Prior studies have noted that inhibitors of MEK1/2 enhanced geldanamycin lethality in malignant hematopoietic cells by promoting mitochondrial dysfunction. The present studies focused on defining the mechanism(s) by which these agents altered survival in carcinoma cells. MEK1/2 inhibitors (PD184352; AZD6244 (ARRY-142886)) interacted in a synergistic manner with geldanamycins (17AAG, 17DMAG) to kill hepatoma and pancreatic carcinoma cells that correlated with inactivation of ERK1/2 and AKT and with activation of p38 MAPK; p38 MAPK activation was ROS-dependent. Treatment of cells with MEK1/2 inhibitors and 17AAG reduced expression of c-FLIP-s that was mechanistically connected to loss of MEK1/2 and AKT function; inhibition of caspase 8 or over-expression of c-FLIP-s abolished cell killing by MEK1/2 inhibitors and 17AAG. Treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent plasma membrane clustering of CD95 without altering the levels or cleavage of FAS ligand. In parallel, treatment of cells with MEK1/2 inhibitors and 17AAG caused a p38 MAPK-dependent association of caspase 8 with CD95. Inhibition of p38 MAPK or knock down of BID, FADD or CD95 expression suppressed MEK1/2 inhibitor and 17AAG lethality. Similar correlative data were obtained using a xenograft flank tumor model system. Our data demonstrate that treatment of tumor cells with MEK1/2 inhibitors and 17AAG induces activation of the extrinsic pathway and that suppression of c-FLIP-s expression is crucial in transduction of the apoptotic signal from CD95 to promote cell death.
CD95; caspase; extrinsic; FLIP
We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills brain cancer cells. OSU toxicity was enhanced by the HSP90 inhibitor 17-N-Allylamino-17-demethoxygeldanamycin (17AAG) that correlated with reduced expression of ERBB1 and ERBB2. Inhibition of the extrinsic apoptosis pathway blocked the interaction between 17AAG and OSU. OSU toxicity was enhanced by the inhibitor of ERBB1/2/4, lapatinib. Knock down of ERBB1/2/4 in a cell line specific fashion promoted OSU toxicity. Combined exposure of cells to lapatinib and OSU resulted in reduced AKT and ERK1/2 activity; expression of activated forms of AKT and to a lesser extent MEK1 protected cells from the lethal effects of the drug combination. Knock down of PTEN suppressed, and expression of PTEN enhanced, the lethal interaction between OSU and lapatinib. Downstream of PTEN, inhibition of mTOR recapitulated the effects of lapatinib. Knock down of CD95, NOXA, PUMA, BIK or AIF, suppressed lapatinib and OSU toxicity. Knock down of MCL-1 enhanced, and overexpression of MCL-1 suppressed, drug combination lethality. Lapatinib and OSU interacted in vivo to suppress the growth of established tumors. Collectively our data argue that the inhibition of ERBB receptor function represents a useful way to enhance OSU lethality in brain tumor cells.
glioblastoma; medulloblastoma; lapatinib; OSU-03012; apoptosis; autophagy; ERBB1; PTEN
The cellular glucose-regulated protein GRP78-BiP is a member of the HSP70 stress family of gene products, and the protein is a resident component of the endoplasmic reticulum, where it is thought to play a role in the folding and oligomerization of secretory and membrane-bound proteins. GRP78-BiP also binds to malfolded proteins, and this may be one mechanism for preventing their intracellular transport. An induction in synthesis of the GRP78-BiP protein occurs in cells infected with paramyxoviruses (R. W. Peluso, R. A. Lamb, and P. W. Choppin, Proc. Natl. Acad. Sci. USA 75:6120-6124, 1978). We have studied the expression and activity of the GRP78-BiP gene and synthesis of the GRP78-BiP protein during infection with the paramyxovirus simian virus 5 (SV5). We wished to identify the viral component capable of causing activation of GRP78-BiP since GRP78-BiP interacts specifically and transiently with the SV5 hemagglutinin-neuraminidase (HN) glycoprotein during HN folding (D. T. W. Ng, R. E. Randall, and R. A. Lamb, J. Cell Biol. 109:3273-3289, 1989). Expression of cDNAs of the SV5 wild-type HN glycoprotein and a mutant form of HN that is malfolded but not the SV5 F glycoprotein or SV5 cytoplasmic proteins P, V, and M caused increased amounts of GRP78-BiP mRNA to accumulate, as detected by nuclease S1 protection assays. As unfolded or malfolded forms of HN cannot be detected to accumulate during SV5 infection, the data suggest that the flux of HN through the ER in SV5-infected cells can cause activation of GRP78-BiP transcription.
The endoplasmic reticulum (ER)-localized chaperone protein, GRP78-BiP, is involved in the folding and oligomerization of secreted and membrane proteins, including the simian virus 5 hemagglutinin-neuraminidase (HN) glycoprotein. To understand this interaction better, we have constructed a series of HN mutants in which specific portions of the extracytoplasmic domain have been deleted. Analysis of these mutant polypeptides expressed in CV-1 cells have indicated that GRP78-BiP binds to selective sequences in HN and that there exists more than a single site of interaction. Mutant polypeptides have been characterized that are competent and incompetent for association with GRP78-BiP. These mutants have been used to show that the induction of GRP78-BiP synthesis due to the presence of nonnative protein molecules in the ER is dependent on GRP78-BiP complex formation with its substrates. These studies have implications for the function of the GRP78-BiP protein and the mechanism by which the gene is regulated.
We have further defined mechanism(s) by which the drug OSU-03012 (OSU) kills tumor cells. OSU lethality was suppressed by knock down of PERK and enhanced by knock down of ATF6 and IRE1α. OSU treatment suppressed expression of the chaperone, BiP/GRP78, and did so through reduced stability of the protein. Knock down of BiP/GRP78 further enhanced OSU lethality. Overexpression of BiP/GRP78 abolished OSU toxicity. Pre-treatment of cells with OSU enhanced radiosensitivity to a greater extent than concomitant or sequential drug treatment with radiation exposure. Expression of a mutant active p110 PI3K, or mutant active forms of the EGFR in GBM cells did not differentially suppress OSU killing. In contrast loss of PTEN function reduced OSU lethality, without altering AKT, p70 S6K or mTOR activity, or the drug's ability to radiosensitize GBM cells. Knock down of PTEN protected cells from OSU and radiation treatment whereas re-expression of PTEN facilitated drug lethality and radiosensitization. In a dose-dependent fashion OSU prolonged the survival of mice carrying GBM tumors and interacted with radiotherapy to further prolong survival. Collectively, our data show that reduced BiP/GRP78 levels play a key role in OSU-3012 toxicity in GBM cells, and that this drug has in vivo activity against an invasive primary human GBM isolate.
OSU-03012; BiP/GRP78; ER stress; PERK; ionizing radiation; ceramide
A specific inhibitor of heat shock protein 90 (Hsp90), 17-AAG, has been shown to inhibit tumor growth through cell cycle arrest, differentiation, or apoptosis. Because angiogenesis is important for tumor growth, we hypothesize that inhibition of angiogenesis by 17-AAG may mediate some of its antitumor effects.
Methods and Results
Because protein kinase Akt and endothelial nitric oxide synthase (eNOS) are critical for angiogenesis, we studied the effects of 17-AAG on the phosphorylation and expression of Akt and eNOS in human umbilical vein endothelial cells. In a concentration- and time-dependent manner, inhibition of Hsp90 by 17-AAG decreased Akt and eNOS expression by 74% and 81%, respectively. Inhibition of eNOS expression by 17-AAG occurred at the transcriptional level as determined by eNOS promoter activity and nuclear run-on assay. Furthermore, treatment with 17-AAG decreased basal and vascular endothelial growth factor-stimulated Akt and eNOS phosphorylation. This corresponded with decreased NO production and inhibition of endothelial cell migration and angiogenesis. The anti-angiogenic effect of 17-AAG was partially reversed by the NO donor, SNAP.
These findings indicate that Hsp90 is important not only for Akt and eNOS phosphorylation but also for eNOS gene transcription and suggests that Hsp90 may be a novel target for anti-angiogenic therapy.
angiogenesis; endothelium; nitric oxide; heat shock protein; protein kinase Akt
The geldanamycin derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) are promising chemotherapeutic drugs that inhibit heat shock protein 90 (HSP90) function. Previous studies have shown that 17-AAG/DMAG treatment induces the degradation of mutant BRAF (V600E) and inhibits the activation of MAP/ERK1/2 (MEK1/2). We have found, however, that HSP90 inhibition alone is not sufficient for efficient BRAF(V600E) degradation in some cells. HSP90 inhibitors structurally unrelated to geldanamycin, radicicol and novobiocin, while inducing the degradation of the HSP90 client protein RAF-1 fail to induce BRAF(V600E) degradation or inhibit MEK1/2 activation in HT29 human colon cancer cells ‥ Moreover, after treatment with 17-DMAG, the kinase activity of residual, un-degraded BRAF(V600E) was also lost. Incubation of cells with a reactive oxygen species (ROS) scavenger, N-acetyl cysteine (NAC), partially restored kinase activity and also partially prevented BRAF(V600E) degradation due to 17-DMAG treatment. Conversely, treatment with the ROS producing drug menadione clearly inhibited MEK1/2 and reduced BRAF(V600E). These results suggest that in addition to direct inhibition of HSP90, the anti-tumor effect of geldanamycin and its derivatives is also mediated though the production of ROS which may directly inactivate tumorigenic mutant BRAF(V600E).
BRAF; MAP kinase; geldanamycin; HSP90; ROS
The Ras/Raf/MEK/ERK signaling has been implicated in uncontrolled cell proliferation and tumor progression in pancreatic cancer. The purpose of this study is to evaluate the antitumor activity of MEK inhibitor U0126 in combination with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in pancreatic cancer cells. Western blotting showed that 17-AAG caused a 2- to 3-fold transient activation of MEK/ERK signaling in pancreatic cancer cells. The activation sustained for 6 h before phospho-ERK (p-ERK) destabilization. The selective MEK inhibitor U0126 completely abolished 17-AAG induced ERK1/2 activation and resulted in more than 80% of phosphor-ERK degradation after only 15 min treatment. Moreover, U0126 had complementary effect on 17-AAG regulated oncogenic and cell cycle related proteins. Although 17-AAG downregulated cyclin D1, cyclin E, CDK4 and CDK6, it led to cyclin A and CDK2 accumulation, which was reversed by the addition of U0126. Anti-proliferation assay showed that combination of U0126 and 17-AAG resulted in synergistic cytotoxic effect. More importantly, 17-AAG alone only exhibited moderate inhibition of cell migration in vitro, while addition of U0126 dramatically enhanced the inhibitory effect by 2- to 5-fold. Taken together, these data demonstrate that MEK inhibitor U0126 potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells. The combination of Hsp90 and MEK inhibition could provide a promising avenue for the treatment of pancreatic cancer.
Hsp90; MEK; ERK; 17-AAG
Malignant pleural mesothelioma (MPM) is an aggressive malignancy for which there is no approved targeted therapy. We examined the therapeutic efficacy of the mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors against human MPM cell lines both in vitro and orthotopically inoculated into severe combined immunodeficient (SCID) mice. In addition, the molecular mechanisms of these agents were confirmed in vitro and in vivo. The MEK or the PI3K inhibitor suppressed MPM cell growth in vitro in a dose-dependent manner via induction of G1 cell cycle arrest and apoptosis. In addition, combined use of the MEK and PI3K inhibitors showed an additive or synergistic inhibitory effect on MPM cell growth compared to treatment with either individual drug. Treatment with MEK or PI3K inhibitor suppressed the production of thoracic tumors and pleural effusion and prolonged the survival time of EHMES-10 cell-bearing SCID mice. The combination therapy more effectively prolonged the survival time compared to treatment with either individual drug. Immunohistochemical and western blot analysis of thoracic tumors suggested that these agents induced cell cycle arrest, apoptosis and inhibition of tumor angiogenesis. Our results suggest that a combination of MEK and PI3K inhibitors is a promising therapeutic strategy for MPM.
malignant pleural mesothelioma; mitogen-activated protein kinase kinase inhibitor; phosphatidylinositol 3-kinase inhibitor; cell cycle; apoptosis; angiogenesis
The trichothecene mycotoxin deoxynivalenol (DON) induces systemic expression of the interleukin-6 (IL-6) and other proinflammatory cytokines in the mouse. The purpose of this study was to test the hypothesis that DON triggers an endoplasmic reticulum (ER) stress response in murine macrophages capable of driving IL-6 gene expression. DON at concentrations up 5000 ng/ml. was not cytotoxic to peritoneal cells. However, DON markedly decreased protein levels but not the mRNA levels of glucose-regulated protein (GRP) 78 (BiP), a chaperone known to mediate ER stress. Inhibitor studies suggested that DON-induced GRP78 degradation was cathepsin and calpain dependent but was proteosome-independent. RNAi-mediated knockdown of GRP78 resulted in increased IL-6 gene expression indicating a potential downregulatory role for this chaperone. GRP78 is critical to the regulation of the two transcription factors, X-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6), which bind to cAMP-response element (CRE) and drive expression of CRE-dependent genes such as IL-6. DON exposure was found to increase IRE1α protein, its modified products spliced XBP1 mRNA and XBP1 protein as well as ATF6. Knockdown of ATF6 but not XBP1 partially inhibited DON-induced IL-6 expression in the macrophages. Three other trichothecenes (satratoxin G, roridin, T-2 toxin) and the ribosome inhibitory protein ricin were also found to induce GRP78 degradation suggesting that other translation inhibitors might evoke ER stress. Taken together, these data suggest that in the macrophage DON induces GRP78 degradation and evokes an ER stress response that could contribute, in part, to DON-induced IL-6 gene expression.
deoxynivalenol (DON); interleukin-6; ER stress, translation inhibition
Immunoglobulin heavy chain binding protein (BiP, GRP 78) coprecipitates with soluble and membrane-associated variants of the T-cell antigen receptor alpha chain (TCR-alpha) which are stably retained within the ER. Chelation of Ca2+ during solubilization of cells leads to the dissociation of BiP from the TCR-alpha variants, which is dependent upon the availability of Mg2+ and hydrolyzable ATP; this suggests that Ca2+ levels can serve to modulate the association/dissociation of these proteins with BiP. In vivo treatment of cells expressing either the soluble or membrane-anchored TCR-alpha variants with the Ca2+ ionophore, A23187, or an inhibitor of an ER Ca(2+)-ATPase, thapsigargin, or the membrane-permeant Ca2+ chelator BAPTA-AM, results in the redistribution of these proteins out of the ER and their subsequent secretion or cell surface expression. Under the same assay conditions, no movement of BiP out of the ER is observed. Taken together, these observations indicate that decreased Ca2+ levels result in the dissociation of a protein bound to BiP, leading to its release from ER retention. These data suggest that the intracellular fate of newly synthesized proteins stably associated with BiP can be regulated by Ca2+ levels in the ER.
The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established.
Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells.
We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect.
MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.
Despite studies that demonstrate the antitumor activity of Hsp90 inhibitors such as geldanamycin (GA) and its derivative 17-allylamino-demethoxygeldanamycin (17-AAG), recent reports indicate that these inhibitors lack significant single-agent clinical activity. Resistance to Hsp90 inhibitors has been previously linked to expression of P-glycoprotein (P-gp), and the multidrug resistant (MDR) phenotype. However, the stress response induced by GA treatment can also cause resistance to Hsp90-targeted therapy. Therefore we chose to further investigate the relative importance of P-gp and the stress response in 17-AAG resistance. Colony forming assays revealed that high expression of P-gp could increase the 17-AAG IC50 6-fold in cells transfected with P-gp as compared with parent cells. A549 cells selected for resistance to GA overexpressed P-gp, but verapamil (VP) did not reverse the resistance. These cells also overexpressed Hsp27, and Hsp70 was induced with 17-AAG treatment. When the GA and 17-AAG resistant cells were transfected with Hsp27 and/or Hsp70 siRNA, the 17-AAG IC50 decreased 10-fold compared to control transfected cells. Transfection with siRNA directed against Hsp27, Hsp70, or Hsp27 and Hsp70 also increased sensitivity to EC78, a purine scaffold-based Hsp90 inhibitor that is not a P-gp substrate. We conclude that P-gp may contribute, in part, to resistance to 17-AAG, but induction of stress response proteins such as Hsp27 and Hsp70 by Hsp90-targeted therapy plays a larger role. Taken together, our results indicate that targeting of Hsp27 and Hsp70 should be exploited to increase the clinical efficacy of Hsp90-directed therapy.
17-AAG; Geldanamycin; P-glycoprotein; Hsp90; Hsp27
The present studies were initiated to determine in greater molecular detail the regulation of CHK1 inhibitor lethality in transfected and infected breast cancer cells and using genetic models of transformed fibrobalsts. Multiple MEK1/2 inhibitors (PD184352, AZD6244 [ARRY-142886]) interacted with multiple CHK1 inhibitors (UCN-01 [7-hydroxystaurosporine], AZD7762) to kill mammary carcinoma cells and transformed fibroblasts. In transformed cells, CHK1 inhibitor-induced activation of ERK1/2 was dependent upon activation of SRC family non-receptor tyrosine kinases as judged by use of multiple SRC kinase inhibitors (PP 2, Dasatinib; AZD0530), use of SRC/FYN/YES deleted transformed fibroblasts or by expression of dominant negative SRC. Cell killing by SRC family kinase inhibitors and CHK1 inhibitors was abolished in BAX/BAK−/− transformed fibroblasts and suppressed by overexpression of BCL-XL. Treatment of cells with BCL-2/BCL-XL antagonists promoted SRC inhibitor + CHK1 inhibitor-induced lethality in a BAX/BAK-dependent fashion. Treatment of cells with [SRC + CHK1] inhibitors radio-sensitized tumor cells. These findings argue that multiple inhibitors of the SRC-RAS-MEK pathway interact with multiple CHK1 inhibitors to kill transformed cells.
CHK1; SRC; apoptosis; breast cancer; kinase; therapeutics; intrinsic; caspase
In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that whereas ER stress-induced autophagy was suppressed by PI3KC3 inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using siRNA, only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells where GRP78 expression was knockdown by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3-Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells where GRP78 was knockdown. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.
autophagy; GRP78; unfolded protein response
The geldanamycin derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG), binds to the amino-terminal ATP binding pocket of the 90 kDa heat shock protein (Hsp-90) and inhibits this chaperone from stabilizing client proteins involved with the malignant phenotype. We examined the effects of a combined modality protocol involving photodynamic therapy (PDT) and 17-AAG in mouse mammary carcinoma cells and tumors. PDT increased the expression of the anti-apoptotic and pro-angiogenic proteins survivin, Akt, HIF-1α, MMP-2 and VEGF in tumor tissue and this expression decreased significantly when 17-AAG was included in the treatment regimen. Tumor bearing mice treated with PDT and 17-AAG had improved long-term tumoricidal responses when compared with individual treatment protocols. We conclude that Hsp-90 plays an active role in modulating tumor responsiveness following PDT and targeting Hsp-90 with 17-AAG enhances the therapeutic effectiveness of PDT.
photodynamic therapy; PDT; apoptosis; Hsp-90; 17-AAG; geldanamycin
Treatment of several types of cancer such as lung, breast, prostate, and pancreas has shown notable progresses in the past decades. However, after an initial response, tumors eventually became resistant to chemotherapy. This phenomenon, known as chemoresistance, accounts for the death of most cancer patients. Several studies in patients refractory to therapy have revealed the upregulation of the molecular chaperone GRP78/Binding Protein, BiP (BiP) both at the RNA and protein expression level. Furthermore GRP78/BiP relocates to the cell membrane in malignant but not in benign cells. In this communication we review studies on the role and the mechanism of action of GRP78/BiP during development of chemoresistance in cancer cells. In addition we discuss the possible role of GRP78 as a biomarker and as a target in cancer therapy.
cell stress; chaperone; unfolded protein response; drug resistance; therapy
17-Allylamino-17-demethoxygeldanamycin (17AAG) is an experimental chemotherapeutic agent believed to form free radicals in vivo, and cancer cell resistance to 17AAG is believed to be a thiol-dependent process. Inhibitors of thiol-dependent hydroperoxide metabolism [l-buthionine-S,R-sulfoximine (BSO) and auranofin] were combined with the glucose metabolism inhibitor 2-deoxy-d-glucose (2DG) to determine if 17AAG-mediated cancer cell killing could be enhanced. When 2DG (20 mM, 24 h), BSO (1 mM, 24 h), and auranofin (500 nM, 3 h) were combined with 17AAG, cell killing was significantly enhanced in three human cancer cell lines (PC-3, SUM159, MDA-MB-231). Furthermore, the toxicity of this drug combination was significantly greater in SUM159 human breast cancer cells, relative to HMEC normal human breast epithelial cells. Increases in toxicity seen with this drug combination also correlated with increased glutathione (GSH) and thioredoxin (Trx) oxidation and depletion. Furthermore, treatment with the thiol antioxidant NAC (15 mM, 24 h) was able to significantly protect from drug-induced toxicity and ameliorate GSH oxidation, Trx oxidation, and Trx depletion. These data strongly support the hypothesis that simultaneous inhibition of GSH- and Trx-dependent metabolism is necessary to sensitize human breast and prostate cancer cells to 2DG+17AAG-mediated killing via enhancement of thiol-dependent oxidative stress. These results suggest that simultaneous targeting of both GSH and Trx metabolism could represent an effective strategy for chemosensitization in human cancer cells.
Glutathione; Thioredoxin; 2-Deoxyglucose; Auranofin; Thioredoxin reductase; Free radicals
Hsp90 is important in the folding, maturation and stabilization of pro-tumorigenic client proteins and represents a viable drug target for the design of chemotherapies. Previously, we reported the development of novobiocin analogues designed to inhibit the C-terminal portion of Hsp90, which demonstrated the ability to decrease client protein expression. We now report the characterization of the novel novobiocin analogue, F-4, which demonstrates improved cytotoxicity in prostate cancer cell lines compared to the N-terminal inhibitor, 17-AAG.
Materials and Methods
LNCaP and PC-3 cells were treated with 17-AAG or F-4 in anti-proliferative, apoptosis, cell cycle and cytotoxicity assays. Western blot and prostate specific antigen (PSA) ELISAs were used to determine client protein degradation, induction of Hsp90 and to assess the functional status of the androgen receptor (AR) in response to F-4 treatment. Surface Plasmon Resonance (SPR) was also used to determine the binding properties of F-4 to Hsp90.
F-4 demonstrated improved potency and efficacy compared to novobiocin in anti-proliferative assays and decreased expression of client proteins. PSA secretion was inhibited in a dose-dependent manner that paralleled a decrease in AR expression. The binding of F-4 to Hsp90 was determined to be saturable with a binding affinity (Kd) of 100 µM. In addition, superior efficacy was demonstrated by F-4 compared to 17-AAG in experiments measuring cytotoxicity and apoptosis
These data reveal distinct modes of action for N-terminal and C-terminal Hsp90 inhibitors, which may offer unique therapeutic benefits for the treatment of prostate cancer.
Hsp90 inhibitors; prostate cancer; novobiocin
The ability of interferon (IFN) to induce the expression of antiviral genes, and therefore suppress viral infection, is dependent on the activity of cellular suppressors. The Ras/MEK pathway is one of these cellular suppressors, since the activation of Ras/MEK permits viral replication in the presence of alpha IFN (IFN-α). Here, we have investigated the mechanism by which activated Ras/MEK inhibits the IFN-α response. We found that the induction of antiviral proteins in response to IFN-α was impaired in Ras-transformed NIH 3T3 (RasV12) cells. The inhibition of the Ras/MEK pathway restored the IFN-mediated induction of antiviral genes, indicating that activated Ras interrupts the IFN pathway upstream of antiviral gene transcription. Indeed, the IFN-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2 was inhibited in RasV12 cells compared to that of vector control cells. In addition, we found that the total amount of STAT2 was reduced in RasV12 cells. To determine if the impaired IFN-α response can be rescued by restoring the overall level of STAT2, we overexpressed STAT2 in RasV12 cells. The IFN-α-induced phosphorylation of STAT1 and STAT2, as well as the expression of antiviral protein, were restored, and IFN-induced antiviral protection was partially restored. Moreover, we demonstrated that the downregulation of STAT2 levels by Ras/MEK was mediated at the transcriptional level. Thus, the activation of the Ras/MEK pathway reduces the amount of STAT2 available for propagating the IFN signal, resulting in the impairment of the IFN-α-induced antiviral response.
Benzoquinone ansamycin antibiotics such as geldanamycin (GA) bind to the N-terminal ATP binding domain of Hsp90 and inhibit its chaperone functions. Despite in vitro and in vivo studies indicating promising antitumor activity, derivatives of GA, including 17-AAG have demonstrated little clinical efficacy as single agents. Thus, combination studies of 17-AAG and several cancer chemotherapeutics, including cisplatin (CDDP), have begun. In colony-forming assays, the combination of CDDP and GA or 17-AAG was synergistic, and caused increased apoptosis compared to each agent alone. One measurable response that results from treatment with Hsp90-targeted agents is the induction of an HSF-1 heat shock response. Treatment with GA + CDDP revealed that CDDP suppresses upregulation of HSF-1 transcription, causing decreased levels of stress-inducible proteins such as Hsp27 and Hsp70. However, CDDP treatment did not prevent trimerization and nuclear localization of HSF-1, but inhibited DNA binding of HSF-1 as demonstrated by chromatin immunoprecipitation. Melphalan, but not camptothecin, caused similar inhibition of GA-induced HSF-1-mediated Hsp70 upregulation. MTS cell survival assays revealed that deletion of Hsp70 caused increased sensitivity to GA (Hsp70+/+ IC50=63.7±14.9 nM and Hsp70−/− IC50=4.3±2.9 nM), which confirmed that a stress response plays a critical role in decreasing GA sensitivity. Our results suggest that the synergy of GA + CDDP is due, in part, to CDDP-mediated abrogation of the heat shock response through inhibition of HSF-1 activity. Clinical modulation of the HSF-1-mediated heat shock response may enhance the efficacy of Hsp90-directed therapy.
17-AAG; Geldanamycin; Cisplatin; Hsp90; HSF-1
We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models.
The activity of ganetespib was compared to that of the geldanamycin 17-AAG in biochemical assays, cell lines and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model.
Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its co-chaperone, p23, more potently than 17-AAG. In genomically-defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC50 values ranging 2–30 nM, substantially lower than those required for 17-AAG (20– 3,500 nM). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t1/2 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, re-expression of mutant EGFR occurred by 72 hours, correlating with reversal of anti-proliferative and pro-apoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also demonstrated activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA.
Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation.
HSP90; ERBB Receptor Tyrosine Kinases; NSCLC; Non-Geldanamycin
We determined how CXC-chemokine signalling and necrosis factor-κB (NF-κB) activity affected heat-shock protein 90 (Hsp90) inhibitor (geldanamycin (GA) and 17-allylamino-demethoxygeldanamycin (17-AAG)) cytotoxicity in castrate-resistant prostate cancer (CRPC).
Geldanamycin and 17-AAG toxicity, together with the CXCR2 antagonist AZ10397767 or NF-κB inhibitor BAY11-7082, was assessed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay in two CRPC lines, DU145 and PC3. Flow cytometry quantified apoptotic or necrosis profiles. Necrosis factor-κB activity was determined by luciferase readouts or indirectly by quantitative PCR and ELISA-based determination of CXCL8 expression.
Geldanamycin and 17-AAG reduced PC3 and DU145 cell viability, although PC3 cells were less sensitive. Addition of AZ10397767 increased GA (e.g., PC3 IC20: from 1.67±0.4 to 0.18±0.2 nM) and 17-AAG (PC3 IC20: 43.7±7.8 to 0.64±1.8 nM) potency in PC3 but not DU145 cells. Similarly, BAY11-7082 increased the potency of 17-AAG in PC3 but not in DU145 cells, correlating with the elevated constitutive NF-κB activity in PC3 cells. AZ10397767 increased 17-AAG-induced apoptosis and necrosis and decreased NF-κB activity/CXCL8 expression in 17-AAG-treated PC3 cells.
Ansamycin cytotoxicity is enhanced by inhibiting NF-κB activity and/or CXC-chemokine signalling in CRPC cells. Detecting and/or inhibiting NF-κB activity may aid the selection and treatment response of CRPC patients to Hsp90 inhibitors.
prostate cancer; CXC-chemokines; CXCL8; NF-κB; Hsp90 inhibitors
Perturbing Hsp90 chaperone function targets hypoxia inducible factor (HIF) function in a von Hippel-Lindau (VHL) independent manner, and represents an approach to combat the contribution of HIF to cell renal carcinoma (CCRCC) progression. However, clinical trials with the prototypic Hsp90 inhibitor 17-AAG have been unsuccessful in halting the progression of advanced CCRCC.
Here we evaluated a novel next generation small molecule Hsp90 inhibitor, EC154, against HIF isoforms and HIF-driven molecular and functional endpoints. The effects of EC154 were compared to those of the prototypic Hsp90 inhibitor 17-AAG and the histone deacetylase (HDAC) inhibitor LBH589.
The findings indicate that EC154 is a potent inhibitor of HIF, effective at doses 10-fold lower than 17-AAG. While EC154, 17-AAG and the histone deacetylase (HDAC) inhibitor LBH589 impaired HIF transcriptional activity, CCRCC cell motility, and angiogenesis; these effects did not correlate with their ability to diminish HIF protein expression. Further, our results illustrate the complexity of HIF targeting, in that although these agents suppressed HIF transcripts with differential dynamics, these effects were not predictive of drug efficacy in other relevant assays.
We provide evidence for EC154 targeting of HIF in CCRCC and for LBH589 acting as a suppressor of both HIF-1 and HIF-2 activity. We also demonstrate that 17-AAG and EC154, but not LBH589, can restore endothelial barrier function, highlighting a potentially new clinical application for Hsp90 inhibitors. Finally, given the discordance between HIF activity and protein expression, we conclude that HIF expression is not a reliable surrogate for HIF activity. Taken together, our findings emphasize the need to incorporate an integrated approach in evaluating Hsp90 inhibitors within the context of HIF suppression.
The purpose of this study is to clarify the effect of a heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in combination with X-rays or carbon-ion beams on cell killing in human oral squamous cell carcinoma LMF4 cells. Cell survival was measured by colony formation assay. Cell-cycle distribution was analyzed by flow cytometry. Expression of DNA repair-related proteins was investigated by western blotting. The results showed 17-AAG to have synergistic effects on cell lethality with X-rays, but not with carbon-ion beams. The 17-AAG decreased G2/M arrest induced by X-rays, but not by carbon-ion beams. Both X-ray and carbon-ion irradiation up-regulated expression of non-homologous end-joining-associated proteins, Ku70 and Ku80, but 17-AAG inhibited only X-ray-induced up-regulation of these proteins. These results show that 17-AAG with X-rays releases G2/M phase arrest; cells carrying misrepaired DNA damage then move on to the G1 phase. We demonstrate, for the first time, that the radiosensitization effect of 17-AAG is not seen with carbon-ion beams because 17-AAG does not affect these changes.
heat shock protein 90; 17-allylamino-17-demethoxygeldanamycin (17-AAG); carbon-ion beam irradiation; radiosensitization