thapsigargin and tanespimycin

Genome-wide transcript profiling to elucidate responses to HSP90 inhibition revealed strong induction of HSPA6 in MCF-7 cells treated with 17-AAG. Time- and dose dependent induction of HSPA6 (confirmed by qPCR and Western Blots) occurred also upon treatment with Radicicol, another HSP90 inhibitor. HSPA6 was not detectable in untreated cells or cells treated with toxins that do not inhibit HSP90, or upon applying oxidative stress. Thus, HSPA6 induction is not a general response to cytotoxic insults. Modulation of HSPA6 levels by siRNA-mediated inhibition or recombinant expression did not influence 17-AAG mediated cell death. HSPA6 induction as a consequence of HSP90 inhibition occurs in various (but not all) cell lines and may be a more specific marker for HSP90 inhibition than induction of other HSP70 proteins.

Topics: Amino Acid Sequence; Benzoquinones; Blotting, Western; Brefeldin A; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hot Temperature; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Leupeptins; Macrolides; MCF-7 Cells; Molecular Sequence Data; RNA Interference; Sequence Homology, Amino Acid; Thapsigargin; Time Factors; Transcriptional Activation

The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Benzoquinones; Boronic Acids; Bortezomib; Carcinoma, Basal Cell; Cell Line; DNA-Binding Proteins; Drosophila melanogaster; Drosophila Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Green Fluorescent Proteins; Hedgehog Proteins; Heterocyclic Compounds, 4 or More Rings; HSP90 Heat-Shock Proteins; Lactams, Macrocyclic; Medulloblastoma; Mice; Mutant Proteins; Pyrazines; Receptors, G-Protein-Coupled; RNA Interference; RNA, Small Interfering; Signal Transduction; Smoothened Receptor; Thapsigargin; Ubiquitin-Protein Ligases; Unfolded Protein Response

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