIP might be implicated in response to oxidative stress. Dephosphorylation of phospho-p38 by CacyBP/SIP in NB2a cells treated with hydrogen peroxide is much more effective than in control ones. In conclusion, involvement of CacyBP/SIP in the regulation of p38 kinase activity, in addition to that of ERK1/2, might point to the function of CacyBP/SIP in pro-survival and pro-apoptotic pathways.

Protein phosphatase Mg2+/Mn2+ dependent 1F promotes smoking-induced breast cancer by inactivating phosphorylated-p53-induced signals.

We previously demonstrated that the activation of α9-nicotinic acetylcholine receptor (α9-nAchR) signaling by smoking promotes breast cancer formation. To investigate the downstream signaling molecules involved in α9-nAChR-induced breast tumorigenesis, we used real-time polymerase chain reactions and Western blotting to assess expression of protein phosphatase Mg2+/Mn2+ dependent 1F (PPM1F), a Ser/Thr protein phosphatase, in human breast cancer samples (n=167). Additionally, stable PPM1F-knockdown and -overexpressing cell lines were established to evaluate the function of PPM1F. The phosphatase activity of PPM1F in nicotine-treated cells was assessed through Western blotting, confocal microscopy, and fluorescence resonance energy transfer. Higher levels of PPM1F were detected in the breast cancer tissues of heavy smokers (n=7, 12.8-fold) greater than of non-smokers (n= 28, 6.3-fold) (**p=0.01). In vitro, nicotine induced PPM1F expression, whereas α9-nAChR knockdown reduced the protein expression of PPM1F. A series of biochemical experiments using nicotine-treated cells suggested that the dephosphorylation of p53 (Ser-20) and BAX (Ser-184) by PPM1F is a critical posttranslational modification, as observed in breast cancer patients who were heavy smokers. These observations indicate that PPM1F may be a mediator downstream of α9-nAChR that activates smoking-induced carcinogenic signals. Thus, PPM1F expression could be used for prognostic diagnosis or inhibited for cancer prevention and therapy.

Abstract 3685: Redox regulation of p53 stability is a function of inhibition of PP2A-mediated dephosphorylation at threonine 55

Abstract Protein Phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase that has critical roles in numerous cellular pathways. The role of PP2A as an essential tumor suppressor in numerous death signaling pathways is well documented. In this regard, recent work from our laboratory has highlighted a novel mechanism of apoptosis inhibition via post-translational redox-modification of PP2A. Increase in intracellular superoxide, either by pharmacological inhibition or siRNA-mediated silencing of superoxide dismutase 1 (SOD1), results in the dissociation of PP2A-AC catalytic core from its B regulatory subunit. Dissociation of the PP2A heterotrimer is a result of the selective nitration [by peroxynitrite derived from the reaction of superoxide with nitric oxide] of a conserved tyrosine residue on B56δ which we identified as Y289 (B56δY289). Importantly, we showed in lymphoma cells that PP2A-AC catalytic core is typically targeted to Bcl-2 by its B56δ regulatory subunit. When O2- is elevated, it signals for the sustained phosphorylation of Bcl-2 at S70 by inducing the release of PP2A-AC catalytic core from B56δ-bound Bcl-2. This results in the sustained phospho-activation of anti-apoptotic Bcl-2 and resistance to apoptotic stimuli. Notably, Bcl-2 is not the only substrate of PP2A, nor is B56δ the only PP2A regulatory subunit susceptible to redox modification. Thus, implications from redox-modification of PP2A may extend beyond the phospho-activation of Bcl-2. In the context of carcinogenesis, another notable substrate of PP2A is the tumor suppressor, p53. p53 stability is negatively influenced by its phosphorylation status at threonine-55 (T55). Phosphorylation of T55, on the other hand, is countered by PP2A harboring the B56γ3 subunit. Here we provide evidence that the B56γ3 sub-unit of PP2A complexes with p53 and this complex is sustained upon an increase in intracellular superoxide. Importantly, redox-mediated inhibition of B56γ3-containing PP2A (PP2AB56γ3) prevents the dephosphorylation of p53 at T55, thereby negatively impacting the stability of p53 in tumor cells with elevated superoxide level. This, we hypothesize, may be a novel death inhibitory mechanism that renders tumor cells resistant to conventional DNA-damaging therapeutic agents that rely heavily on the activation of p53 for death induction. Citation Format: Stephen Chong, Shazib Pervaiz. Redox regulation of p53 stability is a function of inhibition of PP2A-mediated dephosphorylation at threonine 55. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3685.

Mitogen-Dependent Regulation of DUSP1 Governs ERK and p38 Signaling During Early 3T3-L1 Adipocyte Differentiation

Knowledge concerning mechanisms that control proliferation and differentiation of preadipocytes is essential to our understanding of adipocyte hyperplasia and the development of obesity. Evidence has shown that temporal regulation of mitogen-activated protein kinase (MAPK) phosphorylation and dephosphorylation is critical for coupling extracellular stimuli to cellular growth and differentiation. Using differentiating 3T3-L1 preadipocytes as a model of adipocyte hyperplasia, we examined a role for dual-specificity phosphatase 1 (DUSP1) on the timely modulation of MAPK signaling during states of growth arrest, proliferation, and differentiation. Using real-time reverse transcription PCR (qRT-PCR), we report that DUSP1 is induced during early preadipocyte proliferation concomitant with ERK and p38 dephosphorylation. As deactivation of ERK and p38 is essential for the progression of adipocyte differentiation, we further showed that de novo mRNA synthesis was required for ERK and p38 dephosphorylation, suggesting a role for "inducible" phosphatases in regulating MAPK signaling. Pharmacological and genetic inhibition of DUSP1 markedly increased ERK and p38 phosphorylation during early adipocyte differentiation. Based on these findings, we postulated that loss of DUSP1 would block adipocyte hyperplasia. However, genetic loss of DUSP1 was not sufficient to prevent preadipocyte proliferation or differentiation, suggesting a role for other phosphatases in the regulation of adipogenesis. In support of this, qRT-PCR identified several MAPK-specific DUSPs induced during early (DUSP2, -4, -5, & -6), mid (DUSP4 & -16) and late (DUSP9) stages of adipocyte differentiation. Collectively, these data suggest an important role for DUSPs in regulating MAPK dephosphorylation, with an emphasis on DUSP1, during early adipogenesis.

PKC δ and βII regulate angiotensin II-mediated fibrosis through p38: a mechanism of RV fibrosis in pulmonary hypertension.

Pulmonary hypertension (PH) eventually leads to right ventricular (RV) fibrosis and dysfunction that is associated with increased morbidity and mortality. Although angiotensin II plays an important role in RV remodeling associated with hypoxic PH, the molecular mechanisms underlying RV fibrosis in PH largely remain unresolved. We hypothesized that PKC-p38 signaling is involved in RV collagen accumulation in PH and in response to angiotensin II stimulation. Adult male Sprague-Dawley rats were exposed to 3 wk of normoxia or hypoxia (10% FiO2 ) as a model of PH. Hypoxic rats developed RV hypertrophy and fibrosis associated with an increase in PKC βII and δ protein expression and p38 dephosphorylation in freshly isolated RV cardiac fibroblasts. Further mechanistic studies were performed in cultured primary cardiac fibroblasts stimulated with angiotensin II, a key activator of ventricular fibrosis in PH. Angiotensin II induced a reduction in p38 phosphorylation that was attenuated following chemical inhibition of PKC βII and δ. Molecular and chemical inhibition of PKC βII and δ abrogated angiotensin II-induced cardiac fibroblast proliferation and collagen deposition in vitro. The effects of PKC inhibition on proliferation and fibrosis were reversed by chemical inhibition of p38. Conversely, constitutive activation of p38 attenuated angiotensin II-induced increase of cardiac fibroblast proliferation and collagen accumulation. PKC βII- and δ-dependent inactivation of p38 regulates cardiac fibroblast proliferation and collagen deposition in response to angiotensin II, which suggests that the PKC-p38 signaling in cardiac fibroblasts may be involved and important in the pathophysiology of RV fibrosis in PH.

Insulin-like growth factors inhibit dendritic cell-mediated anti-tumor immunity through regulating ERK1/2 phosphorylation and p38 dephosphorylation

Insulin-like growth factors (IGFs) can promote tumorigenesis via inhibiting the apoptosis of cancer cells. The relationship between IGFs and dendritic cell (DC)-mediated immunity were investigated. Advanced-stage ovarian carcinoma patients were first evaluated to show higher IGF-1 and IGF-2 concentrations in their ascites than early-stage patients. IGFs could suppress DCs' maturation, antigen presenting abilities, and the ability to activate antigen-specific CD8+ T cell. IGF-treated DCs also secreted higher concentrations of IL-10 and TNF-α. IGF-treated DCs showed decreased ERK1/2 phosphorylation and reduced p38 dephosphorylation. The percentages of matured DCs in the ascites were significantly lower in the IGF-1 or IGF-2 highly-expressing WF-3 tumor-bearing mice. The IGF1R inhibitor – NVP-AEW541, could block the effects of IGFs to rescue DCs' maturation and to restore DC-mediated antigen-specific immunity through enhancing ERK1/2 phosphorylation and p38 dephosphorylation. IGFs can inhibit DC-mediated anti-tumor immunity through suppressing maturation and function and the IGF1R inhibitor could restore the DC-mediated anti-tumor immunity. Blockade of IGFs could be a potential strategy for cancer immunotherapy.

Abstract 1802: Small molecule compound NCI-8 induces mutant p53 degradation via inhibition of the MDM2-Hsp90 axis

Abstract The tumor suppressor p53 prevents cancer development via regulating cell cycle arrest, cell death, or repair processes. Over 50% of human cancers harbor mutant p53. Mutations in p53 not only abrogate its tumor suppressor function, but also endow mutant p53 with a gain-of-function (GOF), creating a proto-oncogene that contributes to tumorigenesis, tumor progression and chemotherapeutic resistance. Thus, targeting mutant p53 to restore wild-type p53 pathway signaling is an attractive strategy for cancer therapy. We previously reported a novel small molecular compound, NCI-8, which not only restores wild-type p53 signaling, but also depletes mutant p53 protein (Zhang S, Zhou L, Hong B, et al. Cancer Res 2013;73(8 Suppl):Abstract 2171). In this study, we further elucidate the mechanism of the small molecular compound NCI-8-mediated degradation of mutant p53 protein. We found that the small molecule NCI-8 induces mutant p53 protein degradation via the MDM2-ubiquitin-proteasome pathway. NCI-8 exposure leads to phosphorylation of p53 at Thr55. Dephosphorylation of p53 at Thr55 rescues mutant p53 protein from degradation in cells treated with Apigenin, suggesting that phosphorylation at Thr55 is required for mutant p53 protein degradation induced by NCI-8. Hsp90 and hsp70 are two molecular chaperones that stabilize mutant p53 protein by affecting the MDM2 mediated turnover of mutant p53, therefore, we investigated the effect of NCI-8 on the MDM2-hsp chaperone axis. We found that less HSP90 but more MDM2 were bound to mutant p53 in cells treated with NCI-8, suggesting that NCI-8 induces mutant p53 protein degradation through disturbing the MDM2-hsp chaperone axis. Correlated to the degradation of mutant p53 protein, the p73 pathway was activated in response to NCI-8 treatment specifically in mutant p53-expressing colorectal cancer cells. Our results provide a possible mechanism of NCI-8-mediated p73-dependent restoration of the p53 pathway via mutant p53 protein degradation. Citation Format: Shengliang Zhang, Lanlan Zhou, Christina Leah B. Kline, David T. Dicker, Wafik S. El-Deiry. Small molecule compound NCI-8 induces mutant p53 degradation via inhibition of the MDM2-Hsp90 axis. [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 1802. doi:10.1158/1538-7445.AM2014-1802

2′-Hydroxy C16-Ceramide Induces Apoptosis-Associated Proteomic Changes in C6 Glioma Cells

Ceramide is a bioactive sphingolipid involved in regulation of numerous cell signaling pathways. Evidence is accumulating that differences in ceramide structure, such as N-acyl chain length and desaturation of sphingoid base, determine the biological activities of ceramide. Using synthetic (R)-2'-hydroxy-C16-ceramide, which is the naturally occurring stereoisomer, we demonstrate that this ceramide has more potent pro-apoptotic activity compared to its (S) isomer or non-hydroxylated C16-ceramide. Upon exposure to (R)-2'-hydroxy-ceramide, C6 glioma cells rapidly underwent apoptosis as indicated by caspase-3 activation, PARP cleavage, chromatin condensation, and annexin V stain. A 2D gel proteomics analysis identified 28 proteins whose levels were altered during the initial 3 h of exposure. Using the list of 28 proteins, we performed a software-assisted pathway analysis to identify possible signaling events that would result in the observed changes. The result indicated that Akt and MAP kinase pathways are among the possible pathways regulated by (R)-2'-hydroxy-ceramide. Experimental validation confirmed that 2'-hydroxy-ceramide significantly altered phosphorylation status of Akt and its downstream effector GSK3β, as well as p38, ERK1/2, and JNK1/2 MAP kinases. Unexpectedly, robust phosphorylation of Akt was observed within 1 h of exposure to 2'-hydroxy-ceramide, followed by dephosphorylation. Phosphorylation status of MAPKs showed a complex pattern, in which rapid phosphorylation of ERK1/2 was followed by dephosphorylation of p38 and ERK1/2 and phosphorylation of the 46 kDa isoform of JNK1/2. These data indicate that (R)-2'-hydroxy-ceramide regulates multiple signaling pathways by affecting protein kinases and phosphatases with kinetics distinct from that of the extensively studied non-hydroxy-ceramide or its unnatural stereoisomer.