Overexpression of Eukaryotic Initiation Factor 4E Induces Critical Transcription Factors Such As c-Myc in Multiple Myeloma Resulting in Enhanced Clonogenic Tumor Growth in Vitro and in Vivo.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2908-2908
Author(s):  
Shirong Li ◽  
Jing Fu ◽  
Jordan M. Schecter ◽  
Caisheng Lu ◽  
Markus Mapara ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Protein Synthesis ◽  
Tumor Growth ◽  
Myeloma Cell ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Eukaryotic Initiation Factor 4E

Abstract Abstract 2908 Introduction: The eukaryotic initiation factor 4E (eIF4E) is a critical regulator in protein synthesis. It has been shown that overexpression and/or activation of eIF4E is critical for oncogenic protein synthesis. The precise role of protein translation in multiple myeloma (MM) is less clear. Recently it has been shown that eIF4E protein levels are higher in primary CD138+ MM cells than in normal plasma cells (Li, Jin et al. 2011) and that mutations in genes related to mRNA translation are involved in the pathogenesis of multiple (Chapman, Lawrence et al. 2011). Therefore, understanding the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. In this study, we analyzed the effects of introduction of ectopic eIF4E in MM cell lines compared with their parent cells in vitro and in vivo. Results: To examine the effect of overexpressed eIF4E in MM, we transduced MM cell lines with lentiviral particles encoding human eIF4E with GFP as selection marker. Introduction of ectopic eIF4E significantly increased critical factors for myeloma cell growth such as myc, cyclin D1, C/EBP beta and IRF4 as detected by western blotting. Overexpression of eIF4E resulted in a significant (p<0.001) increase of DNA synthesis compared to empty vector control (EV) cells. Cell cycle analysis revealed a decreasing number of cells in G0/G1 phase (62% vs 49%) and cells arresting in the G2/M phase (14% vs 23%), not affecting cell apoptosis. Overexpression of eIF4E further led to the significant increase (p=0.004) of clonogenic tumor growth with expansion of clonogenic colony numbers (22.3 ± 2.5 vs 40.3 ± 2.1) and size. To determine whether overexpressed eIF4E also affects MM tumor growth in vivo, we generated subcutaneous MM xenografts in severe combined immunodeficient x beige (SCID/bg) mice using the EV and eIF4E-OE-U266 cells. In contrast to EV-U266 tumors, animals bearing eIF4E-OE-U266 xenografts showed a significant increase (p<0.001) of tumor growth by 180% after 13 days. Conclusion: Here we show that eIF4E, a key player in translational machinery, promotes multiple myeloma cell growth both in vitro and in vivo. When eIF4E is overexpressed, it enhances protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation. Disclosures: Lentzsch: Celgene: Consultancy, Research Funding.

scholarly journals Phosphorylation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required for protein synthesis in vitro and in vivo

2001 ◽  
Vol 268 (20) ◽  
pp. 5375-5385 ◽  
Author(s):  
Linda McKendrick ◽  
Simon J. Morley ◽  
Virginia M. Pain ◽  
Rosemary Jagus ◽  
Bhavesh Joshi
Keyword(s):  
Protein Synthesis ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Eukaryotic Initiation Factor 4E

Inducible Silencing Of eIF4E Using a Tet-On System Results In Myeloma Growth In Vivo That Correlates With eIF4E Expression

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3164-3164
Author(s):  
Shirong Li ◽  
Jing Fu ◽  
Jordan M. Schecter ◽  
Caisheng Lu ◽  
Markus Y. Mapara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Synthesis ◽  
Cell Growth ◽  
Tumor Growth ◽  
Critical Role ◽  
Myeloma Cell ◽  
Control Shrna

Abstract Introduction Overexpression and/or activation of eukaryotic initiation factor 4E (eIF4E) is critical for oncogenic protein synthesis. Mutations in genes related to mRNA translation are involved in the pathogenesis of multiple myeloma (Chapman, Lawrence et al. 2011). Recently, we found that MM cells express high levels of eIF4E protein compared to normal plasma cells and overexpression of eIF4E induces transcription factors such as c-myc critical for the growth of multiple myeloma cells (Li, Fu et al. 2011,2012). The understanding of the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. Here we show the critical role of eIF4E driven protein synthesis by using an inducible knockdown system to silence eIF4E gene expression and confirm the critical role of eIF4E in multiple myeloma growth in vivo and in vitro. Methods and Results We stably infected U266, RPMI-8226, IM-9 and MM.1S cells with a robust inducible single-lentiviral knockdown vector pLKO-Tet-On containing either control non-targeting shRNA or eIF4E targeting shRNA sequences. Doxycycline-induced eIF4E shRNA expression resulted in significant decrease of eIF4E mRNA and protein in eIF4E-shRNA but not the control shRNA infected MM cells. To determine the effects of eIF4E knockdown on MM cell growth and viability, stably transfected cell lines were grown in the presence or absence of doxycycline. Silencing of eIF4E by doxycycline induction of eIF4E shRNA in RPMI-8226 cells significantly inhibited (>72%,P<0.01) cell growth accompanied by a decrease of c-myc, cyclin D1, C/EBP beta and IRF4 all critical for myeloma cell growth. Cell cycle analysis revealed increased cells population in G0/G1 phase (62% vs 80%) in doxycycline-induced eIF4E shRNA cells with a significant reduction (P<0.001) of clonogenic tumor growth reflected by a decrease in colony numbers (27.6 ± 4.2 vs 5.3 ± 3.4) and size. To determine the role of high expression of eIF4E in MM tumor growth in vivo, we generated subcutaneous MM xenografts in severe combined immunodeficient x beige (SCID/bg) mice using the inducible U266-Tet-CT-shRNA and U266-Tet-eIF4E-shRNA cells. In contrast to vehicle or doxycycline-treated control shRNA tumors, doxycycline treated animals bearing U266-Tet-eIF4E-shRNA xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 21 days. The transient inhibition of tumor growth correlated with the transient doxycycline-induced eIF4E knockdown further confirming the critical role of eIF4E. Immunohistochemical staining of tumors confirmed the decreased of eIF4E expression in doxycycline-treated mice bearing U266-Tet-eIF4E-shRNA tumors compared with tumors of vehicle-treated or non-doxycyclin treated mice. Conclusion Here we show that eIF4E, a key player in the translational machinery, promotes multiple myeloma cell growth. We found that high eIF4E expression is indispensable for the growth of MM cells both in vitro and in vivo. Silencing of eIF4E decreases protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation, and resulted in tumor inhibition. Our study indicated that targeting transcriptional initiating factor eIF4E may represent a novel therapeutic strategy for MM treatment. Disclosures: Schecter: Seattle Genetics: Honoraria, Research Funding. Lentzsch:Celgene: Research Funding.

scholarly journals Targeting CD47/TNFAIP8 by miR-155 overcomes drug resistance and inhibits tumor growth through induction of phagocytosis and apoptosis in multiple myeloma

Haematologica ◽  
2019 ◽  
Vol 105 (12) ◽  
pp. 2813-2823 ◽  
Author(s):  
Nasrin Rastgoo ◽  
Jian Wu ◽  
Mariah Liu ◽  
Maryam Pourabdollah ◽  
Eshetu G. Atenafu ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Tumor Growth ◽  
Myeloma Cell ◽  
Therapeutic Modality ◽  
Drug Resistant ◽  
Myeloma Cells ◽  
Advanced Stages

The mechanisms of drug resistance in multiple myeloma are poorly understood. Here we show that CD47, an integrin-associated receptor, is significantly upregulated in drug resistant myeloma cells in comparison with parental cells, and that high expression of CD47 detected by immunohistochemistry is associated with shorter progression free and overall survivals in multiple myeloma patients. We show that miR-155 is expressed at low levels in drug resistant myeloma cells and is a direct regulator of CD47 through its 3'UTR. Furthermore, low miR-155 levels are associated with advanced stages of disease. MiR-155 overexpression suppressed CD47 expression on myeloma cell surface, leading to induction of phagocytosis of myeloma cells by macrophages and inhibition of tumor growth. MiR-155 overexpression also re-sensitized drug-resistant myeloma cells to bortezomib leading to cell death through targeting TNFAIP8, a negative mediator of apoptosis in vitro and in vivo. Thus, miR-155 mimics may serve as a promising new therapeutic modality by promoting phagocytosis and inducing apoptosis in patients with refractory/relapsed multiple myeloma.

scholarly journals Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in vivo in human multiple myeloma

BMC Cancer ◽  
2014 ◽  
Vol 14 (1) ◽  
Author(s):  
Shigeyuki Yokokura ◽  
Saki Yurimoto ◽  
Akihito Matsuoka ◽  
Osamu Imataki ◽  
Hiroaki Dobashi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Tumor Growth ◽  
Inhibit Tumor Growth ◽  
Calmodulin Antagonists ◽  
Induce Cell Cycle Arrest ◽  
Cycle Arrest ◽  
Human Multiple Myeloma

scholarly journals Evidence that the dephosphorylation of Ser535 in the ∊-subunit of eukaryotic initiation factor (eIF) 2B is insufficient for the activation of eIF2B by insulin

2002 ◽  
Vol 367 (2) ◽  
pp. 475-481 ◽  
Author(s):  
Xuemin WANG ◽  
Maarten JANMAAT ◽  
Anne BEUGNET ◽  
Fiona E.M. PAULIN ◽  
Christopher G. PROUD
Keyword(s):  
Protein Synthesis ◽  
Glycogen Synthase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Ovary Cells

Eukaryotic initiation factor (eIF) 2B is a guanine-nucleotide exchange factor that plays a key role in the regulation of protein synthesis. It is activated by insulin, serum and other agents that stimulate general protein synthesis. The largest (∊) subunit of eIF2B is a substrate for glycogen synthase kinase (GSK)-3 in vitro, and phosphorylation by GSK3 inhibits the activity of eIF2B. The site of phosphorylation has previously been identified as Ser535. GSK3 is inactivated by phosphorylation in response to insulin or serum. In Chinese-hamster ovary cells, insulin and serum bring about the dephosphorylation of Ser535in vivo, concomitantly with the phosphorylation of GSK3, and these effects are mediated through signalling via phosphoinositide 3-kinase. We have made use of inhibitors of GSK3 to determine whether GSK3 is responsible for phosphorylation of Ser535in vivo and to explore the role of phosphorylation of Ser535 in the regulation of eIF2B. Treatment of cells with LiCl or with either of two recently developed GSK3 inhibitors, SB-415286 and SB-216763, brought about the dephosphorylation of Ser535, which strongly indicates that this site is indeed a target for GSK3 in vivo. However, these compounds did not elicit significant activation of eIF2B, indicating, consistent with conclusions from one of our previous studies, that additional inputs are required for the activation of eIF2B. Our results also show that each of the inhibitors used affects overall protein synthesis and have additional effects on translation factors or signalling pathways apparently unrelated to their effects on GSK3, indicating that caution must be exercised when interpreting data obtained using these compounds.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

scholarly journals Highly Differentiated Anti-CD47 Antibody, AO-176, Potently Inhibits Hematologic Malignancies Alone and in Combination

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1844-1844
Author(s):  
John Richards ◽  
Myriam N Bouchlaka ◽  
Robyn J Puro ◽  
Ben J Capoccia ◽  
Ronald R Hiebsch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Combination Therapy ◽  
Tumor Growth ◽  
Growth Inhibition ◽  
Tumor Cells ◽  
Tumor Growth Inhibition ◽  
Employment Equity ◽  
Equity Ownership

AO-176 is a highly differentiated, humanized anti-CD47 IgG2 antibody that is unique among agents in this class of checkpoint inhibitors. AO-176 works by blocking the "don't eat me" signal, the standard mechanism of anti-CD47 antibodies, but also by directly killing tumor cells. Importantly, AO-176 binds preferentially to tumor cells, compared to normal cells, and binds even more potently to tumors in their acidic microenvironment (low pH). Hematological neoplasms are the fourth most frequently diagnosed cancers in both men and women and account for approximately 10% of all cancers. Here we describe AO-176, a highly differentiated anti-CD47 antibody that potently targets hematologic cancers in vitro and in vivo. As a single agent, AO-176 not only promotes phagocytosis (15-45%, EC50 = 0.33-4.1 µg/ml) of hematologic tumor cell lines (acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, and T cell leukemia) but also directly targets and kills tumor cells (18-46% Annexin V positivity, EC50 = 0.63-10 µg/ml) in a non-ADCC manner. In combination with agents targeting CD20 (rituximab) or CD38 (daratumumab), AO-176 mediates enhanced phagocytosis of lymphoma and multiple myeloma cell lines, respectively. In vivo, AO-176 mediates potent monotherapy tumor growth inhibition of hematologic tumors including Raji B cell lymphoma and RPMI-8226 multiple myeloma xenograft models in a dose-dependent manner. Concomitant with tumor growth inhibition, immune cell infiltrates were observed with elevated numbers of macrophage and dendritic cells, along with increased pro-inflammatory cytokine levels in AO-176 treated animals. When combined with bortezomib, AO-176 was able to elicit complete tumor regression (100% CR in 10/10 animals treated with either 10 or 25 mg/kg AO-176 + 1 mg/kg bortezomib) with no detectable tumor out to 100 days at study termination. Overall survival was also greatly improved following combination therapy compared to animals treated with bortezomib or AO-176 alone. These data show that AO-176 exhibits promising monotherapy and combination therapy activity, both in vitro and in vivo, against hematologic cancers. These findings also add to the previously reported anti-tumor efficacy exhibited by AO-176 in solid tumor xenografts representing ovarian, gastric and breast cancer. With AO-176's highly differentiated MOA and binding characteristics, it may have the potential to improve upon the safety and efficacy profiles relative to other agents in this class. AO-176 is currently being evaluated in a Phase 1 clinical trial (NCT03834948) for the treatment of patients with select solid tumors. Disclosures Richards: Arch Oncology Inc.: Employment, Equity Ownership, Other: Salary. Bouchlaka:Arch Oncology Inc.: Consultancy, Equity Ownership. Puro:Arch Oncology Inc.: Employment, Equity Ownership. Capoccia:Arch Oncology Inc.: Employment, Equity Ownership. Hiebsch:Arch Oncology Inc.: Employment, Equity Ownership. Donio:Arch Oncology Inc.: Employment, Equity Ownership. Wilson:Arch Oncology Inc.: Employment, Equity Ownership. Chakraborty:Arch Oncology Inc.: Employment, Equity Ownership. Sung:Arch Oncology Inc.: Employment, Equity Ownership. Pereira:Arch Oncology Inc.: Employment, Equity Ownership.

scholarly journals ID: 1023 All-trans retinoic acid targets gastric cancer stem cells and inhibits patient-derived gastric carcinoma tumor growth

2017 ◽  
Vol 4 (S) ◽  
pp. 98
Author(s):  
P H Nguyen ◽  
J Giraud ◽  
C Staedel ◽  
L Chambonnier ◽  
P Dubus ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Gastric Carcinoma ◽  
Tumor Growth ◽  
Cell Cycle Progression ◽  
3D Culture ◽  
All Trans Retinoic Acid

Gastric carcinoma is the third leading cause of cancer-related death worldwide. This cancer, most of the time metastatic, is essentially treated by surgery associated with conventional chemotherapy, and has a poor prognosis. The existence of cancer stem cells (CSC) expressing CD44 and a high aldehyde dehydrogenase (ALDH) activity has recently been demonstrated in gastric carcinoma and has opened new perspectives to develop targeted therapy. In this study, we evaluated the effects of all-transretinoic acid (ATRA) on CSCs in human gastric carcinoma. ATRA effects were evaluated on the proliferation and tumorigenic properties of gastric carcinoma cells from patient-derived tumors and cell lines in conventional 2D cultures, in 3D culture systems (tumorsphere assay) and in mouse xenograft models. ATRA inhibited both tumorspheres initiation and growth in vitro, which was associated with a cell-cycle arrest through the upregulation of cyclin-dependent kinase (CDK) inhibitors and the downregulation of cell-cycle progression activators. More importantly, ATRA downregulated the expression of the CSC markers CD44 and ALDH as well as stemness genes such as Klf4 and Sox2 and induced differentiation of tumorspheres. Finally, 2 weeks of daily ATRA treatment were sufficient to inhibit gastric tumor progression in vivo, which was associated with a decrease in CD44, ALDH1, Ki67 and PCNA expression in the remaining tumor cells. Administration of ATRA appears to be a potent strategy to efficiently inhibit tumor growth and more importantly to target gastric CSCs in both intestinal and diffuse types of gastric carcinoma.

Expression of mutant eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) reduces inhibition of guanine nucleotide exchange activity of eIF-2B mediated by eIF-2 alpha phosphorylation

1994 ◽  
Vol 14 (7) ◽  
pp. 4546-4553
Author(s):  
K V Ramaiah ◽  
M V Davies ◽  
J J Chen ◽  
R J Kaufman
Keyword(s):  
Protein Synthesis ◽  
Initiation Factor ◽  
Guanine Nucleotide ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Wild Type ◽  
Guanine Nucleotide Exchange ◽  
Initiation Factor 2 ◽  
Exchange Activity

The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.

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