scholarly journals The Gfi1-SphK1 Axis Regulates the Growth and Survival of Myeloma Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5615-5615
Author(s):  
Daniela Nicoleta Petrusca ◽  
Evgeny Berdyshev ◽  
Patrick Mulcrone ◽  
Colin D. Crean ◽  
Judith L Anderson ◽  
...  
Keyword(s):  
Cell Growth ◽  
Protein Expression ◽  
Cell Fate ◽  
Cell Lines ◽  
Cancer Progression ◽  
Specific Inhibitor ◽  
Lipid Mediator ◽  
Growth And Survival ◽  
Protein Levels ◽  
Adhesive Interactions

Abstract In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable hematologic malignancy due to emergence of drug-resistant clones. We previously reported that MM cells upregulate expression of the transcriptional repressor, Growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSC), which induces prolonged inhibition of osteoblast differentiation. We recently showed that Gfi1 levels are also increased in the majority of CD138+ cells from MM patients and cell lines ,and Gfi1 levels significantly correlated with Mcl-1 protein expression. Further, Gfi1 repressed MM cell death by inhibiting expression of apoptosis-inducing genes. Importantly, Gfi1 overexpression in MM cells enhanced MM cell growth and conferred resistance to proteasome-inhibitor- induced apoptosis. However, the mechanisms responsible for these effects of Gfi1are unknown. Sphingosine kinase 1 (SphK1) is overexpressed in many cancers including MM, and catalyzes the phosphorylation of sphingosine (SPH) to sphingosine-1-phosphate (S1P). S1P is a pleiotropic lipid mediator that regulates cell survival, migration, the recruitment of immune cells and angiogenesis, all of which contribute to cancer progression. Therefore, we hypothesized that adhesive interactions between MM cells and BMSC stimulate survival and growth of MM cells in part through the Gfi1-SphK1 axis by modulating their sphingolipid profile (Ceramide/SPH/S1P ratio). We found that SphK1 mRNA is highly expressed in CD138+ cells from MM patients and cell lines compared with normal donors. Further, Gfi1 protein expression correlated significantly with SphK1 protein level in CD138+ cells from MM patients and MM cell lines. Soluble factors (IL-6 and S1P) that are increased in the MM microenvironment, hypoxia (1% O2) and adhesive interactions of MM cells with BMSC further increased Gfi1 and SphK1 mRNA and protein levels in MM cells. Gfi1 Knockdown (KD) in MM cells induced a profound decrease of SphK1 mRNA and protein activity and inhibited MM cell growth and viability. In contrast, over-expression of Gfi1 in MM cells increased SphK1 levels that conferred a survival advantage to MM cells over empty vector-transduced control cells. Increased Gfi1 expression also resulted in increased intracellular S1P and decreased sphingosine levels, as measured by LC-MS/MS. Further, treatment of MM cells with a SphK1 specific inhibitor (SKI2), dose-dependently reduced MM cell viability at 24h, regardless of their p53 status. Interestingly, p53 null MM cells were more resistant to SK12, as compared with p53 replete cells. In p53 replete MM cells, SphK1 inhibition significantly reduced c-Myc protein expression, induced autophagy (as shown by increased LC3 II protein levels) and increased total ceramides levels. Moreover, BMSC protected p53 replete MM cells from the anti-survival effects of SKI2 in 3D cultures. These data suggest that Gfi1 regulates MM growth in part by enhancing the expression and activity of SphK1. Taken together, our results support that Gfi1 acts as a key regulator of MM growth and survival, at least partially through modulation of SphK1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. Disclosures Roodman: Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

scholarly journals GFI1-Dependent SGPP1 Repression Promotes Growth and Survival of Myeloma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4387-4387
Author(s):  
Daniela Nicoleta Petrusca ◽  
Evgeny Berdyshev ◽  
Colin D. Crean ◽  
Judith L Anderson ◽  
G. David Roodman
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Cell Fate ◽  
Cell Lines ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Drug Resistant ◽  
Growth And Survival ◽  
Protein Levels ◽  
P53 Status

Multiple myeloma (MM) remains incurable for the vast majority of patients due to emergence of drug resistant clones and mutations inducing drug resistant relapses. This is despite the fact that new therapies have greatly improved progression-free and overall survival for patients with standard risk myeloma. We recently found that the transcriptional repressor GFI1 is increased in bone marrow stromal cells of MM patients (MM-BMSC) where it causes prolonged suppression of osteoblast differentiation, and in CD138+ cells from MM patients, where GFI1 levels significantly correlate with disease progression. We also found that GFI1 overexpression (o/e) enhances MM cell growth and partially confers resistance to proteasome inhibitors in vitro as well as enhances tumor growth and osteoclastogenesis in vivo. Although the mechanisms responsible for these GFI1 effects in p53wt MM cells were p53-dependent, we found that GFI1 is also essential for MM cell survival regardless of their p53 status. The p53-independent mechanisms responsible for Gfi1 effects on MM cells growth and survival of are unknown. Sphingolipids are bioactive lipids that can control MM cell growth and survival. The balance between the levels of Sphingosine-1-phosphate (S1P) and its metabolic precursors ceramide (Cer) and sphingosine (SPH) form a rheostat that determines whether a cell proliferates or dies. We hypothesized that GFI1 represses SGPP1, the enzyme responsible for degrading S1P via salvage and recycling of sphingosine into long-chain ceramides. This repression changes the intracellular sphingolipid profile (Cer/S1P/SPH ratio) to maintain c-Myc upregulation in a protein phosphatase 2 (PP2A)-dependent manner, thus promoting growth and survival of MM cells. To test this hypothesis we measured S1P, SPH and Cer levels by mass spectrometry (LC-MS/MS). LC-MS/MS evaluation showed that bone marrow plasma of MM patients has significant higher levels of S1P when compared to normal donors. Moreover, intracellular S1P levels of MM.1S GFI1 o/e cells were also significantly higher as compared to those of MM.1S empty vector controls. Knock-down (KD) of Gfi1 in MM.1S cells strikingly increased SGPP1 and decreased SphK1 (the enzyme which catalyzes S1P production) mRNA levels, while GFI1 o/e cells had the opposite effect. We found that CD138+ cells isolated from MM patients expressed elevated levels of SphK1 mRNA compared to MGUS patients, and that SphK1 protein levels directly correlate with GFI1 levels in MM patient CD138+ cells and cell lines (r= 0.527). We also detected an indirect correlation (r= -0.961) between GFI1 and SGPP1 mRNA levels in five different MM cell lines. These results indicate a GFI1-dependent imbalance of the enzymes regulating S1P production. Further, KD GFI1 and SphK1 inhibition (5 μM SK1I) had a profound inhibitory effect on c-Myc protein levels and induced caspase 3 activation as detected by Western blotting, while GFI1 o/e cells had significant higher levels of c-Myc and were more resistant to SK1I treatment. Exogenous ceramide (10 μM Cer 16:0) treatment or SphK1 inhibition (5 μM SK1I), both treatments known to trigger intracellular ceramide production, significantly inhibited MM cell viability (measured by AlamarBlue), regardless of their p53 status (MM1.S p53 +/+ and KMS-11 p53 -/-). This inhibition of MM viability was GFI1-dependent, as GFI1 o/e cells were significantly more resistant to ceramide-induced cell death, which was PP2A dependent, as PP2A inhibition with okadaic acid (OA) restored it. MM.1S cells with KD of GFI1 exhibited significantly higher PP2A activity then control cells, supporting our observation that c-Myc modulation by GFI1 is PP2A-dependent. c-Myc protein levels were significantly decreased in MM.1S control cells treated with ceramide and rescued by OA pre-treatment; thus mimicking the effects of changing GFI1 levels and I2PP2A (the PP2A endogenous inhibitor) and confirms that PP2A mediates the effects of GFI1 on c-Myc. Taken together, our results show that GFI1 acts as a key regulator of MM growth and survival, at least partially through modulation of SGPP1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. Disclosures Roodman: Amgen: Membership on an entity's Board of Directors or advisory committees.

Selective Inhibition of HDAC1 and HDAC2 Is a Potential Therapeutic Option for B-All

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2900-2900 ◽  
Author(s):  
Matthew C. Stubbs ◽  
Won-Il Kim ◽  
Tina Davis ◽  
Jun Qi ◽  
James Bradner ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Hdac Inhibitor ◽  
Therapeutic Option ◽  
Hdac Inhibitors ◽  
Specific Inhibitor ◽  
Selective Inhibition ◽  
Class I ◽  
Protein Levels ◽  
Class I Hdacs

Abstract Abstract 2900 Histone deacetylase inhibitors (HDACi) have emerged as potent anticancer agents, and could open the door for future epigenetic therapies. As our understanding of the importance of epigenetic histone modifications in B-cell acute lymphoblastic leukemia (B-ALL) increases, we hypothesized that HDACi could potentially be a useful therapeutic option. The pan-HDAC inhibitor LAQ824 (Novartis) was toxic to B-ALLs in low nM concentrations in vitro, and treated cells had increased p21 and DNA damage response as indicated by increased γH2A.X protein levels. Additionally, the related compound panobinostat (Novartis) reduced leukemic burden from B-ALL patient samples in primary xenograft models, indicating that pan-HDAC inhibition is a putative B-ALL therapeutic option. To determine HDAC isoform-specific effects, we used a high throughput assay that exposed B-ALL cell lines to a panel of HDAC inhibitors. This screen indicated that tubacin, an HDAC6 specific inhibitor, cannot inhibit B-ALL cell growth within a dose range where HDAC6 is the only HDAC targeted. This finding was further validated using another HDAC6 specific inhibitor, WT-161. The screen also indicated that benzimide compounds such as MGCD-0103 (MethylGene) and MS-275 (Entinostat, Syndax) which only target class I HDACs (HDAC1-3) effectively inhibited growth in the cell lines. These data indicate that inhibiting the class I HDACs is sufficient to suppress B-ALL cell line growth. To determine which HDACs are necessary for cell viability, we lentivirally introduced isoform-specific shRNAs into our ALL cell lines. Knockdown of HDAC1 or HDAC2 resulted in p21 induction, slowed growth rate and resulted in a modest increase in apoptosis. Knockdown of HDAC3 lead to increased p21 and γH2A.X protein levels, along with induction of apoptosis, closely mimicking the results of pan-HDAC inhibitor treatment of the cells. Although depletion of HDAC3 had a more immediate impact on B-ALL viability by comparison to HDAC1/2, concerns about the contribution of HDAC3 inhibition to toxicity led us to further investigate whether specific inhibition of HDAC1/2 might be efficacious in B-ALL. Treatment of B-ALL cells with Merck 60, a tool compound with selectivity for HDAC1/2, was efficacious against was effective against B-ALL lines in the low to mid nM range. The kinetics of growth suppression were slower with this compound than with the pan-HDAC inhibitors. Using this compound, the ALL lines required 72 hours of exposure before cell growth was diminished, and apoptosis ensued. This may be due to the increased time necessary to accumulate acetylated histone marks as observable by western blot (18 hours for Merck 60 vs. 2–4 hours for LAQ824). Increased levels of p21 and γH2A.X were also observed. Interestingly, AML cell lines were much less sensitive to the HDAC1/2 specific inhibitor than were the B-ALL lines (roughly 5–10 fold), whereas pan-HDAC inhibitors were equally effective against AML and ALL. Additionally, non-hematopoietic tumor derived cell lines were insensitive to Merck 60, with EC50 values exceeding 20μM. Our findings indicate that pan-HDAC and class I specific HDAC inhibitors are possible therapeutic options for B-ALL. In contrast to most other cancer cell types studied, selective inhibition of HDAC1 and HDAC2 was sufficient to induce apoptosis in B-ALL lines. Together, these results suggest that small molecules specifically targeting HDAC1/2 may have therapeutic utility in B-ALL, and may provide improved therapeutic index by comparison to pan-HDAC or class I HDAC inhibitors that also target HDAC3. Disclosures: No relevant conflicts of interest to declare.

Combination of the BET Inhibitor GS-5829 and a BCL2 Inhibitor Resulted in Broader Activity in DLBCL and MCL Cell Lines

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5104-5104 ◽  
Author(s):  
Jamie Bates ◽  
Saritha Kusam ◽  
Stacey Tannheimer ◽  
Astrid Clarke ◽  
Thomas Kenney ◽  
...  
Keyword(s):  
Cell Growth ◽  
Protein Expression ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Employment Equity ◽  
Bet Inhibitor ◽  
Protein Levels ◽  
Bcl2 Protein ◽  
Equity Ownership ◽  
Induced Apoptosis

Abstract Introduction: GS-5829 is a potent and selective inhibitor of the bromodomain and extra-terminal (BET) family of proteins. BET proteins regulate the transcription of key oncogenes including MYC, resulting in cancer cell growth (Yang Z. et. al. Mol Cell Biol 2008; LeRoy G. et. al. Mol Cell 2008). Diffuse large B-cell lymphoma (DLBCL) is a diverse and genetically heterogeneous subtype of non-Hodgkin's lymphoma (NHL) (Lenz G. et. al. N Engl J Med 2010). Lymphomas harboring translocation of both MYC and BCL2, the so called "double hit" lymphoma, have a poor prognosis (Li S. et. al. Mod Pathol 2012). High protein expression of MYC and BCL2 together, in the absence of translocation, was also shown to be a poor prognostic indicator for DLBCL patients treated with R-CHOP which consists of rituximab, Cyclophosphamide, Hydroxydaunorubicin, Oncovin (vincristine), and Prednisone (Green T. et. al. J Clin Oncol 2012). Though double hit lymphoma occurs in 5% of DLBCL patients, 29% of DLBCL patients express high levels of both proteins-even in the absence of translocation. ABT-199 is a potent and selective BCL2 inhibitor and sensitivity to ABT-199 in DLBCL cell lines correlates with high basal BCL2 protein expression (Souers A. et. al. Nat Med 2013). We evaluated the combination of GS-5829 and ABT-199 in DLBCL and MCL cell lines in vitro. Methods: The activity of GS-5829 and ABT-199 alone or in combination in DLBCL and Mantle Cell Lymphoma (MCL) cell lines was assessed by evaluating cell growth inhibition and apoptosis. Growth inhibition was evaluated by CellTiterGlo or ATPLite. Apoptosis was assessed by Annexin V positivity by flow cytometry. The protein levels of MYC, BCL2, BIM, BCL-XL, and MCL1 were quantified by Western blotting and normalized to ACTIN levels. Analyses were performed to determine whether the expression of any of the profiled proteins correlated with the activity of GS-5829 or ABT-199. Results: DLBCL cell lines were broadly sensitive to cell growth inhibition by GS-5829, with EC50s ranging from 17 to 330 nM. Growth inhibition correlated with the reduction in MYC protein levels upon treatment with GS-5829. Treatment with GS-5829 induced apoptosis in a subset of DLBCL cell lines (58%). Cell lines with low level expression of BCL2 were more sensitive to GS-5829-induced apoptosis (p < 0.05 by T-Test). Expression levels of the other profiled proteins did not correlate with GS-5829 activity. DLBCL cell lines varied in sensitivity to ABT-199, with EC50s ranging from 1.5 to 7200 nM. Cell lines that were sensitive to ABT-199 in general had high levels of BCL2 protein. The single-agent activity of GS-5829 and ABT-199 in DLBCL cell lines had an apparent reciprocal relationship (Figure 1). The combination of GS-5829 and ABT-199 at clinically relevant concentrations resulted in broader activity and greater suppression of cell growth than either agent alone in a panel of DLBCL and MCL cell lines (Figure 2). Conclusion: The data demonstrated a reciprocal sensitivity of DLBCL cell lines to the BET inhibitor GS-5829 and the BCL2 inhibitor ABT-199. The combination of these two agents resulted in superior growth inhibition in DLBCL and MCL cell lines. These data suggest that GS-5829 and ABT-199 in combination may lead to broader, superior responses in DLBCL and MCL. Figure 1 Growth Inhibition by ABT-199 and Induction of Apoptosis by GS-5829 are Significantly Different in the High and Low Basal BCL2 Protein Expression Groups. Figure 1. Growth Inhibition by ABT-199 and Induction of Apoptosis by GS-5829 are Significantly Different in the High and Low Basal BCL2 Protein Expression Groups. Figure 2 Combination of GS-5829 and ABT-199 Resulted in More Potent Inhibition of Cell Growth in DLBCL Cell Lines at Clinically Relevant Concentrations (42 - 120 nM GS-5829 in Blue and 330 nM ABT-199). Figure 2. Combination of GS-5829 and ABT-199 Resulted in More Potent Inhibition of Cell Growth in DLBCL Cell Lines at Clinically Relevant Concentrations (42 - 120 nM GS-5829 in Blue and 330 nM ABT-199). Disclosures Bates: Gilead Sciences: Employment, Equity Ownership. Kusam:Gilead Sciences: Employment, Equity Ownership. Tannheimer:Gilead Sciences: Employment. Clarke:Gilead Sciences, Inc.: Employment, Equity Ownership. Kenney:Gilead Sciences, Inc.: Employment, Equity Ownership. Breckenridge:Gilead Sciences: Employment, Equity Ownership. Tumas:Gilead Sciences: Employment, Equity Ownership.

scholarly journals Long noncoding RNA LINC00941 promotes pancreatic cancer progression by competitively binding miR-335-5p to regulate ROCK1-mediated LIMK1/Cofilin-1 signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Wang ◽  
Zhiwei He ◽  
Jian Xu ◽  
Peng Chen ◽  
Jianxin Jiang
Keyword(s):  
Pancreatic Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Progression ◽  
Noncoding Rna ◽  
Epithelial Mesenchymal Transition ◽  
Loss Of Function ◽  
Mesenchymal Transition

AbstractAn accumulation of evidence indicates that long noncoding RNAs are involved in the tumorigenesis and progression of pancreatic cancer (PC). In this study, we investigated the functions and molecular mechanism of action of LINC00941 in PC. Quantitative PCR was used to examine the expression of LINC00941 and miR-335-5p in PC tissues and cell lines, and to investigate the correlation between LINC00941 expression and clinicopathological features. Plasmid vectors or lentiviruses were used to manipulate the expression of LINC00941, miR-335-5p, and ROCK1 in PC cell lines. Gain or loss-of-function assays and mechanistic assays were employed to verify the roles of LINC00941, miR-335-5p, and ROCK1 in PC cell growth and metastasis, both in vivo and in vitro. LINC00941 and ROCK1 were found to be highly expressed in PC, while miR-335-5p exhibited low expression. High LINC00941 expression was strongly associated with larger tumor size, lymph node metastasis, and poor prognosis. Functional experiments revealed that LINC00941 silencing significantly suppressed PC cell growth, metastasis and epithelial–mesenchymal transition. LINC00941 functioned as a molecular sponge for miR-335-5p, and a competitive endogenous RNA (ceRNA) for ROCK1, promoting ROCK1 upregulation, and LIMK1/Cofilin-1 pathway activation. Our observations lead us to conclude that LINC00941 functions as an oncogene in PC progression, behaving as a ceRNA for miR-335-5p binding. LINC00941 may therefore have potential utility as a diagnostic and treatment target in this disease.

scholarly journals The Role of Sphingosine-1-Phosphate and Ceramide-1-Phosphate in Inflammation and Cancer

2017 ◽  
Vol 2017 ◽  
pp. 1-17 ◽  
Author(s):  
Nitai C. Hait ◽  
Aparna Maiti
Keyword(s):  
Cell Growth ◽  
Cell Fate ◽  
Cancer Progression ◽  
Tissue Injury ◽  
Cell Types ◽  
Cancer Cell Growth ◽  
Ceramide Level ◽  
Cytokines And Chemokines ◽  
Sphingosine 1 Phosphate ◽  
Ceramide 1

Inflammation is part of our body’s response to tissue injury and pathogens. It helps to recruit various immune cells to the site of inflammation and activates the production of mediators to mobilize systemic protective processes. However, chronic inflammation can increase the risk of diseases like cancer. Apart from cytokines and chemokines, lipid mediators, particularly sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P), contribute to inflammation and cancer. S1P is an important player in inflammation-associated colon cancer progression. On the other hand, C1P has been recognized to be involved in cancer cell growth, migration, survival, and inflammation. However, whether C1P is involved in inflammation-associated cancer is not yet established. In contrast, few studies have also suggested that S1P and C1P are involved in anti-inflammatory pathways regulated in certain cell types. Ceramide is the substrate for ceramide kinase (CERK) to yield C1P, and sphingosine is phosphorylated to S1P by sphingosine kinases (SphKs). Biological functions of sphingolipid metabolites have been studied extensively. Ceramide is associated with cell growth inhibition and enhancement of apoptosis while S1P and C1P are associated with enhancement of cell growth and survival. Altogether, S1P and C1P are important regulators of ceramide level and cell fate. This review focuses on S1P and C1P involvement in inflammation and cancer with emphasis on recent progress in the field.

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.

Protein Expression Patterns of Candidate Genes Involved in Apoptosis and Cell Cycle Control in Genetic Subgroups of Chronic Lymphocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2796-2796
Author(s):  
Christof Schneider ◽  
Dirk Winkler ◽  
Meike Loddenkemper ◽  
Alexander Krober ◽  
Peter Lichter ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Expression ◽  
Cyclin D1 ◽  
Candidate Genes ◽  
Cell Lines ◽  
Normal Karyotype ◽  
Lymphocytic Leukemia ◽  
Expression Levels ◽  
Protein Levels ◽  
Atm Protein

Abstract Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable clinical course. Genomic aberrations (such as 13q−, 11q−, +12q, 17p−) can be found in about 80% of CLL cases and define pathogenic as well as clinical subgroups. Similarly, the mutational status of the variable region of the immunoglobulin heavy-chain gene (VH) identifies subgroups with different maturation stage and clinical outcome. In this study protein expression levels of candidate genes involved in cell cycle and apoptosis control (p53, ATM, Akt1, PI3-K, p21, p27, cdk4, Cyclin-D1, D2, D3, Bax, Bcl-2, Apaf-1, Smac, XIAP, cIAP2, survivin) were examined by Western Blotting. A total of 87 CLL cases derived from the subgroups with 11q- (n=22), 17p-/p53 mutation (n=18), +12q (n=24), 13q- (n=8) or a normal karyotype (n=15) were studied and compared to the cell lines EHEB and JVM-2. VH-mutation status was available for 65 cases (unmutated n=48, mutated n=17). Due to limitations in sample availability not all proteins could be examined in all cases. A highly homogenous expression pattern for all the proteins studied was observed in the CLL subgroup with a normal karyotype. This pattern was independent of the VH-status. CLL samples with normal karyotype, +12q and 13q deletion showed equal levels of ATM as compared to EHEB and JVM-2. As compared to cases with a normal karyotype the ATM level within the 11q- subgroup was reduced in 5 cases and absent in 1 case among 11 evaluable 11q- cases. The 17p- subgroup was comprised of 3 cases with concomitant 17p- and 11q- and 15 cases with 17p- but no 11q-. The latter group showed ATM protein levels comparable to the levels of the normal karyotype group. In the group with 17p- and 11q- there was an ATM expression level similar to the groups with 17p- and normal karyotype in two cases while one case had a reduced ATM protein level comparable to the 11q- subgroup. All cases with 17p- exhibited a stronger expression of p53 as compared to the cell lines and all other cases, except for one case with normal karyotype and one with an 11q-. No p53 mutations could be detected in exons 5–9 by sequencing in these two cases. High levels of survivin protein were found in all cases with 17p- and/or 11q-, 13q-, +12q while the subgroup with a normal karyotype showed lower levels. High levels of cdk4 protein were expressed in cases with 17p-, 11q- and 13q- while cdk4 protein levels were low in the subgroup with +12q and normal karyotype. Regarding p21, p27, Bcl2, Bax, Smac, Apaf-1, Cyclin D1–D3, cIAP2, XIAP, Akt1 and PI3K no variation in the expression levels were observed across the genetic CLL subgroups. Comparing the CLL cases to the cell lines the differences in expression levels were found for the cell cycle regulators Cyclin D1, D2, D3, p21 and p27. While the cell lines showed strong protein levels for Cyclin D1, D2, D3 and p21, they were nearly absent in the CLL cases. Expression of p27 was higher in all CLL cases as compared to JVM-2 and EHEB. In conclusion, the 17q- subgroup was the only group with a high level of p53 protein expression indicating that p53 is the affected gene in this subgroup. In contrast, the ATM protein levels are reduced only in a part of the 11q- cases indicating a possible role of additional candidate genes. Cases with +12q and normal karyotype showed weak expression of cdk4 pointing out a possible function in these subgroups.

Inhibition of ERK1/2 Activity by the MEK1/2 Inhibitor AZD6244 (ARRY-142886) Induces Human Multiple Myeloma Cell Apoptosis in the Bone Marrow Microenvironment: A New Therapeutic Strategy for MM.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3460-3460 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Iris Breitkreutz ◽  
Weihua Song ◽  
Peter Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Caspase 3 ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Cyclin E1 ◽  
Human Multiple Myeloma

Abstract Activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) signaling pathway mediates tumor cell growth in many cancers, including human multiple myeloma (MM). Specifically, this pathway mediates MM cell growth and survival induced by cytokines/growth factors (i.e. IL-6, IGF-1, CD40, BAFF) and adhesion to bone marrow stromal cells (BMSCs), thereby conferring resistance to apoptosis in the bone marrow (BM) milieu. In this study, we therefore examined the effect of the MEK1/2 inhibitor AZD6244 (ARRY-142886), on human MM cell lines, freshly isolated patient MM cells and MM cells adhered to BMSCs. AZD6244, inhibits constitutive and cytokine (IL-6, IGF-1, CD40)-stimulated ERK1/2, but not AKT phosphorylation. Importantly, AZD6244 inhibits the proliferation and survival of human MM cell lines, regardless of sensitivity to conventional chemotherapy, as well as freshly isolated patient MM cells. AZD6244 induces apoptosis in patient MM cells even in the presence of BMSCs, as evidenced by caspase 3 activity and PARP cleavage at concentrations as low as 20 nM. AZD6244 overcomes resistance to apoptosis in MM cells conferred by IL-6 and BMSCs, and inhibits IL-6 secretion induced by MM adhesion to BMSCs. AZD6244 suppresses MM cell survival/growth signaling pathways (i.e., STAT3, Bcl-2, cyclin E1, CDK1, CDK3, CDK7, p21/Cdc42/Rac1-activated kinase 1, casein kinase 1e, IRS1, c-maf) and up-regulates proapoptotic cascades (i.e., BAX, BINP3, BIM, BAG1, caspase 3, 8, 6). AZD6244 also upregulates proteins triggering cell cycle arrest (i.e. p16INK4A, p18INK4C, p21/WAF1 [Cdkn1a], p27 [kip1], p57). In addition, AZD6244 inhibits adhesion molecule expression in MM cells (i.e. integrin a4 [VLA-4], integrin b7, ICAM-1, ICAM-2, ICAM-3, catenin a1, c-maf) associated with decreased MM adhesion to BMSCs. These pleiotropic proapoptotic, anti-survival, anti-adhesion and -cytokine secretion effects of AZD6244 abrogate BMSC-derived protection of MM cells, thereby sensitizing them to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. In contrast, AZD6244 has minimal cytotoxicity in BMSCs and does not inhibit DNA synthesis in CD40 ligand-stimulated CD19 expressing B-cells derived from normal donors at concentrations toxic to MM cells (between 0.02–2 mM). Furthermore, AZD6244 inhibits the expression/secretion of osteoclast (OC)-activating factors (i.e., macrophage inflammatory protein (MIP)-1a, MIP-1b, IL-1b, VEGF) from MM cells. It also downregulates MM growth and survival factors (IL-6, BAFF, APRIL) in OC cultures derived from MM patient peripheral blood mononuclear cells (PBMCs). Significantly, AZD6244 inhibits OC differentiation from MM PBMCs (n=10) in a dose-dependent manner. Together these results provide the preclinical basis for clinical trials with AZD6244 (ARRY-142886) in MM.

Biological Sequelae of TRAF2 Downregulation in Waldenstrom Macroglobulinemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3526-3526
Author(s):  
Xavier Leleu ◽  
Lian Xu ◽  
Zachary R. Hunter ◽  
Sophia Adamia ◽  
Evdoxia Hatjiharissi ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Cell Growth ◽  
Cell Lines ◽  
Genomic Alteration ◽  
Rt Pcr ◽  
Growth And Survival ◽  
Healthy Donors ◽  
Cell Growth And Survival

Abstract Background. Several TNF family members (CD40L and BAFF/BLYS) have been implicated in Waldenstrom’s Macroglobulinemia (WM) cell growth and survival. More recently, abnormalities in the APRIL-TACI pathway have been demonstrated by us in WM cells (Hunter, ASH2006, #228). TRAFs (TNFR-associated factor) are a family of adaptor proteins that mediate signal transduction from multiple members of the TNF receptor superfamily. In particular, TRAFs facilitate pro-apoptotic signaling from the TACI receptor, and TRAF2 is of importance among the TRAF adapter proteins since this protein is required for TNF-alpha-mediated activation of SAPK/JNK MAPK known to be involved in drug-induced death of tumor B cells. We therefore examined the role of TRAF2 in WM growth and survival. Method. We investigated TRAF2, 3 and 5 gene expression in WM patient bone marrow (BM) CD19+ cells and cell lines (BCWM.1, WSU-WM) and compared their expression to BM CD19+ cells from healthy donors. Expression of human TRAF transcripts were determined using real time quantitative RT-PCR (qPCR) based on TaqMan fluorescence methodology. To evaluate the role of TRAF2, a knockdown model was prepared in BL2126 B-cells and BCWM.1 WM cells using electroporation, with resulted ≥50% knockdown efficiency using RT-PCR and immunoblotting. Results. We found that TRAF3 and 5 gene expression was higher in WM versus healthy donors, while TRAF2 expression was lower in 8/13 (60%) patients, using qPCR. TRAFs gene expression did not correlate with tumor burden or WM prognostic markers. We next sought to understand the biological sequelae of TRAF2 deficiency in BL2126 and BCWM.1 cells and found that TRAF2 knockdown induced increased survival at 72 hours in both cell lines. We next studied sequence analysis of 20 WM patients CD19+ BM cells to determine whether there was a TRAF2 genomic alteration, and found heterozygous early termination mutation in exon 5 in 1 (5%) patient. Conclusion. Our data demonstrate that TRAF2 is a commonly dysregulated TNF family adapter protein in patients with WM, with important consequences in WM cell growth and survival.

Recruitment of the SWI/SNF Chromatin Remodeling Complex by NFATc1 in the Transcriptional Regulation of the C-MYC Oncogene in Aggressive B-Cell Lymphomas

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3808-3808 ◽  
Author(s):  
Lan V. Pham ◽  
Archito Tamayo ◽  
Hai-Jun Zhou ◽  
Yen-Chiu Lin-Lee ◽  
Lingchen Fu ◽  
...  
Keyword(s):  
Cell Growth ◽  
Protein Expression ◽  
B Cell ◽  
Binding Site ◽  
Chromatin Remodeling ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Growth And Survival ◽  
Myc Oncogene ◽  
Cell Growth And Survival

Abstract The NFAT (nuclear factor of activated T-cells) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway for involvement in the pathogenesis of various malignancies, including large B-cell lymphoma (LBCL), a non-Hodgkin’s lymphoma subgroup that is generally responsive to conventional cancer therapies (R-CHOP), but relapse is common that subsequently leads to therapeutic resistance. Although we have shown previously that NFAT family member NFATc1 is constitutively activated and has the ability to maintain cell growth and survival in LBCL cell lines and primary cells, the molecular mechanism(s) underlying how NFATc1 regulates cell growth and survival in LBCL is still unclear. In this study, we demonstrate that the well-known oncogene c-myc is transcriptionally regulated by the transcription factor NFATc1 in LBCL, through a chromatin remodeling mechanism that involves the recruitment of the SWI/SNF chromatin-remodeling complex. In aggressive B-cell lymphoma cell lines, c-myc oncogene protein expression was shown to correlate with NFATc1 protein expression. We further showed that NFATc1 binds to a specific DNA binding element within the proximal c-myc promoter and up-regulates c-myc transcription. The SWI/SNF proteins Brg-1 and Brm, chromatin-remodeling proteins that utilize ATP hydrolysis for energy to modify chromatin structure in order to regulate gene expression, also were shown to bind to the NFAT binding site on the c-myc promoter. Confocal microscopic analysis showed that NFATc1 colocalizes with Brg-1, and co-immunoprecipitation assays showed that Brg-1 interacts with NFATc1. Both proteins interact with the c-myc promoter within the NFAT binding site, as demonstrated by chromatin-immunoprecipitation (ChIP) analysis. Induction of a constitutively active mutant of NFATc1 (caNFATc1) in an NFATc1 negative lymphoma cell line induces c-myc protein expression. Constitutively active NFATc1 also enhances Brg-1 binding to the c-myc promoter when analyzed by ChIP-qPCR assays, suggesting that NFATc1 recruits Brg-1 to the NFAT binding site in the c-myc promoter. Down-regulation of NFATc1 by chemical inhibitors (FK-506) or by validated shRNA of NFATc1, inhibited c-myc protein expression and in-vitro lymphoma cell growth. Our data indicates a novel control mechanism for the transcriptional regulation of c-myc in the pathophysiology of aggressive lymphoma B cells and suggests that targeting NFATc1 could have therapeutic value.

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