Brian R. Keppler

The Biochemical Role of the Heat Shock Protein 90 Chaperone Complex in Establishing Human Telomerase Activity

Telomerase is a ribonucleoprotein complex that synthesizes the G-rich DNA found at the 3′-ends of linear chromosomes. Human telomerase consists minimally of a catalytic protein (hTERT) and a template-containing RNA (hTR), although other proteins are involved in regulating telomerase activity in vivo. Several chaperone proteins, including hsp90 and p23, have demonstrable roles in establishing telomerase activity both in vitro and in vivo, and previous reports indicate that hsp90 and p23 are required for the reconstitution of telomerase activity from recombinant hTERT and hTR. Here we report that hTERT and hTR associate in the absence of a functional hsp90-p23 heterocomplex. We also report that hsp90 inhibitors geldanamycin and novobiocin inhibit recombinant telomerase even after telomerase is assembled. Inhibition by geldanamycin could be overcome by allowing telomerase to first bind its primer, suggesting a role for hsp90 in loading telomerase onto the telomere. Inhibition by novobiocin could not similarly be overcome but instead resulted in destabilization of the hTERT polypeptide. We propose that the hsp90-p23 complex fine tunes and stabilizes a functional telomerase structure, allowing primer loading and extension.

Chromatin-modifying enzymes as therapeutic targets – Part 1

Background: Part 1 of this review described the importance of histone acetylases, deacetylases, methylases and demethylases in transcriptional control and their potential as therapeutic targets. However, precise gene regulation requires the involvement of more than just the addition or removal of acetyl and methyl groups on histones. Histone phosphorylation, ubiquitylation, SUMOylation and poly-ADP-ribosylation, as well as ATP-dependent nucleosome remodeling complexes, play equally pivotal roles in the maintenance of transcriptional fidelity. Accordingly, the enzymes responsible for these modifications are also misregulated in various disease states. Objective: To review the complex roles of chromatin-modifying enzymes in gene regulation and to highlight their potential as therapeutic targets. Methods: This review is based on recent published literature and online resources. Results/conclusion: In this second and final part of the review, we discuss the importance of these other histone and nucleosome modifying enzymes in gene transcription as well as their therapeutic potential.

A strategically designed small molecule attacks alpha-ketoglutarate dehydrogenase in tumor cells through a redox process

BackgroundTargeting cancer cell metabolism is recognized as a promising arena for development of cancer chemotherapeutics. Moreover, redox metabolism is also systematically altered in tumor cells. Indeed, there is growing reason to believe that tumor-specific alteration of redox control of metabolism will be central to understanding and attacking malignancy. We report here that lipoate analog CPI-613 attacks a gate-keeping, lipoate-using metabolic enzyme, alpha-ketoglutarate dehydrogenase (KGDH), by a redox mechanism selectively in tumors cells.ResultsCPI-613 inhibited KGDH function strongly and rapidly, selectively in tumor cells. Moreover, CPI-613 induced a correspondingly rapid, powerful redox signal in tumor cell mitochondria. This signal was associated with redox modification of KGDH (including extensive enzyme glutathionylation and redox blockage of enzyme lipoate sulfhydryls), correlating with KGDH inactivation. The source of this tumor-specific mitochondrial redox modulatory signal was not electron transport complexes (I or III), but was largely or entirely the E3 (dihydrolipoamide dehydrogenase) component of dehydrogenases, including KGDH. Finally, we demonstrated that KGDH activity was redox regulated (in tumor cells), as expected if a tumor-specific redox process (auto)regulates KGDH.ConclusionsOur data demonstrate that lipoate analog CPI-613 attacks redox control of KGDH activity in tumor cells, perhaps by modulation of an existing lipoate-sensitive allosteric process normally governing tumor cell KGDH activity. Together with its previously reported, mechanistically distinct (non-redox) effects on the other major, lipoate-using mitochondrial metabolic enzyme, pyruvate dehydrogenase, CPI-613’s KGDH effects indicate that this agent simultaneously attacks multiple central, essential components of tumor cell metabolic regulation.

Ubiquitin-dependent and Ubiquitin-independent Control of Subunit Stoichiometry in the SWI/SNF Complex

The mammalian SWI/SNF chromatin remodeling complex is a key player in multiple chromatin transactions. Core subunits of this complex, including the ATPase, Brg-1, and various Brg-1-associated factors (BAFs), work in concert to maintain a functional remodeling complex. This intra-complex regulation is supervised by protein-protein interactions, as stoichiometric levels of BAF proteins are maintained by proteasomal degradation. We show that the mechanism of BAF155-mediated stabilization of BAF57 involves blocking its ubiquitination by preventing interaction with TRIP12, an E3 ubiquitin ligase. Consequently, as opposed to complexed BAF57, whose principal lysines are unavailable for ubiquitination, uncomplexed BAF57 can be freely ubiquitinated and degraded by the proteasome. Additionally, a BAF57 mutant, which contains no lysine residues, was found to retain its ability to be stabilized by interaction with BAF155, suggesting that in addition to the ubiquitin-dependent mechanism of BAF57 degradation, there exists a ubiquitin-independent mechanism that may involve the direct interaction of BAF57 with the proteasome. We propose that this regulatory mechanism exists to ensure functional fidelity of the complex and prevent the accumulation of uncomplexed proteins, which may disrupt the normal activity of the complex.

BRACO19 analog dimers with improved inhibition of telomerase and hPot 1.

Human chromosomes terminate with telomeres, which contain double-stranded G-rich, repetitive DNA followed by a single-stranded overhang of the G-rich sequence. Single-stranded oligonucleotides containing G-rich telomeric repeats have been observed in vitro to fold into a variety of G-quadruplex topologies depending on the solution conditions. G-quadruplex structures are notable in part because G-quadruplex ligands inhibit both the enzyme telomerase and other telomere-binding proteins. Because telomerase is required for growth by the majority of cancers, G-quadruplex-stabilizing ligands have become an attractive platform for anticancer drug discovery. Here, we present the preparation and biochemical activities of a novel series of 3,6-disubstituted acridine dimers modeled after the known G-quadruplex ligand BRACO19. These BRACO19 Analog Dimer (BAD) ligands were shown to bind to human telomeric DNA and promote the formation of intramolecular G-quadruplexes in the absence of monovalent cations. As expected, the BAD ligands bound to telomeric DNA with a 1:1 stoichiometry, whereas the parent compound BRACO19, a monomer, bound with a 2:1 stoichiometry. The BAD ligands exhibited potent inhibition of human telomerase with IC(50) values similar to or lower than those of BRACO19. Furthermore, the BAD ligands displayed greater potency in the inhibition of hPot1 and increased selectivity for G-quadruplex DNA when compared to BRACO19. Collectively, these experiments support the hypothesis that there is an increased potency and selectivity to be gained in the design of G-quadruplex-stabilizing agents that incorporate multiple interactions.

ortho-Quinone tanshinones directly inhibit telomerase through an oxidative mechanism mediated by hydrogen peroxide.

The tanshinone natural products possess a variety of pharmacological properties including anti-bacterial, anti-inflammatory, anti-oxidant, and anti-neoplastic activity. The molecular basis of these effects, however, remains largely unknown. In the present study, we explored the direct effect of tanshinones on the enzyme telomerase. Telomerase is up-regulated in the majority of cancer cells and is essential for their survival, making it a potential anti-cancer drug target. We found that the ortho-quinone tanshinone II-A inhibits telomerase in a time- and DTT-dependent fashion, and the hydrogen peroxide scavenger catalase protected telomerase from inactivation. These findings demonstrate that ortho-quinone containing tanshinones can inhibit telomerase owing to their ability to generate reactive oxygen species. The results also provide evidence that telomerase is directly and negatively regulated by reactive oxygen species.

Emerging Roles of the 26S Proteasome in Nuclear Hormone Receptor-Regulated Transcription

The mechanisms by which nuclear hormone receptors (NHRs) regulate transcription are highly dynamic and require interplay between a myriad of regulatory protein complexes including the 26S proteasome. Protein degradation is the most well-established role of the proteasome; however, an increasing body of evidence suggests that the 26S proteasome may regulate transcription in proteolytic and nonproteolytic mechanisms. Here we review how these mechanisms may apply to NHR-mediated transcriptional regulation. This article is part of a Special Issue entitled The 26S Proteasome: When degradation is just not enough!

Palmatine suppresses glutamine-mediated interaction between pancreatic cancer and stellate cells through simultaneous inhibition of survivin and COL1A1

Reciprocal interaction between pancreatic stellate cells (PSCs) and cancer cells (PCCs) in the tumor microenvironment (TME) promotes tumor cell survival and progression to lethal, therapeutically resistant pancreatic cancer. The goal of this study was to test the ability of Palmatine (PMT) to disrupt this reciprocal interaction in vitro and examine the underlying mechanism of interaction. We show that PSCs secrete glutamine into the extracellular environment under nutrient deprivation. PMT suppresses glutamine-mediated changes in GLI signaling in PCCs resulting in the inhibition of growth and migration while inducing apoptosis by inhibition of survivin. PMT-mediated inhibition of (glioma-associated oncogene 1) GLI activity in stellate cells leads to suppression (collagen type 1 alpha 1) COL1A1 activation. Remarkably, PMT potentiated gemcitabine’s growth inhibitory activity in PSCs, PCCs and inherently gemcitabine-resistant pancreatic cancer cells. This is the first study that shows the ability of PMT to inhibit growth of PSCs and PCCs either alone or in combination with gemcitabine. These studies warrant additional investigations using preclinical models to develop PMT as an agent for clinical management of pancreatic cancer.

Abstract 5392: Metabolic profiling of castrate-resistant prostate cancer reveals novel role for bile acids in driving castration resistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The 5-year survival for metastatic castrate-resistant prostate cancer patients is less than 30% despite significant progress in the understanding of prostate cancer biology and development of novel therapeutic agents. A major contributing factor for the observed low survival rate of patients with castrate resistant disease is the lack of knowledge regarding metabolic alterations and their underlying contributions during development of castrate-resistant phenotype. Studies conducted in our laboratory and others identified a potential role for 2-methoxyestradiol (2-ME2) to prevent prostate cancer development and progression through inhibition of the anti-apoptotic protein FLIP. The goal of the current study was to identify biochemical changes in response to castration and treatment with 2-ME2 in serum from transgenic adenocarcinoma of mouse prostate (TRAMP) mice using mass spectrometry based global profiling. We identified a total of 54 biochemicals of which 16 increased and 38 decreased in castrated animals compared to sham-castration. Treatment of sham-castrated animals with low and high doses of 2-ME2 altered 91 and 145 biochemicals respectively. On the other hand treatment of castrated animals modulated 89 and 106 biochemicals. Cumulative analysis of these data also identified alteration of 60 biochemicals associated with castration effect, 149 with treatment and 70 interactions between castration and treatment effects. Castration affected metabolites involved in variety of metabolic pathways including lipid, oxidative stress, energetics and bile acid. Given the data showing enhanced expression of FLIP in castrate-resistant prostate tumors and upregulation of bile acids in patients undergoing androgen deprivation therapy, we examined the activation of FLIP in prostate cancer cells in response to deoxycholic acid (DCA). Our data suggests that transcriptional activity of FLIP was higher in PC-3 cells treated with DCA. To the best of our knowledge, this is the first report demonstrating global metabolomic profiling of serum in response to castration and provide a framework for therapeutic targeting of bile acid metabolism. Supported by NIH CA 135451 (APK). Citation Format: Divya Chakravarthy, Paul Rivas, Brian Keppler, Jianhua Ruan, Rita Ghosh, Addanki Pratap Kumar. Metabolic profiling of castrate-resistant prostate cancer reveals novel role for bile acids in driving castration resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5392. doi:10.1158/1538-7445.AM2013-5392 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.