Benzoquinone ansamycins are antibiotics with anticancer potential. First described as tyrosine kinase inhibitors, they are now frequently used to target HSP90 chaperone function. While herbimycin A ...and geldanamycin (GA) have been widely used in preclinical studies, both drugs are poor candidates for clinical trials owing to their in vivo toxicity and lack of stability. We therefore examined the biologic effects of 17-allylamino-17-demethoxygeldanamycin (17-AG), an ansamycin derivative with lower in vivo toxicity than GA.
Binding of 17-AG to HSP90 was studied in vitro using a GA-affinity beads competition assay. We analyzed the drug-induced destabilization of p185erbB2, Raf-1 and mutant p53 in SKBR3 breast cancer cells by Western blotting. The antiproliferative activities of 17-AG and GA were compared using the MTT assay.
We found that, in a similar manner to GA itself, 17-AG bound specifically to HSP90. It also led to degradation of the receptor tyrosine kinase p185erbB2, the serine/threonine kinase Raf-1 and mutant p53. Both GA and 17-AG displayed comparable antiproliferative effects in SKBR3 and MCF7 cells. Even though HSP90 binding by 17-AG was weaker than by GA, 17-AG and GA caused biologic effects in tumor cells at similar doses.
17-AG shares the important biologic features of its parent compound GA. Since 17-AG has a better toxicity profile than GA, it is an interesting candidate benzoquinone ansamycin for clinical development.
BRCA1, a tumor suppressor protein implicated in hereditary forms of breast and ovarian cancer, is transcriptionally regulated in a proliferation-dependent manner. In this study, we demonstrate a ...substantial role for proteolysis in regulating the BRCA1 steady-state protein level in several cell lines. N-acetyl-leu-leu-norleucinal (ALLN), an inhibitor of the proteasome, calpain, and cathepsins, caused BRCA1 protein to accumulate in the nucleus of several human breast, prostate, and melanoma cell lines which express low or undetectable basal levels of BRCA1 protein, but not in cells with high basal expression of BRCA1. Protease inhibition did not increase BRCA1 synthesis, nor change its mRNA level, but it dramatically prolonged the protein's half-life. In contrast to ALLN, lactacystin and PS341, two specific proteasome inhibitors, as well as calpastatin peptide and PD150606, two selective calpain inhibitors, had no effect on BRCA1 stability, whereas ALLM, an effective calpain and cathepsin inhibitor but weak proteasome inhibitor, did stimulate accumulation of BRCA1. Moreover, three inhibitors of acidic cysteine proteases, chloroquine, ammonium chloride and bafilomycin, were as effective as ALLN. These results demonstrate that degradation by a cathepsin-like protease in fine balance with BRCA1 transcription is responsible for maintaining the low steady-state level of BRCA1 protein seen in many cancer cells.
Abstract
Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, ...effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. To uncover cell type-specific dependencies to LDH, we screened a diverse panel of 94 cancer cell lines for responsiveness to two novel LDH A/B inhibitors developed through the NCI Experimental Therapeutics Program (NExT). We found that Ewing sarcoma (EWS) cell lines were exquisitely sensitive, with IC50 values approximately ten-fold below the median IC50 of the panel. To understand the mechanism behind this sensitivity, we genetically knocked down LDHA and LDHB using siRNA, and discovered that EWS cell lines were sensitive to loss of LDHA only, which inhibited proliferation and induced apoptosis. Notably, treatment of EWS cells with the LDH inhibitors phenocopied these effects. Additionally, genetic knockdown of EWS-FLI1, the oncogenic driver of EWS, resulted in loss of LDHA, but not LDHB. Analysis of publicly available ChIP-seq data generated using shFLI1-transfected EWS cells revealed that LDHA, but not LDHB, is directly regulated by EWS-FLI1. Functional mechanistic studies of glycolytic intermediates and cellular bioenergetics in EWS cells treated with the LDH inhibitors demonstrated that loss of viability was due to impairment of glycolysis, which occurred both in vitro and in vivo, and perturbation of the NAD+/NADH ratio. The translational potential of these compounds was next evaluated using in vivo analyses of pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Intravenous administration of the LDH inhibitors resulted in diminished LDH activity, reduction of the lactate-to-pyruvate ratio, tumor cell necrosis, and a decrease in tumor growth rate in aggressive xenograft models of EWS. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, our data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as EWS that exhibit oncogene-dependent expression of LDH and increased glycolytic activity.
This abstract is also being presented as Poster B33.
Citation Format: Choh Yeung, Anna E. Gibson, Sameer H. Issaq, Nobu Oshima, Marielle E. Yohe, Haiyan Lei, Ganesha Rai, Daniel J. Urban, Michelle S. Johnson, Gloria A. Benevides, Giuseppe L. Squadrito, Sandy Eldridge, John Hamre III, Arnulfo Mendoza, Jack F. Shern, Lee J. Helman, Murali C. Krishna, Matthew D. Hall, Victor M. Darley-Usmar, Leonard M. Neckers, Christine M. Heske. Lactate dehydrogenase A is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma abstract. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR08.
Abstract
Background
In order to survive bladder cancer (BCa) cells deregulate key pathways - including the inhibition of apoptosis - while developing “oncogene addiction” to molecular chaperones such ...as Heat Shock Protein 90 (HSP90). While successful in vitro, monotherapies targeting such pathways have failed in clinical trials. We aim to show the superiority of combination therapy using proteostatic and targeted cytotoxic agents. We hypothesize that these combinations will synergize to increase cytotoxicity (CT) in BCa by amplifying the apoptosis via simultaneous activation of both intrinsic and extrinsic apoptotic pathways.
Methods
The proteostatic agents tested were the HSP90 inhibitor STA9090 (9090) and the proteasome inhibitor Marizomib (NPI). The cytotoxic agent used was a synthetic TRAIL ligand. These agents were tested in multiple ATCC BCa cell lines and the NCI patient tumor-derived BCa cell line UOBL 101 (UOBL). CT was determined via MTT assay. Mechanism of cytotoxic response was investigated using a series of immunoblots targeting the components of intrinsic and extrinsic apoptotic pathways and nuclear degradation proteins. All the assays were performed in triplicates for validation.
Results
9090 (IC50 7.4-10 nM) was effective in all cell lines, as was NPI (IC50 10-20 nM). TRAIL caused 100% CT in UOBL (IC50 2.5 ng/mL, 95% CI 1.0, 7.0) and a maximal 50% CT in T24, however the cytotoxicity of individual agents was not sustained. Combination effects of 9090 + TRAIL and NPI + TRAIL demonstrated an increase from 50% CT to 100% CT at the IC50 of both 9090 and NPI, and this effect was sustained over the course of the experiment. The increased cytotoxicity of combination therapy had corresponded to rapid cytoplasmic release of cytochrome c, additive increase in cleaved caspase 3 and caspase 8, with the corresponding increase in cleaved PARP and γ-H2ax.
Conclusions
Combination of a pro-apoptotic agent with a molecular chaperone or proteasome inhibitor holds promise in BCa therapyby affecting intracellular anti-apoptotic signaling and compromising efficient proteostasis in cancer cells lines, and should be investigated further.
Citation Format: Raju Chelluri, Piyush K. Agarwal, Leonard M. Neckers, Armine K. Smith. Synergistic effect of targeted combination therapy in bladder cancer model using HSP90 inhibitors. abstract. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3796. doi:10.1158/1538-7445.AM2015-3796
Protein ubiquitination is crucial to many diverse and critical functions of cells. Although it has been long known that conjugation of ubiquitin to proteins results in their destruction by the ...proteasome, recently it has become apparent that reversible protein ubiquitination, particularly monoubiquitination, performs regulatory functions in cells, analogous to protein phosphorylation. The most powerful and sensitive technique for measuring specific protein ubiquitination is antiubiquitin immunoblotting of the immunoprecipitated protein after gel electrophoresis. Efficient antibodies recognizing ubiquitinated proteins are now available, making ubiquitin immunoblotting a practical tool for research into the many and varied aspects of this extremely interesting posttranslational protein modification. Here, we describe in detail the steps to follow in order to determine whether a particular protein might become ubiquitinated, or deubiquitinated, and we offer warnings about pitfalls to avoid in antiubiquitin immunoblotting.
The benzoquinone ansamycin geldanamycin (GA) stimulates proteasome-mediated degradation of plasma membrane–associated ErbB2, a receptor tyrosine kinase. Drug sensitivity is mediated by ErbB2's kinase ...domain and occurs subsequent to the disruption of Hsp90 interaction with this domain. Full-length ErbB2 is efficiently processed via the endoplasmic reticulum (ER) and Golgi network, so that at steady state most of the detectable protein is plasma membrane associated. However, previous studies have also demonstrated the GA sensitivity of newly synthesized ErbB2, normally a minor component of the total cellular pool of the kinase. Drug sensitivity of nascent ErbB2 is distinguished by 2 characteristics—protein instability and inability to traverse the ER. As nascent ErbB2 can associate with both cytoplasmic Hsp90 and its ER luminal homolog Grp 94, also a GA-binding protein, the purpose of this study was to examine the relative contributions of the cytoplasmic and ER luminal domains of ErbB2 to the GA sensitivity of the nascent kinase. By studying the drug sensitivity of ErbB2/DK, a construct lacking ErbB2's cytoplasmic kinase domain, and by examining the activity of a GA derivative that preferentially binds Hsp90, we conclude that both the stability and the maturation of nascent ErbB2 are regulated by its cytoplasmic, Hsp90-interacting domain.
Abstract
Increased conversion of pyruvate to lactate by Lactate Dehydrogenase A (LDHA) is a feature of many neoplasms. Therefore, LDHA inhibition is considered a promising approach toward developing ...a new therapeutic strategy against cancers expressing the Warburg phenotype. To develop this strategy for clinical use, a feasible and sensitive noninvasive imaging approach that can dynamically evaluate LDHA activity in vivo would be highly beneficial since in vitro metabolic profiling dose not always predict in vivo cancer metabolism.
Hyperpolarized (HP) 13C Magnetic Resonance Imaging (MRI) can be used to perform dynamic 13C metabolic flux analysis in vivo. In particular, use of 1-13Cpyruvate (13C-pyr) with 13C MRI permits real-time monitoring of intratumoral LDHA activity through dynamic observation of conversion of 13C-pyr to 1-13Clactate (13C-lac).
This study aimed to apply 13C MRI technology in support of a therapeutic strategy to explore efficacy of a newly developed and highly potent LDH inhibitor (LDHi) in a glycolytic tumor model using MiaPaCa2 xenografts in mice.
In vitro analysis showed that the LDHi dose-dependently inhibited human LDH activity and suppressed in vitro cell growth in MiaPaCa2 cells. By ex vivo assay, LDH activity in MiaPaCa2 xenografts was significantly suppressed (82.2 ± 5.6 % decrease compared to vehicle controls) following a single intravenous(IV) injection of 50 mg/kg LDHi.
Next, 13C MRI imaging of HP 13C-pyr metabolism was performed before and after a single LDHi IV injection to assess inhibitor impact on intratumoral metabolic flux in vivo. 13C MR spectroscopy confirmed that LDHi suppressed intratumor LDHA activity dose- and time-dependently. The maximum effective dose was 50 mg/kg and inhibitor impact in the tumor persisted for 10-12 hrs after a single injection. In addition, lactate production in the tumor was suppressed 30 minutes after IV administration of LDHi, as was the 13C-lac to 13C-pyr ratio decreased by 83.3 ± 4.4 % compared to vehicle controls. Importantly, the close correlation of these data with the results of the ex vivo LDH activity assay, suggests that 13C MRI can reliably monitor in vivo on target effects of LDHi without need for tissue sampling. Based on the data using 13C MRI, we developed a therapeutic strategy for using LDHi in an efficacy study of MiaPaCa2 xenografts. Intermittent IV administration of LDHi significantly suppressed tumor growth in this glycolytic model.
In conclusion, intratumoral inhibition of LDHA in vivo upon IV administration of a novel LDHi was readily visualized by HP 13C MRI, confirming the utility of this noninvasive method. This methodology can be of great value in developing new therapeutic strategies using LDHi and perhaps other metabolic inhibitors to treat cancers characterized by the Warburg phenotype. Further, HP 13C MRI should allow for selection of those patients likely to respond to such treatments.
Citation Format: Nobu Oshima, Shun Kishimoto, Kristin Beebe, Dan Crooks, Michael Moses, Kazutoshi Yamamoto, Jeffry R. Brender, Anastasia Sowers, Ganesha Rai, Daniel Urban, Goria Benavides, Giuseppe Squadrito, Victor Darley-Usmar, Matthew Hall, James B. Mitchell, Murali C. Krishna, Leonard M. Neckers. Evaluation of a novel LDH inhibitor efficacy in vivo in a glycolytic cancer model using hyperpolarized 13C magnetic resonance imaging abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4104.
N-myc is a short-lived transcription factor, frequently amplified in human neuroblastomas. The ubiquitin-proteasome system is involved in the degradation of many short-lived cellular proteins and ...previous studies have shown that ubiquitin-dependent proteolysis is implicated in the turn-over of N-myc in vitro. However, calpain has also been implicated in N-myc degradation in vitro. Here we report that, in vivo, N-myc is a sensitive substrate for the 26S proteasome in N-myc amplified neuroblastoma cells. We observed that inhibition of the 26S proteasome with two inhibitors, ALLnL and lactacystin, led to an elevation of the N-myc protein steady-state and increased N-myc protein polyubiquitination, as revealed by ubiquitin Western blotting. Pulse-chase experiments have shown that the increased N-myc levels resulted from stabilization of the protein. In contrast treatment with several calpain and cathepsin inhibitors failed to block N-myc degradation in vivo. Furthermore, fluorescence microscopy of ALLnL-treated cells localized N-myc exclusively to the nuclear compartment, suggesting the absence of a requirement for transport to the cytoplasm prior to degradation.
Abstract
OBJECTIVE
Esophageal adenocarcinoma (EAC) is a lethal disease for which novel therapies are needed. Malignant cell metabolism often shifts from mitochondrial respiration to aerobic ...glycolysis (Warburg effect). However, glucose alone does not meet the metabolic requirements of cancer cells; therefore, glutamine becomes an essential nutrient. A critical step in glutamine metabolism is the conversion of glutamine to glutamate by the enzyme glutaminase-1 (Gls-1), a potential therapeutic target. The present study was undertaken to identify the effect of targeting glutamine utilization by the mitochondrion in EAC.
METHODS
Assays were performed in 3 EAC and 1 Barrett's cell lines: Flo-1, NCI-SB-ESC2 (Esc2), OE33, and CP-C. qRT-PCR and immunoblot were used for Gls-1 expression. Cyquant® and Millipore Invasion Kit measured proliferation and invasion. Flow cytometry, SA-β-galactosidase assay, and immunoblot assessed cell cycle, apoptosis, senescence, and autophagy. Seahorse Extracellular Flux Analyzer was used to quantitate mitochondrial respiration and glycolytic capacity. Cells were treated with bis-2 5-phenylacetamido-1,2,4-thiadiazol-2-yl ethylsulfide (BPTES), an inhibitor of Gls-1, and/or metformin, a mitochondrial electron transport chain inhibitor. Glycolysis was inhibited with 2-deoxyglucose (2-DG). Flo-1 and Esc2 were transduced with shRNA targeting Gls-1.
RESULTS
Glutamine withdrawal decreased proliferation of Flo-1, OE33, and CP-C more than Esc2. Similarly, BPTES caused a more significant decrease in proliferation in Flo-1, OE33, and CP-C than in Esc2. BPTES growth inhibition was reversed by α-ketoglutarate, a metabolite of glutamate. Unlike BPTES, metformin decreased proliferation in all cell lines which was augmented with 2-DG inhibition of glycolysis. Metabolic and knockdown experiments were performed in Flo-1 and Esc2. Glucose partially blocked mitochondrial spare capacity in Flo-1, but completely abrogated the spare capacity in Esc2 suggesting a higher glycolytic dependency in Esc2. Also, knockdown of Gls-1 blocked mitochondrial spare capacity and increased glycolysis in both cell lines. Knockdown of gls-1 decreased proliferation and invasion. Glutamine withdrawal induced autophagy as evidenced by increased LC3 and pAMPK and decreased p70 S6 Kinase in both cell lines. Apoptosis and senescence were not observed. To test glutamine's effect on glycolysis, glutamine repressed the negative regulator of glycolysis, thioredoxin interacting protein (TXNIP), suggesting that glutamine can increase glycolysis while simultaneously fueling the mitochrondria.
CONCLUSIONS
Mitochondrial respiration is mediated by both glucose and glutamine in EAC cells. Glutamine exerts an effect on glycolysis through repression of TXNIP. Whereas EAC cells have unique metabolic profiles, targeting common metabolic steps such as Gls-1 may be novel strategies to treat EAC.
Citation Format: Deborah R. Depew, Paul L. Feingold, Kate Brown, Yuan Xu, Mahadev Rao, Michael Moses, Leonard M. Neckers, David S. Schrump, R Taylor Ripley. Mitochondrial inhibition decreases the malignant phenotype of esophageal adenocarcinoma cells through the induction of autophagy. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1005.
Abstract
aim Increased lactate production is a feature of many neoplasms, and Lactate Dehydrogenase A (LDH-A) plays a key role in conversion of pyruvate to lactate. LDHA inhibition, therefore, is ...considered to be a promising approach toward developing a new therapeutic strategy for cancer treatment focused on targeting cancer metabolism. Non-invasive imaging approaches able to monitor metabolic fluxes in vivo will be useful for this purpose.
Hyperpolarized 13C Magnetic Resonance Imaging (MRI) has been well known as a valuable technology to investigate metabolic processes in tumor xenografts, allowing us to perform dynamic 13C-metabolic flux analysis in vivo. Use of 1-13Cpyruvate with this technology provides the ability to monitor LDHA activity in real time through dynamic observation of conversion of 1-13Cpyruvate to 1-13Clactate.
This study aimed to monitor drug efficacy of a newly developed LDH inhibitor (LDHI, obtained from National Cancer Institute Experimental Therapeutics Program, NExT) in a xenograft tumor model using 13C MRI technology with hyperpolarized 13C-labeled pyruvate.
Results Hyperpolarized 1-13Cpyruvate MR studies were performed before and after LDHI administration to assess the impact on metabolic flux in vivo. Using hyperpolarized 1-13Cpyruvate MR Spectroscopy (MRS), we found that lactate production was significantly suppressed by LDHI administration in MiaPaca (a glycolytic pancreatic cancer cell line) tumors, as was the 1-13Clactate to 1-13Cpyruvate ratio (1-13C-Lac/Pyr), which was calculated from the areas under the curves (AUC) using time-intensity data. This ratio decreased from 1.08 to 0.128 (88.1% decrease) 30 minutes after intravenous administration of the LDHI. In addition, hyperpolarized 1-13Cpyruvate MRS revealed that LDHI significantly suppressed lactate production in a dose dependent manner. Furthermore, Chemical Shift Imaging with 13C MRI demonstrated that the 1-13Clactate signal in each voxel clearly decreased, compared to that before LDHI administration. The sum of 1-13Clactate signals in the tumor region decreased after LDHI administration, resulting in a significant decrease in the tumor-specific 1-13C Lac/Pyr ratio (1.463±0.31 before LDHI to 0.134±0.036 30 minutes after LDHI administration, a 90.67±2.56% decrease, n=3, p<0.01).
Conclusions These results indicate that hyperpolarized 13C-MRI is a useful method to evaluate on-target efficacy of novel LDH inhibitors in vivo, and this technique can be used to determine optimum dose and exposure time of the LDHI in the tumor region. The current method can be of great value in providing an in vivo pharmacodynamic biomarker for this novel anti-cancer therapeutic targeting deregulated tumor metabolism.
Citation Format: Nobu Oshima, Shun Kishimoto, Kristin Beebe, Keita Saito, Kazutoshi Yamamoto, Jeffery Brender, Anastasia Sowers, Ganesha Rai, Bryan T. Mott, David J. Maloney, James B. Mitchell, Murali K. Cherukuri, Leonard M. Neckers. Monitoring the impact on metabolic flux in vivo of a newly developed LDH inhibitor using hyperpolarized 13C magnetic resonance spectroscopic imaging abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2017-2852
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