USP18 promotes cell proliferation and suppressed apoptosis in cervical cancer cells via activating AKT signaling pathway.

BACKGROUND: The deubiquitinating (DUB) enzyme ubiquitin-specific protease 18 (USP18), also known as UBP43, is an ubiquitin-specific protease linked to several human malignancies. However, USP18's underlying function in human cervical cancer remains unclear. In the current study, we aimed to analyse the role of USP18 and its signalling pathways in cervical cancer. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemical staining were performed to analyse USP18 levels in cervical cancer and matched to adjacent normal tissues. Moreover, RNA interference (RNAi) and lentiviral-mediated vector transfections were performed to silence and overexpress USP18, respectively, in cervical cancer cells. Further, Cell Counting Kit-8 (CCK-8) and Annexin V/PI staining assays were used to assess its biological function in cell proliferation and apoptosis, respectively. A xenograft model was used to examine USP18's function in vivo. RESULTS: The present findings demonstrated that USP18 was overexpressed in cervical cancer specimens and cell lines. Silencing USP18 in SiHa and Caski cervical cancer cell lines inhibited cell proliferation, induced apoptosis, and promoted cleaved caspase-3 expression. In contrast, USP18 overexpression showed the opposite effects in human HcerEpic cells. A Gene Set Enrichment Analysis revealed that USP18 was enriched in the PI3K/AKT signalling pathway in cervical cancer. Hence, the PI3K/AKT inhibitor LY294002 was used to determine the relationship between USP18 and AKT in cervical cancer cells. Importantly, LY294002 significantly abolished the effects of USP18 overexpression in cervical cancer cells. In vivo, USP18 silencing inhibited human cervical cancer cells' tumorigenicity. CONCLUSIONS: The current study indicates that USP18 is an oncogenic gene in cervical cancer. Our findings not only deepened the understanding of USP18's biological function in cervical cancer pathogenesis, but we also provided novel insight for cervical cancer therapy. TRIAL REGISTRATION: Retrospectively registered.

ZNF479 downregulates metallothionein-1 expression by regulating ASH2L and DNMT1 in hepatocellular carcinoma.

Decreased expression of metallothionein-1 (MT-1) is associated with a poor prognosis in hepatocellular carcinoma (HCC). Here, we found that MT-1 expression was suppressed by 14-3-3ε, and MT-1 overexpression abolished 14-3-3ε-induced cell proliferation and tumor growth. We identified that 14-3-3ε induced expression of ZNF479, a novel potential transcriptional regulator by gene expression profiling and ZNF479 contributed to 14-3-3ε-suppressed MT-1 expression. ZNF479 induced the expression of DNMT1, UHRF1, and mixed-lineage leukemia (MLL) complex proteins (ASH2L and Menin), and increased tri-methylated histone H3 (H3K4me3) levels, but suppressed H3K4 (H3K4me2) di-methylation. ZNF479-suppressed MT-1 expression was restored by silencing of ASH2L and DNMT1. Furthermore, ZNF479 expression was higher in HCC tissues than that in the non-cancerous tissues. Expression analyses revealed a positive correlation between the expression of ZNF479 and DNMT1, UHRF1, ASH2L, and Menin, and an inverse correlation with that of ZNF479, ASH2L, Menin, and MT-1 isoforms. Moreover, correlations between the expression of ZNF479 and its downstream factors were more pronounced in HCC patients with hepatitis B. Here, we found that ZNF479 regulates MT-1 expression by modulating ASH2L in HCC. Approaches that target ZNF479/MLL complex/MT-1 or related epigenetic regulatory factors are potential therapeutic strategies for HCC.

The comparison between dual inhibition of mTOR with MAPK and PI3K signaling pathways in KRAS mutant NSCLC cell lines.

KRAS mutations are found in 15-25 % of patients with lung adenocarcinoma, and they lead to constitutive activation of KRAS signaling pathway that results in sustained cell proliferation. Currently, there are no direct anti-KRAS therapies available. Therefore, it is rational to target the downstream molecules of KRAS signaling pathway, which are mitogen-activated protein kinase (MAPK) signaling pathway (RAF-MEK-ERK) and PI3K pathway (PI3K-AKT-mTOR). Here, we examined the inhibition of both these pathways alone and in combination and analyzed the anti-proliferative and apoptotic events in KRAS mutant NSCLC cell lines, A549 and Calu-1. Cytotoxicity was determined by MTT assay after the cells were treated with LY294002 (PI3K inhibitor), U0126 (MEK inhibitor), and RAD001 (mTOR inhibitor) for 24 and 48 h. The expression levels of p-ERK, ERK, AKT, p-AKT, p53, cyclinD1, c-myc, p27(kip1), BAX, BIM, and GAPDH were detected by western blot after 6 and 24 h treatment. Although PI3K/mTOR inhibition is more effective in cytotoxicity in A549 and Calu-1 cells, MEK/mTOR inhibition markedly decreases cell proliferation protein marker expressions. Our data show that combined targeting of MEK and PI3K-AKT with mTOR is a better option than single agents alone for KRAS mutant NSCLC, thus opening the possibility of a beneficial treatment strategy in the future.

[New therapeutical strategies in metastatic hormone-dependent breast cancer]. / Nouvelles stratégies thérapeutiques dans le cancer du sein hormono-dépendant métastatique.

Hormone-dependent breast cancer is the first example of cancer treated by targeted therapy for more than 30 years. Blocking estrogen pathway was the first therapeutical strategy for this subtype of breast cancer, and remains the principle of current standard treatment. Despite the efficacy of drugs used in endocrine therapy, hormone resistance is a major problem for the management of patients with hormone-dependent breast cancer. In this review, we will discuss the development of strategies targeting the PI3K/Akt/mTOR pathway, CDK4/6 (Cyclin Dependent Kinase 4/6) and FGFR (Fibroblast Growth Factor Receptor) in hormone-dependent metastatic breast cancer (ER+). Recent results of clinical trials showed that combination of endocrine therapy with such pharmacological inhibitors is a promising strategy to overcome endocrine resistance. Mutated forms and isoforms of ERα have been recently discovered and its targeting could represent an therapeutic alternative. Future progress will focus on the identification of new compounds and combinations with other targeted therapies to improve the efficacy of such inhibitors in clinical practice.

Adaptive mitochondrial reprogramming and resistance to PI3K therapy.

BACKGROUND: Small molecule inhibitors of phosphatidylinositol-3 kinase (PI3K) have been developed as molecular therapy for cancer, but their efficacy in the clinic is modest, hampered by resistance mechanisms. METHODS: We studied the effect of PI3K therapy in patient-derived tumor organotypic cultures (from five patient samples), three glioblastoma (GBM) tumor cell lines, and an intracranial model of glioblastoma in immunocompromised mice (n = 4-5 mice per group). Mechanisms of therapy-induced tumor reprogramming were investigated in a global metabolomics screening, analysis of mitochondrial bioenergetics and cell death, and modulation of protein phosphorylation. A high-throughput drug screening was used to identify novel preclinical combination therapies with PI3K inhibitors, and combination synergy experiments were performed. All statistical methods were two-sided. RESULTS: PI3K therapy induces global metabolic reprogramming in tumors and promotes the recruitment of an active pool of the Ser/Thr kinase, Akt2 to mitochondria. In turn, mitochondrial Akt2 phosphorylates Ser31 in cyclophilin D (CypD), a regulator of organelle functions. Akt2-phosphorylated CypD supports mitochondrial bioenergetics and opposes tumor cell death, conferring resistance to PI3K therapy. The combination of a small-molecule antagonist of CypD protein folding currently in preclinical development, Gamitrinib, plus PI3K inhibitors (PI3Ki) reverses this adaptive response, produces synergistic anticancer activity by inducing mitochondrial apoptosis, and extends animal survival in a GBM model (vehicle: median survival = 28.5 days; Gamitrinib+PI3Ki: median survival = 40 days, P = .003), compared with single-agent treatment (PI3Ki: median survival = 32 days, P = .02; Gamitrinib: median survival = 35 days, P = .008 by two-sided unpaired t test). CONCLUSIONS: Small-molecule PI3K antagonists promote drug resistance by repurposing mitochondrial functions in bioenergetics and cell survival. Novel combination therapies that target mitochondrial adaptation can dramatically improve on the efficacy of PI3K therapy in the clinic.

Vulnerabilities of PTEN-TP53-deficient prostate cancers to compound PARP-PI3K inhibition.

UNLABELLED: Prostate cancer is the most prevalent cancer in males, and treatment options are limited for advanced forms of the disease. Loss of the PTEN and TP53 tumor suppressor genes is commonly observed in prostate cancer, whereas their compound loss is often observed in advanced prostate cancer. Here, we show that PARP inhibition triggers a p53-dependent cellular senescence in a PTEN-deficient setting in the prostate. Surprisingly, we also find that PARP-induced cellular senescence is morphed into an apoptotic response upon compound loss of PTEN and p53. We further show that superactivation of the prosurvival PI3K-AKT signaling pathway limits the efficacy of a PARP single-agent treatment, and that PARP and PI3K inhibitors effectively synergize to suppress tumorigenesis in human prostate cancer cell lines and in a Pten/Trp53-deficient mouse model of advanced prostate cancer. Our findings, therefore, identify a combinatorial treatment with PARP and PI3K inhibitors as an effective option for PTEN-deficient prostate cancer. SIGNIFICANCE: The paucity of therapeutic options in advanced prostate cancer displays an urgent need for the preclinical assessment of novel therapeutic strategies. We identified differential therapeutic vulnerabilities that emerge upon the loss of both PTEN and p53, and observed that combined inhibition of PARP and PI3K provides increased efficacy in hormone-insensitive advanced prostate cancer.

HIV-1 Nef induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors.

The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes.

Noninfectious pneumonitis with the use of mTOR inhibitors in breast cancer.

The mammalian target of rapamycin (mTOR) inhibitor class of drugs represents the newest addition to the armamentarium of therapies for hormonally driven breast cancer. It has recently been shown that the addition of mTOR inhibitor everolimus to aromatase inhibitors in hormone receptor-positive breast cancers improves progression-free survival. However, a clinically significant toxicity associated with this class of drugs is the development of noninfectious pneumonitis (NIP). Although generally mild and manageable, everolimus-induced NIP requires prompt diagnosis and management. This article will provide a brief overview of data relating to dysregulation of the phosphatidylinositol-3-kinase/protein kinase B/mTOR pathway in breast cancer; review the literature relating to the efficacy and safety of mTOR inhibitors in breast cancer; and evaluate the incidence, severity, and optimal management of mTOR inhibitor-related NIP in breast cancer.

Exploiting the head and neck cancer oncogenome: widespread PI3K-mTOR pathway alterations and novel molecular targets.

SUMMARY: Two studies published in this issue of Cancer Discovery describe the emerging mutational landscape of head and neck squamous cell carcinomas (HNSCC) and their genomic and epigenetic alterations, thus identifying novel actionable cancer drivers and predictive biomarkers for targeted therapies. Most genomic alterations in HNSCC converge in a handful of molecular pathways, resulting in cell-cycle deregulation, genomic instability, cell differentiation defects, and persistent mitogenic signaling, the latter involving aberrant phosphoinositide 3-kinase (PI3K)/mTOR pathway activation, thereby rendering HNSCC responsive to PI3K/mTOR inhibitors. Cancer Discov; 3(7); 722-5. ©2013 AACR.

Cap-translation inhibitor, 4EGI-1, restores sensitivity to ABT-737 apoptosis through cap-dependent and -independent mechanisms in chronic lymphocytic leukemia.

PURPOSE: The lymph node microenvironment promotes resistance to chemotherapy in chronic lymphocytic leukemia (CLL), partly through induction of BCL2 family prosurvival proteins. Currently available inhibitors do not target all BCL2 family prosurvival proteins and their effectiveness is also modified by proapoptotic BCL2 homology domain 3 (BH3) only protein expression. The goal of this study was to evaluate synergy between the eIF4E/eIF4G interaction inhibitor, 4EGI-1, and the BH3 mimetic, ABT-737. EXPERIMENTAL DESIGN: CLL cells were cultured in conditions to mimic the lymph node microenvironment. Protein synthesis and cap-complex formation were determined. Polysome association of mRNAs from BCL2 family survival genes was analyzed by translational profiling. The effects of 4EGI-1 and the BCL2/BCL2L1 antagonist, ABT-737, on CLL cell apoptosis were determined. RESULTS: Protein synthesis was increased approximately 6-fold by stromal cell/CD154 culture in a phosphoinositide 3-kinase α (PI3Kα)-specific manner and was reduced by 4EGI-1. PI3K inhibitors and 4EGI-1 also reduced cap-complex formation but only 4EGI-1 consistently reduced BCL2L1 and BCL2A1 protein levels. 4EGI-1, but not PI3K inhibitors or rapamycin, induced an endoplasmic reticulum stress response including proapoptotic NOXA and the translation inhibitor phosphorylated eIF2α. 4EGI-1 and ABT-737 synergized to cause apoptosis, independent of levels of prosurvival protein expression in individual patients. CONCLUSIONS: Overall protein synthesis and cap-complex formation are induced by microenvironment stimuli in CLL. Inhibition of the cap-complex was not sufficient to repress BCL2 family prosurvival expression, but 4EGI-1 inhibited BCL2A1 and BCL2L1 while inducing NOXA through cap-dependent and -independent mechanisms. 4EGI-1 and ABT-737 synergized to produce apoptosis, and these agents may be the basis for a therapeutically useful combination.

Perifosine downregulates MDR1 gene expression and reverses multidrug-resistant phenotype by inhibiting PI3K/Akt/NF-κB signaling pathway in a human breast cancer cell line.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is the major clinical impediment to chemotherapy of breast cancers. Down-regulation of PI3K/Akt pathway has been described as related to reversal of MDR in cancer cells. Here, we investigated the reversal effect on MDR phenotype of perifosine, a new Akt inhibitor, in breast cancer cell lines. In this study, MCF-7/ADM cells and MCF-7 cells were treated with different concentrations of perifosine. Our results suggested that perifosine reversed MDR partially by downregulation of P-gp expression and inhibition of PI3K/Akt/NF-κB pathway in the MCF-7/ADM cell line. The novel Akt inhibitor perifosine may be a promising new drug due to its ability to reverse MDR in human breast cancer cells.

PI3K-independent AKT activation in cancers: a treasure trove for novel therapeutics.

AKT/PKB serine threonine kinase, a critical signaling molecule promoting cell growth and survival pathways, is frequently dysregulated in many cancers. Although phosphatidylinositol-3-OH kinase (PI3K), a lipid kinase, is well characterized as a major regulator of AKT activation in response to a variety of ligands, recent studies highlight a diverse group of tyrosine (Ack1/TNK2, Src, PTK6) and serine/threonine (TBK1, IKBKE, DNAPKcs) kinases that activate AKT directly to promote its pro-proliferative signaling functions. While some of these alternate AKT activating kinases respond to growth factors, others respond to inflammatory and genotoxic stimuli. A common theme emerging from these studies is that aberrant or hyperactivation of these alternate kinases is often associated with malignancy. Consequently, evaluating the use of small molecular inhibitors against these alternate AKT activating kinases at earlier stages of cancer therapy may overcome the pressing problem of drug resistance surfacing especially in patients treated with PI3K inhibitors.

The susceptibility of trypanosomatid pathogens to PI3/mTOR kinase inhibitors affords a new opportunity for drug repurposing.

BACKGROUND: Target repurposing utilizes knowledge of "druggable" targets obtained in one organism and exploits this information to pursue new potential drug targets in other organisms. Here we describe such studies to evaluate whether inhibitors targeting the kinase domain of the mammalian Target of Rapamycin (mTOR) and human phosphoinositide-3-kinases (PI3Ks) show promise against the kinetoplastid parasites Trypanosoma brucei, T. cruzi, Leishmania major, and L. donovani. The genomes of trypanosomatids encode at least 12 proteins belonging to the PI3K protein superfamily, some of which are unique to parasites. Moreover, the shared PI3Ks differ greatly in sequence from those of the human host, thereby providing opportunities for selective inhibition. METHODOLOGY/PRINCIPAL FINDINGS: We focused on 8 inhibitors targeting mTOR and/or PI3Ks selected from various stages of pre-clinical and clinical development, and tested them against in vitro parasite cultures and in vivo models of infection. Several inhibitors showed micromolar or better efficacy against these organisms in culture. One compound, NVP-BEZ235, displayed sub-nanomolar potency, efficacy against cultured parasites, and an ability to clear parasitemia in an animal model of T. brucei rhodesiense infection. CONCLUSIONS/SIGNIFICANCE: These studies strongly suggest that mammalian PI3/TOR kinase inhibitors are a productive starting point for anti-trypanosomal drug discovery. Our data suggest that NVP-BEZ235, an advanced clinical candidate against solid tumors, merits further investigation as an agent for treating African sleeping sickness.

Effect of hyperglycemia on human monocyte activation.

Our recent study defined the chemokine-induced human monocyte signaling under normoglycemic condition. To explore the hyperglycemia-induced monocyte signaling, we performed adhesion, migration, and transmigration assays on human monocytes obtained from THP-1 cell line in the presence of normal (5 mM) and high (10 and 20 mM) glucose concentrations without chemokines. We observed augmented (P < 0.01) monocyte adhesion to human umbilical vein endothelial cell monolayer at 10 than 5 mM glucose with no further increase at 20-mM glucose concentration (P < 0.07 vs 10 mM; P < 0.01 vs 5 mM). But incremental increases in monocyte migration (P < 0.01), transmigration (P < 0.01), and stress fiber response (P < 0.01) were observed at 10- and 20-mM glucose concentrations in comparison to 5-mM glucose concentrations. We found gradational increase (P < 0.01) in phosphorylation of Akt(S473) and glycogen synthase kinase (GSK3ß(S9)) in hyperglycemia (10 and 20 mM) when compared with 5 mM glucose. Furthermore, hyperglycemia (both 10 and 20 mM)-treated monocyte showed up-regulated phosphorylation of p101 and p110γ subunits of PI-3 kinase in comparison to 5 mM glucose. Hyperglycemia-induced monocyte migration was restored to basal levels in the presence of PI-3 kinase inhibitor, LY. These observations imply that modest hyperglycemia per se, as is commonly observed in diabetic individuals, is a potent stimulator of monocyte activity even without chemokines.

Inhibition of CK2{alpha} and PI3K/Akt synergistically induces apoptosis of CD34+CD38- leukaemia cells while sparing haematopoietic stem cells.

BACKGROUND/AIM: The CD34(+)CD38(-) leukaemia cell population contains leukaemia stem cells (LSCs) responsible for treatment failure in acute myeloid leukaemia (AML) and, thus, novel therapies are required to eradicate LSCs without harming healthy haematopoietic stem cells (HSC). MATERIALS AND METHODS: The present study evaluated the effects of co-treatment with LY294002 (a PI3K/Akt inhibitor) and apigenin (a CK2 inhibitor) (LY/Api) at subtoxic concentrations on leukaemia cell lines and primary AML cells. RESULTS: LY/Api synergistically induced apoptosis in leukaemia cells, especially CD34(+)CD38(-) leukaemia cells. However, these effects were negligible in HSCs. LY/Api-induced apoptosis was accompanied by activation of caspase cascades and disruption of mitochondrial membrane potential. Caspase inhibitor or Akt overexpression abrogated this synergistic induction in apoptosis by LY/Api. LY/Api also led to remarkable down-regulation of anti-apoptotic proteins including Bcl-xL and NF-κB in CD34(+)CD38(-) leukaemia cells, but not in healthy hematopoietic stem cells. CONCLUSION: Inhibition of both CK2 and PI3K/Akt pathways may be a promising LSCs-targeted therapeutic strategy for AML.

Involvement of the PI3K/Akt pathway in estrogen-mediated regulation of human CYP7B1: identification of CYP7B1 as a novel target for PI3K/Akt and MAPK signalling.

The steroid hydroxylase CYP7B1 metabolizes neurosteroids, cholesterol derivatives, and estrogen receptor (ER) ligands. Previous studies identified CYP7B1 as a target for regulation by estrogen. The present study examines the mechanism for estrogen-mediated regulation of the human CYP7B1 gene promoter. Treatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), abolished ER-mediated up-regulation of a CYP7B1 promoter-luciferase reporter in HepG2 cells, whereas overexpression of PI3K or Akt significantly increased estrogenic up-regulation of CYP7B1. Overexpression of dominant-negative mutant Akt abolished ER-mediated stimulation of CYP7B1 in HepG2 cells. Data indicated no binding of ER to CYP7B1 promoter sequences, suggesting that ER interacts with the PI3K/Akt pathway without binding to the gene. At low ER levels, overexpression of Akt suppressed CYP7B1 promoter activity, suggesting that its effect on CYP7B1 is different when estrogens are absent. In HEK293 cells, CYP7B1 transcription was much less affected by Akt, indicating that the mechanism for up-regulation of CYP7B1 is different in different cell types. Other experiments indicated that MAPK signalling may affect basal CYP7B1 levels. The current results, indicating that regulation of CYP7B1 by ER can be mediated via the PI3K/Akt signal pathway, a regulatory pathway important for cellular survival and growth, suggest an important role for CYP7B1 in cellular growth, particularly in connection with estrogenic signalling.

Implication of NAG-1 in synergistic induction of apoptosis by combined treatment of sodium salicylate and PI3K/MEK1/2 inhibitors in A549 human lung adenocarcinoma cells.

Aspirin is used as chemopreventive agents in a variety of human cancer cells including those of colon, lung, breast, and leukemia. Sodium salicylate (NaSal, the natural deacetylated form of aspirin) induced cell cycle arrest and apoptosis in a dose-dependent manner in A549 cells; high dose (20mM) of NaSal-induced apoptosis, whereas low dose (2-10mM) induced cell cycle arrest. We found that NaSal-activated Akt/PKB, ERK1/2, and p38MAPK signal cascades. Twenty micromolar of NaSal-induced apoptotic response of A549 cells was enhanced by the PI3K inhibitors (LY294002 and wortmannin) and in a less extent by the MEK1/2 inhibitors (U0126 and PD98059), whereas it was suppressed by the p38MAPK inhibitor (SB203580). Furthermore, simultaneous inhibition of the Akt/PKB and ERK1/2 signal cascades could lower the dose of NaSal to induce apoptosis to 2mM in A549 lung cancer cells. Similar enhancement was observed in cells treated with 2mM NaSal and 100muM genistein, an inhibitor of receptor tyrosine kinases (RTKs) that are upstream of PI3K and MEK1/2 signaling. We further demonstrated that NAG-1 plays a key role in apoptosis by NaSal-based combined treatment. Collectively, our findings indicate that inhibition of the pro-survival Akt/PKB and ERK1/2 signaling may increase the chemopreventive effects of NaSal and combined treatment of two natural compounds (NaSal and genistein) results in a highly synergistic induction of apoptosis, thereby increasing the chemopreventive effects of NaSal against cancer.

Neuropeptides activate human mast cell degranulation and chemokine production.

During neuronal-induced inflammation, mast cells may respond to stimuli such as neuropeptides in an FcepsilonRI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to immunoglobulin E (IgE)/anti-IgE and compound 48/80. Primary cultured mast cells, generated from CD34(+) progenitors in the presence of stem cell factor and interleukin-6 (IL-6), and human cultured mast cells (LAD2) were stimulated with these and other stimuli (gastrin, concanavalin A, radiocontrast media, and mannitol) and their degranulation and chemokine production was assessed. VIP and SP stimulated primary human mast cells and LAD cells to degranulate; gastrin, concanavalin A, radiocontrast media, mannitol, CGRP and NGF did not activate degranulation. While anti-IgE stimulation did not induce significant production of chemokines, stimulation with VIP, SP or compound 48/80 potently induced production of monocyte chemoattractant protein-1, inducible protein-10, monokine induced by interferon-gamma (MIG), RANTES (regulated on activation, normal, T-cell expressed, and secreted) and IL-8. VIP, SP and compound 48/80 also activated release of tumour necrosis factor, IL-3 and granulocyte-macrophage colony-stimulating factor, but not IL-4, interferon-gamma or eotaxin. Human mast cells expressed surface neurokinin 1 receptor (NK1R), NK2R, NK3R and VIP receptor type 2 (VPAC2) but not VPAC1 and activation of human mast cells by IgE/anti-IgE up-regulated expression of VPAC2, NK2R, and NK3R. These studies demonstrate the pattern of receptor expression and activation of mast cell by a host of G-protein coupled receptor ligands and suggest that SP and VIP activate a unique signalling pathway in human mast cells. These results are likely to have direct relevance to neuronally induced inflammatory diseases.

Shp2E76K mutant confers cytokine-independent survival of TF-1 myeloid cells by up-regulating Bcl-XL.

Shp2 has been known to mediate growth factor-stimulated cell proliferation, but its role in cell survival is less clear. Gain-of-function Shp2 mutants such as Shp2E76K are associated with myeloid leukemias. We found that Shp2E76K could transform cytokine-dependent human TF-1 myeloid cells into cytokine independence and further characterized the Shp2E76K-induced cell survival mechanism in this study. Expression of Shp2E76K suppressed the cytokine withdrawal-induced intrinsic/mitochondrial apoptosis pathway, which is controlled by the Bcl-2 family proteins. Analysis of Bcl-2 family proteins showed that Bcl-XL and Mcl-1 were up-regulated in Shp2E76K-transformed TF-1 (TF-1/Shp2E76K) cells. Knockdown of Bcl-XL but not Mcl-1 with short hairpin RNAs prevented Shp2E76K-induced cytokine-independent survival. Roscovitine, which down-regulated Mcl-1, also did not prevent cytokine-independent survival of TF-1/Shp2E76K cells, whereas the Bcl-XL inhibitor HA14-1 did. Ras and mitogen-activated protein kinases Erk1 and Erk2 (Erk1/2) were constitutively activated in TF-1/Shp2E76K cells, whereas little active Akt was detected under cytokine-free conditions. Shp2E76K-induced Bcl-XL expression was suppressed by Mek inhibitors and by a dominant-negative Mek1 mutant but not by the phosphoinositide 3-phosphate inhibitor LY294002 and the Akt inhibitor API-2. Inhibition of Erk1/2 blocked cytokine-independent survival of TF-1/Shp2E76K cells, whereas inhibition of Akt had a minimal effect on cytokine-independent survival of TF-1/Shp2E76K cells. These results show that Shp2E76K can evoke constitutive Erk1/2 activation in TF-1 cells. Furthermore, Shp2E76K induces cytokine-independent survival of TF-1 cells by a novel mechanism involving up-regulation of Bcl-XL through the Erk1/2 pathway.