thapsigargin and Multiple-Myeloma

Protein translation is inhibited by the unfolded protein response (UPR)-induced eIF-2α phosphorylation to protect against endoplasmic reticulum (ER) stress. In addition, we found additional inhibition of protein translation owing to diminished mTORC1 (mammalian target of rapamycin complex1) activity in ER-stressed multiple myeloma (MM) cells. However, c-myc protein levels and myc translation was maintained. To ascertain how c-myc was maintained, we studied myc IRES (internal ribosome entry site) function, which does not require mTORC1 activity. Myc IRES activity was upregulated in MM cells during ER stress induced by thapsigargin, tunicamycin or the myeloma therapeutic bortezomib. IRES activity was dependent on upstream MAPK (mitogen-activated protein kinase) and MNK1 (MAPK-interacting serine/threonine kinase 1) signaling. A screen identified hnRNP A1 (A1) and RPS25 as IRES-binding trans-acting factors required for ER stress-activated activity. A1 associated with RPS25 during ER stress and this was prevented by an MNK inhibitor. In a proof of principle, we identified a compound that prevented binding of A1 to the myc IRES and specifically inhibited myc IRES activity in MM cells. This compound, when used alone, was not cytotoxic nor did it inhibit myc translation or protein expression. However, when combined with ER stress inducers, especially bortezomib, a remarkable synergistic cytotoxicity ensued with associated inhibition of myc translation and expression. These results underscore the potential for targeting A1-mediated myc IRES activity in MM cells during ER stress.

Topics: Antineoplastic Agents; Bortezomib; Cell Line; Drug Delivery Systems; Endoplasmic Reticulum Stress; Genes, myc; Humans; Internal Ribosome Entry Sites; Mechanistic Target of Rapamycin Complex 1; Multiple Myeloma; Multiprotein Complexes; Protein Biosynthesis; Thapsigargin; TOR Serine-Threonine Kinases; Tunicamycin

To assess the role of the serum and glucocorticoid-regulated kinase (SGK) kinase in multiple myeloma, we ectopically expressed wild type or a phosphomimetic version of SGK into multiple myeloma cell lines. These cells were specifically resistant to the ER stress inducers tunicamycin, thapsigargin, and bortezomib. In contrast, there was no alteration of sensitivity to dexamethasone, serum starvation, or mTORC inhibitors. Mining of genomic data from a public database indicated that low baseline SGK expression in multiple myeloma patients correlated with enhanced ability to undergo a complete response to subsequent bortezomib treatment and a longer time to progression and overall survival following treatment. SGK overexpressing multiple myeloma cells were also relatively resistant to bortezomib in a murine xenograft model. Parental/control multiple myeloma cells demonstrated a rapid upregulation of SGK expression and activity (phosphorylation of NDRG-1) during exposure to bortezomib and an SGK inhibitor significantly enhanced bortezomib-induced apoptosis in cell lines and primary multiple myeloma cells. In addition, a multiple myeloma cell line selected for bortezomib resistance demonstrated enhanced SGK expression and SGK activity. Mechanistically, SGK overexpression constrained an ER stress-induced JNK proapoptotic pathway and experiments with a SEK mutant supported the notion that SGK's protection against bortezomib was mediated via its phosphorylation of SEK (MAP2K4) which abated SEK/JNK signaling. These data support a role for SGK inhibitors in the clinical setting for myeloma patients receiving treatment with ER stress inducers like bortezomib.. Enhanced SGK expression and activity in multiple myeloma cells contributes to resistance to ER stress, including bortezomib challenge.

Topics: Animals; Apoptosis; Bortezomib; Cell Line, Tumor; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Female; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Mice; Multiple Myeloma; Protein Serine-Threonine Kinases; Thapsigargin; Tunicamycin; Up-Regulation; Xenograft Model Antitumor Assays

As myeloma cells actively produce and secrete immunoglobulins, they are prone to ER stress, which if unresolved leads to apoptosis. We found that myeloma cell death induced by the ER stressor Thapsigargin was highly variable, ranging from 2 to 89%. Induction of ATF4 and CHOP was observed in myeloma cells under Thapsigargin independently of cell death. The decrease in Mcl-1 was associated with protein translation inhibition and identified as a crucial factor in Thapsigargin sensitivity, since it was the only Bcl-2 family protein differentially modified between sensitive and resistant myeloma cells. Bak but not Bax was found to contribute to Thapsigargin-induced apoptosis. Appropriately, a basal Mcl-1/Bak interaction was demonstrated in Thapsigargin-sensitive cells. Of note, the only pro-apoptotic protein freed from Mcl-1 under Thapsigargin was Bak, whereas Mcl-1/Noxa or Mcl-1/Bim complexes were simultaneously increased. Thus, the disruption of the basal Mcl-1/Bak complex in Thapsigargin-sensitive cells seemed to be an essential event in cell death induction, probably favored by the induced Noxa and Bim BH3-only proteins. These findings underscore the implication of the Mcl-1/Bak axis in myeloma cell death triggered by Thapsigargin.

Topics: Antineoplastic Agents; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Bcl-2-Like Protein 11; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Protein Binding; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Signal Transduction; Sulfonamides; Thapsigargin; Transfection

Protein kinase CK2 promotes multiple myeloma cell growth by regulating critical signaling pathways. CK2 also modulates proper HSP90-dependent client protein folding and maturation by phosphorylating its co-chaperone CDC37. Because the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is central in myeloma pathogenesis, we tested the hypothesis that the CK2/CDC37/HSP90 axis could be involved in UPR in myeloma cells.. We analyzed CK2 activity upon ER stress, the effects of its inactivation on the UPR pathways and on ER stress-induced apoptosis. The consequences of CK2 plus HSP90 inhibition on myeloma cell growth in vitro and in vivo and CK2 regulation of HSP90-triggered UPR were determined.. CK2 partly localized to the ER and ER stress triggered its kinase activity. CK2 inhibition reduced the levels of the ER stress sensors IRE1α and BIP/GRP78, increased phosphorylation of PERK and EIF2α, and enhanced ER stress-induced apoptosis. Simultaneous inactivation of CK2 and HSP90 resulted in a synergic anti-myeloma effect (combination index = 0.291) and in much stronger alterations of the UPR pathways as compared with the single inhibition of the two molecules. Cytotoxicity from HSP90 and CK2 targeting was present in a myeloma microenvironment model, on plasma cells from patients with myeloma and in an in vivo mouse xenograft model. Mechanistically, CK2 inhibition led to a reduction of IRE1α/HSP90/CDC37 complexes in multiple myeloma cells.. Our results place CK2 as a novel regulator of the ER stress/UPR cascades and HSP90 function in myeloma cells and offer the groundwork to design novel combination treatments for this disease.

Topics: Animals; Apoptosis; Benzoquinones; Blotting, Western; Casein Kinase II; Cell Line, Tumor; Cells, Cultured; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mice; Mice, SCID; Microscopy, Confocal; Multiple Myeloma; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Thapsigargin; Unfolded Protein Response; Xenograft Model Antitumor Assays

Because accumulation of potentially toxic malfolded protein may be extensive in immunoglobulin-producing multiple myeloma (MM) cells, we investigated the phenomenon of autophagy in myeloma, a physiologic process that can protect against malfolded protein under some circumstances. Autophagy in MM cell lines that express and secrete immunoglobulin and primary specimens was significantly increased by treatment with the endoplasmic reticulum stress-inducing agent thapsigargin, the mammalian target of rapamycin inhibitor rapamycin, and the proteasome inhibitor bortezomib. Inhibition of basal autophagy in these cell lines and primary cells by use of the inhibitors 3-methyladenine and chloroquine resulted in a cytotoxic effect that was associated with enhanced apoptosis. Use of small interfering RNA to knock down expression of beclin-1, a key protein required for autophagy, also inhibited viable recovery of MM cells. Because the data suggested that autophagy protected MM cell viability, we predicted that autophagy inhibitors would synergize with bortezomib for enhanced antimyeloma effects. However, the combination of these drugs resulted in an antagonistic response. In contrast, the autophagy inhibitor 3-methyladenine did synergize with thapsigargin for an enhanced cytotoxic response. These data suggest that autophagy inhibitors have therapeutic potential in myeloma but caution against combining such drugs with bortezomib.

Topics: Adenine; Antifungal Agents; Antimalarials; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Boronic Acids; Bortezomib; Cell Proliferation; Chloroquine; Drug Therapy, Combination; Enzyme Inhibitors; Humans; Immunoblotting; Membrane Proteins; Microscopy, Fluorescence; Multiple Myeloma; Pyrazines; RNA, Small Interfering; Sirolimus; Thapsigargin; Tumor Cells, Cultured

Plasma cells producing high levels of paraprotein are dependent on the unfolded protein response (UPR) and chaperone proteins to ensure correct protein folding and cell survival. We hypothesized that disrupting client-chaperone interactions using heat shock protein 90 (Hsp90) inhibitors would result in an inability to handle immunoglobulin production with the induction of the UPR and myeloma cell death. To study this, myeloma cells were treated with Hsp90 inhibitors as well as known endoplasmic reticulum stress inducers and proteasome inhibitors. Treatment with thapsigargin and tunicamycin led to the activation of all 3 branches of the UPR, with early splicing of XBP1 indicative of IRE1 activation, upregulation of CHOP consistent with ER resident kinase (PERK) activation, and activating transcription factor 6 (ATF6) splicing. 17-AAG and radicicol also induced splicing of XBP1, with the induction of CHOP and activation of ATF6, whereas bortezomib resulted in the induction of CHOP and activation of ATF6 with minimal effects on XBP1. After treatment with all drugs, expression levels of the molecular chaperones BiP and GRP94 were increased. All drugs inhibited proliferation and induced cell death with activation of JNK and caspase cleavage. In conclusion, Hsp90 inhibitors induce myeloma cell death at least in part via endoplasmic reticulum stress and the UPR death pathway.

Topics: Apoptosis; Caspases; Cell Line, Tumor; Endoplasmic Reticulum; HSP90 Heat-Shock Proteins; Humans; Inclusion Bodies; Multiple Myeloma; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Folding; Thapsigargin; Tunicamycin

The endoplasmic reticulum (ER) is an organelle in which proteins are modified. When unfolded proteins accumulate in the ER under various stresses, ER stress (ERS) pathways, including the induction of chaperones, are activated to protect the cell. However, when ERS is excessive, the cell undergoes apoptosis. This study investigated ERS in multiple myeloma cells (MMCs) because they contain a well-developed ER due to M-protein production. The myeloma cell line 12-PE underwent apoptosis via caspase-3 after treatment with thapsigargin (thap), an ERS inducer, while another cell line, U266, did not. To understand the mechanism regulating this heterogeneity, the induction of chaperones by thap was analysed. Chaperones were up-regulated in U266 cells but down-regulated in 12-PE cells, suggesting that chaperones contribute to cell survival under ERS. Analysis of XBP-1, a transcriptional inducer of chaperones, in freshly isolated MMCs from 22 myeloma cases revealed 10 cases with active XBP-1, who also showed significantly poorer survival (p < 0.05), suggesting that chaperone expression protects MMCs from apoptosis, thereby allowing tumor cell expansion. These results suggest that MMCs are subjected to ERS under certain circumstances and that chaperones are induced to protect the cells against such ERS. Inhibition of chaperones could be a new target for myeloma therapy.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Cell Line, Tumor; Cell Survival; DNA-Binding Proteins; Drug Resistance, Neoplasm; Endoplasmic Reticulum; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Molecular Chaperones; Multiple Myeloma; Nuclear Proteins; Oxidative Stress; Prognosis; Regulatory Factor X Transcription Factors; Retrospective Studies; RNA, Messenger; Survival Analysis; Thapsigargin; Transcription Factors; X-Box Binding Protein 1

Quality control in the endoplasmic reticulum must discriminate nascent proteins in their folding process from terminally unfolded molecules, selectively degrading the latter. Unassembled Ig-mu and J chains, two glycoproteins with five N-linked glycans and one N-linked glycan, respectively, are degraded by cytosolic proteasomes after a lag from synthesis, during which glycan trimming occurs. Inhibitors of mannosidase I (kifunensine), but not of mannosidase II (swainsonine), prevent the degradation of mu chains. Kifunensine also inhibits J chain dislocation and degradation, without inhibiting secretion of IgM polymers. In contrast, glucosidase inhibitors do not significantly affect the kinetics of mu and J degradation. These results suggest that removal of the terminal mannose from the central branch acts as a timer in dictating the degradation of transport-incompetent, glycosylated Ig subunits in a calnexin-independent way. Kifunensine does not inhibit the degradation of an unglycosylated substrate (lambda Ig light chains) or of chimeric mu chains extended with the transmembrane region of the alpha T cell receptor chain, implying the existence of additional pathways for extracting proteins from the endoplasmic reticulum lumen for proteasomal degradation.

Topics: Alkaloids; Cysteine Endopeptidases; Cytosol; Endoplasmic Reticulum; Enzyme Inhibitors; Glycoproteins; Glycosylation; Homeostasis; Humans; Immunoglobulin J-Chains; Immunoglobulin mu-Chains; Kinetics; Mannosidases; Multienzyme Complexes; Multiple Myeloma; Proteasome Endopeptidase Complex; Protein Subunits; Receptors, Antigen, T-Cell, alpha-beta; Recombinant Fusion Proteins; Thapsigargin; Tumor Cells, Cultured

This study examined the hypothesis that certain immunoglobulin light chains directly altered mesangial cell calcium homeostasis. Intracellular Ca2+ concentration (intracellular [Ca2+]) signaling was determined in suspensions of rat mesangial cells using the acetoxymethyl ester of fura 2 with a calcium removal/replacement protocol. Pretreatment of cultured rat mesangial cells with a glomerulopathic kappa-light chain (gle) produced reversible dose- and time-dependent attenuation of ATP- and thrombin-evoked [Ca2+] transients (189 +/- 24 vs. 126 +/- 10 nM, P < 0.05 with ATP; 198 +/- 5 vs. 117 +/- 3 nM, P < 0.05 with thrombin) and capacitative calcium influx (199 +/- 14 vs. 142 +/- 17 nM, P < 0.05 for ATP; 252 +/- 19 vs. 198 +/- 18 nM, P < 0.05 for thrombin). Mesangial cells treated with gle and supplemented with myo-inositol (450 microM) did not demonstrate the attenuation of the ATP-evoked [Ca2+] transient and capacitative calcium influx. Gle also decreased mean [Ca2+] transient (80 +/- 7 vs. 56 +/- 1 nM, P < 0.05) and capacitative calcium influx (306 +/- 10 vs. 241 +/- 4 nM, P < 0.05) in response to thapsigargin, a Ca2+-adenosinetriphosphatase inhibitor. This inhibition was not reversed by exogenous myo-inositol. Another kappa-light chain (10 microg/ml) did not affect mesangial cell calcium signaling. Deranged mesangial cell calcium homeostasis by certain light chains may play a central pathogenetic role in glomerulosclerosis associated with deposition of immunoglobulin light chains.

Topics: Adenosine Triphosphate; Animals; Aorta; Calcium; Cells, Cultured; Egtazic Acid; Glomerular Mesangium; Homeostasis; Humans; Immunoglobulin kappa-Chains; Inositol; Kidney Cortex; Kinetics; Male; Multiple Myeloma; Muscle, Smooth, Vascular; Rats; Rats, Sprague-Dawley; Thapsigargin; Time Factors