Knockdown Clones Research Articles

RBM10 Modulates Apoptosis and Influences TNF-α Gene Expression.

Recent evidence suggests that protein encoded by the RNA Binding Motif 10 (RBM10) gene has the ability to modulate apoptosis. The objective of this study was to test this hypothesis by manipulating RBM10 expression levels and examining the downstream consequences. The results showed that transient overexpression of RBM10 correlated with significantly elevated levels of tumour necrosis factor alpha (TNF-α) mRNA and soluble TNF-α (sTNF-α) protein, and increased apoptosis (phosphatidyl serine exposure on the outer cell membrane and nuclear condensation). Stable RNA interference-mediated RBM10 knockdown clones were less susceptible to TNF-α-mediated apoptosis, and had decreased sTNF-α protein levels. Elevated levels of TNF-α associated with RBM10 overexpression resulted from increased TNF-α transcription, not TNF-α mRNA stabilization. These results suggest that RBM10 has the ability to modulate apoptosis, and that it does so via a mechanism involving alterations to TNFR super family-mediated signaling. These data provide the first direct evidence that human RBM10 can function as an apoptosis modulator and cytokine expression regulator.

Loss of EZH2 and Aberrant DNA Methylation Account for PKC412 Drug Resistance in the FLT3-ITD Positive Acute Myeloid Leukemia (AML) Cell Line MV4-11,

Abstract 3468The development of drug resistance is a common feature in AML that occurs towards classical cytotoxic drugs as well as novel kinase inhibitors. Mutations in primary drug targets explain a significant fraction of acquired drug resistance but the mechanisms of resistance still remain unknown in most patients. About 20–30% of all AML patients possess the mutant FLT3-ITD leading to a constitutive activation of the FLT3 kinase. Flt3-mutations can be targeted by treatment with tyrosine kinase inhibitors. Resistance to these kinase inhibitors is an increasing phenomenon whose mechanisms are not entirely understood today. As epigenetic mechanisms are shown to play an important role in leukemia pathogenesis they are also likely to influence drug resistance. To elucidate the potential role of epigenetic mechanisms in the development of drug resistance towards kinase inhibitors we used a PKC412 partially resistant clone (MV4-11R) of the AML cell line MV4-11, which harbors a homozygous FLT3 internal tandem duplication (ITD) mutation.An initial screening for histone modifying enzymes revealed a downregulation of EZH2 on mRNA as well as protein level compared with the parental cell line. The reduction of EZH2, a H3K27 methyltransferase, in MV4-11R is furthermore correlating with globally diminished H3K27me3 levels. ChIP-Seq experiments using H3K27me3 antibody revealed differences in histone 3 K27 methylation at specific promoter sites between the parental and resistant MV4-11. To test for an increased drug resistance due to reduced EZH2 protein levels lentiviral knock-down of EZH2 was performed in the MV4-11 parental cell line and three individual knock-down cell clones were investigated for their drug resistance potential. These knock downs all showed elevated IC50 values as well as resistance towards the apoptosis-inducing effects of PKC412 compared with the scrambled shRNA cells. Furthermore, EZH2 protein levels of 5 FLT3-ITD-positive AML patient samples were determined by Western Blot, samples were treated with PKC412 for 3 days and cell survival was assayed. Using this approach higher EZH2 levels in patients could also be associated with a higher sensitivity to PKC412 pointing to a putative role of EZH2 in the development of PKC412 resistance in vivo.As EZH2 has been shown to interact with DNMTs in the context of the Polycomb Repressive Complex 2 and 3 (Viré et al., Nature 2006), we analyzed whether parental and resistant MV4-11 differ in their DNA methylation pattern. To identify hyper-/hypomethylated genes in MV4-11R, we applied the Illumina 27k Methylation BeadChip approach as well as Reduced Representation Bisulfite Sequencing (RRBS) for a genome wide CpG methylation analysis. In particular genes associated with apoptosis pathways and signal transduction were hypermethylated in MV4-11R cells compared to the parental cell line. Based on the observation of DNA methylation changes between the parental cell line and MV4-11R, treatment with the demethylating agent 2-deoxy-5-azacytidine (Aza dC) was conducted to investigate recovery of drug sensitivity. Incubation with 250 nM Aza dC for 5 days could restore the sensitivity of MV4-11R towards PKC412 as shown in proliferation and apoptosis assays. Using the Affymetrix Human Gene 1.0 ST Array platform we identified 110 genes whose expression was reactivated after treatment of MV4-11R with Aza dC, predominantly genes playing a role in signal transduction, apoptosis pathways and cell cycle. A cell cycle analysis of the MV4-11 and MV4-11R cells indeed showed that the resistant cell line is cycling less than the parental one, which possibly also favors the resistance development towards PKC412.Summarized, our data show that a loss of EZH2 accompanied by DNA methylation changes lead to PKC412 resistance in a FLT3-ITD AML cell line model possibly reflecting a way of acquisition of drug resistance in patients. Disclosures:Thiede:Novartis: Lectures, Research Funding. Müller-Tidow:Novartis: Research Funding.

Bone Marrow Stromal Cells Inhibit Apoptosis of Chronic Lymphocytic Leukemia Cells (CLL) by Expressing Erythroid Differentiation Regulator 1 (ERDR1)

Abstract 1764 Background:Chemotherapy resistance in chronic lymphocytic leukemia (CLL) is in part mediated by anti-apoptotic signals produced by bone marrow stromal cells. Identifying these signals is the first step to overcoming this resistance. ERDR1 was initially described as an inducer of hemoglobinization. We now present evidence that it inhibits apoptosis of CLL cells. Methods/Results:Previously, we showed that wild type (WT) but not CCR5−/− mesenchymal cells increase pulmonary melanoma metastasis in CCR5−/− mice. This observation led us to compare gene expression in the lungs of these mice. Using an Affymetrix expression array, we found Erdr1 was differentially expressed in the wild type mice. To show that the increase in metastasis was mediated by Erdr1, we transferred WT pulmonary mesenchymal cells transfected with Erdr1 shRNA or non-targeted control shRNA. CCR5−/− mice receiving Erdr1 knockdown mesenchymal cells had a 27.3% to 37% decrease in metastasis compared to animals receiving control transfected cells (p<0.01).One explanation for the decrease in metastasis would be a failure of the Erdr1 knockdown cells to survive in the lung. Since the knockdown and control vectors express EGFP, we were able to compare the quantity of transfected cells surviving in the lung by applying an EGFP ELISA to lung homogenates. These experiments showed no difference in the number of surviving mesenchymal cells. These results suggested Erdr1 was acting as a pro-survival factor for the melanoma cells.Since ERDR1 expression is the highest in the bone marrow, we compared the survival of CLL cells co-cultured with control and Erdr1 knockdown cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. Peripheral blood samples were then collected from 10 untreated CLL patients and co-cultured with these stromal cell lines. After 72 and 96 hours, total cell counts and apoptosis were measured using Annexin V/PI. At both time points, the cell counts were higher when the CLL cells were co-cultured with control cell lines compared to Erdr1 knockdown lines (OR 1.88 ± 0.27, 2.52 ± 0.66 respectively). The increase in total cell number was associated with a decrease in the percentage of apoptotic cells (OR 0.69 ± 0.18, 0.58 ± 0.12 respectively).Since Erdr1 was differentially expressed in WT compared to CCR5−/− mice, we considered the regulation of this gene by chemokine agonists. In these experiments, 100 ng/ml of CCL4 was added to WT and CCR5−/− PMCs and mRNA was harvested at 12, 24, and 48 hours. Using real-time PCR, we found that Erdr1 expression was increased compared to baseline in the WT mesenchymal cells by 1.33 fold ± 0.06 (p < 0.05) after 24 hours. By 48 hours, expression had increased by 3.36 fold ± 0.14 (p < 0.001). As expected, CCL4 did not increase expression of Erdr1 in CCR5−/− mesenchymal cells.Since Erdr1 is associated with hemoglobinization, we also investigated the effect of hypoxia on Erdr1 expression. In these experiments, deferoxamine was added to the stromal cell line M2-10B4 at varying concentrations. Significant fold increases in Erdr1 expression were seen after 48 hours of 1.43 ± 0.10 (50nM), 1.96 ± 0.08, (100nM), and 2.44 ± 0.01 (200nM). Implications for the treatment of human disease:Other investigators have shown that stromal cells such as nurse-like cells and CLL cells can interact to induce CCL4 and promote CLL cell survival. Our work suggests a novel pathway by which this may take place. Disclosures:No relevant conflicts of interest to declare.

A Unique Role of GATA1s in Down Syndrome Acute Megakaryocytic Leukemia Biology and Therapy

BackgroundAcute megakaryocytic leukemia (AMkL) in Down syndrome (DS) children is uniformly associated with somatic GATA1 mutations, which result in the synthesis of a shorter protein (GATA1s) with altered transactivation activity compared to the wild-type GATA1. It is not fully established whether leukemogenesis and therapeutic responses in DS AMkL patients are due to loss of the wild-type GATA1 or due to a unique function of GATA1s.MethodologyStable clones of CMK cells with decreased GATA1s or Bcl-2 levels were generated by using GATA1- or BCL-2-specific lentivirus shRNAs. In vitro ara-C, daunorubicin, and VP-16 cytotoxicities of the shRNA stable clones were determined by using the Cell Titer-blue reagent. Apoptosis and cell cycle distribution were determined by flow cytometry analysis. Changes in gene transcript levels were determined by gene expression microarray and/or real-time RT-PCR. Changes in protein levels were measured by Western blotting. In vivo binding of GATA1s to IL1A promoter was determined by chromatin immunoprecipitation assays.ResultsLentivirus shRNA knockdown of the GATA1 gene in the DS AMkL cell line, CMK (harbors a mutated GATA1 gene and only expresses GATA1s), resulting in lower GATA1s protein levels, promoted cell differentiation towards the megakaryocytic lineage and repressed cell proliferation. Increased basal apoptosis and sensitivities to ara-C, daunorubicin, and VP-16 accompanied by down-regulated Bcl-2 were also detected in the CMK GATA1 shRNA knockdown clones. Essentially the same results were obtained when Bcl-2 was knocked down with lentivirus shRNA in CMK cells. Besides Bcl-2, down-regulation of GATA1s also resulted in altered expression of genes (e.g., IL1A, PF4, and TUBB1) related to cell death, proliferation, and differentiation.ConclusionOur results suggest that GATA1s may facilitate leukemogenesis and potentially impact therapeutic responses in DS AMkL by promoting proliferation and survival, and by repressing megakaryocytic lineage differentiation, potentially by regulating expression of Bcl-2 protein and other relevant genes.

Novel function of keratins 5 and 14 in proliferation and differentiation of stratified epithelial cells

Keratins are cytoplasmic intermediate filament proteins preferentially expressed by epithelial tissues in a site-specific and differentiation-dependent manner. The complex network of keratin filaments in stratified epithelia is tightly regulated during squamous cell differentiation. Keratin 14 (K14) is expressed in mitotically active basal layer cells, along with its partner keratin 5 (K5), and their expression is down-regulated as cells differentiate. Apart from the cytoprotective functions of K14, very little is known about K14 regulatory functions, since the K14 knockout mice show postnatal lethality. In this study, K14 expression was inhibited using RNA interference in cell lines derived from stratified epithelia to study the K14 functions in epithelial homeostasis. The K14 knockdown clones demonstrated substantial decreases in the levels of the K14 partner K5. These cells showed reduction in cell proliferation and delay in cell cycle progression, along with decreased phosphorylated Akt levels. K14 knockdown cells also exhibited enhanced levels of activated Notch1, involucrin, and K1. In addition, K14 knockdown AW13516 cells showed significant reduction in tumorigenicity. Our results suggest that K5 and K14 may have a role in maintenance of cell proliferation potential in the basal layer of stratified epithelia, modulating phosphatidylinositol 3-kinase/Akt-mediated cell proliferation and/or Notch1-dependent cell differentiation.

CA125 (MUC16) gene silencing suppresses growth properties of ovarian and breast cancer cells

The tumour-associated antigen CA125 (mucin 16, MUC16) is commonly expressed in ovarian cancer, and can also be detected in other tumour of epithelial origin, but its physiological role is largely unknown. The aim of the present study was to investigate the impact of MUC16 gene silencing on the growth properties of ovarian and breast cancer cells. We analysed cellular effects linked to oncogenesis, such as proliferation, cell cycle and apoptosis, after transient and stable transfection with MUC16 short hairpin RNA (shRNA) in diverse epithelial cancer cell lines with different MUC16 expression. Furthermore, alterations in cell adhesion, migration and invasion were evaluated in stable MUC16 knockdown clones. The growth of all tested MUC16+ tumour cells was significantly suppressed by induction of caspase-dependent apoptosis after transient transfection with MUC16 shRNA, irrespective of the initial MUC16 expression level and cancer origin. Growth inhibition could be confirmed in stable MUC16 knockdown clones, albeit caspase-dependent death pathways seemed no longer be activated. In MUC16low+ ovarian cancer cells, stable MUC16 gene silencing resulted in a substantial blockade of colony formation, cell adhesion, migration and invasiveness associated with reduced activation of metalloproteinases-2 (MMP-2). By contrast, the tested MUC16high+ cell lines displayed a non-motile and non-invasive phenotype which was not affected by MUC16 knockdown, probably due to the expression of different MUC16 isoforms with divergent functions in individual cell lines. Our results provide evidence for a central role of MUC16 in cancer cell survival pathways. Additionally, MUC16 might also be involved in adhesion, migration and invasion depending on the type of cancer cell.

Knockdown of ornithine decarboxylase antizyme 1 causes loss of uptake regulation leading to increased N 1, N 11-bis(ethyl)norspermine (BENSpm) accumulation and toxicity in NCI H157 lung cancer cells

Ornithine decarboxylase antizyme 1 (AZ1) is a major regulatory protein responsible for the regulation and degradation of ornithine decarboxylase (ODC). To better understand the role of AZ1 in polyamine metabolism and in modulating the response to anticancer polyamine analogues, a small interfering RNA strategy was used to create a series of stable clones in human H157 non-small cell lung cancer cells that expressed less than 5-10% of basal AZ1 levels. Antizyme 1 knockdown clones accumulated greater amounts of the polyamine analogue N (1),N (11)-bis(ethyl)norspermine (BENSpm) and were more sensitive to analogue treatment. The possibility of a loss of polyamine uptake regulation in the knockdown clones was confirmed by polyamine uptake analysis. These results are consistent with the hypothesis that AZ1 knockdown leads to dysregulation of polyamine uptake, resulting in increased analogue accumulation and toxicity. Importantly, there appears to be little difference between AZ1 knockdown cells and cells with normal levels of AZ1 with respect to ODC regulation, suggesting that another regulatory protein, potentially AZ2, compensates for the loss of AZ1. The results of these studies are important for the understanding of both the regulation of polyamine homeostasis and in understanding the factors that regulate tumor cell sensitivity to the anti-tumor polyamine analogues.

Glucosylceramide synthase in the fat body controls energy metabolism in Drosophila

Glucosylceramide synthase (GlcT-1) catalyzes the synthesis of glucosylceramide (GlcCer), the core structure of major glycosphingolipids (GSLs). Obesity is a metabolic disorder caused by an imbalance between energy uptake and expenditure, resulting in excess stored body fat. Recent studies have shown that GSL levels are increased in obese rodents and that pharmacologically reducing GSL levels by inhibiting GlcCer synthesis improves adipocyte function. However, the molecular mechanism underlying these processes is still not clearly understood. Using Drosophila as a model animal, we report that GlcT-1 expression in the fat body, which is equivalent to mammalian adipose tissue, regulates energy metabolism. Overexpression of GlcT-1 increases stored nutrition (triacylglycerol and carbohydrate) levels. Conversely, reduced expression of GlcT-1 in the fat body causes a reduction of fat storage. This regulation occurs, at least in part, through the activation of p38-ATF2 signaling. Furthermore, we found that GlcCer is the sole GSL of the fat body, indicating that regulation of GlcCer synthesis by GlcT-1 in the fat body is responsible for regulating energy homeostasis. Both GlcT-1 and p38-ATF2 signaling are evolutionarily conserved, leading us to propose an evolutionary perspective in which GlcT-1 appears to be one of the key factors that control fat metabolism.

Abstract 3597: Pazopanib potentiates paclitaxel-induced mitotic catastrophe in association with inhibition of aurora kinases

Abstract Background: We recently reported a 49% RECIST response rate in conjunction with a phase 2 clinical trial of the multi-targeted kinase inhibitor pazopanib in differentiated thyroid cancers (DTC; Lancet Oncology 2010, 11:962-72), prompting a search for synergistic pazopanib-containing doublets in thyroid cancer cell lines. Methods and Results: Among all explored pazopanib-containing combinations, paclitaxel and pazopanib produced the greatest combined cytotoxic effects (continuous exposure pazopanib, 24 h paclitaxel; Combination Index, CI, at IC90=0.3). Interestingly, analogous synergy resulted when combining pazopanib with other antimicrotubule agents including other taxanes (docetaxel), Vinca alkyloids (vincristine) and epothilones (ixabepilone) – suggesting a class effect. Further, we found this combination to be synergistic in multiple cancer cell lines including DTC, medullary and anaplastic thyroid cancer lines, and in the A549 lung cancer line. In pursuit of underlying mechanism(s), we noted that the combination produced a greater fraction of cells in G2/M phase of the cell cycle and increased apoptosis in comparison to paclitaxel alone, prompting us to examine whether pazopanib might potentiate the effects of paclitaxel on mitosis. Analysis of cell fate resulting from pazopanib and paclitaxel alone and in combination using time-lapsed video microscopy demonstrated that as low as 2.5 μM pazopanib augmented paclitaxel-induce mitotic catastrophe by 2.4 fold, while inducing no appreciable cell death alone. As pazopanib is somewhat promiscuous in inhibition of kinases, we hypothesized that observed combined effects with antimicrotubule agents might be attributed to its inhibition of cell cycle by targeting CDKs or mitosis by targeting aurora kinases. Inhibition of CDKs 1 and 2 required pazopanib concentrations 1000-times those required to inhibit its primary kinase targets, VEGF-Rs; whereas aurora kinases A and B were inhibited at much lower concentrations (IC50s, 500-1000 nM). Hence, we pursued shRNA aurora A knockdown experiments: potentiation of pazopanib effects were observed in knockdown clones, suggesting that pazopanib single-agent effects are at least in part mediated via aurora A inhibition. Moreover, augmentation of paclitaxel effects were achieved in knockdown clones that rivaled those observed for the pazopanib/paclitaxel combination. Additionally, pazopanib/paclitaxel synergy was reduced in aurora A knockdown clones (CI at IC80: vector&amp;lt;0.5, knockdown clones &amp;gt;1.0), indicating that aurora A inhibition by pazopanib is contributory to paclitaxel/pazopanib synergy. Conclusions: Pazopanib combines with antimicrotubule agents to produce cytotoxic synergy associated with augmentation of mitotic catastrophe that appears at least in part mediated by its ability to inhibit aurora kinases. Supported in part by CA125750. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3597. doi:10.1158/1538-7445.AM2011-3597

Abstract 2422: Epithelial interleukin-4 receptor promotes metastasis of mammary tumor cells

Abstract Interleukin-4 (IL4) is known primarily for its immune system functions where it can stimulate lymphocytes and promote macrophage activation. Various cancer cells, including breast, are now known to express functionally active IL4 receptors, which appear to play a role in cell survival. We believe that blocking IL4 signaling specifically in epithelial cells will attenuate metastasis because of reduced cell survival in secondary sites. To test this hypothesis, we have generated multiple clones of two murine mammary tumor cell lines that show efficient knockdown of an essential component of the IL4 receptor, IL4-receptor [IL4R] alpha. Initial characterization of knockdown and control clones showed a significantly slower growth rate in vitro as assessed both by metabolic (MTT) assays and direct cell counting. Flow cytometric analysis using either annexin V staining or sub G1 measurement showed that clones lacking IL4Ralpha had 2-fold higher basal levels of apoptosis. To test effects in vivo, we have performed experimental and spontaneous metastasis assays using clones of the 4T1 cell line. Both pulmonary and hepatic metastatic lesions were significantly decreased [2.4-fold lower] in mice injected with IL4Ralpha knockdown clones compared to controls. There was however no significant effect on the primary mammary tumors. Together these results suggest that IL4 signaling in epithelial tumor cells is pro-metastatic, and blocking it may be a useful therapeutic approach for preventing metastatic spread. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2422. doi:10.1158/1538-7445.AM2011-2422

Abstract 1443: RhoE is underexpressed in hepatocellular carcinoma and suppresses cell motility and invasiveness

Abstract Hepatocellular carcinoma (HCC) is a major malignancy worldwide and is the second commonest fatal cancer in Southeast Asia and Mainland China including Hong Kong, due to the high prevalence of hepatitis B viral infection. Despite definite improvements in the outcome of patients with HCC, the overall prognosis of this cancer is still unsatisfactory because of late presentation and frequent tumor recurrence after surgical resection. In this regard, knowledge of the molecular and cellular targets underlying the development and progression of liver cancer may provide novel opportunities for therapeutic interventions for this cancer. As part of our continual study on the Rho/ROCK pathway in HCC, in this current study, we aimed to find out the role of RhoE (also known as Rnd3), a member of the Rho family of small GTP-binding proteins, in HCC. We have found that RhoE was frequently underexpressed in 43/71 (60.6%) of our human HCCs at mRNA level using qPCR, as compared with their corresponding non-tumorous livers (p &amp;lt;0.001). Moreover, RhoE knockdown in HCC cells by lentiviral infection of shRhoE enhanced cell migration and invasion in vitro. Using transwell migration assay, we showed that stable shRNA knockdown of RhoE in SMMC7721 and BEL7402 HCC cell lines resulted in faster migration of the cells than their corresponding non-target controls. Moreover, stable RhoE knockdown clones also invaded through the matrigel faster in matrigel invasion assay. Previous studies have indicated that phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at Thr850 was essential for phosphorylation of myosin II and crucial for cell contractility. To further delineate the roles of RhoE on actomyosin contractility, the MYPT1 phosphorylation levels in the Rho stable knockdown transfectants in SMMC, BEL and Hep3B HCC cells were assessed by Western blotting and found to be increased as compared with their non-target control. This increased phosphorylation was reversed by ROCK inhibitor Y27632. Furthermore, the ROCK inhibitor Y27632 was able to suppress the accelerated cell migration brought by the knockdown of RhoE in SMMC, suggesting that RhoE may exert its effects by inhibiting the activity of ROCK1. Since MYPT1 is specifically phosphorylated by ROCK at Thr850, the finding suggests that RhoE interacts with and suppresses the activity of ROCK1 in HCC cells. To conclude, our data suggest that RhoE in HCC suppresses both cell motility and invasiveness of HCC cells and that RhoE may exert its effects by inhibiting the activity of ROCK1. (This study was funded by Hong Kong Research Grants Council CRF grants (RGC CRF HKU1/06C and HKU 7/CRG/09) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1443. doi:10.1158/1538-7445.AM2011-1443

Abstract 1054: S100B affects cell signaling in malignant melanoma

Abstract Establishing the mechanism by which S100B effects ERK and its downstream proteins will provide valuable insight into the role of S100B in the progression of melanoma and potentially aid in developing new pharmacological drugs. S100B is a 21.5 kDa symmetric homodimer that is highly conserved and expressed in a number of tissues and cell lines, including melanocytes. Generally, low levels of S100B have trophic effects, while higher levels are problematic, as is the case in human malignant melanoma. S100B is an effective and widely used prognostic marker for malignant melanoma, with increased levels of serum S100B being predictive of disease stage, increased recurrence, and low overall survival. It has previously been found that S100B directly interacts with the tumor suppressor protein p53, down-regulating its protein levels, thereby allowing damaged cells to proliferate and mutations to propagate. S100B has also been implicated in several other signaling pathways, including the MAPK pathway (BRAF-MEK-ERK). The initial hypothesis tested was that S100B inhibits the phosphorylation of ERK targets in a calcium-dependent manner. To evaluate the significance of S100B in melanoma cell lines, stable S100B knock-down clones were created using lentiviral shRNA particles. The extent of the knock-down varies among the clones and shows a correlation between the level of remaining S100B and the level of phosphorylated ERK, where the clone with the least amount of S100B also has the lowest level of phosphorylated ERK. The clones also display a decreased rate of proliferation measured with both an MTT based assay, as well as with electrical impedance. Over-expression studies were also conducted where normal human S100B, but not a mutant S100B incapable of binding calcium, results in increased phosphorylated ERK, but decreased phosphorylated RSK at Thr573. To determine if S100B was interacting with RSK directly, pull-down assays were performed, and RSK was detected in S100B pull-downs in the presence of calcium; however, it was not detected in the presence of the calcium chelator EDTA. In conclusion, we demonstrate that the calcium-binding protein S100B affects the MAPK pathway by simultaneously increasing phosphorylated ERK and decreasing phosphorylated RSK, together which are consistent with the increased growth rates and cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1054. doi:10.1158/1538-7445.AM2011-1054

Abstract 2290: Crosstalk between ER and PI3K signaling pathways in human luminal type breast cancer and its therapeutic implications

Abstract Background: We have recently shown that the luminal B subtype breast cancer (BC) is associated with PI3K molecular signature. Breast tumors of the luminal B subtype generally have lower ER expression levels and/or activity and are more aggressive and less sensitive to endocrine therapy when compared to the luminal A tumors. To further investigate the role of PI3K signaling in BC luminal types and endocrine resistance, we have characterized an array of luminal A and B cell lines, as well as their endocrine resistant derivatives, to examine how modulation of the PI3K pathway can influence ER activity and endocrine sensitivity. Materials and Methods: We focused on endocrine resistant derivatives of two luminal A (ZR75-1 and BT483) and two luminal B (ZR75-B and CAMA-1) cell lines. Resistant clones were developed by long-term (&amp;gt;6 months) endocrine treatment [estrogen deprivation (ED) or ED plus Tamoxifen (Tam)]. IC50 values of the PI3K inhibitor BEZ-235 were determined in the parental and the endocrine resistant lines, and qRT-PCR was performed to measure the effect of the inhibitor on mRNA levels of ER and its regulated genes (e.g. PR, CAV1, IGF1R and IRS1). Cells were also treated (4 days) with Tam, BEZ-235, or the combination and growth inhibition assays were conducted to assess the drugs efficacy. Inducible PTEN knockdown clones were established in MCF7L and BT483 cell lines. Endocrine sensitivity of these clones was studied in vitro and in vivo, using xenograft mouse models. Results: Ten days of treatment showed that all parental lines were sensitive to endocrine therapy. In contrast, stabilized endocrine resistant cell lines showed similar cell growth rate under Tam or ED treatment compared to control E2 treatment (p&amp;gt;0.05). ZR75-1 endocrine resistant derivatives acquired higher sensitivity to BEZ-235 treatment compared to the parental (p&amp;lt;0.05); a finding that is currently being confirmed in other cell line models. BEZ-235 increased expression of ER and its targeted genes in all 4 parental cell lines (p&amp;lt;0.05). BEZ-235 and Tam administered together additively suppressed cell growth in all 4 parental cell lines (p&amp;lt;1E-5). PTEN knockdown significantly increased the levels of pAkt and decreased the expression of ER and its regulated genes (PR and IGF1R). In vivo xenograft mouse models showed that PTEN knockdown renders tumors continued growth under ED, in contrast to the regression of parental/control tumors under this endocrine regimen (p&amp;lt;0.05). Discussion: The inverse association between PI3K and ER pathways in luminal BC cell lines validates the relevance of these models for further studies. Endocrine resistant phenotype may be partially caused by activated PI3K signaling. Our data suggest that inhibition of PI3K signaling can restore ER levels and/or activity, thereby reversing the aggressive and hormonal-refractory phenotype of the tumors, and achieving improved endocrine therapy response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2290. doi:10.1158/1538-7445.AM2011-2290

Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases

Large-scale fed-batch cell culture processes of CHO cells are the standard platform for the clinical and commercial production of monoclonal antibodies. Lactate is one of the major by-products of CHO fed-batch culture. In pH-controlled bioreactors, accumulation of high levels of lactate is accompanied by high osmolality due to the addition of base to control pH of the cell culture medium, potentially leading to lower cell growth and lower therapeutic protein production during manufacturing. Lactate dehydrogenase (LDH) is an enzyme that catalyzes the conversion of the substrate, pyruvate, into lactate and many factors including pyruvate concentration modulate LDH activity. Alternately, pyruvate can be converted to acetyl-CoA by pyruvate dehydrogenases (PDHs), to be metabolized in the TCA cycle. PDH activity is inhibited when phosphorylated by pyruvate dehydrogenase kinases (PDHKs). In this study, we knocked down the gene expression of lactate dehydrogenase A (LDHa) and PDHKs to investigate the effect on lactate metabolism and protein production. We found that LDHa and PDHKs can be successfully downregulated simultaneously using a single targeting vector carrying small inhibitory RNAs (siRNA) for LDHa and PDHKs. Moreover, our fed-batch shake flask evaluation data using siRNA-mediated LDHa/PDHKs knockdown clones showed that downregulating LDHa and PDHKs in CHO cells expressing a therapeutic monoclonal antibody reduced lactate production, increased specific productivity and volumetric antibody production by approximately 90%, 75% and 68%, respectively, without appreciable impact on cell growth. Similar trends of lower lactate level and higher antibody productivity on average in siRNA clones were also observed from evaluations performed in bioreactors.

Proteome-wide Dysregulation by PRA1 Depletion Delineates a Role of PRA1 in Lipid Transport and Cell Migration

We have previously identified prenylated Rab acceptor 1 (PRA1) as a novel cellular interacting partner for Epstein-Barr virus-encoded oncoprotein, latent membrane protein 1 (LMP1). The intracellular trafficking and full signaling of LMP1 requires its interaction with PRA1. To further explore the role of PRA1 in Epstein-Barr virus-associated nasopharyngeal carcinoma (NPC) cells, we generated several PRA1-knockdown cell clones, which exhibited altered cell morphology and increased cell motility. We identified proteins differentially expressed in the knockdown clones by means of isobaric mass tags labeling coupled with multidimensional liquid chromatography-mass spectrometry. We validated a panel of proteins, which showed consistent up-regulation in PRA1-knockdown clones and participated in regulating lipid homeostasis and cell migration. Immunofluorescence staining further revealed altered localization of these proteins and accumulation of intracellular cholesterol in PRA1-knockdown clones. These effects were phenocopied by treatment with a cholesterol transport inhibitor, U18666A. Moreover, overexpressed PRA1 was able to alleviate the dysregulation of these affected proteins either from PRA1 knockdown or U18666A treatment, implying a role for PRA1 in regulating the levels of these affected proteins in response to altered cholesterol homeostasis. We further demonstrated that LMP1 expression caused PRA1 sequestration in NPC cells, leading to a consequence reminiscent of PRA1 knockdown. Finally, the immunohistochemistry showed a physiological relevance of the PRA1-associated proteome-wide changes in NPC biopsy tissues. In sum, our findings delineated novel roles of PRA1 in lipid transport and cell migration, and provided additional insights into the molecular basis of NPC morphogenesis, namely a consequence of LMP1-PRA1 interaction.

Intertissue Mechanical Stress Affects Frizzled-Mediated Planar Cell Polarity in the Drosophila Notum Epidermis

Frizzled/planar cell polarity (Fz/PCP) signaling controls the orientation of sensory bristles and cellular hairs (trichomes) along the anteroposterior axis of the Drosophila thorax (notum). A subset of the trichome-producing notum cells differentiate as "tendon cells," serving as attachment sites for the indirect flight muscles (IFMs) to the exoskeleton. Through the analysis of chascon (chas), a gene identified by its ability to disrupt Fz/PCP signaling under overexpression conditions, and jitterbug (jbug)/filamin, we show that maintenance of anteroposterior planar polarization requires the notum epithelia to balance mechanical stress generated by the attachment of the IFMs. chas is expressed in notum tendon cells, and its loss of function disturbs cellular orientation at and near the regions where IFMs attach to the epidermis. This effect is independent of the Fz/PCP and fat/dachsous systems. The chas phenotype arises during normal shortening of the IFMs and is suppressed by genetic ablation of the IFMs. chas acts through jbug/filamin and cooperates with MyosinII to modulate the mechanoresponse of notum tendon cells. These observations support the notion that the ability of epithelia to respond to mechanical stress generated by one or more interactions with other tissues during development and organogenesis influences the maintenance of its shape and PCP features.

Regulation of Ca2+-entry in pancreatic α-cell line by transient receptor potential melastatin 4 plays a vital role in glucagon release

Elevation in the intracellular Ca2+ concentration stimulates glucagon secretion from pancreatic α-cells. The Transient Receptor Potential Melastatin 4 channel (TRPM4) is critical for Ca2+ signaling. However, its role in glucagon secreting α-cells has not been investigated. We identified TRPM4 gene expression and protein in the αTC1-6 cell line using RT-PCR and immunocytochemistry. Furthermore, we performed a detailed biophysical characterization of the channel using the patch-clamp technique to confirm that currents typical for TRPM4 were present in αTC1-6 cells. To investigate TRPM4 function, we generated a stable knockdown clone using shRNA and a lentiviral vector. Inhibition of TRPM4 significantly reduced the responses to different agonists during Ca2+ imaging analysis with Fura-2AM. The reduction in the magnitude of Ca2+ signals resulted in decreased glucagon secretion. These results suggested that depolarization by TRPM4 may play an important role in controlling glucagon secretion from α-cells and perhaps glucose homeostasis.

Erythroid Differentiation Regulator 1 (ERDR1) From Nurse-Like Cells Promotes the Survival of Chronic Lymphocytic Leukemia Cells

AbstractAbstract 1372 Background:Chemotherapy resistance in chronic lymphocytic leukemia (CLL) can be mediated by anti-apoptotic signals produced by stromal or nurse-like cells. Developing strategies to overcome this resistance is hindered by the lack of suitable “stromal” targets responsible for these signals. We have discovered that erythroid differentiation regulator 1 (ERDR1) may be a candidate target for such a strategy. In this study, we show Erdr1 is generated by several stromal cell types including bone marrow stromal cells, fibrocytes, and nurse-like cells. Furthermore, inhibition of stroma-generated Erdr1 results in increased apoptosis of co-cultured CLL cells. Methods/Results:We initially identified Erdr1 on an Affymetrix array that compared the gene expression of wild type and CCR5-/- mice with pulmonary metastasis. The increased expression of Erdr1 in the wild type mice was particularly pronounced in the pulmonary mesenchymal cells. Therefore, these cells were transfected with one of two shRNAs (shRNA #9 or shRNA#11) and the survival of these cells was compared with mesenchymal cells transfected with a non-targeted control vector. After 15 days in culture, the control cells expanded normally; however, no significant expansion was seen in either the shRNA#9 or shRNA#11 transfected cells. These differences in cellular expansion were associated with differences in apoptosis. 21.4+1.6% of the Erdr1 knockdown cells were annexin V+ compared to 11.2+1.9% of the non-targeted control (p<0.03). Using GFP as a marker for transfection, we were also able to show that knockdown of Erdr1 increased the apoptosis of surrounding non-transfected mesenchymal cells. Thus, Erdr1 is a critical protein for the survival of stromal cells.Further analysis of the mesenchymal cell subpopulations revealed the greatest expression of Erdr1 in the CD45+, thy1.1+/− fibrocytes. When compared to CD45- fibroblasts, the fibrocytes expressed CCR5 and increased Erdr1 expression by 14.2+/−2.9 fold when treated with the CCR5 ligand CCL4. Given the similarities between fibrocytes and nurse-like cells, we went on to measure the effect of Erdr1 inhibition on CLL cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. These clones were then co-cultured with primary CLL cells. At 96 hours, leukemia cells co-cultured with the control lines had expanded by 1.33 + 0.9 compared to 0.74 + 0.22 fold in the knock-down lines (p<0.03). As before, the lack of cellular expansion was associated with an increase in apoptosis.To further show the relevance of these findings to CLL, we demonstrated that human fibrocytes and nurse-like cells expressed mRNA and protein for ERDR1 in all patient samples tested. Implications for the treatment of human disease:Our data demonstrate that ERDR1 is a critically important protein for the survival of nurse-like cells. These data suggest that targeting ERDR1 or the upstream pathway through CCR5 might be a novel approach for the treatment of CLL. Disclosures:No relevant conflicts of interest to declare.

Targeting Histone Deacetylase 6 (HDAC6) as a Novel Approach to Overcome T-Cell Anergy.

AbstractAbstract 1483The anti-inflammatory molecules IL-10 and STAT3 are major hurdles in our efforts to harness effective anti-tumor immunity1. Previously, we have unveiled a role for histone deacetylase 11 (HDAC11) as a negative regulator of IL-10 gene transcriptional activity in antigen-presenting cells (APCs)2. In an effort to better characterize the epigenetic influences upon IL-10, we screened other HDACs and found that over-expression of HDAC6 in the macrophage cell line RAW264.7 was able to transcriptionally upregulate IL-10 in response to LPS. Next, using lentiviral vectors encoding shRNA for HDAC6, we generated stable knockdown clones of HDAC6 (HDAC6KD). In contrast to HDAC6 overexpression, HDAC6KD cells produced less IL-10 both at the mRNA and protein levels. Further analysis of HDAC6KD clones revealed elevated expression of the co-stimulatory molecule B7.2. Functionally, LPS-stimulated HDAC6KD cells were better able to activate anti-HA (hemagglutinin-influenza) transgenic CD4+ T cells to produce IL-2 and IFN-γ. More relevant to the tumor setting, anti-HA CD4+ anergic T cells isolated from animals bearing HA-expressing A20 B-cell lymphoma regained their ability to produce IL-2 and IFN-γ when cultured in vitro with HDAC6KD cells in the presence of cognate HA-antigen. These same T-cells remained anergic when cultured with control APCs displaying normal levels of HDAC6. Mechanistically, we have found that APCs lacking HDAC6 display a significant abrogation of STAT3 activation in response to LPS stimulation. Given the demonstration that HDACs, other than HDAC6, can regulate STAT3 activity3, we are actively investigating whether the effect we have observed is a result of direct HDAC6-STAT3 interaction or a diminished autocrine signaling through IL-10. Taken together, through abrogation of two well-known tolerogenic mechanisms in APCs, IL-10 and STAT3, HDAC6 inhibition represents an attractive therapeutic approach to overcome tumor-induced T-cell anergy and tip the balance towards effective anti-lymphoma immunity.1. Cheng F., et al. Immunity 2003 Sep;19(3):425-36.2. Villagra A., et al. Nat Immunol. 2009 Jan;10(1):92-100.3. Yuan Z., et al. Science. 2005 Jan 14;307(5707):269-73. Disclosures:No relevant conflicts of interest to declare.

UHRF1‐mediated tumor suppressor gene inactivation in nonsmall cell lung cancer

The UHRF1 gene possesses an essential role in DNA methylation maintenance, but its contribution to tumor suppressor gene hypermethylation in primary human cancers currently remains unclear. mRNA expression levels of UHRF1, DNMT1, DNMT3A, DNMT3B, and E2F1 were evaluated in 105 primary nonsmall cell lung carcinomas by quantitative polymerase chain reaction. The methylation status of CDKN2A and RASSF1 promoters was examined by pyrosequencing. UHRF1 was knocked down by short hairpin RNA in A549 lung adenocarcinoma cells. All 4 genes were overexpressed in a coordinated manner in the lung tumor tissues, and their expression correlated with that of E2F1. Higher UHRF1 expression in tumor tissues correlated with the hypermethylation of CDKN2A (P = .005) and RASSF1 promoters (P = .034), and the relationship with a combined epigenotype was even stronger (P = 2.3 × 10(-4) ). When UHRF1 was knocked down in A549 lung adenocarcinoma cells, lower methylation levels of RASSF1, CYGB, and CDH13 promoters were observed. Also, UHRF1 knockdown clones demonstrated reduced proliferation and decreased cell migration properties. Our data demonstrate that UHRF1 is a key epigenetic switch, which controls cell cycle in nonsmall cell lung carcinoma through its ability to sustain the transcriptional silencing of tumor suppressor genes by maintaining their promoters in a hypermethylated status. Thus, UHRF1 should be considered, along with DNMTs, among the potential targets for cancer treatment and/or therapeutic stratification.