Blockage Of Activity Research Articles

RETRACTED ARTICLE: Human leptin triggers proliferation of A549 cells via blocking endoplasmic reticulum stress-related apoptosis

Lung cancer is a disease characterized by uncontrolled cell growth in tissues of the lung. Leptin is a pleiotropic hormone with antiapoptotic and proliferative roles involved in several systems. However, there is no known antiapoptotic mechanism of leptin in non-small cell lung cancer (NSCLC). So, we investigated the antiapoptotic mechanism of leptin in NSCLC. Proliferation, apoptosis, and the specific mechanism of leptin-transfected cells were analyzed in this study. Leptin, p-Perk, IRE1, cleaved ATF6, spliced XBP1, eIF2-α, TRAF2, CHOP, and caspase 12 proteins were detected by Western blot, and endoplasmic reticulum (ER) stress-related mRNA was detected by semi-quantitative reverse transcription PCR (RT-PCR). Leptin in A549 and transfected cells inhibited cisplatin-activated ER stress-associated mRNA transcription and activation of proteins. ER stress unfolded protein response (UPR) proteins, PERK and ATF6, were involved in leptin-triggered apoptosis. XBP1 and TRAF2 were increased significantly when treated with cisplatin in A549-siLPT and non-transfected cells. CHOP expression was blocked in A549 and transfected cells (LPT-PeP and LPT-EX cells). In conclusion, leptin can promote the proliferation of A549 cells through blocking ER stress-mediated apoptosis. This blocking is mediated by the p-Perk and ATF6 pathway through blocking activation of CHOP.

Characterization of the Molecular Mechanism of the Bone-Anabolic Activity of Carfilzomib in Multiple Myeloma

Carfilzomib, the next generation of proteasome inhibitor, may increase osteoblast-related markers in patients with multiple myeloma, but the molecular mechanism of its effect on mesenchymal stem cell differentiation to osteoblasts remains unknown. Herein, we demonstrated that carfilzomib significantly promoted mesenchymal stem cell differentiation into osteoblasts. In osteoprogenitor cells and primary mesenchymal stem cells from patients with myeloma, carfilzomib induced increases in alkaline phosphatase activity, matrix mineralization, and calcium deposition via Wnt-independent activation of β-catenin/TCF signaling. Using affinity pull-down assays with immunoblotting analysis and immunofluorescence, we found that carfilzomib induced stabilization of both free and active forms of β-catenin in a time- and dose-dependent manner that was not associated with β-catenin transcriptional regulation. Nuclear translocation of β-catenin protein was associated with TCF transcriptional activity that was independent of the effects of GSK3β-activation and of signaling induced by 19 Wnt ligands, 10 Frizzled receptors, and LRP5/6 co-receptors. Blocking activation of β-catenin/TCF signaling by dominant negative TCF1 or TCF4 attenuated carfilzomib-induced matrix mineralization. Thus, carfilzomib induced osteoblast differentiation via Wnt-independent activation of the β-catenin/TCF pathway. These results provide a novel molecular mechanism critical to understanding the anabolic role of carfilzomib on myeloma-induced bone disease.

Stimulation of Sigma Receptors with Afobazole Blocks Activation of Microglia and Reduces Toxicity Caused by Amyloid-β25–35

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the leading cause of senile dementia in the United States. Accumulation of amyloid-β (Aβ) and the effects of this peptide on microglial cells contribute greatly to the etiology of AD. Experiments were carried out to determine whether the pan-selective σ-receptor agonist afobazole can modulate microglial response to the cytotoxic Aβ fragment, Aβ25-35. Treatment with afobazole decreased microglial activation in response to Aβ, as indicated by reduced membrane ruffling and cell migration. The effects of afobazole on Aβ25-35-evoked migration were concentration dependent and consistent with σ-receptor activation. When afobazole was coapplied with either BD-1047 [N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide] or rimcazole, which are σ-1- and σ-2-selective antagonists, respectively, the inhibition of Aβ25-35-induced migration by afobazole was reduced. Prolonged exposure of microglia to Aβ25-35 resulted in glial cell death that was associated with increased expression of the proapoptotic protein Bax and the death protease caspase-3. Coapplication of afobazole with Aβ25-35 decreased the number of cells expressing both Bax and caspase-3 and resulted in a concomitant enhancement in cell survival. Although afobazole inhibited activation of microglia cells by Aβ25-35, it preserved normal functional responses in these cells after exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24-hour exposure to Aβ25-35. However, coincubation in afobazole returned these responses to near control levels. Therefore, stimulation of σ-1 and σ-2 receptors by afobazole prevents Aβ25-35 activation of microglia and inhibits Aβ25-35-associated cytotoxicity, suggesting that afobazole may be useful for AD therapeutics.

MicroRNA-216a/217-Induced Epithelial-Mesenchymal Transition Targets Pten and SMAD7 to Promote Drug Resistance and Recurrence of Liver Cancer

Tumor recurrence and metastases are the major obstacles to improving the prognosis of patients with hepatocellular carcinoma (HCC). To identify novel risk factors associated with HCC recurrence and metastases, we have established a panel of recurrence-associated microRNAs (miRNAs) by comparing miRNA expression in recurrent and nonrecurrent human HCC tissue samples using microarrays (recurrence is defined as recurrent disease occurring within a 2-year time point of the original treatment). Among the panel, expression of the miR-216a/217 cluster was consistently and significantly up-regulated in HCC tissue samples and cell lines associated with early tumor recurrence, poor disease-free survival, and an epithelial-mesenchymal transition (EMT) phenotype. Stable overexpression of miR-216a/217-induced EMT increased the stem-like cell population, migration, and metastatic ability of epithelial HCC cells. Phosphatase and tensin homolog (PTEN) and mothers against decapentaplegic homolog 7 (SMAD7) were subsequently identified as two functional targets of miR-216a/217, and both PTEN and SMAD7 were down-regulated in HCC. Ectopic expression of PTEN or SMAD7 partially rescued miR-216a/217-mediated EMT, cell migration, and stem-like properties of HCC cells. Previously, SMAD7 was shown to be a transforming growth factor beta (TGF-β) type 1 receptor antagonist. Here, we further demonstrated that overexpression of miR-216a/217 acted as a positive feedback regulator for the TGF-β pathway and the canonical pathway involved in the activation of phosphoinositide 3-kinase/protein kinase K (PI3K/Akt) signaling in HCC cells. Additionally, activation of the TGF-β- and PI3K/Akt-signaling pathways in HCC cells resulted in an acquired resistance to sorafenib, whereas blocking activation of the TGF-β pathway overcame miR-216a/217-induced sorafenib resistance and prevented tumor metastases in HCC. Overexpression of miR-216a/217 activates the PI3K/Akt and TGF-β pathways by targeting PTEN and SMAD7, contributing to hepatocarcinogenesis and tumor recurrence in HCC.

The promotion of osteochondral repair by combined intra-articular injection of parathyroid hormone-related protein and implantation of a bi-layer collagen-silk scaffold

The repair of osteochondral defects can be enhanced with scaffolds but is often accompanied with undesirable terminal differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Parathyroid hormone-related protein (PTHrP) has been shown to inhibit aberrant differentiation, but administration at inappropriate time points would have adverse effects on chondrogenesis. This study aims to develop an effective tissue engineering strategy by combining PTHrP and collagen-silk scaffold for osteochondral defect repair. The underlying mechanisms of the synergistic effect of combining PTHrP administration with collagen-silk scaffold implantation for rabbit knee joint osteochondral defect repair were investigated. In vitro studies showed that PTHrP treatment significantly reduced Alizarin Red staining and expression of terminal differentiation-related markers. This is achieved in part through blocking activation of the canonical Wnt/β-catenin signaling pathway. For the in vivo repair study, intra-articular injection of PTHrP was carried out at three different time windows (4–6, 7–9 and 10–12 weeks) together with implantation of a bi-layer collagen-silk scaffold. Defects treated with PTHrP at the 4–6 weeks time window exhibited better regeneration (reconstitution of cartilage and subchondral bone) with minimal terminal differentiation (hypertrophy, ossification and matrix degradation), as well as enhanced chondrogenesis (cell shape, Col2 and GAG accumulation) compared with treatment at other time windows. Furthermore, the timing of PTHrP administration also influenced PTHrP receptor expression, thus affecting the treatment outcome. Our results demonstrated that intra-articular injection of PTHrP at 4–6 weeks post-injury together with collagen-silk scaffold implantation is an effective strategy for inhibiting terminal differentiation and enhancing chondrogenesis, thus improving cartilage repair and regeneration in a rabbit model.

Toluquinol, a marine fungus metabolite, is a new angiosuppresor that interferes the Akt pathway

Toluquinol, a methylhydroquinone produced by a marine fungus, was selected in the course of a blind screening for new potential inhibitors of angiogenesis. In the present study we provide the first evidence that toluquinol is a new anti-angiogenic-compound. In a variety of experimental systems, representing the sequential events of the angiogenic process, toluquinol treatment of activated endothelial cells resulted in strong inhibitory effect. Toluquinol inhibited the growth of endothelial and tumor cells in culture in the micromolar range. Our results indicate that the observed growth inhibitory effect could be due, at least in part, to an induction of apoptosis. Toluquinol induced endothelial cell death is mediated via apoptosis after a cell cycle block and caspase activation. Capillary tube formation on Matrigel and migratory, invasive and proteolytic capabilities of endothelial cells were inhibited by addition of toluquinol at subtoxic concentrations. Inhibition of the mentioned essential steps of in vitro angiogenesis agrees with the observed inhibition of the in vivo angiogenesis, substantiated by using the chick chorioallatoic membrane assay and confirmed by the murine Matrigel plug, the zebrafish embryo neovascularization and the zebrafish caudal fin regeneration assays. Data here shown altogether indicate that toluquinol has antiangiogenic effects both in vitro and in vivo that are exerted partly by suppression of the VEGF and FGF-induced Akt activation of endothelial cells. These effects are carried out at lower concentrations to those required for other inhibitors of angiogenesis, what makes toluquinol a promising drug candidate for further evaluation in the treatment of cancer and other angiogenesis-related pathologies.

Controlling Natural Killer Cell Responses: Integration of Signals for Activation and Inhibition

Understanding how signals are integrated to control natural killer (NK) cell responsiveness in the absence of antigen-specific receptors has been a challenge, but recent work has revealed some underlying principles that govern NK cell responses. NK cells use an array of innate receptors to sense their environment and respond to alterations caused by infections, cellular stress, and transformation. No single activation receptor dominates; instead, synergistic signals from combinations of receptors are integrated to activate natural cytotoxicity and cytokine production. Inhibitory receptors for major histocompatibility complex class I (MHC-I) have a critical role in controlling NK cell responses and, paradoxically, in maintaining NK cells in a state of responsiveness to subsequent activation events, a process referred to as licensing. MHC-I-specific inhibitory receptors both block activation signals and trigger signals to phosphorylate and inactivate the small adaptor Crk. These different facets of inhibitory signaling are incorporated into a revocable license model for the reversible tuning of NK cell responsiveness.

Nuclear receptors and their selective pharmacologic modulators.

Nuclear receptors are ligand-activated transcription factors and include the receptors for steroid hormones, lipophilic vitamins, sterols, and bile acids. These receptors serve as targets for development of myriad drugs that target a range of disorders. Classically defined ligands that bind to the ligand-binding domain of nuclear receptors, whether they are endogenous or synthetic, either activate receptor activity (agonists) or block activation (antagonists) and due to the ability to alter activity of the receptors are often termed receptor "modulators." The complex pharmacology of nuclear receptors has provided a class of ligands distinct from these simple modulators where ligands display agonist/partial agonist/antagonist function in a tissue or gene selective manner. This class of ligands is defined as selective modulators. Here, we review the development and pharmacology of a range of selective nuclear receptor modulators.

A clinical feasibility study of atrial and ventricular electromechanical wave imaging

Cardiac resynchronization therapy (CRT) and atrial ablation procedures currently lack a noninvasive imaging modality for reliable treatment planning and monitoring. Electromechanical wave imaging (EWI) is an ultrasound-based method that has previously been shown to be capable of noninvasively and transmurally mapping the activation sequence of the heart in animal studies by estimating and imaging the electromechanical wave, that is, the transient strains occurring in response to the electrical activation, at both high temporal and spatial resolutions. To demonstrate the feasibility of transthoracic EWI for mapping the activation sequence during different cardiac rhythms in humans. EWI was perfor`med in patients undergoing CRT and a left bundle branch block (LBBB) during sinus rhythm, left ventricular pacing, and right ventricular pacing, as well as in patients with atrial flutter (AFL) before intervention, EWI findings from patients with AFL were subsequently correlated with results from invasive intracardiac electrical mapping studies during intervention. In addition, the feasibility of single-heartbeat EWI at 2000 frames/s is demonstrated in humans for the first time in a patient with both AFL and right bundle branch block (RBBB). The electromechanical activation maps demonstrated the capability of EWI to localize the pacing sites and characterize the bundle branch block activation sequence transmurally in patients with CRT. In patients with AFL, the EWI propagation patterns obtained with EWI were in excellent agreement with those obtained from invasive intracardiac mapping studies. Our findings demonstrate the potential capability of EWI to aid in the assessment and follow-up of patients undergoing CRT pacing therapy and atrial ablation, with preliminary validation in vivo.

Type 3 Muscarinic Receptors Modulate Macrophage Phenotype in the Presence of Th2 Cytokines

Purpose: Macrophages (Mφ) play a critical role in IBD. Resident Mφs within the intestinal lamina propria are exposed to many antigens, but are tolerant to stimulation and prevent inflammation (Heinsbroek & Gordon, 2009). We showed previously that stimulation of type 3 (M3R) muscarinic receptors (MR) encoded by Chrm3 promoted the development of pro-inflammatory classically activated Mφs (CAM) (McLean, et al, 2012). Whether M3R plays a role in the development of alternatively activated Mφs (AAM), which promote wound healing and tissue repair, is unclear. Our aim was to test the hypothesis that M3R plays a role in AAM development. Methods: Bone marrow cells were isolated from WT and Chrm3-/- mice and differentiated in the presence of monocyte colony stimulating factor (M-CSF) for 7 days. Flow cytometry was used to analyze cell populations. After 24 hour treatment with a muscarinic receptor-selective agonist, bethanechol (BTH), +/- the Th2 cytokine IL-4, cells were harvested and mRNA expression of the AAM marker Arg-1 assess by real-time PCR. Results: We confirmed that ˜95% of our cell population was positive for both CD11b and F4/80, indicating that these cells are Mφs. These Mφs express mRNA for M1R, M2R, M3R and M4R. In WT Mφs, BTH treatment significantly attenuated Arg-1 expression, indicating a constitutive bias against AAM development. Treatment of naïve WT Mφs with IL-4 significantly up-regulated Arg-1 expression; addition of BTH to IL-4 did not enhance this effect. In contrast, in Chrm3-/- Mφs up-regulation of Arg-1 in response to IL-4 was significantly greater than that observed in WT Mφs. Moreover, addition of BTH to IL-4 potentiated this up-regulation of Arg-1 in Chrm3-/- Mφs. Conclusion: Treatment with BTH alone attenuated the development of AAMs from naïve WT Mφs. As expected, treatment of WT Mφs with IL-4 induced the development of AAMs. In WT Mφs, addition of BTH to IL-4 did not modulate this effect. In M3R-deficient Mφs, IL-4 further polarized Mφs to the AAM phenotype. Notably, exposing M3R-deficient Mφs to both IL-4 & BTH further enhances AAM polarization. We previously reported that M3R expression exerts pro-inflammatory effects on Mφs. Our findings suggest that in the presence of IL-4, M3R activation attenuates the development of AAMs and, in the presence of IL-4, further polarizes M3R-deficient Mφs to an AAM phenotype, possibly by acting at M1R, M2R, and/or M4R. This effect is not observed in WT Mφs, likely due to M3R activity. These data further support a pro-inflammatory role of M3R. Pharmacologic attempts to manipulate Mφ phenotypes should focus on blocking M3R expression or activation to promote AAMs, a strategy that may reduce disease activity or maintain remission in IBD patients.

Interactions with WNK (With No Lysine) Family Members Regulate Oxidative Stress Response 1 and Ion Co-transporter Activity

Two of the four WNK (with no lysine (K)) protein kinases are associated with a heritable form of ion imbalance culminating in hypertension. WNK1 affects ion transport in part through activation of the closely related Ste20 family protein kinases oxidative stress-responsive 1 (OSR1) and STE20/SPS1-related proline-, alanine-rich kinase (SPAK). Once activated by WNK1, OSR1 and SPAK phosphorylate and stimulate the sodium, potassium, two chloride co-transporters, NKCC1 and NKCC2, and also affect other related ion co-transporters. We find that WNK1 and OSR1 co-localize on cytoplasmic puncta in HeLa and other cell types. We show that the C-terminal region of WNK1 including a coiled coil is sufficient to localize the fragment in a manner similar to the full-length protein, but some other fragments lacking this region are mislocalized. Photobleaching experiments indicate that both hypertonic and hypotonic conditions reduce the mobility of GFP-WNK1 in cells. The four WNK family members can phosphorylate the activation loop of OSR1 to increase its activity with similar kinetic constants. C-terminal fragments of WNK1 that contain three RFXV interaction motifs can bind OSR1, block activation of OSR1 by sorbitol, and prevent the OSR1-induced enhancement of ion co-transporter activity in cells, further supporting the conclusion that association with WNK1 is required for OSR1 activation and function at least in some contexts. C-terminal WNK1 fragments can be phosphorylated by OSR1, suggesting that OSR1 catalyzes feedback phosphorylation of WNK1.

Circulating VEGF levels as a biomarker to clinical benefit to PTC299, the first inhibitor of VEGF synthesis.

e13588 Background: Solid tumor growth is dependent on angiogenesis, a process mediated primarily by vascular endothelial growth factor (VEGF). Currently approved anti-angiogenic agents sequester VEGF or block activation of VEGF receptors. Despite extensive investigations, no reliable biomarkers have been identified for these agents. PTC299 is an investigational oral small molecule that differs from existing therapies by inhibiting VEGF synthesis at the translational level while conserving physiologic VEGF expression. Methods: A phase 1b clinical trial is evaluating PTC299 monotherapy (36 pts) or in combination with docetaxel (10 pts) from 0.3 mg/kg to 600 mg bid in patients with advanced incurable cancer. Serum VEGF was assessed at baseline and during therapy. The maximum on‑treatment reduction in VEGF at any post-baseline visit was used for a stratified analysis. Decreases in VEGF, defined as <10% (low) and ≥10% (high), identified the strata. Duration of therapy was used as a surrogate for stable disease in a Kaplan‑Meier analysis. Results: The study enrolled 46 patients (20 males; 26 females; median age: 62 years). Patients with lower VEGF reductions while on therapy (<10%, n=12) had a median duration of stable disease of 5.3 weeks while patients with greater maximal VEGF reductions (≥10%, n=31) had significantly longer stable disease (median 12.0 weeks, log-rank p<0.001). VEGF decreases were greater in patients with higher baseline values. No Grade 3 or 4 hypertension, bleeding, or proteinuria occurred. One patient had an acute liver injury. Conclusions: Reductions in circulating VEGF are associated with prolonged duration of anti-angiogenic therapy and stable disease. Results are consistent with preclinical experiments and support the hypothesis that PTC299 does not affect physiologic VEGF but inhibits pathologic VEGF. In this study, oral administration of PTC299 as monotherapy or in combination with docetaxel therapy has been generally well tolerated at all doses tested.

Simple regional strain pattern analysis to predict response to cardiac resynchronization therapy: Rationale, initial results, and advantages

A classical strain pattern of early contraction in one wall and prestretching of the opposing wall followed by late contraction has previously been associated with left bundle branch block (LBBB) activation and short-term response to cardiac resynchronization therapy (CRT). Aims of this study were to establish the long-term predictive value of an LBBB-related strain pattern and to identify changes in contraction patterns during short-term and long-term CRT. Sixty-seven patients with standard CRT criteria were prospectively enrolled between early 2009 and late 2010. Echocardiography including regional strain analysis by 2-dimensional speckle tracking was performed 1 week before implantation, at day 1, and 6 months after. Response was defined as a decrease in left ventricular end-systolic volume ≥ 15%. The predictive ability of a classical pattern was compared with time-to-peak measurements from velocity and deformation analysis. Forty-three patients (65%) were classified as responders. The presence of a classical pattern showed 91% specificity and 95% sensitivity for response and performed significantly better than time-to-peak parameters in prediction of response to CRT (P < .001, all). In responders, CRT acutely increased septal longitudinal peak systolic strain (-8.7% ± 3.6% to -11.1% ± 3%, P < .001) but not in nonresponders. The classical pattern is highly predictive of response to CRT and superior to time-to-peak methods. Patients who obtain long-term reverse remodeling are characterized by short-term reversal of the classical strain pattern. These findings emphasize the value of recognizing potentially reversible strain patterns in selection of CRT candidates.

Vitamin D Blocks Estrogen Signaling through PKA

The hormone estrogen (E2) activates a CaM KK, CaM KI, and ERK pathway to promote proliferation of MCF‐7 breast cancer cells. ERK activation is inhibited by cAMP and PKA in certain cells. PKA has numerous cellular substrates including CREB and CaM KK. PKA phosphorylation of CaM KK inhibits its activity and promotes its association with 14‐3‐3γ. Therefore hormones that stimulate PKA activation may in turn block activation of CaM KK and ERK as well as cell proliferation. Our goal was to evaluate PKA's ability to phosphorylate CaM KK and promote its subsequent binding to 14‐3‐3γ in Vitamin D‐treated cells. In addition we examined whether Vitamin D's inhibition of E2‐stimulated ERK activation in MCF‐7 cells utilized PKA using the inhibitor, H‐89. Our results suggest that activation of PKA with Vitamin D inhibits E2 stimulation of ERK activation and increases CaM KK phosphorylation. Vitamin D treatment of MCF‐7 cells, but not E2, increased CaM KK association with endogenous 14‐3‐ 3. Interestingly, Vitamin D induced phosphorylation of CaM KK and ERK inhibition were both reversed by H‐89. Our results suggest that Vitamin D activation of PKA triggers the subsequent phosphorylation and inhibition of CaM KK through its association with 14‐3‐3 preventing CaM KK from activating ERK in MCF‐7 cells.

Hypoxia Blocks Activation of JAK2V617F Through Suppression of SHP-2 Expression in MPN Cells

Abstract 126Oxygen tension regulates the function of hematopoietic stem cells (HSCs). The hypoxic microenvironment in bone marrow, known as the hypoxic niche, supports a quiescent state of HSCs and maintains long-term repopulating activity. In addition to normal HSCs, hypoxia also affects the fate of leukemic stem cells. Giuntoli et al. reported that hypoxia selects for cells with high-repopulating activity in BCR-ABL-positive leukemia cell lines that have reduced sensitivity to imatinib. The V617F-activating mutation in JAK2 plays a major role in BCR-ABL-negative myeloproliferative neoplasms (MPN). JAK2V617F activates a variety of signal transduction molecules and induces cytokine-independent growth while suppressing apoptosis in HSCs.In this study, we investigated the effects of hypoxia on JAK2V617F-positive cells. To this end, we used JAK2V617F-harboring human leukemia cell lines, HEL and SET-2. We found that culturing under hypoxic conditions (1% O2) significantly suppressed growth of these cells. Interestingly, we found that hypoxia reduced the autophosphorylation of JAK2V617F. Phosphorylation of STAT5, a major downstream target of JAK2V617F, was also highly suppressed in the hypoxic condition. Furthermore, expression of several target genes of STAT5, including cyclin D and Bcl-xL, was drastically decreased after exposure to hypoxia. For further study, after obtaining informed consent, we performed an endogenous erythroid colony (EEC) formation assay on peripheral blood mononuclear cells from JAK2V617F-positive polycythemia vera patients (n=7). We found that hypoxia suppressed EEC formation (Normoxia: 43.8±10.0; Hypoxia: 25.9±4.7, P < 0.05) and suppressed phosphorylation of JAK2 in these cells. Next, we investigated the molecular mechanisms of how hypoxia suppressed activity of JAK2V617F. To elucidate these mechanisms, we initially determined whether hypoxia induced the expression of several negative regulators for JAK2. Hypoxia did not change protein levels of SOCS1 or SOCS3, suppressors of the cytokine signaling (SOCS) family of proteins. We also confirmed that the expression of the tyrosine phosphatases PTP1B and CD45, which could dephosphorylate JAK2, were not altered in hypoxia-treated cells. In contrast, the expression of SHP-2, an SH-2 domain-containing protein tyrosine phosphatase, was drastically diminished after exposure to hypoxia in both HEL and SET2 cells. Importantly, hypoxia also suppressed the expression of SHP-2 proteins in peripheral blood mononuclear cells derived from polycythemia vera patients. To confirm that SHP-2 is required for activation of JAK2V617F, we treated HEL and SET-2 cells with an inhibitor of SHP-2. The inhibitor clearly diminished auto-phosphorylation of JAK2V617F in both cell lines. Our observations show that hypoxia suppressed activity of JAK2V617F and induced growth arrest in JAK2V617F-positive MPN cells. SHP-2 plays important roles in these processes. Interestingly, several studies have reported that SHP-2 is required for the activation of JAK2 by cytokines. In addition, recent studies have revealed that the activity of SHP-2 is increased in platelets from MPN patients. In conclusion, a hypoxic environment may modulate the fate of JAK2-positive MPN cells through suppression of SHP-2 levels and through subsequent suppression of JAK2V617F activity. Disclosures:No relevant conflicts of interest to declare.

Circulation: Clinical Summaries

Circulation: Clinical Summaries

Roles of the Polycomb group proteins in stem cells and cancer

Polycomb group proteins have long been linked to the occurrence of different forms of cancer. Polycomb proteins form at least two distinct complexes, the Polycomb-repressive complexes 1 and 2 (PRC1 and PRC2). Some of the PRC complex subunits have been found to be overexpressed in a variety of different tumors. Epigenetic perturbations are likely to be the cause for transcriptional misregulation of tumor suppressor genes and of certain cell fates. It is especially critical for stem cells that their potential to self-renewal and to differentiate is tightly controlled and properly orchestrated. Misregulation of Polycomb protein levels often leads to either a block or unscheduled activation of developmental pathways, thereby enhancing the proliferation capability of a cell. The consequences of this misregulation have been linked to the establishment of cancer stem cells, which can produce tumors through a combination of increased self-renewal and the lack of complete cellular differentiation. Cancer stem cells are believed to persist within tumors and to elicit relapse and metastasis. In this review, we recapitulate the roles of Polycomb proteins in stem cell biology, and the impact their misregulation can have on cancer.

A Novel Role for Bruton's Tyrosine Kinase in Hepatocyte Growth Factor-mediated Immunoregulation of Dendritic Cells

The limited success of dendritic cell (DC)-based immunotherapy in multiple myeloma is partly due to hepatocyte growth factor (HGF)-induced DC dysfunction. From a therapeutic standpoint, it is important to understand the molecular events involved in inhibition of DC activation/maturation by HGF. Because Bruton's tyrosine kinase (Btk) negatively regulates maturation and immunostimulatory function of DCs, a role for Btk in HGF-induced inhibition of both murine and human DCs was investigated. We demonstrate that Btk is a novel proximal component of HGF-induced c-MET (HGF receptor) signaling. Following HGF treatment, Btk binds to c-MET and becomes activated. Btk activation in turn blocks the NF-κB pathway and subsequent DC activation via the c-Src-PI3K-AKT-mammalian target of rapamycin (mTOR) pathway. Notably, Btk activation is necessary for HGF-induced association of c-Src and PI3K with c-MET. Furthermore, we provide the first evidence that HGF inhibits DC activation by inducing autocrine interleukin (IL)-10 secretion, which requires activation of Btk. Blocking activation of Btk and its downstream the c-Src-PI3K-AKT-mTOR pathway prevents HGF-induced IL-10 secretion by DCs. In addition, neutralization of IL-10 secretion from DCs impaired the inhibitory effect of HGF on DCs. Thus, our study identifies a novel role for Btk in HGF-induced DC inhibition.

Truncated ErbB2 Expressed in Tumor Cell Nuclei Contributes to Acquired Therapeutic Resistance to ErbB2 Kinase Inhibitors

ErbB2 tyrosine kinase inhibitors (TKI) block tyrosine autophosphorylation and activation of the full-length transmembrane ErbB2 receptor (p185(ErbB2)). In addition to p185(ErbB2), truncated forms of ErbB2 exist in breast cancer cell lines and clinical tumors. The contribution of these truncated forms, specifically those expressed in tumor cell nuclei, to the development of therapeutic resistance to ErbB2 TKIs has not been previously shown. Here, we show that expression of a 95-kDa tyrosine phosphorylated form of ErbB2, herein referred to as p95L (lapatinib-induced p95) was increased in ErbB2(+) breast cancer cells treated with potent ErbB2 TKIs (lapatinib, GW2974). Expressed in tumor cell nuclei, tyrosine phosphorylation of p95L was resistant to inhibition by ErbB2 TKIs. Furthermore, the expression of p95L was increased in ErbB2(+) breast cancer models of acquired therapeutic resistance to lapatinib that mimic the clinical setting. Pretreatment with proteasome inhibitors blocked p95L induction in response to ErbB2 TKIs, implicating the role of the proteasome in the regulation of p95L expression. In addition, tyrosine phosphorylated C-terminal fragments of ErbB2, generated by alternate initiation of translation and similar in molecular weight to p95L, were expressed in tumor cell nuclei, where they too were resistant to inhibition by ErbB2 TKIs. When expressed in the nuclei of lapatinib-sensitive ErbB2(+) breast cancer cells, truncated ErbB2 rendered cells resistant to lapatinib-induced apoptosis. Elucidating the function of nuclear, truncated forms of ErbB2, and developing therapeutic strategies to block their expression and/or activation may enhance the clinical efficacy of ErbB2 TKIs.

Hepatocyte growth factor-induced c-Src-phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway inhibits dendritic cell activation by blocking IκB kinase activity

Hepatocyte growth factor modulates activation and antigen-presenting cell function of dendritic cells. However, the molecular basis for immunoregulation of dendritic cells by hepatocyte growth factor is undefined. In the current study, we demonstrate that hepatocyte growth factor exhibits inhibitory effect on dendritic cell activation by blocking IκB kinase activity and subsequent nuclear factor-κB activation. Inhibition of IκB kinase is mediated by hepatocyte growth factor-induced activation of c-Src. Proximal signaling events induced in dendritic cells by hepatocyte growth factor include a physical association of c-Src with the hepatocyte growth factor receptor c-MET and concomitant activation of c-Src. Activation of c-Src in turn establishes a complex consisting of phosphatidylinositol 3-kinase and c-MET, and promotes downstream activation of the phosphatidylinositol 3-kinase/AKT pathway and mammalian target of rapamycin. Blocking activation of c-Src, phosphatidylinositol 3-kinase and mammalian target of rapamycin prevents hepatocyte growth factor-induced inhibition of IκB kinase, nuclear factor-κB and dendritic cell activation. Notably, hepatocyte growth factor-stimulated c-Src activation results in induction of phosphatidylinositol 3-kinase complexes p85α/p110α and p85α/p110δ, which is required for activation of mammalian target of rapamycin, and consequent inhibition of IκB kinase and nuclear factor-κB activation. Our findings, for the first time, have identified the c-Src-phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway that plays a pivotal role in mediating the inhibitory effects of hepatocyte growth factor on dendritic cell activation by blocking nuclear factor-κB signaling.