SHIP1 Protein Research Articles

Activated Src kinases downstream of BCR-ABL and Flt3 induces proteasomal degradation of SHIP1 by phosphorylation of tyrosine 1021

Within the various subtypes of ALL, patients with a BCR-ABL-positive background as well as with a genetic change in the KMT2A gene have by far the worst survival probabilities. Interestingly, both subtypes are characterized by highly activated tyrosine kinases. SHIP1 serves as an important negative regulator of the PI3K/AKT signaling pathway, which is often constitutively activated in ALL. The protein expression of SHIP1 is decreased in most T-ALL and in some subgroups of B-ALL. In this study, we analyzed the expression of SHIP1 protein in detail in the context of groups with aberrant activated tyrosine kinases, namely BCR-ABL (Ph+) and Flt3 (KMT2A translocations). We demonstrate that constitutively activated Src kinases downstream of BCR-ABL and receptor tyrosine kinases reduce the SHIP1 expression in a SHIP1-Y1021 phosphorylated-dependent manner with subsequent ubiquitin marked proteasomal degradation. Inhibition of BCR-ABL (Imatinib), Flt3 (Quizartinib) or Src-Kinase-Family (Saracatinib) leads to significant reconstitution of SHIP1 protein expression. These results further support a functional role of SHIP1 as tumor suppressor protein and could be the basis for the establishment of a targeted therapy form.

Multistep IgE Mast Cell Desensitization Is a Dose- and Time-Dependent Process Partially Regulated by SHIP-1

Multistep mast cell desensitization blocks the release of mediators following IgE crosslinking with increasing doses of Ag. Although its invivo application has led to the safe reintroduction of drugs and foods in IgE-sensitized patients at risk for anaphylaxis, the mechanisms of the inhibitory process have remained elusive. We sought to investigate the kinetics, membrane, and cytoskeletal changes and to identify molecular targets. IgE-sensitized wild-type murine (WT) and FcεRIα humanized (h) bone marrow mast cells were activated and desensitized with DNP, nitrophenyl, dust mites, and peanut Ags. The movements of membrane receptors, FcεRI/IgE/Ag, actin, and tubulin and the phosphorylation of Syk, Lyn, P38-MAPK, and SHIP-1 were assessed. Silencing SHIP-1 protein was used to dissect the SHIP-1 role. Multistep IgE desensitization of WT and transgenic human bone marrow mast cells blocked the release of β-hexosaminidase in an Ag-specific fashion and prevented actin and tubulin movements. Desensitization was regulated by the initial Ag dose, number of doses, and time between doses. FcεRI, IgE, Ags, and surface receptors were not internalized during desensitization. Phosphorylation of Syk, Lyn, p38 MAPK, and SHIP-1 increased in a dose-response manner during activation; in contrast, only SHIP-1 phosphorylation increased in early desensitization. SHIP-1 phosphatase function had no impact on desensitization, but silencing SHIP-1 increased β-hexoxaminidase release, preventing desensitization. Multistep IgE mast cell desensitization is a dose- and time-regulated process that blocks β-hexosaminidase, impacting membrane and cytoskeletal movements. Signal transduction is uncoupled, favoring early phosphorylation of SHIP-1. Silencing SHIP-1 impairs desensitization without implicating its phosphatase function.

Endothelial cell damage is the central part of COVID-19 and a mouse model induced by injection of the S1 subunit of the spike protein

Neurologic complications of symptomatic COVID-19 are common. Brain tissues from 13 autopsies of people who died of COVID-19 were examined. Cultured endothelial and neuronal cells were incubated with and wild type mice were injected IV with different spike subunits. In situ analyses were used to detect SARS-CoV-2 proteins and the host response. In 13/13 brains from fatal COVID-19, pseudovirions (spike, envelope, and membrane proteins without viral RNA) were present in the endothelia of microvessels ranging from 0 to 14 positive cells/200× field (mean 4.3). The pseudovirions strongly co-localized with caspase-3, ACE2, IL6, TNFα, and C5b-9. The surrounding neurons demonstrated increased NMDAR2 and neuronal NOS plus decreased MFSD2a and SHIP1 proteins. Tail vein injection of the full length S1 spike subunit in mice led to neurologic signs (increased thirst, stressed behavior) not evident in those injected with the S2 subunit. The S1 subunit localized to the endothelia of microvessels in the mice brain and showed co-localization with caspase-3, ACE2, IL6, TNFα, and C5b-9. The surrounding neurons showed increased neuronal NOS and decreased MFSD2a. It is concluded that ACE2+ endothelial damage is a central part of SARS-CoV2 pathology and may be induced by the spike protein alone. Thus, the diagnostic pathologist can use either hematoxylin and eosin stain or immunohistochemistry for caspase 3 and ACE2 to document the endothelial cell damage of COVID-19.

Ectopic Expression of Hematopoietic SHIP1 in Human Colorectal Cancer.

Colorectal cancer (CRC) is a heterogeneous disease that results from the accumulation of mutations in colonic mucosa cells. A subclass of CRC is characterized by microsatellite instability, which is thought to occur mainly through inactivation of the DNA mismatch repair genes MLH1 and MSH2. The inositol 5-phosphatase SHIP1 is expressed predominantly in hematopoietic cells. In this study, the expression of SHIP1 in carcinomas and its putative correlation with clinicopathologic parameters, expression of DNA repair genes and microsatellite instability was investigated. By analyzing a multi-tumor tissue microarray, expression of SHIP1 was detected in 48 out of 72 cancer entities analyzed. The expression of SHIP1 protein of 145 kDa was confirmed by Western blot analysis in 7 out of 14 carcinoma cell lines. Analysis of a large colorectal cancer tissue microarray with 1009 specimens revealed SHIP1 expression in 62% of the samples analyzed. SHIP1 expression was inversely correlated with lymph node metastasis, vascular invasion and tumor grade, and it was positively associated with left-sided tumor localization. Interestingly, a strong relationship between the expression of SHIP1 and nuclear and membranous beta-catenin and the DNA repair genes MLH1 and MSH2 was observed.

Anti-inflammatory effect of interleukin-35 in mice with colitis and its mechanism

To investigate the anti-inflammatory effect and mechanisms of interleukin-35 (IL-35) in inflammatory bowel disease. BALB/c mice were divided into three groups with 10 mice in each group:control group, model group (oral administration of 4% glucan sodium sulfate for 7 d) and IL-35-treated group (oral administration of 4% glucan sodium sulfate for 7 d, intraperitoneal injection of 2 μg IL-35 at d2-5). Disease activity index (DAI) was scored every day. After 7 d, the mice were sacrificed, and the serum and intestinal tissue samples were collected. The gross morphology of the colon was observed; HE staining was used to observe the pathological changes of colon tissue; flow cytometry was employed to detect the change of macrophage polarization ratio in colon tissue; the mRNA expression levels of cytokines IL-6, TNF-α, IFN-γ, IL-10 and SHIP1 in colon tissue were determined by real-time quantitative RT-PCR; the expression and distribution of SHIP1 in colon tissue was measured by immunohistochemistry; Western blotting was adopted to detect the expression level of SHIP1 protein in colonic intestinal tissues of each group. The DAI scores of the mice in the model group were higher than those in the control group, while the DAI scores in the IL-35-treated group were lower than those in the model group (all P<0.01). Compared with the control group, the colon length was significantly shortened in the model group (P<0.05), while the colon length of the IL-35-treated group had an increasing trend compared with the model group, but the difference was not statistically significant (P>0.05). Compared with the model group, microscopic inflammatory infiltration score was decreased and microscopic crypt destruction and score was significantly lower in IL-35-treated group (all P<0.05). The relative expression of proinflammatory cytokines IL-6, TNF-α and IFN-γ in the colon tissue of IL-35-treated group was decreased compared with the model group, while the relative expression of IL-10 mRNA was higher than that of the model group (all P<0.05). Compared with the control group, the proportion of M1 macrophages in the model group increased (P<0.05), while the proportion of M1 macrophages in the IL-35-treated group was lower than that in the model group (P<0.05). The relative expression of SHIP1 mRNA and protein in the colon tissue of IL-35-treated group was higher than that in the model group (all P<0.05). IL-35 can inhibit the polarization of M1 macrophages and regulate inflammatory cytokines to promote anti-inflammatory effect on mice with colitis.

Time-course analysis of SHIP-1 modulation and expression in a pancreatic cancer model

Abstract Pancreatic cancer (PC) is an aggressive disease with minimally effective therapies due to immunosuppression. Src Homology Inositol Phosphatase (SHIP-1) expression is essential in regulating myeloid derived suppressor cell (MDSC) and T regulatory cell (Treg) homeostasis and function, which can positively or negatively impact antitumor immunity. Inflammatory factors play an important role in the regulation of SHIP-1 expression. We demonstrated in our pancreatic cancer tumor bearing mice (TB) that SHIP-1 gene and protein expression is diminished in lymphoid tissues, correlating with an expansion of MDSC and Treg percentages compared to control mice (CTRL). These TB mice are generated via subcutaneous injection with Panc02 cells. Furthermore, we showed that inflammatory factors were elevated in TB mice compared to CTRL mice. In this study we sought to pinpoint the exact time when SHIP-1 expression was reduced, in correlation with the expansion of MDSC and Treg, along with the increase in production of inflammatory factors in TB mice. To evaluate the loss of SHIP-1, weekly time-course experiments were performed via collection of peripheral blood (PB) and lymphoid tissues for a total of 4 weeks. Preliminary data indicates a reduction of SHIP-1 expression two weeks post tumor inoculation, which correlated with the expansion of MDSC and Treg percentages and an increase in tumor and spleen weights in TB mice compared to CTRL mice. Also, PB from TB mice showed a weekly increase in the production of inflammatory factors compared to CTRL mice. The results from this study may further establish SHIP-1 as a potential therapeutic target that may help combat PC in humans.

P-176 SHIP1 Deficiency in Human IBD

The causes of IBD are not fully known. Significant insights into the etiology of human IBD have come from Genome-Wide Association Studies (GWAS), which have identified more than 100 different inherited mutations that influence the risk of IBD in affected families. Only about 10% to 15% of all IBD adult patients have any of these genetic mutations. This means that risk factors for approximately 85% of IBD patients have yet to be defined. We have previously shown that mutation of SHIP1 leads to a Crohn's-like ileitis in mice. We hypothesized that loss of SHIP1 might potentially contribute to IBD in humans. SHIP1 expression was analyzed by qRT-PCR, Western blot, intracellular flow cytometry (icFlow) and RNAseq in-patient PBL. In order to determine if SHIP1/INPP5D deficiency might also contribute to human IBD, we analyzed SHIP1 expression in more than 100 adult IBD patients. We find that ∼15% of the adult IBD population demonstrates a profound degree of SHIP1 deficiency at the protein level (<10% of normal). However, both qRT-PCR and RNAseq analysis indicate these SHIP1-deficient patients have normal SHIP1 mRNA expression. Exome sequencing of the entire SHIP1/INPP5D gene in 8 separate SHIP1-deficient patients revealed no coding changes in the SHIP1/INPP5D gene that could account for the profound decrease in SHIP1 protein expression. Treatment of blood leukocytes with a proteasome inhibitor significantly increases SHIP1 protein expression in SHIP1 deficient patient PBL, but has the opposite effect in SHIP1-sufficient patients PBL. Flow cytometric analysis showed that SHIP1-deficient patients have a significant reduction in circulating CD4+ T cells, as do CD4CreSHIPflox/flox mice that have a selective ablation of SHIP1 expression in T lineage cells. In order to identify mutations or changes in gene expression at other loci that could adversely impact SHIP1 protein expression we performed RNAseq analysis on 10 separate SHIP1 deficient patients. Comparison of their transcriptomes to normal healthy controls indicates profound disruption of gene expression in SHIP1 deficient IBD patients. We also recently assessed disease severity in SHIP1 deficient IBD patients. We find that 63% of SHIP1-deficient CD patients have had surgical resection of their GI tract versus 23% in the SHIP1-sufficient cohort (P = 0.024) and have required on average 1.9 resections versus 0.3 in the SHIP1-sufficient CD cohort (P = 0.009). We also performed a computational drug screen that led to the identification of a novel SHIP1 agonist, K204. Using a human T cell model of SHIP1 protein deficiency, we show that K204 can significantly increase SHIP1 protein expression in intact cells. We find that a significant proportion of IBD patients have a profound reduction of SHIP1 expression. SHIP1 deficiency appears to have an adverse impact on disease progression as more than 60% of CD patients that are SHIP1-deficient require surgical resection of their GI tract, including multiple resections. We also find that SHIP1 protein is preferentially degraded by the proteasome system in SHIP1-deficient patients. Importantly, proteasome inhibitors and a novel SHIP1 agonist can partially restore SHIP1 protein expression suggesting a novel therapeutic approach in IBD.

P-193 Altered SHIP1 Protein Stability in a Subset of IBD Patients

P-193 Altered SHIP1 Protein Stability in a Subset of IBD Patients

Efects of Inhibiting miR-155 Expression on the Proliferation and Apoptosis of Leukemia THP-1 Cells

The aim of this study was to investigate the effects of miR-155 inhibitor transfection on the proliferation and apoptosis of THP-1 cells. The miR-155 inhibitor was transfected into THP-1 cells (THP-1I) by using X-treme GENE siRNA transfection reagent. Cells without transfection (THP-1C) and cells with negative transfection (THP-1IC) were used as controls. Quantitative real-time polymerase chain reaction (RT-PCR) was performed to detect the expression of miR-155 and relative expression of SHIP1 mRNA in the cells. Cell proliferation was assayed using CCK-8 method. Cell apoptosis were detected by flow cytometry. The expression of SHIP1, TAKT and pAKT in THP-1 cells were detected by Western blot. The results indicated that compared with THP-1C and THP-1IC, the expression of miR-155 in THP-1I cells was significantly reduced; miR-155 inhibition significantly increased apoptosis rate in THP-1 cells (P < 0.05) ; miR-155 inhibition in THP-1 cells caused no significant alteration in SHIP1 mRNA level but significantly increased its protein content, indicating some post-transcriptional modulations might exist underlying the modulation of miR-155 to SHIP1, the miR-155 caused significantly reduced protein level of pAKT (P < 0.05) without interfering TAKT protein content. It is concluded that the miR-155 inhibition may promote THP-1 cell apoptosis through increasing SHIP1 protein content and impairing its downstream PI3K/AKT signaling pathway. This study suggests that miR-155 inhibition may be a promising therapy strategy for treating acute myeloid leukemia (AML).

Integrated metagenomic and metaproteomic analyses of marine biofilm communities

Metagenomic and metaproteomic analyses were utilized to determine the composition and function of complex air–water interface biofilms sampled from the hulls of two US Navy destroyers. Prokaryotic community analyses using PhyloChip-based 16S rDNA profiling revealed two significantly different and taxonomically rich biofilm communities (6,942 taxa) in which the majority of unique taxa were ascribed to members of the Gammaproteobacteria, Alphaproteobacteria and Clostridia. Although metagenomic sequencing indicated that both biofilms were dominated by prokaryotic sequence reads (> 91%) with the majority of the bacterial reads belonging to the Alphaproteobacteria, the Ship-1 metagenome harbored greater organismal and functional diversity and was comparatively enriched for sequences from Cyanobacteria, Bacteroidetes and macroscopic eukaryotes, whereas the Ship-2 metagenome was enriched for sequences from Proteobacteria and microscopic photosynthetic eukaryotes. Qualitative liquid chromatography-tandem mass spectrometry metaproteome analyses identified 678 unique proteins, revealed little overlap in species and protein composition between the ships and contrasted with the metagenomic data in that ~80% of classified and annotated proteins were of eukaryotic origin and dominated by members of the Bacillariophyta, Cnidaria, Chordata and Arthropoda (data deposited to the ProteomeXchange, identifier PXD000961). Within the shared metaproteome, quantitative 18O and iTRAQ analyses demonstrated a significantly greater abundance of structural proteins from macroscopic eukaryotes on Ship-1 and diatom photosynthesis proteins on Ship-2. Photosynthetic pigment composition and elemental analyses confirmed that both biofilms were dominated by phototrophic processes. These data begin to provide a better understanding of the complex organismal and biomolecular composition of marine biofilms while highlighting caveats in the interpretation of stand-alone environmental ‘-omics’ datasets.

Abstract 975: MicroRNA-155 In chronic lymphocytic leukemia influences B-cell receptor signaling

Abstract MicroRNAs (miRNAs) can influence the expression levels of genes that can affect the clinical outcome of patients with chronic lymphocytic leukemia (CLL). In a well-defined cohort of 86 patients, we observed heterogeneity in the relative leukemia-cell expression of miR-155. Cases that expressed high-levels of miR-155 more frequently expressed zeta-associated protein of 70 kD (ZAP-70), used unmutated Ig heavy-chain-variable-region genes (IGHV), and had shorter treatment-free survival (TFS) and overall survival (OS) than cases with low-level expression of miR-155. Recursive partitioning allowed us to define a threshold for “high-level” expression of miR-155 (in copy numbers per CLL cell) that best associates with adverse clinical outcome. We examined the potential basis for this association. One of the genes targeted by miR-155 encodes the phosphatidylinositol polyphosphate 5-phosphatase SHIP-1, which can dephosphorylate proteins that had been activated in response to B-cell receptor (BCR) ligation. We found that CLL cells with “high-level” miR-155 expressed significantly lower levels of SHIP-1 protein and were more sensitive to ligation of surface-µ than CLL cells with low-levels of miR-155. Transfecting CLL cells with miR-155 reduced their expression of SHIP-1 and enhanced the stimulatory-response to surface-µ ligation. Conversely, transfection of CLL cells with a miR-155 inhibitor had the opposite effects. The enhanced sensitivity to BCR-ligation of cells expressing “high-level” miR-155 may account for its association with adverse clinical outcome in patients with CLL. However, within any one patient we also find heterogeneity in the expression levels of miR-155. By GEO data analysis (GSE21029) we found that CLL cells in blood expressed lower levels of miR-155 than did CLL cells in lymphoid tissues, where they interact with supportive cells, such as Nurselike cells or activated T cells, which can provide survival- or growth-promoting signals via expression of BAFF/APRIL or CD40L, respectively. We found that stimulation of CLL cells in vitro via CD40 ligation or exogenous BAFF could induce higher expression of miR-155, leading to reduced leukemia-cell expression of SHIP-1 and enhanced sensitivity to surface-µ ligation. Moreover, we found that the “proliferative” subgroup of blood CD5brightCXCR4dim CLL cells, which represent CLL cells newly released from the tissue microenvironment, expressed higher level of miR-155 and lower levels of SHIP-1 protein, and were more sensitive to surface-µ ligation than the “resting” subgroup of blood CD5dimCXCR4bright CLL cells, which represent cells that may be due to enter the tissue microenvironment. As such, this study also demonstrates that miR-155 could be upregulated by signals in the CLL microenvironment, leading to lower-level expression of SHIP-1, and enhanced stimulation in response to BCR ligation. Citation Format: Liguang Chen, Bing Cui, Suping Zhang, Marek Mraz, Jessie-F. Fecteau, Jian Yu, Ling Zhang, Lei Bao, Laura Rassenti, Karen Messer, Carlo Croce, Thomas Kipps. MicroRNA-155 In chronic lymphocytic leukemia influences B-cell receptor signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 975. doi:10.1158/1538-7445.AM2014-975

SHIP1 is targeted by miR-155 in acute myeloid leukemia.

The SH2 domain-containing inositol 5'-phosphatase1(SHIP1) has been implicated as a suppressor of hematopoietic transformation as its activity can inhibit the PI3K/Akt signaling pathway. Reduced activity of SHIP1 has been observed in acute myeloid leukemia(AML). SHIP1 is a target of microRNA-155(miR-155). Therefore, the aim of the present study was to investigate the role of miR-155/SHIP1 in the pathogenesis of AML. We examined the levels of SHIP1 protein and miR-155 in tissue samples of patients with AML and in AML cell lines. In addition, we investigated cell proliferation, apoptosis and expression of SHIP1/PI3K/AKT pathway molecules in the THP-1 and U937 cell lines after miR-155 inhibitor or mimics were transfected. We showed that the levels of SHIP1 protein were significantly decreased in tissue samples of patients with some subtypes of AML(M4 or M5) and in AML cell lines with concomitant overexpression of miR-155. In addition, we demonstrated that decreased expression of SHIP1 in the AML cell lines was a consequence of increased levels of miR-155 and can therefore be reversed invitro through inhibition of miR-155, with subsequent inhibition of cell proliferation and promotion of cell apoptosis. In conclusion, expression of the SHIP1 protein is targeted by miR-155 in AML. miR-155 acts as an onco-miR, and the miR-155/SHIP1/PI3K/AKT signaling pathway could play an important role in the pathogenesis of AML.

O-018 SHIP1-deficiency in a Subset of IBD Patients

SHIP1 is a 5'-inositol phosphatase that is ubiquitously expressed in cells of the hematopoietic lineage. Previous work in the Kerr lab has demonstrated that germline or induced SHIP1 deficiency in mice causes profound and severe ileitis similar to human Crohn’s Disease (CD) (Kerr et al., GUT 2011). Considering these observations, we hypothesized that SHIP1 and/or the gene encoding it, INPP5D, may contribute to CD susceptibility and pathology in the human population. To determine the prevalence of SHIP1-deficiency in CD and Ulcerative Colitis (UC) patients, we are examining patients from an adult IBD cohort from San Francisco Veteran’s Affairs Hospital, University of California San Francisco (SFVA/UCSF). Whole blood samples from consenting patients are shipped overnight to the Kerr lab, where a multifaceted approach is employed to simultaneously quantify SHIP mRNA expression, protein level and activity in peripheral blood mononuclear cells (PBMC). These assays are designed to identify SHIP1-deficient patients and determine the prevalence of SHIP1 deficiency in this ≈300 patient cohort. Follow-up analysis of SHIP1/INPP5D expression, function, and genetic polymorphisms are then preformed to define molecular and genetic mechanisms that lead to SHIP1-deficiency in IBD. In the 7 months since this study was initiated, we examined samples from 35 patients and have observed that up to 30% of IBD patients exhibit some level of SHIP1 protein deficiency in PBMC. SHIP1 deficiency is observed in both a subset of CD and UC patients. Furthermore, secondary blood draws have been performed on a subset of SHIP1-deficient patients and demonstrated that the degree of SHIP1 deficiency seen in initial blood draws made ∼4 months earlier is still present. Finally, analysis of relative SHIP1 mRNA levels did not correlate to the reduced protein levels suggesting that SHIP1 deficiency is not the result of aberrant gene-specific transcription but rather due to altered translational or post-translational regulation of SHIP1 protein expression in PBMC. Overall, based on preliminary results, we have observed that SHIP1-deficiency may be a contributing factor to disease in a subset of IBD patients, including both CD and UC. While the exact molecular/genetic mechanisms leading to SHIP1-deficiency remain to be determined, we have evidence that it likely results from deregulation of translational or post-translational control mechanisms that impact steady state SHIP1 protein expression. Further analysis of the cause and nature of SHIP1-deficiency in this patient cohort could lead to new therapeutic approaches in the treatment of a substantial proportion of IBD patients.

Targeting Mir-155 via The NEDD8-Activating Enzyme Inhibitor MLN4924: A Novel Therapeutic Approach For Acute Myeloid Leukemia (AML)

microRNA-155 (miR-155) is a short non-coding RNA that is associated with aggressive cancers and known to promote leukemogenesis. Recently, we have reported that aberrant miR-155 upregulation independently identifies high-risk cytogenetically normal AML patients, suggesting that this miR may also serve as a novel therapeutic target in AML. We and others have shown that miR-155 is positively regulated by NF-kB, a transcription factor that is constitutively activated in leukemic blasts and contributes to their aberrant proliferation and survival. MLN4924 (Millennium Pharmaceuticals Inc) is a novel drug that blocks neddylation and subsequent degradation of the NFkB inhibitor, IkBa, thereby inhibiting translocation of NF-kB to the nucleus. MLN4924 has demonstrated promising activity in early clinical trials for AML. We postulated that downregulation of miR-155 via NF-kB inhibition is at least in part responsible for the antileukemic activity of MLN4924. MethodsAML cell lines and primary blasts were treated with 100-1000nM MLN4924 for 3-72 hrs. Messenger RNA and protein levels were determined by quantitative RT-PCR and immunoblotting, respectively. NF-kB activity was measured by luciferase reporter assays. Binding of NF-kB to the miR-155 promoter was detected by electromobility shift assay and Chromatin Immunoprecipitation. Transfection of miR-155 was performed using the siPORT TM NeoFXTM method. Apoptosis was assessed by Annexin V staining. For in vivo studies, we used NOD/SCID/g mice engrafted with MV4-11 cells. Two weeks after transplantation, the engrafted mice received intraperitoneal treatments of 180 mg/kg of MLN4924 every other day for 21 days. Mice in the control group were treated similarly with the vehicle alone (20% 2-hydroxypropyl-betacyclodextrin). ResultsIn AML cell lines and primary AML patient blasts 12hr treatment with MLN4924 resulted in a ∼50% decrease of miR-155 expression at 300nM in THP-1 and MV4-11 cells and at 500nM in AML blasts (p<0.01). This was concomitant with a ∼ 50% and 70% decrease in NF-kB activity and binding to miR-155 promoter, respectively (p<0.01). These results correlated with a significant upregulation of mRNA levels of the key miR-155 target gene, SHIP1 [6-fold (p<0.05), 9-fold (p<0.01), and 2-fold (p<0.05) in THP-1 cells, MV4-11 cells, and AML patient blasts, respectively]. SHIP1 protein levels were increased in all samples as well. SHIP1 is a tyrosine phosphatase that blocks PI3K-mediated membrane localization of AKT, which is often aberrantly activated in human cancers, including leukemia. Thus, we postulated that MLN4924-induced upregulation of SHIP1 via miR-155 downregulation would also result in PI3K/AKT pathway inhibition. As predicted, MLN4924 treatment of AML cell lines and primary blasts resulted in inhibition of the active AKT, as evidenced by a decline of phospho-AKTThr308 levels. Furthermore, the pharmacologic activity of MLN4924 was inhibited by forced expression of miR-155 in THP-1 cells and AML blasts, as shown by a partial loss of SHIP1 upregulation and caspase-3 activation, thus preventing MLN4924-triggered induction of apoptosis (p<0.01) and decrease in cell viability (p<0.05). In vivo, mature miR-155 levels in the peripheral blood of xenografted mice decreased by 50% after 24hrs and 80% after 48hrs (p<0.01) from the first dose of MLN4924. Moreover, 21 days from the start of MLN4924 treatment, the average white blood cell count was significantly lower in the MLN4924-treated group (5,333 cells/ul ± 1040) compared with the vehicle-treated group (36,166 cells/ul ± 10,598; p< 0.01). The average spleen weight was also dramatically reduced in the MLN4924-treated group (58.06 mg ±12.74) compared with the control group (305.66 mg ±51.1; p<0.01). Importantly, MLN4924 significantly prolonged the survival of leukemic mice; median survival was 45.5 vs. 31 days for MLN4924-treated vs. control groups (p<0.0001, n=10 per group), respectively. ConclusionsWe showed that MLN4924 treatment of AML cells in vitro and in vivo resulted in decreased miR-155 expression, reactivation of its target gene, SHIP1, and concomitant inhibition of PI3K/AKT pathway. Our data also support that miR-155 downregulation is a critical component of MLN4924's antileukemic activity. Thus, our work provides novel insight into MLN4924's mechanism of action and the rationale for combining this drug with emerging anti-microRNA compounds. Disclosures:No relevant conflicts of interest to declare.

TanshinoneIIA ameliorates inflammatory microenvironment of colon cancer cells via repression of microRNA-155

TanshinoneIIA, an active component derived from a traditional Chinese medicine, has anti-inflammatory and anti-cancer effect. However, the mechanisms underlying the interaction between anti-inflammation and anti-cancer of TanshinoneIIA remain elusive. In the present study, a cell model of inflammation between macrophages and colon cancer cells was used. The results showed that TanshinoneIIA inhibited the proliferation of inflammation-related colon cancer cells HCT116 and HT-29 by decreasing the production of inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6), which generated by macrophage RAW264.7 cell line. We identified Phosphatidylinositol-3, 4, 5-trisphosphate 5-phosphatase 1 (SHIP1) was a bona fide target of miR-155. TanshinoneIIA restored the down-regulated level of SHIP1 protein after lipopolysaccharide (LPS)-stimulation in RAW264.7 cells. MicroRNA-155 (miR-155) was up-regulated in macrophages, possibly due to the concomitant increase of PU.1, a transcriptional activator of miR-155, accounting for decreased SHIP1. Treatment with TanshinoneIIA prevented increased PU.1 and hence increased miR-155, whereas aspirin could not. These findings support that the interruption of signal conduction between activated macrophages and colon cancer cells could be considered as a new therapeutic strategy and miR-155 could be a potential target for the prevention of inflammation-related cancer.

P049 A possible role of mucosal cytokine profile for predicting response to infliximab therapy in ulcerative colitis

leads to altered cytokine production and the development of Crohn’s like ileitis, which in fact presents one of the few mouse models in which intestinal inflammation develops spontaneously. SHIP1 is expressed exclusively in hematopoietic lineage and bone marrow parenchymal cells, and colitis can be transferred to wild type mice by SHIP1 / splenocytes. These data strongly suggest a role for SHIP1 in immune cells in the pathogenesis of CD. We therefore investigated SHIP1 activity and expression in peripheral blood mononuclear cells (PBMCs) from CD patients. Methods: We developed a SHIP1 activity assay by precipitating endogenous SHIP1 from PBMC lysates and incubating precipitates with PtdIns(3,4,5)P3. The accumulation of free phosphate as a result of SHIP1 activity was measured by Malachite green assay. The specificity and quantitative range of this assay were confirmed by using SHIP1 deficient cells, and testing different concentrations of protein. Total SHIP1, SHIP2 and PTEN levels were determined by quantitative Western blot analysis (Odyssey). Results: We performed SHIP1 activity assays on PBMCs of 34 CD patients. In every individual measurement, SHIP1 activity in PBMCs from healthy donors was taken as control (n = 25). We identified 5 patients with SHIP1 expression below detection limit and hence low SHIP1 activity. The remaining patients showed a significant upregulation of SHIP1 protein expression levels, but reduced SHIP1 activity after correction for SHIP1 expression. Differences in SHIP1 activity and expression patterns were not caused by altered PBMC composition as no significant differences in B-cell, T-cell or monocyte percentages were observed between CD patients (n = 15) and healthy controls (n = 9). SHIP2 and PTEN protein levels were normal in all CD patients. Conclusions: Our results demonstrate for the first time that immune cells from 15% of CD patients have decreased SHIP1 expression and activity levels, which may affect immune cell function through interference with phospholipid levels. The remaining 85% of CD patients show a decreased activity of SHIP1, although this may be compensated for by an increased SHIP1 expression. Together, these data suggest a role for impaired SHIP1 activity in the development of disease in a subset of CD patients.

Loss of SHIP-1 protein expression in high-risk myelodysplastic syndromes is associated with miR-210 and miR-155.

The myelodysplastic syndromes (MDSs) comprise a group of disorders characterized by multistage progression from cytopenias to acute myeloid leukemia (AML). They display exaggerated apoptosis in early stages, but lose this behavior during evolution to AML. The molecular basis for loss of apoptosis is unknown. To investigate this critical event, we analyzed phosphatidylinositol (PI) 3'kinase signaling, implicated as a critical pathway of cell survival control in epithelial and hematological malignancies. PI 3'kinase activates Akt through its production of 3' phosphoinositides. In turn, the phosphoinositides are dephosphorylated by two lipid phosphatases, PTEN and SHIP-1, in myeloid cells. We studied primary MDS-enriched bone marrow cells and bone marrow sections by western blotting, immunohistochemistry, immunocytochemistry and quantitative PCR for components of the SHIP/PTEN/PI 3'kinase signaling circuit. We reported constitutively activated Akt, variable levels of PTEN and uniformly decreased SHIP-1 expression in MDS progenitor cells. Overexpression of SHIP-1, but not the phosphatase-deficient form, inhibited myeloid leukemic growth. Levels of microRNA (miR)-210 and miR-155 transcripts, which target SHIP-1, were increased in CD34(+) MDS cells compared with their normal counterparts. Direct binding of miR-210 to the 3' untranslated region of SHIP-1 was confirmed by luciferase reporter assay. Transfection of a myeloid cell line with miR-210 resulted in loss of SHIP-1 protein expression. These data suggest that miR-155 and miR-210/SHIP-1/Akt pathways could serve as clinical biomarkers for disease progression, and that miR-155 and miR-210 might serve as novel therapeutic targets in MDS.

Murine Pancreatic Adenocarcinoma Dampens SHIP-1 Expression and Alters MDSC Homeostasis and Function

BackgroundPancreatic cancer is one of the most aggressive cancers, with tumor-induced myeloid-derived suppressor cells (MDSC) contributing to its pathogenesis and ineffective therapies. In response to cytokine/chemokine receptor activation, src homology 2 domain-containing inositol 5′-phosphatase-1 (SHIP-1) influences phosphatidylinositol-3-kinase (PI3K) signaling events, which regulate immunohomeostasis. We hypothesize that factors from murine pancreatic cancer cells cause the down-regulation of SHIP-1 expression, which may potentially contribute to MDSC expansion, and the suppression of CD8+ T cell immune responses. Therefore, we sought to determine the role of SHIP-1 in solid tumor progression, such as murine pancreatic cancer.Methodology and Principal FindingsImmunocompetent C57BL/6 mice were inoculated with either murine Panc02 cells (tumor-bearing [TB] mice) or Phosphate Buffer Saline (PBS) (control mice). Cytometric Bead Array (CBA) analysis of supernatants of cultured Panc02 detected pro-inflammatory cytokines such as IL-6, IL-10 and MCP-1. TB mice showed a significant increase in serum levels of pro-inflammatory factors IL-6 and MCP-1 measured by CBA. qRT-PCR and Western blot analyses revealed the in vivo down-regulation of SHIP-1 expression in splenocytes from TB mice. Western blot analyses also detected reduced SHIP-1 activity, increased AKT-1 and BAD hyper-phosphorylation and up-regulation of BCL-2 expression in splenocytes from TB mice. In vitro, qRT-PCR and Western blot analyses detected reduced SHIP-1 mRNA and protein expression in control splenocytes co-cultured with Panc02 cells. Flow cytometry results showed significant expansion of MDSC in peripheral blood and splenocytes from TB mice. AutoMACS sorted TB MDSC exhibited hyper-phosphorylation of AKT-1 and over-expression of BCL-2 detected by western blot analysis. TB MDSC significantly suppressed antigen-specific CD8+ T cell immune responses in vitro.Conclusion/SignificanceSHIP-1 may regulate immune development that impacts MDSC expansion and function, contributing to pancreatic tumor progression. Thus, SHIP-1 can be a potential therapeutic target to help restore immunohomeostasis and improve therapeutic responses in patients with pancreatic cancer.

Mir-155 Contributed to Chronic Lymphocytic Leukemia Survival by Modulation of BCR-Signalling

Abstract 620The microRNA miR-155 is encoded within the B-cell integration cluster bic, which is a frequent integration site for avian leucosis virus. This microRNA is over-expressed in certain B cell lymphomas, including chronic lymphocytic leukemia (CLL), suggesting miR-155 contributes to lymphoma development. Consistent with this are observations that mice made transgenic for miR-155 under a B-cell-specific promoter develop pre-B cell lymphomas. One of the target genes regulated by miR-155 is SHIP-1, a phosphatase that plays a critical role in down-modulating B-cell receptor (BCR) signaling, which appears to provide a growth and/or survival stimulus for CLL B cells. To test for this, we examined for relationships between the CLL-cell expression of miR-155, SHIP-1, magnitude of BCR-signaling, and time from diagnosis to initial therapy (TFS) in 33 pts with CLL. The relative level of miR-155 was determined by real-time PCR. SHIP-1 protein was determined by flow cytometry and immunoblot analyses. We noted variations in expression levels of miR-155 among patient samples studied. CLL cells that expressed levels of miR-155 higher than the median level of miR-155 for all patients studied had significantly lower expression levels of SHIP-1 protein than CLL cells that expressed levels of miR-155 lower than the median level of miR-155 had (7.0 ± 0.2 vs. 8.3 ± 0.47, mean ± SEM, P<0.05). CLL B cells were stimulated with anti-μ or control Ig and then examined for relative protein phosphorylation and calcium influx. CLL cases were segregated into groups with high-BCR signaling versus low BCR-signaling based on their responses to anti-μ stimulation. We found that CLL cells with high-BCR signaling expressed significantly higher levels of miR-155 (1.54 ± 0.22) than did CLL cells with low-BCR signaling (0.90 ± 0.13, p<0.05). CLL cells with high BCR-signaling had significantly lower amounts of SHIP-1 protein (7.1 ± 0.39) than did CLL cells with low BCR-signaling (9.12 ± 0.7, p<0.05). Moreover, Kaplan-Meier survival analysis revealed an association between higher expression levels or miR-155 and shorter survival times from diagnosis to initial therapy (TFS) (P<0.05). CLL patients with greater miR-155 expression had a median TFS of 25.9 months that was significantly shorter than the median TFS of 112.8 months for patients with low miR-155 expression. To further investigate its function role, CLL cells were transfected with miR155 mimic or a miR-155 inhibitor and examined for SHIP-1 expression, BCR signaling, and cell survival, with or without stromal cells. Introduction of miR-155 to CLL cells that had low expression levels of miR-155 significantly reduced SHIP-1 protein expression, enhanced BCR signaling and improved cell survival relative to that of mock transfect CLL cells or CLL cells tranfected with micro RNA. Conversely, transfection of CLL cells that had high expression levels of miR-155 with miR-155 inhibitor resulted in significantly increased SHIP-1 expression, reduced BCR signaling, and poor survival than mock transfected CLL cells. Moreover, transfection of miR-155 inhibitor significantly reduced the capacity of stromal cells to enhance CLL cell survival. We found that the viability of cells transfected with miR-155 inhibitor was significantly lower than that noted for cells transfected with control miRs when cocultured with stromal cells. These results demonstrate that expression of miR-155 can modulate CLL expression of SHIP-1, CLL cell BCR signaling, and survival. Disclosures:No relevant conflicts of interest to declare.

Induction of endotoxin tolerance in vivo inhibits activation of IRAK4 and increases negative regulators IRAK-M, SHIP-1, and A20

TLRs mediate host defense against microbial pathogens by eliciting production of inflammatory mediators and activating expression of MHC, adhesion, and costimulatory molecules. Endotoxin tolerance limits excessive TLR-driven inflammation during sepsis and reprograms macrophage responses to LPS, decreasing expression of proinflammatory cytokines without inhibiting anti-inflammatory and antimicrobial mediators. Molecular mechanisms of reprogramming of TLR4 signaling upon in vivo induction of endotoxin tolerance are incompletely understood. We used an in vivo model of endotoxin tolerance, whereby C57BL/6 mice were i.p.-inoculated with LPS or PBS, followed by in vitro challenge of peritoneal or splenic macrophages with LPS to examine activation of IRAK4 and expression of negative regulatory molecules. Administration of LPS in vivo-induced endotoxin tolerance in peritoneal and splenic macrophages, as evidenced by decreased degradation of IκBα, suppressed phosphorylation of p38 and reduced expression of TNF-α, IL-6, and KC mRNA upon in vitro LPS challenge. Macrophages from control and endotoxin-tolerant mice exhibited comparable TLR4 mRNA levels and similar expression of IL-1RA and IL-10 genes. Endotoxin tolerization in vivo blocked TLR4-driven IRAK4 phosphorylation and activation in macrophages, while increasing expression of IRAK-M, SHIP-1, A20 mRNA, and A20 protein. Thus, induction of endotoxin tolerance in vivo inhibits expression of proinflammatory mediators via impaired activation of IRAK4, p38, and NF-κB and increases expression of negative regulators of TLR4 pathways.