Phosphorylation Status Of ERK Research Articles

Secreted protein NFA47630 from Nocardia farcinica IFM10152 induces immunoprotective effects in mice

PurposeNocardia is emerging as a common and easily neglected cause of both healthcare- and occupation-associated infections worldwide, however, human vaccines for Nocardia prevention are not yet available. In this study, we aimed to evaluate the immunoprotective effect of the NFA47630 protein, a secreted protein abundant in the N. farcinica IFM10152 supernatant.MethodsConservation and characteristics of nfa47630 were analyzed by PCR and bioinformatics. Then recombinant NFA47630 protein was cloned, expressed and purified for further antigenicity analysis. Subsequently, the ability to activate innate immunity was evaluated by examining the phosphorylation status of the MAPK signaling pathway and cytokine levels. Finally, the protective effect was evaluated on rNFA47630-immunized mice.Resultsnfa47630 was conserved in N. farcinica strains with good antigenicity. The rNFA47630 protein was expressed under the optimal conditions of 0.2 mM IPTG, 28 °C, and it can be recognized by anti-N. farcinica and anti-N. cyriacigeorgica sera, but not anti-N. asteroids, anti-N. brasiliensis, anti-N. nova and anti-Mycobacterium bovis sera. It can upregulate the phosphorylation status of ERK, JNK, P38 and the cytokine levels of TNF-α, IL-10, IL-12, and IFN-γ. In addition, mice immunized with rNFA47630 protein exhibited higher antibody titers, greater bacterial clearance ability, milder organ infection, and higher survival rates than PBS-immunized mice.ConclusionsOur data demonstrate that NFA47630 is a potential vaccine candidate for defending against N. farcinica infection.

FAM83G-based Peptide Induces Apoptosis on Cultured Liver Cancer Cell.

Previously, AF-956, which contains S356 of FAM83G and an N-terminal antenna peptide for entry into colon cancer cells, is markedly antiproliferative compared to a control peptide (AF-859), which lacks the N-terminal antenna peptide, by inducing apoptosis via the inhibition of HSP27 phosphorylation at residues S15 and S82. Because FAM83G-derived peptides are promising lead compounds for colon cancer treatment, we reanalyzed the effect of AG-066, which contains S356 of FAM83G and an N-terminal antenna peptide for entry into the liver cancer cells. HepG2 liver cancer cells were incubated with either AF-859 or AG-066 at a concentration of 54 μM at 37 °C for 24, 48, and 72 h. The effects of AF-859 and AG-066 on the cultured HepG2 cells were estimated using an inverted light microscope. Furthermore, the DNA ladder method and the dead cell assay were performed by applying Live/Dead Cell Staining Kit II. Erk phosphorylation was estimated by western blotting. Treatment with AG-066 markedly reduced HepG2 viable cell counts compared to the AF- 859-treated HepG2 cells, as evident from the significantly increased number of dead cells in the culture medium. Additionally, AG-066 treatment increased cellular DNA laddering. We found no difference in Erk phosphorylation status between the AG-066- and AF-859-treated groups. This study illustrated that the peptide with a structure based on FAM83G functions as a spontaneous apoptosis inducer for liver cancer cells. Hence, it is a promising lead compound for the treatment of liver cancer.

SUN-213 The Role of Filamin A (FLNA) in the Regulation of Insulin-Like Growth Factor System in Adrenocortical Carcinomas

Adrenocortical carcinomas (ACCs) are rare endocrine tumors with poor prognosis. They overexpress insulin-like growth factor 2 (IGF2), that drives a proliferative autocrine loop by binding to IGF1R and IR, with molecular dynamics still poorly identified. Although promising, IGF1R/IR-targeted therapies have demonstrated a limited efficacy in clinical trials in ACC patients. The cytoskeleton actin-binding protein filamin A (FLNA) was shown to impair IR and IGF1R signalling in melanoma and neural progenitor cells, respectively. The aims of this study were to test in ACC cells: 1) FLNA involvement in regulating IGF1R and IR expression and signalling; 2) FLNA role in modulating responsiveness to IGF1R and IGFR/IR inhibitors; 3) FLNA expression in ACCs and correlation with IGF system. In ACC cells we found by immunoprecipitation that both IGF1R and IR interacted with FLNA in basal condition, with an increased or decreased FLNA recruitment to IGF1R and to IR, respectively, after IGF2 stimulation. Genetic silencing of FLNA in ACC cell lines H295R and SW13 induced a significant increase of IGF1R expression (1.4- and 2.3-fold, respectively) and a reduction of IR (-85.5±9.1%, p<0.001 and -32±19.1%, respectively, p<0.05), with a downstream effect of increased cell proliferation (130±13.4%, p<0.01 in H295R and 144.3±23.3%, p<0.01 in SW13 cells) accompanied by an enhanced ERK phosphorylation. Accordingly, in ACC primary cultured cells FLNA silencing increased IGF1R levels (2.9-fold) and enhanced IGF2 effects on ERK phosphorylation by 2.2-fold. In addition, FLNA knockdown potentiated the antiproliferative effects of IGF1R/IR inhibitor Linsitinib and IGF1R specific inhibitor NVP-ADW742 in H295R cells and SW13. This key role of FLNA was even more evident in A7/M2 melanoma cell model, since IGF2 and Linsitinib exerted the expected effects on ERK phosphorylation in M2 cells, lacking FLNA, but not in FLNA-expressing counterpart (A7 cells). Finally, western blot analysis showed significantly lower, although variable, FLNA expression in ACCs (n=10) than in adrenocortical adenomas (ACAs) (n=10) (FLNA/GAPDH ratio 0.37±0.38 and 0.90±0.63, respectively, p<0.05). Interestingly, FLNA/IGF1R ratio inversely correlated with ERK phosphorylation status in ACCs (p<0.05) but not in ACA. In conclusion, we demonstrated that low levels of FLNA enhance both IGF2 proliferative effects and IGF1R/IR inhibitors efficacy in ACC cells, suggesting FLNA as a new factor possibly influencing tumor clinical behavior and the response to the therapy with IGF1R/IR-targeted drugs.

Evaluating the Specificity and Potency of Activators and Inhibitors of the cAMP Sensors PKA, Epac2/Rapgef4, and NCS/Rapgef2 Using Cell‐Based Assays for Activation of CREB, p38 MAPK, and ERK

Cyclic AMP elevation is well known to cause dividing neuroendocrine (e.g. PC12) cells to differentiate through a process that includes growth arrest, neurite extension, and induction of neuronal genes. Using the PC12 subclone NS‐1, we have previously shown that downstream of cAMP, signaling for differentiation is mediated by three insulated signaling pathways, initiated by cAMP activation of Epac2/Rapgef4, NCS/Rapgef2, and PKA, which regulate the activation of p38 MAP kinase, ERK, and CREB, respectively. These, in turn, are individually necessary for growth arrest (Epac2/Rapgef4), neurite extension (NCS/Rapgef2), and neuron‐specific gene expression (PKA) (Emery et al., 2014, J Biol Chem, 289:10126–39). Specific inhibitors and activators of PKA, Epac, and NCS/Rapgef2 would be useful to further explore the unique physiological functions of these three cAMP sensors in the CNS. Several compounds that show differential activity with respect to PKA and Epac exist, however, the selectivity of these agents across all three neuroendocrine cAMP sensors is not yet known. The goal of the work reported here was to identify cAMP analogs that provide specific activation and inhibition of each of these cAMP sensors. To this end, we have used a medium‐throughput assay platform that allows for quantitative measurement of the phosphorylation status of ERK, CREB, and p38 MAPK, which serve as read‐outs for activation of NCS/Rapgef2, PKA, and Epac2, respectively. 8‐CPT‐cAMP was found to be a full agonist with a similar potency at each sensor, while 8‐CPT‐2′‐O‐Me‐cAMP was highly specific for Epac relative to PKA (as reported by Enserink et al., 2002, Nat Cell Biol, 4:901–6) and also NCS/Rapgef2. We examined previously characterized PKA site‐specific agonists, i.e. compounds that stimulate PKA holoenzyme activity only when applied in combination, to determine if any of these provide SAR entree to further development of Epac or NCS/Rapgef2‐specific agonists. Our preliminary evidence suggests that several B site‐specific compounds, when administered singly, show significant activity at NCS/Rapgef2 without stimulating either Epac or the PKA holoenzyme. None of the cyclic nucleotide antagonists (Rp diastereomeric cAMP analogs) that we tested effectively inhibited cAMP‐dependent NCS/Rapgef2 signaling. Based on our previous work indicating that the nucleoside adenylate cyclase inhibitor SQ22,536 inhibits NCS/Rapgef2 (Emery et al., 2013, Mol Pharmacol, 83:95–105), along with new insights into structure‐activity relationships for NCS/Rapgef2 gleaned from our survey of B‐site‐specific activators, we propose that the development of a cell‐permeant, highly specific inhibitor of NCS/Rapgef2 is feasible.Support or Funding InformationSupported by NIMH‐IRP project ZO1‐MH002386

MEK inhibition induces apoptosis in osteosarcoma cells with constitutive ERK1/2 phosphorylation.

Conventional high-grade osteosarcoma is the most common primary bone cancer with relatively high incidence in young people. Recurrent and metastatic tumors are difficult to treat. We performed a kinase inhibitor screen in two osteosarcoma cell lines, which identified MEK1/2 inhibitors. These inhibitors were further validated in a panel of six osteosarcoma cell lines. Western blot analysis was performed to assess ERK activity and efficacy of MEK inhibition. A 3D culture system was used to validate results from 2D monolayer cultures. Gene expression analysis was performed to identify differentially expressed gene signatures in sensitive and resistant cell lines. Activation of the AKT signaling network was explored using Western blot and pharmacological inhibition. In the screen, Trametinib, AZD8330 and TAK-733 decreased cell viability by more than 50%. Validation in six osteosarcoma cell lines identified three cell lines as resistant and three as sensitive to the inhibitors. Western blot analysis of ERK activity revealed that sensitive lines had high constitutive ERK activity. Treatment with the three MEK inhibitors in a 3D culture system validated efficacy in inhibition of osteosarcoma viability. MEK1/2 inhibition represents a candidate treatment strategy for osteosarcomas displaying high MEK activity as determined by ERK phosphorylation status.

Osteoprotegerin activates osteosarcoma cells that co-express RANK and RANKL

BackgroundOsteosarcoma (OS) is an aggressive and often fatal cancer that afflicts over 1000 humans and 10,000 dogs per year in the United States. Recent evidence suggests deregulation in the signaling triad, receptor activator of nuclear factor kappa B (RANK), its activating ligand (RANKL), and the RANKL inhibitor, osteoprotegerin (OPG) plays a key role in the pathogenesis of OS. This study investigated the expression of RANK and RANKL in osteosarcoma tumors and cell lines and describes an activating effect of OPG on OS cells in vitro. ResultsCanine OS tumors and cell lines co-express mRNA for both RANK and RANKL. Expression of these proteins in OS cell lines was confirmed by Western blot and immunofluorescence microscopy. Expression of the soluble form of RANKL was not detected in media from OS cells. OPG-Fc incubation increased the phosphorylation status of ERK, AKT and the p65 subunit of nuclear factor kappa B (NFκB) and induced NFκB translocation from the cytoplasm to the nucleus in canine OS cells. OPG increased proliferation in both canine and human derived OS cell lines. ConclusionRANKL is produced by OS tumors and cell lines that also express RANK. This data provides preliminary evidence for a potential autocrine and or paracrine activation pathway in canine OS. An activating effect of exogenous OPG on signal transduction proteins, NFκB and proliferation in OS is described. These data provide new information concerning aberrant signaling in OS and could be important to those considering OPG as a therapeutic agent for osteosarcoma.

Asporin participates in gastric cancer cell growth and migration by influencing EGF receptor signaling.

Asporin (ASPN), a novel member of the small leucine-rich proteoglycan (SLRP) family, serves as a key component of the tumor stroma and has been reported to be abnormally expressed in certain types of tumors. Specifically, the proteoglycan was proven to activate the coordinated invasion of scirrhous gastric cancer and cancer-associated fibroblasts. However, the role of ASPN in cancer cell growth and metastasis has not yet been addressed. In the present study, we aimed to evaluate the tumoricidal benefits of ASPN on tumorigenesis and progression of gastric cancer. Firstly, it was demonstrated that ASPN was overexpressed in gastric carcinoma tissues when compared to the corresponding non‑cancerous tissues, and it had varied levels of expression in gastric cancer epithelial cell lines. Additionally, we assessed the effects of transient siRNA‑mediated ASPN knockdown on gastric cancer cells. ASPN silencing inhibited proliferation and suppressed the migration of immortalized neoplastic epithelial cells. Furthermore, at the molecular level, we found that downregulation of ASPN blocked the anti-apoptotic molecule Bcl-2, increased the expression of pro-apoptotic molecule Bad, reduced the expression of migration-related proteins CD44 and matrix metalloproteinase (MMP)-2, and abrogated the activation of the phosphorylation status of ERK and epidermal growth factor (EGF) and its receptor (EGFR). Collectively, our findings indicate that ASPN is upregulated and plays an oncogenic role in gastric cancer progression and metastasis by influencing the EGFR signaling pathway.

Heterogeneous expression and biological function of ubiquitin carboxy-terminal hydrolase-L1 in osteosarcoma

Ubiquitin carboxyl terminal hydrolase 1 (UCHL1), a member of the UCH class of DUBs, has been reported as either an oncogene or a tumor suppressor. However, the molecular mechanism underlying the biological function of UCHL1 in osteosarcoma is still unclear. This study was aimed at elucidating the roles of UCHL1 in regulating the biological behavior of osteosarcoma cells. In this study, we found that UCHL1 was elevated in osteosarcoma compared with normal bone tissue. Moreover, UCHL1 expression level was correlated with tumor maximum diameter, high rate of lung metastases and short survival time. Then, we found that knockdown of UCHL1 in osteosarcoma cell MG63 inhibited cell proliferation and significantly increased cell population in the G1 phase. Several cyclins promoting G1/S phase transition were reduced after UCHL1 knockdown, including cell cycle regulator cyclin D1, cyclin E1 and CDK6. Moreover, inhibition of UCHL1 in MG63 cells dramatically induced cell apoptosis. We also found that down-regulation of UCHL1 in MG63 significantly inhibited cell invasion. Then, we found that there was a positive correlation between UCHL1 expression level and the Akt and ERK phosphorylation status. Finally, in vivo data showed that knockdown of UCHL1 inhibited osteosarcoma growth in nude mice. These results indicate that UCHL1 could work as an oncogene and may serve as a promising therapeutic strategy for osteosarcoma.

Oxygen- and temperature-dependent expression of survival protein kinases in crucian carp (Carassius carassius) heart and brain.

Living without oxygen is limited to very few vertebrates, one species being the fresh water fish crucian carp (Carassius carassius), which can survive months of anoxia at low temperatures. Mammalian heart and brain are particularly intolerant to oxygen deprivation, yet these organs can be conditioned to display increased resistance, possibly due to activation of several protein kinases. We hypothesized increased phosphorylation status of these kinases in hypoxic and anoxic crucian carp heart and brain. Moreover, we wanted to investigate whether the kinases showing the strongest phosphorylation during hypoxia/anoxia, ERK 1/2, p38-MAPK, JNK, PKCε, and PKCδ, also had increased expression and phosphorylation at cold temperatures, to better cope with the anoxic periods during winter. We found small differences in the phosphorylation status of ERK 1/2, p38-MAPK, JNK, PKCε, and PKCδ during 10 days of severe hypoxia in both heart and brain (0.3 mg O₂/l) and varying responses to reoxygenation. In contrast, 7 days of anoxia (<0.01 mg O₂/l) markedly increased phosphorylation of ERK 1/2, p38-MAPK, JNK in the heart, and p38-MAPK and PKCε in the brain. Similarly, varying acclimation temperature between 4, 10 and 20°C induced large changes in phosphorylation status. Total protein expression in heart and brain neither changed during different oxygen regimes nor with different acclimation temperatures, except for ERK 1/2, which slightly decreased in the heart at 4°C compared with 20°C. A phylogenetic analysis confirmed that these protein kinases are evolutionarily conserved across a wide range of vertebrate species. Our findings indicate important roles of several protein kinases during oxygen deprivation.

Crk-like adapter protein is required for TGF-β-induced AKT and ERK-signaling pathway in epithelial ovarian carcinomas.

Crk-like adapter protein (CrkL) was identified as an important biomarker in epithelial ovarian carcinomas. At the same time, the transforming growth factor β (TGF-β) pathway plays a key role in oncogenesis of advanced cancers. However, more detailed regulation mechanisms are still unclear. So we investigated the role of CrkL in TGF-β pathways in epithelial ovarian carcinomas. The small interfering RNA (siRNA) was used to suppress CrkL in serous papillary cystic adenocarcinoma (SKOV-3) cell line, TGF-β downstream signal molecules AKT and ERK phosphorylation status was tested using the Western blot. Wound healing assay was used to evaluate the capacity of cell migration and proliferation. In this study, CrkL can be activated by TGF-β1 treatment and inhibited by siCrkL. CrkL knockdown markedly suppressed the phosphorylated ERK (p-ERK) as well as the phosphorylated AKT (p-AKT) (p < 0.001) compared with control or TGF-β1 alone. On the other hand, CrkL knockdown could significantly affect SKOV3 wound closure (p < 0.001) using wound healing assay compared to siControl. In conclusion, CrkL protein is required for TGF-β signal pathways through AKT and ERK pathway, which can mediate the development of epithelial ovarian carcinomas. CrkL plays a key regulation role in TGF-β signaling pathway of epithelial ovarian carcinomas, and this study suggested CrkL could be suggested as an efficient target in ovarian cancer treatment.

Abstract 5628: Overcoming cetuximab resistance in HNSCC: the role of AURKB and DUSP6.

Abstract Introduction: As the epidermal growth factor receptor (EGFR) is expressed in up to 90% of all HNSCC tumors and initiates important signaling pathways in carcinogenesis, cetuximab, an EGFR targeting agent, is a promising therapeutic agent. However, many tumors remain non-responsive. Therefore, unraveling the underlying mechanism of resistance is of major importance. Previously, we performed gene expression studies of cetuximab sensitive (LICR-HN2 and SC263) and cetuximab resistant (LICR-HN1 and Cal27) cell lines. These results indicated an upregulation of Aurora kinase B (AURKB) in LICR-HN1 cells and a downregulation of dual specificity phosphatase 6 (DUSP6) in Cal27 cells. Methods: Growth inhibitory experiments for barasertib, an AURKB targeting agent, and apigenin, a MAPK inhibitor, were performed as single-agent (barasertib: 0-25nM and apigenin: 0-40μM), and in combination with 15nM cetuximab for 168h using simultaneous and sequential treatment schedules. Induction of apoptotic cell death was investigated in LICR-HN1 cells using the Annexin V-FITC apoptosis detection kit for all treatment schedules. Immunohistochemical staining for AURKB was performed on formalin fixed, paraffin-embedded tissues from 52 newly diagnosed HNSCC patient samples, using an automated IHC staining platform and will be correlated with clinicopathological parameters. The phosphorylation status of ERK was investigated using the NanoPro technology and the methylation status of DUSP6 will be analyzed using the Pyrosequencing method. Results: The mitosis-targeting drug barasertib induced cytotoxicity in the cetuximab resistant HNSCC cell lines, Cal27 and LICR-HN1, with IC50 values of 13.15 ± 0.60nM and 17.6 ± 1.38nM, respectively. In combination therapy with cetuximab, no additional benefit over barasertib as single agent was observed. However, preliminary results showed that pretreatment of LICR-HN1 cells with 15nM cetuximab followed by barasertib (10 or 20nM) resulted in less viable cells compared to cetuximab or barasertib treatment alone. AURKB expression was present in HNSCC patients, with intensities ranging from 0 - 100%. Apigenin induced cytotoxicity in the two cetuximab resistant cell lines, Cal27 and LICR-HN1, with IC50 values of 22.29 ± 0.60μM and 30.66 ± 0.97μM respectively. Preliminary results showed that the combination of cetuximab with apigenin had a synergistic effect in the Cal27 cell line. Furthermore, increased phosphorylation of ERK was present in the cetuximab-resistant cell lines compared to the cetuximab-sensitive cell lines. Conclusion: These results seem to confirm that overexpression of AURKB and/or downregulation of DUSP6 are involved in mechanisms of cetuximab resistance in HNSCC cell lines and inhibition of these pathways might, therefore, be a new strategy for anticancer therapy. Citation Format: Carolien Boeckx, Vanessa Deschoolmeester, An Wouters, Ken Op de Beeckx, Patrick Pauwels, Olivier Vanderveken, Guy Van Camp, Wim Vanden Berghe, Pol Specenier, Jan B. Vermorken, Marc Peeters, Marc Baay, Filip Lardon. Overcoming cetuximab resistance in HNSCC: the role of AURKB and DUSP6. [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 5628. doi:10.1158/1538-7445.AM2013-5628

Lectin fromAgaricus bisporusInhibited S Phase Cell Population and Akt Phosphorylation in Human RPE Cells

Lectin from the edible mushroom Agaricus bisporus (ABL) was found to inhibit cell proliferation of some ocular and cancer cell lines. To elucidate how ABL inhibited RPE cell proliferation, we investigated the changes in cell cycle distribution and cell proliferation-related signaling pathways after ABL treatment. Primary human RPE cells were isolated and grown in DMEM/F12 with or without the ABL (20 or 90 μg/mL) for 3 days. Analysis of cell cycle was performed by flow cytometry. Phosphorylation status of Erk, Jnk, p38, and Akt as well as p53 expression levels were investigated by Western blotting. The role of phosphorylated-Akt in RPE cell proliferation was further evaluated using LY294002. After ABL treatment (90 μg/mL), the amount of cells present in the S phase was found to be reduced. These changes were not apparent in cells treated with 20 μg/mL ABL. In addition, Erk and Akt were found to be hyperphosphorylated and hypophosphorylated, respectively. The expression levels of phosphorylated-Jnk, phosphorylated-p38, and p53 were not altered when compared with those of the control cells. When RPE cells were treated with LY294002 and deprived from phosphorylated-Akt expression, cell proliferation rate was reduced. Reduction in the amount of cells present in S phase was also observed. Our results showed that ABL hypophosphorylated Akt and this observation is in line with the fact that ABL attenuates cell proliferation. As the level of p53 was not significantly altered by ABL, this indicated that ABL-arrested cell cycle progression was independent of p53 activation.

Abstract 2164: S100B affects MAPK signaling in malignant melanoma via a direct interaction with RSK

Abstract Establishing the mechanism by which S100B affects ERK and its downstream signaling provided insight into how S100B affects the progression of malignant melanoma and could aid in developing new pharmacological drugs. S100B is a 21.5 kDa symmetric homodimer that is highly conserved and expressed in a number of tissues and cell lines, including melanocytes. Generally, low levels of S100B have trophic effects, while higher levels can have dire consequences, as is the case in human malignant melanoma. S100B is an effective and widely used prognostic marker for malignant melanoma, with its increased level in serum being predictive of disease stage, increased recurrence, and low overall survival. More recently, S100B has been investigated as a potential contributor to cancer progression, which may be related to how it impacts cell signaling, including the MAPK pathway (BRAF-MEK-ERK). To further evaluate its significance, S100B knock-down clones were created from the WM115 melanoma cell line, and a positive correlation was found between S100B expression and cell viability, as measured by MTT assays. It was also discovered that cells with suppressed S100B expression showed significantly lower levels of ERK phosphorylation. Likewise, over-expression of S100B in the human melanoma cell line, 501-MEL, showed the reciprocal effect, with the introduction of high levels of S100B leading to increased cell viability and ERK phosphorylation. However, the phosphorylation status of ERK does not translate to all of its downstream targets. For example, increased RSK phosphorylation was observed in the S100B knock-down clones, and correspondingly, RSK phosphorylation was decreased with over-expression of S100B. Additionally, over-expression of a mutant S100B construct (E31A + E72A) that was incapable of binding calcium yielded neither effect, indicating that the effect of S100B on RSK phosphorylation was calcium-dependent. To determine if S100B interacted directly with RSK, pull-down assays were performed next. Consistent with the calcium-mutant data, RSK was detected in S100B pull-downs in the presence of calcium, but not in the presence of the calcium chelator EDTA. Changes in RSK localization was also observed, where RSK was enriched in the nucleus of WM115 cells when S100B was knocked down and diffuse in control cells. Together these data are consistent with a mechanism in which elevated S100B binds directly to RSK, preventing its phosphorylation by ERK and its subsequent translocation to the nucleus. Thus, the calcium-binding protein S100B affects MAPK signaling by increasing levels of phosphorylated ERK while simultaneously decreasing phosphorylated RSK. Together, these two effects of S100B on MAPK signaling could impact cancer progression. 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 2164. doi:1538-7445.AM2012-2164

Gene expression and phosphoprotein profile of certain key neuronal signaling proteins following soman intoxication

Nerve agents irreversibly inhibit acetylcholinesterase (AChE), leading to cholinergic crisis and death at acute exposure levels. The complexity, delayed onset, and persistent nature of nerve agent induced CNS effects need to be elucidated to block their multiple effects. In the present study gene expression and phosphoprotein profile of certain key neuronal proteins were studied after soman exposure. Quantitative real time PCR analysis of c-Fos, Bax, CREB and caspase 3 genes in the hippocampus, cortex and cerebellum showed that only c-Fos and Bax mRNA expression was increased significantly. Western blot analysis also confirmed the induction of c-Fos at early time points both at 0.5 and 1.0 LD 50 dose of soman exposure. Acute soman exposure caused perturbations in the phosphorylation status of ERK, JNK, p38 MAPK, CREB, c-Jun and NF-κB in all the three brain regions. The primary target for soman toxicity, AChE was inhibited in blood and brain up to 90%. Therapeutic treatment comprising of HI-6, atropine and diazepam has completely protected animals from death and reactivated soman inhibited AChE up to 40% in the plasma and RBC. This therapeutic regime also reduced soman induced Bax expression to near control levels, but could not reverse the soman induced changes in c-Fos expression and phosphorylation levels completely. Results suggest that exposure to soman caused persistent changes in these key brain proteins, which could lead to the development of complex neurotoxic effects and there is an urgent need for development of better drugs to stop multiple effects of nerve agents poisoning.

Abstract 684: The difference between gefitinib and serum deprivation in cancer cells

Abstract Gefitinib is a selective inhibitor of epidermal growth factor receptor tyrosine kinase. The serum free condition could resemble to gefitinib exposure since it blocks EGF stimuli. In this study, we examined distinction between gefitinib exposure and serum deprivation in cultured cancer cells. At first, we verified that ERK phosphorylation was downregulated by gefitinib exposure in A431 and A549 cells. We examined the phosphorylation status of ERK under the serum deprivation condition. The phosphorylation of ERK in A431 cells was downregulated after 1hr culture in serum free medium, while after 3-6hr ERK was phosphorylated again. We hypothesized that an auto- or paracrine mechanism would work to phosphorylate ERK. In the cells cultured with the conditioned serum free medium from the dish in which A431 cultured for 24hr, the downregulation of ERK phosphorylation was weaker than with the fresh serum free medium. We confirmed that gefitinib could inhibit the ERK phosphorylation under the serum free condition. Exogenous EGF attenuated the inhibitory effect on ERK phosphorylation with serum free medium. These results suggest EGF may be one of the auto- or paracrine substance that induces ERK phosphorylation. Since the ERK phosphorylation status affects the stability of c-MYC, we also examined whether “gefitinib” and “serum deprivation” affect the expression of c-MYC. 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 684. doi:10.1158/1538-7445.AM2011-684

Abstract LB-230: Automating sample preparation for single-cell protein phosphorylation analysis by flow cytometry

Abstract Protein phosphorylation plays essential roles in regulating cellproliferation, differentiation and other cellular processes. Aberrant regulation of protein kinases, can drive malignancy through inappropriate cell growth and survival signals. Understanding the phosphorylation status of specific cellular proteins in cancer has proven valuable in developing targeted therapies and assays to identify specific drug responses in individual cancer patients. The ability to analyze cells at the single cell level allows flow cytometry to measure protein phosphorylation in rare subsets of cells in complex populations such as blood or bone marrow specimens. However, sample preparation for protein phosphorylation analysis by flow cytometry can be technically challenging, time-consuming, and subject to error when performed manually. Furthermore, the procedure demands strict control of experimental conditions such as time and temperature due to the dynamic nature of protein phosphorylation. In this report, we describe an automated sample preparation method using a @Biomek® NXP Span-8 work-station to monitor the phosphorylation status of ERK and p38 MAPK in human peripheral blood monocytes. The method allows automation of sample preparation steps, including 1) ERK/p38 MAPK stimulation, 2) fixation, 3) erythrocyte lysis, 4) cell permeabilization, and, 5) staining with phospho-specific and cell surface marker antibodies. Using this approach, we were able to observe p38 MAPK and ERK activation in CD14+ monocytes following lipopolysaccharide (LPS) or phorbol-12-myristate-13 acetate (PMA) treatment. The automated sample preparation workstation is capable of processing 48 samples in approximately 2.5 hours. Note: *Method cited Not for Clinical purposes. @For Laboratory Use Only; not for use in diagnostic procedures. 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 LB-230.

Abstract 5569: Neointimal Hyperplasia After Mechanical Endovascular Injury is Augmented Through Angiotensin II-mediated Regional Vascular Inflammation via Activation of the JNK Pathway in Angiotensin-converting Enzyme 2-deficient Mice

Background: Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of the renin-angiotensin system, although its role on vascular inflammation and atherosclerosis remains unclear. Methods and Results: Male ACE2-deficient (ACE2 − /y ) and wild-type (ACE2 + /y ) mice underwent wire-mediated endovascular injury in femoral artery. After 4 weeks, the injured artery in ACE2 − /y mice exhibited marked neointimal hyperplasia compared with that in ACE2 + /y mice. The intima/media ratio in the vascular lesion was 1.97±0.31 in ACE2 + /y mice and 2.78±0.49 in ACE2 − /y mice ( P &lt;0.05). There were no significant differences in blood pressure between both mice. In histology, the accumulation of macrophages in lesions was significantly augmented in ACE2 − /y mice. ACE2 protein expression in vascular cells in wild-type mice was detected by immunohistochemistry and western blotting, and the concentration of angiotensin II (Ang-II) in the injured artery was higher in ACE2 − /y mice than in ACE2 + /y mice. In addition, in the injured artery, quantitative mRNA expressions of vascular inflammatory molecules such as VCAM-1, MCP-1, TNF α , and MMP9 were increased more markedly in ACE2 − /y mice. In contrast, an ARB, olmesartan, attenuated the lesion formation and mRNA expressions of these molecules in the injured artery in ACE2 − /y mice. Next, we analyzed phosphorylation status of ERK and JNK in aortic vascular smooth muscle cells (VSMCs), which were derived from ACE2 + /y or ACE2 − /y mice and were stimulated with Ang-II. Although Ang-II increased ERK and JNK phosphorylation in VSMCs of both mice, the increase was greater in VSMCs of ACE2 − /y mice. Moreover, we evaluated which signaling pathway mediated Ang-II-induced upregulation of VCAM-1 and MMP9 in VSMCs. Although an ERK inhibitor PD98059 had no impact on Ang-II-induced upregulation of VCAM-1 and MMP9 mRNA expressions, a JNK inhibitor SP600125 significantly attenuated them, suggesting the central role of the JNK pathway in transducing the impact of Ang-II on vascular inflammation. Conclusions: ACE2 deletion induces marked neointimal formation in femoral artery after vascular injury through Ang-II-mediated regional vascular inflammation via activation of the JNK pathway. ACE2 may confer protection against vascular diseases.

Abstract 5681: GPR30 is Required for the Pro-apoptotic Effects of Estrogen in Vascular Smooth Muscle Cells

Rapid and non-genomic effects of estradiol (EST) may be mediated through the G-Protein-coupled receptor dubbed GPR30 receptor. The present studies tested the hypothesis that GPR30 expression, which we demonstrate is readily detectable in freshly isolated vascular tissue but is decreasingly detectable in cultured VSMCs, mediates the effects of EST to alter ERK phosphorylation status and apoptosis. In freshly isolated aortic tissue, EST-stimulated ERK phosphorylation whereas in cultured VSMCs, EST inhibits ERK phosphorylation in a dose-dependent manner. However, gene transfer of GPR30 resulted in EST-mediated stimulation of ERK phosphorylation (138±4%, n=10), which is in contrast to the effects of EST in GFP-expressing primary culture of VSMCs (68±3%, n=14). EST-mediated stimulation of ERK subsequent to heterologous GPR30 expression was pertussis toxin-sensitive and PI3 kinase-dependent; under these conditions, EST also inhibited PKA activation. In contrast, in the absence of GPR30 expression in cultured VSMC, EST stimulated PKA activity and inhibited ERK phosphorylation. We also assessed the impact of GPR30 expression on EST-mediated regulation of VSMCs apoptosis. In cultured VSMC transduced with GFP vector (control conditions), EST (10 nM) mediated a decrease in VSMC apoptosis (80±2%, n=7), an effect that was marginally but significantly enhanced by gene transfer of ER α (71±3%, n=7). The anti-apoptotic effect of EST was attenuated by co-incubation with the PKA inhibitor Rp-cAMP. However, PI3 kinase inhibition did not affect estradiol’s anti-apoptotic actions. In contrast, gene transfer of GPR30 into the VSMC resulted in enhancement of apoptotic rates following EST treatment (169±2%, n=7), and this GPR30-mediated pro-apoptotic effect of EST was attenuated by the PI3 kinase inhibitor, but not by Rp-cAMP. Thus, the effect of EST on vascular smooth muscle cell apoptosis is likely dependent on the balance between ER-mediated PKA activation and GPR30-mediated PKA inhibition and PI3 kinase activation. Taken together, we postulate that modulation of GPR30 expression and/or activity may be an important determinant of the effects of estradiol in the vasculature.

A phase II study of AZD6244 in advanced or metastatic hepatocellular carcinoma

e15574 Background: HCC is a deadly disease with few effective treatments. It is characterized by frequent MEK/ERK activation in the absence of RAS or RAF mutation. AZD6244 is a specific inhibitor of MEK that can be administered orally. Methods: Multi-center, single-arm study with a two-stage design utilizing RR as primary endpoint. Patients had advanced or metastatic HCC not amenable to surgical therapy or who failed locoregional therapy. Pts with Childs-Pugh A and compensated B were eligible. No prior systemic therapy was allowed. A pre-treatment biopsy was performed for p-ERK analysis. AZD6244 was administered orally at a dose of 100 mg bid. Results: 19 patients were enrolled, completing the first planned stage. One patient withdrew during cycle 1 with grade 2 AEs and is not evaluable. One patient is too early for evaluation. Another was removed from study for toxicity prior to evaluation. Two patients exhibited clinical progression and were removed from study without imaging (both assigned response of PD). Of 16 evaluable patients, PR + CR was 0%, SD 37.5%, PD 62.5%. Toxicities were generally low grade and included nausea (75%), transaminitis (63%), Alkaline phosphatase (50%), vomiting (50%), fatigue (44%), diarrhea (44%) and skin rash (44%). Grade 3–4 toxicity included transaminitis (38%), hyperbilirubinema (25%), and hypokalemia (13%). Median TTP was 8 weeks (95% CI: 6.6, 11.1). Conclusions: The study has been stopped at the interim analysis due to lack of radiographic response. Time to progression is not suggestive of significant prolonged stable disease. AZD6244 is well tolerated, but appears to have minimal activity in advanced HCC. Analysis of ERK phosphorylation status in biopsy samples is ongoing. No significant financial relationships to disclose.

Suppressive Effect of Orthovanadate on Hepatic Stellate Cell Activation and Liver Fibrosis in Rats

Orthovanadate (OV), an inhibitor of protein tyrosine phosphatases, affects various biological processes in a cell-type-specific manner. In this study, we investigated the effect of OV on hepatic stellate cells (HSCs). When primary rat HSCs were cultured in the presence of 10% serum, they spontaneously lost characteristic stellate morphology, proliferated, and were transformed into an activated state with the formation of abundant stress fibers and increased expression of both alpha-smooth muscle actin and collagen type I mRNA. OV treatment inhibited proliferation and activation of HSCs and partially reversed the phenotype of activated HSCs. Among the signaling molecules investigated, phosphorylation of the Src protein at tyrosine 416 was the most striking in OV-treated HSCs. Treatment of cells with Src family inhibitors partially abrogated the effects of OV. Furthermore, transfection of v-Src into activated HSCs induced a stellate morphology similar to that in the quiescent state. We then examined whether OV could effectively suppress HSC activation in vivo after liver injury induced by either carbon tetrachloride or dimethylnitrosamine. OV significantly reduced the appearance of alpha-smooth muscle actin-positive cells and decreased collagen deposition, concomitant with an improvement in liver function. Our study showed for the first time that OV was able to suppress the activation of HSCs, possibly through the modulation of Src activity, and attenuated fibrosis after chronic liver injury.