Activation Of ERK Pathways Research Articles

USO1 promotes tumor progression via activating Erk pathway in multiple myeloma cells.

This study aimed to explore the influence of USO1 on multiple myeloma (MM) cell proliferation and apoptosis and the related molecular mechanism. The expression of USO1 and MIF in MM tissues and cells, normal bone marrow tissues and cells were determined by qRT-PCR and western blot assay. The cell proliferation and apoptosis of MM cells before and after knockdown of USO1 were determined by MTT assay and flow cytometry, respectively. Before and after knockdown of USO1, the expression of the proliferation-related genes cyclin D1, Mcm2 and PCNA in MM cells was determined by qRT-PCR and western blot assay. The protein level of p-Erk1/2 and MIF was determined by western blot assay and ELISA, respectively. The expression levels of USO1 and MIF in MM tissues and cells were much higher than those in normal bone marrow tissues and cells. Knockdown of USO1 resulted in the inhibited ability of cell proliferation and induced cell apoptosis. The expression of cyclin D1, Mcm2, PCNA and p-Erk1/2 decreased significantly after knockdown of USO1 as well as the decreased MIF secretion. USO1 gene may be a promising target for the therapy of human MM and its diagnosis marker.

Gambogic acid potentiates clopidogrel-induced apoptosis and attenuates irinotecan-induced apoptosis through down-regulating human carboxylesterase 1 and -2

1. In this study, we report that gambogic acid (GA), a promising anticancer agent, potentiates clopidogrel-induced apoptosis and attenuates CPT-11-induced apoptosis by down-regulating human carboxylesterase (CES) 1 and -2 via ERK and p38 MAPK pathway activation, which provides a molecular explanation linking the effect of drug combination directly to the decreased capacity of hydrolytic biotransformation.2. The expression levels of CES1 and CES2 decreased significantly in a concentration- and time-dependent manner in response to GA in Huh7 and HepG2 cells; hydrolytic activity was also reduced.3. The results showed that pretreatment with GA potentiated clopidogrel-induced apoptosis by down-regulating CES1. Moreover, the GA-mediated repression of CES2 attenuated CPT-11-induced apoptosis.4. Furthermore, the ERK and p38 MAPK pathways were involved in the GA-mediated down-regulation of CES1 and CES2.5. Taken together, our data suggest that GA is a potent repressor of CES1 and CES2 and that combination with GA will affect the metabolism of drugs containing ester bonds.

Enhancement of hepatocyte differentiation from human embryonic stem cells by Chinese medicine Fuzhenghuayu

Chinese medicine, Fuzhenghuayu (FZHY), appears to prevent fibrosis progression and improve liver function in humans. Here we found that FZHY enhanced hepatocyte differentiation from human embryonic stem cells (hESC). After treatment with FZHY, albumin expression was consistently increased during differentiation and maturation process, and expression of metabolizing enzymes and transporter were also increased. Importantly, expression of mesenchymal cell and cholangiocyte marker was significantly reduced by treatment with FZHY, indicating that one possible mechanism of FZHY’s role is to inhibit the formation of mesenchymal cells and cholangiocytes. Edu-labelled flow cytometric analysis showed that the percentage of the Edu positive cells was increased in the treated cells. These results indicate that the enhanced proliferation involved hepatocytes rather than another cell type. Our investigations further revealed that these enhancements by FZHY are mediated through activation of canonical Wnt and ERK pathways and inhibition of Notch pathway. Thus, FZHY not only promoted hepatocyte differentiation and maturation, but also enhanced hepatocyte proliferation. These results demonstrate that FZHY appears to represent an excellent therapeutic agent for the treatment of liver fibrosis, and that FZHY treatment can enhance our efforts to generate mature hepatocytes with proliferative capacity for cell-based therapeutics and for pharmacological and toxicological studies.

Soybean Oil-Based Lipid Emulsion Increases Intestinal Permeability of Lipopolysaccharide in Caco-2 Cells by Downregulation of P-Glycoprotein via ERK-FOXO 3a Pathway

Background and Aims: Elevated intestinal permeability of lipopolysaccharide (LPS) is a major complication for patients with parenteral nutrition (PN), but the pathogenesis is poorly understood. Intestinal P-glycoprotein (P-gp) is one of the efflux transporters that contribute to restricting the permeability of lipopolysaccharide via transcellular route. P-gp expression may be regulated by PN ingredients, and thus this study sought to investigate the effect of PN on the expression of P-gp and to elucidate the underlying mechanism in vitro. Methods: Caco-2 cells were treated with PN ingredients. Changes in P-gp expression and function were determined and the role of ERK-FOXO 3a pathway was studied. Transport studies of FITC-lipopolysaccharide (FITC-LPS) across Caco-2 cell monolayers were also performed. Results: Among PN ingredients, soybean oil-based lipid emulsion (SOLE) exhibited significant inhibitory effect on P-gp expression and function. This regulation was mediated via activation of ERK pathway with subsequent nuclear exclusion of FOXO 3a. Importantly, P-gp participated in antagonizing the permeation of FITC-LPS (apical to basolateral) across Caco-2 cell monolayers. SOLE significantly increased the permeability of FITC-LPS (apical to basolateral), which was associated with impaired P-gp function. Conclusions: The expression and function of intestinal P-gp is suppressed by SOLE in vitro.

Controlling keratinocyte activity by LL-37 conjugated nanoparticles

Controlling keratinocyte activity by LL-37 conjugated nanoparticles

Ursodeoxycholic acid upregulates ERK and Akt in the protection of cardiomyocytes against CoCl2.

Ursodeoxycholic acid (UDCA) is used to treat liver diseases and demonstrates cardioprotective effects. Accumulation of the plasma membrane sphingolipid sphingomyelin in the heart can lead to atherosclerosis and coronary artery disease. Sphingomyelinases (SMases) break down sphingomyelin, producing ceramide, and inhibition of SMases activity can promote cell survival. We hypothesized that UDCA regulates activation of ERK and Akt survival signaling pathways and SMases in protecting cardiac cells against hypoxia. Neonatal cardiomyocytes were isolated from 0- to 2-day-old Sprague Dawley rats, and given 100 μM CoCl2, 150 μM H2O2, or placed in a hypoxia chamber for 24 h. The ameliorative effects of 100-μM UDCA treatment for 12 h were then assessed using MTS, QuantiGene Plex (for Smpd1 and Smpd2), and SMase assays, beating rate assessment, and western blotting (for ERK and Akt). Data were analyzed by the paired Student t-tests and one-way analyses of variance. Cell viability decreased significantly after H2O2 (85%), CoCl2 (50%), and hypoxia chamber (52%) treatments compared to the untreated control (100%). UDCA significantly counteracted the effects of chamber- and CoCl2- induced hypoxia on viability and beating rate. However, no significant differences were observed in acid SMase gene and protein expression between the untreated, CoCl2, and UDCA-CoCl2 groups. In contrast, neutral SMase gene and protein expression did significantly differ between the latter two groups. ERK and Akt phosphorylation was higher in hypoxic cardiomyocytes treated with UDCA than those given CoCl2 alone. In conclusion, UDCA regulates the activation of survival signaling proteins and SMases in neonatal rat cardiomyocytes during hypoxia.

Epigenomic reprogramming in peripheral nerve injury.

Epigenomic reprogramming in peripheral nerve injury.

Erythropoietin Protects Retinal Cells in Diabetic Rats Through Upregulating ZnT8 via Activating ERK Pathway and Inhibiting HIF-1α Expression

Zinc transporter 8 (ZnT8) was downregulated in hypoxic retina, which could be rescued by hypoxia-inducible factor-1α (HIF-1α) inhibition. Erythropoietin (EPO) protects retinal cells in diabetic rats through inhibiting HIF-1α as one of its mechanisms. We hence tried to explore the effect of EPO in regulating ZnT8 and protecting retinal cells in diabetic rats and possible mechanisms. Diabetes was induced in Sprague-Dawley rats. Intravitreal injection of EPO was performed 1 month after diabetes onset. The CoCl2-treated rat Müller cell line (rMC-1) was cotreated with EPO, soluble EPO receptor (sEPOR), digoxin, or U0126. Cell viability, cell death, and intracellular zinc level were examined. The expression of ZnT8, HIF-1α, AKT, and ERK was studied. In diabetic rat retinas, EPO significantly decreased HIF-1α expression and increased ZnT8 expression. In CoCl2-treated rMC-1 cells, EPO increased cell viability and decreased intracellular zinc. Erythropoietin or digoxin could activate ERK pathway, downregulate HIF-1α, and upregulate ZnT8. The effect of EPO was abolished by sEPOR and U0126. Transient knockdown of ZnT8 increased intracellular zinc level, but not to a degree that would decrease cell viability or cause cell death. In diabetic retinas, EPO maintains zinc homeostasis through activating the ERK pathway and downregulating HIF-1α, and thus upregulating ZnT8 expression. This work proposed a possible new protective mechanism for EPO in, and indicated a potential target for, the treatment of diabetic retinopathy.

Nrf2 antioxidant defense is involved in survival signaling elicited by 27-hydroxycholesterol in human promonocytic cells

Cholesterol oxidation products such as oxysterols are considered critical factors in the atherosclerotic plaque formation since they induce oxidative stress, inflammation and apoptotic cell death. 27-hydroxycholesterol (27-OH) is one of the most represented oxysterols in atherosclerotic lesions. We recently showed that relatively low concentrations of 27-OH generated a strong survival signaling through an early and transient increase of cellular ROS level, that enhanced MEK-ERK/PI3K-Akt phosphorylation, in turn responsible of a sustained quenching of ROS production. It remains to identify the link between ERK/Akt up-regulation and the consequent quenching effect on ROS intracellular level that efficiently and markedly delay the pro-apoptotic effect of the oxysterol. Here we report on the potent activation of Nrf2 redox-sensitive transcription factor by low micromolar amount of 27-OH added to U937 promonocytic cells. The 27-OH-exerted induction of Nrf2 and subsequently of the target genes, HO-1 and NQO-1, was proved to be: (i) dependent upon the activation of ERK and Akt pathways, (ii) directly responsible for the quenching of intracellular oxidative stress and by this way (iii) ultimately responsible for the observed oxysterol-induced pro-survival response.

Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants.

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf(AVKA) mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf(AVKA) allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf(AVKA) mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf(AVKA). Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.

Novel Mechanisms of Growth Inhibition By Histone Deacetylase Inhibitors in MPN

Histone deacetylase inhibitors (HDACi) are being evaluated in several clinical trials for the treatment of hematological malignancies and 2 of them have been approved for the treatment of T cell lymphoma. We have investigated the effectiveness of using 2 HDACi - Vorinostat (SAHA) and Panobionstat for treatment of systemic mastocytosis (SM) for which no treatments are available. SAHA and Panobinostat showed concentration dependent inhibition of proliferation in mastocytosis patient derived cell lines, HMC1.1 and HMC1.2 bearing either single mutation in the juxtamembrane domain KITG560V or double mutation also including the activation loop KITG560V,D816V, respectively. Interestingly HMC1.2 cells with 2 mutations in KITG560V, D816V were more sensitive to treatment with both SAHA (IC50 ~ 260 nM) and Panobinostat (IC50 ~11 nM) as compared to HMC1.1 cells (IC50 ~ 600 nM for SAHA and IC50 ~ 23 nM with Panobionstat) that have a single mutation in KIT. The inhibition of proliferation by HDACi treatment appeared to be associated with modulation of cell cycle and differentiation rather than induction of apoptosis. We further tested the effect of combining HDACi treatment with tyrosine kinase inhibitor, Dasatinib. Combination of Dasatinib (5 - 20 nM) with either SAHA or Panbionostat was more effective than any of the single drugs in HMC1.1 cells. However such a combination effect was not seen in HMC1.2 cells. Over 90% patients with aggressive systemic mastocytosis carry KITD816V mutation. This particular mutation of KIT is highly resistant to therapeutics and is associated with poor survival. Thus patients carrying the KITD816V mutation are likely to benefit from treatment with HDACi while juxtamembrane mutation bearing patients would benefit more from a combination of HDACi with kinase inhibitors such as Dasatinib. To further understand the mechanism behind HDACi inhibition of KITD816V mediated oncogenic signals, we expressed KITD814V (murine equivalent of human D816V) in myeloid cells and treated them with SAHA and Panobinostat. Cells expressing KITD814V showed cytokine independent proliferation and interestingly, cytokine independent proliferation was more sensitive to inhibition by both SAHA and Panbinostat as compared to the cytokine dependent proliferation. In contrast to HMC cells, inhibition of cytokine independent proliferation by HDACi in KITD814V oncogene expressing cells was associated with significant increase in apoptosis. The percent cells negative for Annexin V stain decreased from 74% in control to ~46% in 24h upon treatment with 500 nM SAHA or 10 nM Panbinostat (p< 0.001; n=3). Induction of apoptosis by HDACi in KITD814V oncogene expressing cells was significantly suppressed in presence of cytokine. Consistent with the in vitro data, treatment with SAHA prolonged the survival of mice transplanted with myeloid cells expressing KITD814V mutation as compared to the vehicle treated group (Median survival - 21d (veh), 34d (SAHA), p=0.0015, n=5). In addition to increased histone acetylation, treatment of cells with HDACi also leads to increase in acetylation of chaperone proteins such as HSP90 which can mediate proteasomal degradation of oncogenes and has been suggested as one of the mechanisms by which HDACi mediates growth inhibition. In contrast, we found no significant decrease in mutant KIT protein expression or AKT expression and phosphorylation upon treatment with either of the HDACi. Though expression of Pim1 a pro survival serine/threonine kinase was down-regulated by SAHA and Panobinostat. Treatment with HDACi was also associated with differential modulation of STAT3, STAT5 and ERK pathways. Treatment with both SAHA and Panobinostat down-regulated STAT5 phosphorylation and up-regulated ERK1/2 phosphorylation with more increase in ERK1/2 phosphorylation observed in cells with higher sensitivity to inhibition by HDACi. Though increased activation of the ERK pathway has been associated with leukemogenesis; there is some evidence to indicate that hyper activation of ERK pathway in absence of additional leukemogenic signals activates a pro senescence program. Differential expression of Pim1 and along with differential modulation of STAT and ERK pathways may contribute to differential sensitivity of the cells to HDACi and are likely to be of therapeutic value in aggressive systemic mastocytosis that has so far been associated with poor prognosis. DisclosuresNo relevant conflicts of interest to declare.

Abstract A11: Radio-sensitivities and angiogenic signaling pathway of irradiated endothelial cells isolated from cancer and normal tissue of human breasts in vitro

Abstract Despite the strong possibility that endothelial cells of tumors and normal tissues may be different in various aspects, most of the previous studies on endothelial cells have been conducted using normal endothelial cells. Therefore, we have developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and further investigated radio-sensitivities and angiogenic signaling pathway in the alternation of angiogenic process observed after the purified cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) were irradiated in vitro. When human breast tissues including cancer were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth factors, endothelial cells grew out of the tissues. The endothelial cells were harvested, cultured as monolayer cells in plates coated with gelatin, and exposed to radiation, and then the changes in clonogenic cell survival, tube formation capacity, γH2AX foci, gene expression, and cellular signaling pathways were determined. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothelial cells. CECs were significantly more radiosensitive in treatment of single dose (4 Gy) and hyperfractionation (4 Gy in 2 or 4 equal fractions) of ionizing radiation (IR) than their normal counterparts although hyperfractionated IR reduced clonogenic cell death in both NECs and CECs than single dose of IR. In addition, we found that IR-induced foci formation of γH2AX in NECs increased 1 hr and declined afterwards when the cells were treated with 4 Gy of IR. On the contrary, it was remained up to 4 hr in CECs. In NECs, 4 Gy of IR induced an increase in the capillary-like tube formation, the expression of matrix metalloproteinase-2 (MMP-2), and the activation of ERK pathway. However, in CECs, 4 Gy of IR significantly reduced the capillary-like tube formation, and induced the expression of angiostatin and the activation of both AKT and JNK pathways. Additionally, inhibition of ERK with a pharmacological inhibitor or a small interfering RNA markedly suppressed the capillary-like tube formation and the expression of MMP-2 caused by IR in NECs. In CECs, inhibition of either AKT or JNK with a pharmacological inhibitor or a small interfering RNA clearly attenuated inhibition of the capillary-like tube formation, and the expression of angiostatin caused by IR. Furthermore, hypoxia increased the capillary-like tube formation and radioresistance in both NECs and CECs, but the delineation of the mechanisms remains to be studied. We have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue. Moreover, our results collectively demonstrate that there are distinct differences in the radiation responses of NECs and CECs, and might provide important clues for improving the efficacy of radiation therapy. [This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (MRC: NRF-2014R1A5A2009392 and NRF-2013M2A2A7043703] Citation Format: Eun-Taex Oh, Min-Jeong Song, Hyemi Lee, Yun-Jeong Choi, Heon Joo Park. Radio-sensitivities and angiogenic signaling pathway of irradiated endothelial cells isolated from cancer and normal tissue of human breasts in vitro. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A11.

Breaking the Paradox: Next-Generation BRAF Inhibitors

A curious biochemical consequence of BRAF inhibition (BRAFi) in BRAF -normal cells is paradoxical ERK pathway activation. This is a major mechanism by which RAS -mutant tumors are accelerated, as seen in BRAFi-driven cutaneous squamous cell carcinoma (SCC) and in the acceleration of mutant RAS -driven leukemia. This effect appears to be universal with the first-generation RAF inhibitors vemurafenib and dabrafenib, which facilitate RAF …

Lysophosphatidic acid enhances survival of human CD34(+) cells in ischemic conditions.

Several clinical trials are exploring therapeutic effect of human CD34+ cells in ischemic diseases, including myocardial infarction. Unfortunately, most of the cells die few days after delivery. Herein we show that lysophosphatidic acid (LPA)-treated human umbilical cord blood-derived CD34+ cells cultured under hypoxic and serum-deprived conditions present 2.2-fold and 1.3-fold higher survival relatively to non-treated cells and prostaglandin E2-treated cells, respectively. The pro-survival effect of LPA is concentration- and time-dependent and it is mediated by the activation of peroxisome proliferator-activator receptor γ (PPARγ) and downstream, by the activation of pro-survival ERK and Akt signaling pathways and the inhibition of mitochondrial apoptotic pathway. In hypoxia and serum-deprived culture conditions, LPA induces CD34+ cell proliferation without maintaining the their undifferentiating state, and enhances IL-8, IL-6 and G-CSF secretion during the first 12 h compared to non-treated cells. LPA-treated CD34+ cells delivered in fibrin gels have enhanced survival and improved cardiac fractional shortening at 2 weeks on rat infarcted hearts as compared to hearts treated with placebo. We have developed a new platform to enhance the survival of CD34+ cells using a natural and cost-effective ligand and demonstrated its utility in the preservation of the functionality of the heart after infarction.

The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways

The membrane-bound mucin MUC4 is a high molecular weight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the post-transcriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.

Beneficial effects of growth hormone-releasing hormone agonists on rat INS-1 cells and on streptozotocin-induced NOD/SCID mice

Agonists of growth hormone-releasing hormone (GHRH) have been previously reported to promote growth, function, and engraftment of islet cells following transplantation. Here we evaluated recently synthesized GHRH agonists on the proliferation and biological functions of rat pancreatic β-cell line (INS-1) and islets. In vitro treatment of INS-1 cells with GHRH agonists increased cell proliferation, the expression of cellular insulin, insulin-like growth factor-1 (IGF1), and GHRH receptor, and also stimulated insulin secretion in response to glucose challenge. Exposure of INS-1 cells to GHRH agonists, MR-356 and MR-409, induced activation of ERK and AKT pathways. Agonist MR-409 also significantly increased the levels of cellular cAMP and the phosphorylation of cAMP response element binding protein (CREB) in INS-1 cells. Treatment of rat islets with agonist, MR-409 significantly increased cell proliferation, islet size, and the expression of insulin. In vivo daily s.c. administration of 10 μg MR-409 for 3 wk dramatically reduced the severity of streptozotocin (STZ)-induced diabetes in nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice. The maximal therapeutic benefits with respect to the efficiency of engraftment, ability to reach normoglycemia, gain in body weight, response to high glucose challenge, and induction of higher levels of serum insulin and IGF1 were observed when diabetic mice were transplanted with rat islets preconditioned with GHRH agonist, MR-409, and received additional treatment with MR-409 posttransplantation. This study provides an improved approach to the therapeutic use of GHRH agonists in the treatment of diabetes mellitus.

Dual-faced SH3BGRL: oncogenic in mice, tumor suppressive in humans.

Despite abundant data supporting c-Src as a metastasis-promoting oncogene, activating mutations of c-Src are rare. This suggests that trans-interacting proteins may have a critical role in regulating c-Src activation. Here, we first report the discovery of Src homology 3 (SH3) domain-binding glutamic acid-rich-like protein (SH3BGRL), a novel c-Src activator in mice. Ectopic expression of murine SH3BGRL (mSH3BGRL) strongly promoted both tumor cell invasion and lung metastasis. Molecularly, mSH3BGRL specifically bound the inactive form of c-Src phosphorylated at Tyr527, promoting Tyr416 phosphorylation of c-Src and subsequent FAK-mediated activation of ERK and AKT signaling pathways. Targeting endogenous c-Src alone was sufficient to abolish mSH3BGRL-induced cancer metastasis in vivo. Unexpectedly, human SH3BGRL (hSH3BGRL) in turn suppressed tumorigenesis and metastasis in nature. We attempted site-specific reversion of hSH3BGRL amino-acid sequence to mSH3BGRL and found V108A substitution sufficient to restore SH3BGRL function as a c-Src activator and metastasis promoter. Notably, the somatic mutation R76C of hSH3BGRL can similarly act as hSH3BGRL-V108A and mSH3BGRL in tumorigenesis and metastasis. Our results uncover an evolutionarily controversial role of SH3BGRL in driving tumor metastasis through c-Src activation, and suggests that hSH3BGRL mutation status could be relevant to cancer diagnosis and therapy.

Tgfbi/Bigh3 silencing activates ERK in mouse retina

BIGH3 is a secreted protein, part of the extracellular matrix where it interacts with collagen and integrins on the cell surface. BIGH3 can play opposing roles in cancer, acting as either tumor suppressor or promoter, and its mutations lead to different forms of corneal dystrophy. Although many studies have been carried out, little is known about the physiological role of BIGH3. Using the cre-loxP system, we generated a mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing did not result in any apparent phenotype modifications, the mice remained viable and fertile.We were able to determine the presence of BIGH3 in the retinal pigment epithelium (RPE). In the absence of BIGH3, a transient decrease in the apoptotic process involved in retina maturation was observed, leading to a transient increase in the INL thickness at P15. This phenomenon was accompanied by an increased activity of the pro-survival ERK pathway.

Phosphoflow-Based Evaluation of Mek Inhibitors as Small-Molecule Therapeutics for B-Cell Precursor Acute Lymphoblastic Leukemia

Upstream mutations that lead to constitutive activation of Erk in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are relatively common. In the era of personalized medicine, flow cytometry could be used as a rapid method for selection of optimal therapies, which may include drugs that target the Erk pathway. Here, we evaluated the utility of phospho-flow, compared to Western blotting, to monitor Erk pathway activation and its inhibition by targeted Mek kinase inhibitors in human BCP ALL. Because the Erk pathway is not only activated endogenously, by mutations, but also by normal extracellular stimulation through stromal contact and serum growth factors, we compared Erk activation ex vivo in ALL cells in the presence and absence of stroma and serum. Phospho-flow was able to readily detect changes in the pool of pErk1/2 that had been generated by normal microenvironmental stimuli in patient-derived BCP-ALL cells passaged in NSG mice, in viably frozen primary patient samples, and in fresh patient samples. Treatment with the Mek1/2 inhibitor selumetinib resulted in a rapid, complete and persistent reduction of microenvironment-generated pErk1/2. Imaging flow cytometry confirmed reduction of nuclear pErk1/2 upon selumetinib treatment. An ALL relapsing with an activating KRasG12V mutation contained higher endogenous as well as serum/stromal-stimulated levels of pErk1/2 than the matched diagnosis sample which lacked the mutation, but selumetinib treatment reduced pErk1/2 to the same level in both samples. Selumetinib and trametinib as Mek inhibitors were mainly cytostatic, but combined treatment with the PI3K∂ inhibitor CAL101 increased cytotoxicity. Thus phospho-flow cytometry could be used as a platform for rapid, individualized in vitro drug sensitivity assessment for leukemia patients at the time of diagnosis.

Leptin-dependent neurotoxicity via induction of apoptosis in adult rat neurogenic cells.

Adipocyte-derived hormone leptin has been recently implicated in the control of neuronal plasticity. To explore whether modulation of adult neurogenesis may contribute to leptin control of neuronal plasticity, we used the neurosphere assay of neural stem cells derived from the adult rat subventricular zone (SVZ). Endogenous expression of specific leptin receptor (ObRb) transcripts, as revealed by RT-PCR, is associated with activation of both ERK and STAT-3 pathways via phosphorylation of the critical ERK/STAT-3 amino acid residues upon addition of leptin to neurospheres. Furthermore, leptin triggered withdrawal of neural stem cells from the cell cycle as monitored by Ki67 labeling. This effect was blocked by pharmacological inhibition of ERK activation thus demonstrating that ERK mediates leptin effects on neural stem cell expansion. Leptin-dependent withdrawal of neural stem cells from the cell cycle was associated with increased apoptosis, as detected by TUNEL, which was preceded by cyclin D1 induction. Cyclin D1 was indeed extensively colocalized with TUNEL-positive, apoptotic nuclei. Cyclin-D1 silencing by specific shRNA prevented leptin-induced decrease of the cell number per neurosphere thus pointing to the causal relationship between leptin actions on apoptosis and cyclin D1 induction. Leptin target cells in SVZ neurospheres were identified by double TUNEL/phenotypic marker immunocytofluorescence as differentiating neurons mostly. The inhibition of neural stem cell expansion via ERK/cyclin D1-triggered apoptosis defines novel biological action of leptin which may be involved in adiposity-dependent neurotoxicity.