Expression of Lipoprotein Lipase In Chronic Lymphocytic Leukemia Cells: Is There a Biological Function?

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3580-3580
Author(s):  
Edit anna Porpaczy ◽  
Stefanie Tauber ◽  
Martin Bilban ◽  
Gerhard Kostner ◽  
Michaela Gruber ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Cell Survival ◽  
Mrna Expression ◽  
Lipoprotein Lipase ◽  
Cell Lines ◽  
Knock Down ◽  
Protein Levels ◽  
Healthy Donors

Abstract Abstract 3580 The expression of lipoprotein lipase (LPL) in CLL cells is an established mRNA surrogate marker for immunoglobulin heavy chain (IgVH) mutational status. High expression of LPL correlates with poor prognosis. However, the possible functional role of LPL in CLL is still unclear. LPL is normally expressed in muscle cells, adipose tissue and macrophages, transported to the luminal surface of endothelial cells where it is bound heparan sulfate-proteoglycans (HSPG). Heparin competes with HSPG for the binding sites and intravenous injection leads to elevated plasma LPL protein levels and enzymatic activity (“heparin release test”). LPL mRNA levels correlate with intracellular protein expression (Heintel et al. Leukemia. 2005; Mansouri et al. Leuk Res. 2010). Moreover cellular lysates from CLL patients contain elevated LPL enzymatic activity compared to healthy donors (Pallasch et al. Leukemia. 2008.). In this study, we investigated the basal (pre-heparin) LPL protein levels by enzyme-linked immunosorbent assay in the serum of 42 CLL patients, 14 non-CLL patients (lymphoma in remission), and 4 healthy donors (HD): Median pre-heparin LPL protein levels were 40.10 ng/ml (range: 5.66–108.44), 44.11 ng/ml (18.26-84.08), and 68.14 ng/ml (33.28-174.38), respectively. Among CLL patients there were no significant differences between those with high (N=16; median LPL protein in serum: 38.10 ng/ml (8.72-73.49)) and low (N=26; 43.12 ng/ml (5.66-108.44) (p=0.354) LPL mRNA expression. Thirteen patients with known LPL mRNA expression were investigated for LPL protein “release” after heparin injection. Ten and twenty minutes after 50 U/kg heparin injection, the elevation of both parameters, LPL protein amount in serum and enzymatic activity in plasma, was similar to those of HD normal values. In detail, medium serum protein levels in samples with high LPL mRNA (N=5) increased from 16.11 to 214.33 and 332.78 ng/ml and in the samples with low mRNA (N=8) from 13.08 to 219.68 and 386.65 ng/ml, respectively. The corresponding median values of the LPL enzymatic activities in high vs. low expressors were: 7.25/15.52/20.01 and 7.45/19.13/20.57 μ M/ml/h. In addition, release of LPL from peripheral mononuclear cells (PBMC) of CLL patients (N=3) by heparin in vitro was absent. Cell viability and LPL mRNA expression remained unaffected in both in vivo and in vitro samples after heparin addition. In order to assess the impact of LPL on cell survival, CLL cells were cultured (N=3) for up to 72 hours with different doses of purified LPL protein. There was no positive effect on cell survival irrespective of primary LPL mRNA expression or culture conditions (with or without FCS). Since these results point to an intracellular effect of LPL, we aimed to identify downstream targets by knock down with siRNA against LPL in 7 CLL samples and 5 cell lines (hepatocellular carcinoma, cervix carcinoma, colon carcinoma, multiple myeloma and acute monocytic leukemia) with high LPL mRNA expression. Gene expression changes were analyzed by microarrays (GeneChip® Human Gene 1.0 ST Array, Affymetrix). Fifteen genes were up- (N=4) or downregulated (N=11) in at least 3 of 5 cell lines by more than 1.5-fold (e.g. GSTP1, COROC1). Nine genes were at least 1.5-fold downregulated in parallel with LPL in the CLL samples only. These genes belong to various pathways (e.g. cell cycle, signaling in immune system, metabolism of carbohydrates) and seem to be specific for CLL. Cross-validation of individual genes is under way. Our data suggest that (1) neither basal serum LPL protein levels nor heparin-induced LPL release in CLL patients are suitable clinical prognostic markers; (2) Stimulation with external LPL protein does not affect CLL cell survival; (3) siRNA knock-down of LPL induces changes in various functional pathways. We conclude that the key role of LPL expression in high-risk CLL is related to its (intra)cellular expression. Disclosures: No relevant conflicts of interest to declare.

GLI1 Directly Regulates the Transcription of AKT Genes in Diffuse Large B-Cell Lymphoma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2399-2399
Author(s):  
Nitin K Agarwal ◽  
Changju Qu ◽  
Kranthi Kunkalla ◽  
Yadong Liu ◽  
Francisco Vega
Keyword(s):  
Cell Viability ◽  
Cell Survival ◽  
Mrna Expression ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Luciferase Reporter ◽  
Knock Down ◽  
Hh Signaling

Abstract Abstract 2399 Activation of the Hedgehog (Hh)/glioma-associated oncogene (GLI) pathway has been found in a growing number of malignancies. We have provided evidence that canonical Hh signaling is required for cell survival and proliferation of DLBCL cell lines. To confirm the pathogenic role of GLI1 in DLBCL, we established GLI1 knock down DLBCL cell lines (OCI-Ly19, HBL-1 and BJAB) using a lentiviral shRNA system and performed cell viability and apoptosis assays. Cell viability assays demonstrated that GLI1 knockdown DLBCL cells experienced a statistically significantly decrease in the number of viable cells in comparison with control cells harboring scramble shRNA. To examine whether decreases number of cell viability in GLI1 knock down cells were due to apoptosis, we performed annexin V and PI assays. We observed marked increase of apoptosis in GLI1 knock down DLBCL cells versus controls (2.5 fold increase for OCI-Ly10, and 5 fold for HBL1 and BJAB). To investigate the mechanism by which GLI1 regulates tumorigenesis and cell survival, we searched for whole genome GLI1-target genes in DLBCL cells using CHIP sequencing technique and identified AKT genes as potential targets of GLI1. Using pharmacological and silencing approaches, we observed that Hh signaling modulates the expression of AKT genes in DLBCL cells. We further identified two putative binding sites for GLI1 in the AKT1 promoter region and confirmed their functionality using chromatin immunoprecipitation, luciferase reporter and site-directed mutagenesis assays. To investigate whether there is any correlation between AKT1 and GLI1 mRNA expression in human DLBCL tumors, we performed quantitative real-time PCR analyses in 17 frozen DLBCL specimens including apharesis samples from pleural effusions. The real time PCR analysis revealed a strong Spearmen correlation coefficient (R2=0.9) between GLI1 and AKT1 mRNA expression. In summary, we provide evidence of the role of GLI1 in the pathobiology of DLBCL and demonstrated a cross talk, at the transcriptional level, between Hh signaling and AKT in DLBCL. A link between these 2 pathways at the trasncriptional level was not previoulsy documented. This finding is of clinical interest as AKT has a key role in lymphoma cell survival and constitutive activation of AKT has been described in DLBCL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TIMP-2 regulates proliferation, invasion and STAT3-mediated cancer stem cell-dependent chemoresistance in ovarian cancer cells

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Ruth M. Escalona ◽  
Maree Bilandzic ◽  
Patrick Western ◽  
Elif Kadife ◽  
George Kannourakis ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Lines ◽  
Cancer Progression ◽  
Mrna Level ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Ovarian Cancer Cells ◽  
Knock Down ◽  
Protein Levels ◽  
Response To Chemotherapy

Abstract Background The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines. Methods FT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence. Results Sixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers. Conclusions The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance.

scholarly journals Olanzapine inhibits hepatic apolipoprotein A5 secretion inducing hypertriglyceridemia in schizophrenia patients and mice

2021 ◽  
Author(s):  
Xiansheng Huang ◽  
Yiqi Zhang ◽  
Wenqiang Zhu ◽  
Piaopiao Huang ◽  
Jingmei Xiao ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Plasma Triglyceride ◽  
High Dose ◽  
Mrna Levels ◽  
Apolipoprotein A5 ◽  
Protein Levels ◽  
Olanzapine Treatment ◽  
Primary Mouse

Olanzapine, an antipsychotic drug, was reported to induce hypertriglyceridemia, whereas the underlying mechanism remains incompletely understood. This study was to determine the role of apolipoprotein A5 (apoA5) in olanzapine-induced hypertriglyceridemia. In this study, 36 drug-naive and first-episode schizophrenic adult patients (aged 18-60 years) in a multi-center clinical trial (ClinicalTrials.gov NCT03451734) were enrolled. Before and after olanzapine treatment, plasma lipid and apoA5 levels were detected. Moreover, 21 female C57BL/6 J mice (8 weeks old) were divided into 3 groups (n = 7/each group): low-dose olanzapine (3 mg/kg/day), high-dose olanzapine (6 mg/kg/day) and control group. After 6 weeks, plasma glucose, lipids and apoA5 as well as hepatic apoA5 protein and mRNA expression in these animals were detected. In our study in vitro, primary mouse hepatocytes and HepG2 cells were treated with olanzapine of 25, 50, 100 μmol/L, respectively. After 24 hours, apoA5 protein and mRNA levels in hepatocytes were detected. Our study showed that olanzapine treatment significantly increased plasma triglyceride levels and decreased plasma apoA5 levels in these schizophrenic patients. A significant negative correlation was indicated between plasma triglyceride and apoA5 levels in these patients. Consistently, olanzapine dose-dependently increased plasma triglyceride levels and decreased plasma apoA5 levels in mice. Surprisingly, an elevation of hepatic apoA5 protein levels was detected in mice after olanzapine treatment, with no changes of APOA5 mRNA expression. Likewise, olanzapine increased apoA5 protein levels in hepatocytes in vitro, without changes of hepatocyte APOA5 mRNA. Therefore, our study provides the first evidence about the role of apoA5 in olanzapine-induced hypertriglyceridemia. Furthermore, plasma apoA5 reduction, resulting in hypertriglyceridemia, could be attributed to olanzapine-induced inhibition of hepatic apoA5 secretion.

scholarly journals Smoothened Stabilizes and Protects TRAF6 from Proteosomal Degradation: A Novel Non-Canonical Role of Smoothened with Implications in Lymphoma Biology

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 646-646
Author(s):  
Changju QU ◽  
Amineh Vaghefi ◽  
Kranthi Kunkalla ◽  
Jennifer R Chapman ◽  
Yadong Liu ◽  
...  
Keyword(s):  
Cell Survival ◽  
Cell Lines ◽  
E3 Ligase ◽  
B Cell Lymphoma ◽  
Mrna Levels ◽  
Protein Levels ◽  
Multiple Cancers ◽  
Proteosomal Degradation ◽  
Hh Signaling

Abstract Tumor necrosis factor receptor-associated factor 6 (TRAF6), an (K63) E3-ligase, plays a crucial role in many biological processes and its activity is relevant in the biology of multiple cancers including diffuse large B cell lymphoma (DLBCL). Although molecules that trigger TRAF6 activation have been defined, those that stabilize TRAF6 levels and/or enhance TRAF6 function remain largely unclear. Previously, we found that activation of smoothened (SMO) with recombinant Hedgehog (Hh) ligand increased the binding between SMO with TRAF6, as well as TRAF6 protein levels (Blood 2013; 121:4718-28). In addition, transient overexpression of SMO resulted in increased K63-Ub of both TRAF6 and NEMO indicating stabilization of these proteins resulting in NF-kB activation. This is relevant, as more recently we found that TRAF6 amplifies pAKT signaling in DLBCL and that TRAF6 is the dominant E3 ligase for the K63-Ub of AKT in DLBCL. Moreover, TRAF6 recruitment to the cell membrane, and stabilization of its ubiquitination profile are facilitated by SMO. SMO is a member of the Frizzled-class G-protein-coupled receptor (GPCRs) and is traditionally known for its role as signal transducer in canonical Hedgehog (Hh) signaling. These observations prompted us to investigate whether the ability of SMO to increase TRAF6 levels is limited to ligand induced signaling, whether it contributes to chemoresistance in DLBCL cells, and whether SMO directly participates in controlling TRAF6 levels. To confirm the regulatory role of SMO in the TRAF6/AKT axis in DLBCL cells (HBL1 and HT) and further outline the nature of the underlying regulation, we measured the impact of activation of the Hh pathway with recombinant Shh ligand on TRAF6 levels, with and without SMO knockdown or recombinant SMO overexpression. Canonical Hh signaling results in the activation of the GLI1 transcription factor and the subsequent elevation of GLI1 mRNA levels is an established indicator of activation of the Hh pathway. However, neither SMO activation nor the knockdown of GLI1 had a significant impact on TRAF6 mRNA levels. These findings indicate that TRAF6 is not transcriptionally regulated by SMO signaling through GLI1 (canonical Hh signaling). In contrast, overexpression of SMO or siRNA knockdown of SMO resulted in an increase or decrease of TRAF6 protein levels, respectively. Consistent with the decrease of AKT activation (pAKT T308 and S473) after TRAF6 knockdown, the increase in TRAF6 levels that follows SMO overexpression resulted in an increase in the levels of AKT phosphorylation. Altogether, these observations suggest a post-translational regulation of TRAF6 by SMO. Indeed, stable knockdown of SMO dramatically reduces the half-life of TRAF6 in both HBL1 and HT cells in the presence of cyclohexamide. Furthermore, overexpression of SMO increases K63-Ub of both TRAF6 and AKT. In contrast, the SMO induced decrease in K48-Ub occurred only for TRAF6 but not for AKT. These data link the SMO-stimulated activation of TRAF6 to the enhancement of AKT signaling and protection of TRAF6 from proteasomal degradation. Mechanistically, we found that SMO, through its C-terminal tail, stabilizes TRAF6 and protects TRAF6 from proteosomal degradation, an effect mediated by ubiquitin-specific protease-8 (USP8). Importantly, this functional link between SMO and TRAF6 is reflected in DLBCL patient samples where high expression of both molecules correlates with poor prognosis. Resistance to DXR is a serious challenge in the treatment of DLBCL, and activated AKT is known to contribute to DXR resistance in multiple cancers including DLBCL. We evaluated whether SMO and TRAF6 support resistance to DXR in DLBCL cell lines. We exposed HT and HBL1 cells as well as their counterparts with stable knockdown of TRAF6 or SMO to DXR for 96hrs. Cell viability after exposure to DXR was determined by an Annexin V and PI staining assay. Silencing SMO or TRAF6 dramatically decreased cell survival after treatment with DXR. In summary, we report that SMO is needed to facilitate and maintain TRAF6-dependent elevated pAKT levels in DLBCL cell lines of germinal (GC) and non-GC subtypes, and that the SMO/TRAF6 axis contributes to DXR resistance in DLBCL. Our study reveals a novel and potential central cell survival signaling mechanism in which SMO stabilizes and protects TRAF6 from proteosomal degradation. Disclosures Lossos: Affimed: Research Funding.

Targeting of CD317 by the immunotoxin HM1.24-ETA’ to allow immunotherapy in glioblastoma patients.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13560-e13560
Author(s):  
Dorothee Gramatzki ◽  
Emese Szabo ◽  
Martin Gramatzki ◽  
Matthias Peipp ◽  
Michael Weller
Keyword(s):  
Mrna Expression ◽  
Cell Lines ◽  
Dependent Manner ◽  
Glioblastoma Cell ◽  
Single Chain ◽  
Human Glioblastoma ◽  
Human Glioblastoma Cell ◽  
Protein Levels ◽  
Glioblastoma Cell Lines

e13560 Background: Glioblastoma is the most common primary malignant brain tumor with a poor prognosis. CD317 (HM1.24) is a transmembrane protein and may exist in differently spliced variants. It is highly expressed on plasma cells in multiple myeloma, as well as in certain solid tumor types. While several antibody drug conjugates are already in clinical practice, small immunotoxins with a different intracellular mode of action are only established in hairy cell leukemia. The immunotoxin HM1.24-ETA’ protein is a CD317 single chain Fv (scFv) antibody fused to a truncated version of Pseudomonas aeruginosa exotoxin A (ETA’). Methods: In vivo CD317 mRNA expression in human glioma of different grades and survival probabilities of glioblastoma patients based on CD317 mRNA expression were analyzed using the database of the Cancer Genome Atlas network (TCGA). CD317 protein expression was analyzed by immunohistochemistry in a human tissue microarray (TMA). In vitro CD317 mRNA expression was assessed by RT-PCR and CD317 protein levels by flow cytometry in several human glioblastoma cell lines. A cytotoxicity assay after treatment with HM1.24-ETA’ immunotoxin was performed in human glioblastoma cell lines. Results: Data on mRNA expression from the TCGA database demonstrated, that CD317 was upregulated in human glioblastomas compared to lower grade gliomas. In the group of glioblastoma patients increased CD317 mRNA expression was associated with decreased probability of survival ( p< 0.001). CD317 protein levels correlated directly with the tumor grade of astrocytic gliomas in the TMA. CD317 was expressed heterogeneously on mRNA and protein levels in the tested cell-lines in vitro. HM1.24-ETA’ induced cytotoxicity in CD317-positive glioblastoma cells in a concentration-dependent manner. Animal experiments currently performed suggest activity in glioblastoma xenografted mice. Conclusions: These data highlight CD317 as an interesting target antigen and HM1.24-ETA’ immunotoxin as a strategy for immunotherapy of glioblastoma patients.

Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5080-5080
Author(s):  
Shankaranarayana Paneesha ◽  
Raghu Adya ◽  
Hemali Khanji ◽  
Ed Leung ◽  
C. Vijayasekar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Mrna Expression ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Human Myeloma Cell

Abstract Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection. Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE). Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

scholarly journals Acquiring Metastatic Competence by Oral Squamous Cell Carcinoma Cells Is Associated with Differential Expression ofα-Tubulin Isoforms

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Becky Lou ◽  
David Engler ◽  
William Dubinsky ◽  
Jean Wu ◽  
Nadarajah Vigneswaran
Keyword(s):  
Cell Lines ◽  
Cell Shape ◽  
Quantitative Difference ◽  
Distribution Patterns ◽  
Carcinoma Cells ◽  
Protein Levels ◽  
Distinct Cell ◽  
Tubulin Isoforms

We performed comparative global proteomics analyses of patient-matched primary (686Tu) and metastatic (686Ln) OSCC cells. The metastatic OSCC 686Ln cells showed greaterin vitromigratory/invasive potential and distinct cell shape from their parental primary 686Tu cells. Ettan DIGE analysis revealed 1316 proteins spots in both cell lines with >85% to be quantitatively similar (<2 folds) between the two cell lines. However, two protein spots among four serial spots were highly dominant in 686Ln cells. Mass spectrometry sequencing demonstrated all four spots to beα-tubulin isotypes. Further analysis showed no significant quantitative difference in theα-tubulin between the two cell lines either at mRNA or protein levels. Thus, two distinct isoforms ofα-tubulin, probably due to posttranslational modification, were associated with metastatic 686Ln cells. Immunofluorescence demonstrated remarkable differences in the cytosolicα-tubulin distribution patterns between the two cells. In 686Tu cells,α-tubulin proteins formed a normal network composed of filaments. In contrast,α-tubulin in 686Ln cells exhibited only partial cytoskeletal distribution with the majority of the protein diffusely distributed within the cytosol. Sinceα-tubulin is critical for cell shape and mobility, our finding suggests a role ofα-tubulin isoforms in acquisition of metastatic phenotype and represents potential target for therapeutic intervention.

scholarly journals Cell-based siRNA screens highlight triple-negative breast cancer cell epigenetic vulnerability

2021 ◽  
Vol 7 (4) ◽  
pp. 196
Author(s):  
Albane Gaudeau ◽  
Coralie Clua Provost ◽  
Thierry Dorval ◽  
Andrew Walsh ◽  
Michael Hannus ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Viability ◽  
Cell Survival ◽  
Cell Lines ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Knock Down ◽  
Tnbc Cell ◽  
Low Efficiency

<p><strong>Background:</strong> Triple-negative breast cancer (TNBC) is a heterogeneous disease defined by ER-, PR- and HER2-negative phenotype and in most cases, a relatively aggressive clinical behaviour. The lack of specific targeted therapies and low efficiency of currently available chemotherapies spurred several clinical trials in the last few years. Despite encouraging results, TNBC still remains a major unmet medical need that prompted us to explore the role of 863 epigenetic modulators in TNBC cell survival.</p><p><strong>Methods:</strong> A comprehensive siRNA library was screened to explore the role of known epigenetic modulators in TNBC cell viability and growth. The knock-down effect was evaluated for 863 epigenetic genes using 4 siRNAs/gene in two TNBC and a non-TNBC cell lines using ATP-based luminescence and nuclei count image-based assays. Considering siRNA off-target effects, four analysis methods including a classical threshold-based analysis and three ranking methods were applied to determine on-target hits for each screen readout. Hit genes common to both phenotypic readouts highlighted strong epigenetic players involved in TNBC cell survival.</p><p><strong>Results:</strong> Overall, knock-down of many epigenetic modulator genes mitigates cell survival in TNBC and a non-TNBC cell lines depicted from both phenotypic readouts. Interestingly, ranking-based analysis confirmed hit genes identified in threshold-based analysis and also revealed additional hits enabling us to confirm CDK1 and KMT5A as important regulators in TNBC cell viability and growth. Surprisingly, CHAF1A appeared as a new candidate gene involved in TNBC cell survival.</p><p><strong>Conclusions:</strong> Taken together, siRNA epigenetic screening results identified CHAF1A as a novel regulator of TNBC cell survival.</p>

Role of Selective HDAC6 Inhibition On Multiple Myeloma Bone Disease

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 328-328
Author(s):  
Loredana Santo ◽  
Diana Cirstea ◽  
Bin Wang ◽  
Tso-Pang Yao ◽  
Joy Y. Wu ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Board Of Directors ◽  
Cell Lines ◽  
Bone Disease ◽  
Research Funding ◽  
Advisory Committees ◽  
Vitro Data ◽  
In Vitro Data

Abstract Abstract 328 In multiple myeloma (MM), deregulated osteoclast (OC)/osteoblast (OB) cross-talk induces osteolytic bone lesions. The HDAC6 selective inhibitor, rocilinostat (ACY-1215), in combination with bortezomib has shown potent anti myeloma activity in preclinical studies, which provided the rationale for a clinical trial that is currently recruiting relapsed/refractory MM patients (NCT01323751). However, while the beneficial role of bortezomib in tumor-related bone disease has been previously described, the effect of HDAC6 inhibition is not known. Evidence suggests a positive effect on bone turnover as pan HDAC inhibitors accelerate OB maturation and suppress OC maturation in vitro. Here, we evaluated effects of the selective HDAC6 inhibitor rocilinostat (Acetylon Pharmaceuticals, Inc), alone and in combination with bortezomib in MM bone disease. Rocilinostat (1 μM) alone and in combination with bortezomib (2.5 nM) inhibited OC differentiation, evidenced by a decreased number of TRAP positive multinucleated cells and bone-resorbing activity. In addition, rocilinostat (1 μM) significantly decreased cell growth of mature OC in co-culture with MM cell lines and in combination with bortezomib inhibited transcription factors implicated in OC differentiation including p-ERK, p-AKT, c-FOS, and NFATC1. Importantly, such an effect was cytokine (RANKL and M-CSF) dependent. Further, rocilinostat, alone and in combination, enhanced OB differentiation, evidenced by increased alkaline phosphatase (ALP) enzyme activity and alizarin red staining. In addition, we found increased mRNA expression of beta-catenin, osteocalcin, ALP, and RUNX2. Based on this promising in vitro data, we used the xenograft model of disseminated human MM in SCID mice to study the effect of rocilinostat, alone and in combination with bortezomib, on MM bone disease. MM.1S-GFP-Luc cells were injected intravenously, and MM disease progression was followed by bioluminescence imaging. A significant decrease in tumor burden was observed in mice following three weeks of treatment with rocilinostat, alone or in combination with bortezomib. Isolating serum from control and treated mice, we also observed a significant decrease of TRAPc5b levels, a marker of bone resorption, as well as a significant increase in osteocalcin levels, a marker of bone formation, in the serum of the combination treated cohort. Cells isolated from the calvaria from the combination treated group compared to the control group showed a significant increase in the mRNA expression of ALP, RUNX2, and osterix, as well as a significant decrease in the mRNA expression ratio of RANKL/OPG. To elucidate the role of HDAC6 inhibition on bone turnover, we used HDAC6 knockout mice. Cells were isolated from femurs, tibia, and spine of 2 month-old wild type (WT) and HDAC6 knockout (KO) mice and mRNA expression for osteocalcin, ALP, RUNX2 and osterix was assessed by qPCR. We observed a significant increase in osteocalcin mRNA expression without significant changes in the mRNA expression of ALP, RUNX2 and osterix. Bone marrow stromal cells (BMSCs) differentiated from WT and KO mice were co-cultured with MM murine cell lines and, notably, the proliferative advantage conferred by BMSC isolated from HDAC6 KO mice to MM cell lines was significantly decreased compared to WT BMSCs. These data suggest that a microenviroment lacking HDAC6 reduces MM cell proliferation. Moreover, treatment with rocilinostat (1mM) for 24 h inhibited proliferation of MM cells cocultured with WT BMSCs to levels observed in MM cells cultured with KO BMSC lacking endogenous HDAC6. Finally, the effect of co-treatment with rocilinostat (1μM) and bortezomib (2.5 nM) on proliferation of MM cells co-cultured with WT-BMSC was similar to that observed when bortezomib was added to MM cells in cocultures with KO BMSC. In conclusion, the in vitro data and the in vivo results from the xenograft models of human MM in SCID mice, as well as data in the HDAC6 KO mice, indicate a potential beneficial role of HDAC6 inhibition on MM-related bone disease. We are currently performing dynamic and static histomorphometric analysis to confirm this effect on bone remodeling at the tissue level. These effects on bone remodeling are an added benefit for MM patients and will be assessed prospectively in our ongoing clinical trial. Disclosures: Hideshima: Acetylon: Consultancy. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Jones:Acetylon Pharmaceuticals, Inc.: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millennium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document