Hormone-dependent transactivation by several of the steroid hormone receptors is potentiated by the Hsp90-associated cochaperone FKBP52, although not by the closely related FKBP51. Here we analyze the mechanisms of potentiation and the functional differences between FKBP51 and FKBP52. While both have peptidyl-prolyl isomerase activity, this is not required for potentiation, as mutations abolishing isomerase activity did not affect potentiation. Genetic selection in Saccharomyces cerevisiae for gain of potentiation activity in a library of randomly mutated FKBP51 genes identified a single residue at position 119 in the N-terminal FK1 domain as being a critical difference between these two proteins. In both the yeast model and mammalian cells, the FKBP51 mutation L119P, which is located in a hairpin loop overhanging the catalytic pocket and introduces the proline found in FKBP52, conferred significant potentiation activity, whereas the converse P119L mutation in FKBP52 decreased potentiation. A second residue in this loop, A116, also influences potentiation levels; in fact, the FKBP51-A116V L119P double mutant potentiated hormone signaling as well as wild-type FKBP52 did. These results suggest that the FK1 domain, and in particular the loop overhanging the catalytic pocket, is critically involved in receptor interactions and receptor activity.Multiple cellular factors influence hormone-dependent activation of steroid receptors and cellular responses to hormone exposure. Our interest has focused on molecular chaperones that assemble with steroid receptors and alter receptor activity. More than a dozen chaperone and cochaperone proteins have been identified in steroid receptor complexes (23, 28); some chaperones are restricted to different stages of receptor assembly, and others compete for common assembly sites in the receptor complex. In vitro studies have identified five chaperones that are minimally necessary for efficient maturation and maintenance of the ability of the receptor to bind hormone (9,11,17). These are the major heat shock proteins Hsp40, Hsp70, and Hsp90 plus the cochaperone Hop, which can act as an adaptor by simultaneously binding both Hsp70 and Hsp90 (3,30), and the cochaperone p23, which stabilizes the association of Hsp90 with receptor (15, 16). Of the multiple other cochaperones observed in receptor complexes, some have unknown functions, although others are involved in the proteolytic stability of the receptor and yet others have been shown to modulate the receptor response to hormone. Most notable of the latter are two Hsp90 cochaperones in the FK506 binding protein (FKBP) family of peptidyl-prolyl isomerase (PPIase) that have been shown to alter hormonal potency (5, 24, 25). FKBP51 was identified as a cellular factor contributing to glucocorticoid resistance in cells from New World primates (24) by inhibiting glucocorticoid receptor (GR) response to hormone (8). In contrast, FKBP52 was found to enhance GR response to hormone (25) and to similarly enhance the receptors for androgens (AR) ...
Abstract. Mitochondria are central to a variety of cellular processes, from metabolism to cell death. In this study, we demonstrated that an increase in the critical mitochondrial protein, cytochrome c, correlated with drug resistance in a cell culture model of aggressive lymphoma. Increased cytochrome c expression was also correlated with decreased survival in the aggressive diffuse large B-cell and mantle cell lymphomas, but not in the indolent follicular lymphoma. this suggests that an increased reliance on the mitochondria for energy allows tumor cells to be metabolic opportunists and contribute to tumor development and drug resistance. IntroductionApoptosis resistance and altered metabolism are common cancer cell traits. In a murine lymphoma model, selection for oxidative stress resistance results in concomitant resistance to apoptosis and the acquisition of a more-aggressive tumor phenotype. WeHI7.2 thymic lymphoma cells transfected with catalase (cAt2, cAt38) or selected for resistance to hydrogen peroxide (200r) demonstrate resistance to glucocorticoid-induced apoptosis (1,2), one component of the cHoP lymphoma chemotherapy regimen. the oxidative stress-resistant cells are also resistant to the cyclophosphamide, doxorubicin and vincristine components of cHoP (tome et al, unpublished data). catalase-overexpressing cells exhibit increased tumor growth compared to WeHI7.2 parental cells in a mouse xenograft model (2). Additionally, the oxidative stress-resistant cells demonstrate an altered metabolic profile, including the ability to generate AtP from alternative carbon sources such as glutamine (3,4). When treated with glucocorticoids, the oxidative stress-resistant cells are better able to maintain AtP levels as compared to WeHI7.2 cells (3). experiments with an uncoupler of mitochondrial respiration showed that the cAt38 and 200r cells produce more AtP from mitochondria than the WeHI7.2 cells (4).Mitochondria are central to cellular metabolism and the decision to undergo apoptosis (5). thus, changes in critical mitochondrial proteins may explain the more-aggressive tumor phenotype of the resistant lymphoma variants. In particular, cytochrome c plays an integral role as an electron carrier in the mitochondrial respiratory chain, and the release of this protein from the mitochondrial intermembrane space results in commitment to death via apoptosis (5). In a previous study, we showed that cytochrome c release is delayed in the resistant variants (1), and that mitochondria isolated from these cells demonstrate an intrinsic resistance to cytochrome c release (Wilkinson et al, unpublished data). the present study examined whether alterations in cytochrome c protein levels correlate with the previously characterized more-aggressive tumor phenotype of the resistant cells. to test the potential clinical relevance of these findings, gene expression data from lymphoma specimens were analyzed. Materials and methodsMitochondrial protein levels in cell lines. WeHI7.2 murine thymic lymphoma cells and variants were main...
Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin (mTOR) inhibitors. While cyclin D1 can be regulated through the mTOR pathway, treatment with mTOR inhibitors often inhibits MCL growth without altering cyclin D1 levels of expression. Thus, the anti-tumor activity of mTOR inhibitors is likely mediated through other key targets. In other malignancies, recent data suggests that increased oxidative stress plays a role in the growth suppressive mechanisms of mTOR inhibitors. Initial studies in our lab indicated that a 48 hour rapamycin treatment of MCL cell lines increased overall levels of reactive oxygen species (ROS). Specifically, we found evidence of increased hydrogen peroxide (H2O2) levels in the cells. One potential source of H2O2 is manganese superoxide dismutase (MnSOD), which is a known tumor suppressor in several cell types. MnSOD catalyzes the dismutation of superoxide to H2O2 in the mitochondria. Thus, it removes one form of ROS while generating another. We hypothesized that the anti-tumor effect of mTOR inhibitors in MCL is mediated by an accumulation of ROS, specifically H2O2, due to an increase in MnSOD protein expression. We investigated the effect of a 48 hour rapamycin treatment on MnSOD in two MCL cell lines and genetic manipulation of MnSOD in Jurkat cells, a T lymphocyte cell line. Rapamycin treatment increased MnSOD in the MCL cell lines as well as Jurkat cells. Overall, sensitivity of cells to rapamycin correlated with high MnSOD levels. Analysis of the results showed that rapamycin treatment increased MnSOD expression in parallel with H2O2. Over-expression of MnSOD in Jurkat cells elevated the level of H2O2 and resulted in a cytotoxic, rather than cytostatic, effect of rapamycin. Treatment with the general ROS scavenger, N-acetyl cysteine, reduced overall ROS, cellular levels of H2O2, and the growth inhibitory effect of rapamycin. These findings indicate that the rapamycin-induced cytostatic effect is characterized by increased levels of MnSOD and H2O2, which is necessary for the full growth inhibitory effect of rapamycin. Rapamycin and its derivatives are generally well accepted as inhibitors of the mTORC1 complex upon initial treatment, but recent studies in other cell lines have shown that long term treatment can have an inhibitory effect on the mTORC2 complex and its downstream targets, such as Akt. Our results found that treatment with rapamycin resulted in a loss of S473 phosphorylation of Akt in the MCL cell lines. Increased levels of MnSOD following treatment of Jurkat cells were found to be due to inhibition of the mTORC2 complex. These results suggest that in MCL cells treated with rapamycin for 48 hours the mTORC2 complex is inhibited. Multiple transcription factors that have been linked to MnSOD regulation are affected by the Akt pathway. By understanding the key signaling molecules and affected pathways in the anti-tumor effects of rapamycin, we may be able to identify additional predictive markers to improve the therapeutic value of mTOR inhibitors, or study drug combinations that will enhance the effect of ROS-induced cytotoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A175.
Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. MCLs are aggressive lymphomas, usually characterized by over-expression of cyclin D1. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin (mTOR) inhibitors. While cyclin D1 can be regulated through the mTOR pathway, treatment with mTOR inhibitors often inhibit MCL growth without altering cyclin D1. Thus, the anti-tumor activity of mTOR inhibitors is likely mediated through other key targets. One potential target is C/EBPβ-LIP, a transcription factor regulated through the mTOR pathway. One target of C/EBPβ gene regulation is manganese superoxide dismutase (MnSOD), an antioxidant defense enzyme with tumor suppressor activity in some cell types. MnSOD catalyzes the dismutation of superoxide to hydrogen peroxide. Thus, it removes one form of reactive oxygen species (ROS) while generating another. Previous studies in our laboratory have shown that C/EBPβ-LIP levels decrease with mTOR inhibitor treatment in proportion to increasing MnSOD levels. We hypothesize that the anti-tumor effect of mTOR inhibitors in MCL is mediated by the increase in MnSOD protein expression and accumulation of ROS, specifically hydrogen peroxide. Here we investigated the effect of rapamycin on MnSOD and the levels of ROS in two human MCL-derived cell lines. High MnSOD levels correlated with increased sensitivity of cells treated with rapamycin. To investigate the biological effects of increased MnSOD expression, we measured overall ROS levels (DCFH) and the rate of hydrogen peroxide efflux with the fluorescent probe Amplex Red, which is indicative of changes to hydrogen peroxide levels in the cells. These results showed that both overall ROS and hydrogen peroxide levels increased in parallel with MnSOD expression. These increases in hydrogen peroxide were not due to decreased levels of catalase or glutathione peroxidase. Treatment with the general ROS scavenger, N-acetyl cysteine, reduced overall ROS, cellular levels of hydrogen peroxide, and the growth inhibitory effect of rapamycin. These findings indicate that rapamycin-induced cytostatic effect is characterized by generation of ROS, specifically hydrogen peroxide. This is likely due to the increased levels of MnSOD. By understanding the key signaling molecules in the anti-proliferative effects of rapamycin, we may be able to identify additional predictive markers to improve the therapeutic value of mTOR inhibitors, or study drug combinations that will enhance the effect of ROS-induced cytotoxicity. Citation Information: Cancer Res 2009;69(23 Suppl):C55.
321 Gene expression profiling of newly diagnosed diffuse large B-cell lymphomas shows a correlation between the expression of oxidative stress-related genes and patient prognosis following chemotherapy. Anthracyclines and cyclophosphamide used in lymphoma therapy are known to work, at least in part, through oxidative stress. We hypothesized that single nucleotide polymorphisms (SNPs) in oxidative stress-related genes may contribute to clinical outcomes and/or the development of hematologic toxicities for patients treated with curative intent anthracycline-based therapies for aggressive B-cell Non-Hodgkin lymphomas. Our study involved 473 patients enrolled in 7 prior Phase II or Phase III Southwest Oncology Group clinical trials. DNA for the SNP analyses was obtained from banked, paraffin-embedded diagnostic tissue. Genotyping was performed for 72 SNPs in 33 oxidative stress-related genes. After excluding pathologically or clinically ineligible patients and those with SNP call rates <85%, 345 patients were included in the analysis. All analyses were stratified on clinical trial and adjusted by IPI score. We found polymorphic alleles in aldo-keto reductase family 1 member C3 (AKR1C3), inducible nitric oxide synthase (NOS2) and myeloperoxidase (MPO) were associated with patient outcomes (see table below). AKR1C3 is a member of a superfamily of oxidoreductases. The diverse reactions catalyzed by these enzymes include the conversion of steroid hormones (e.g., glucocorticoids) to more potent forms and the repair of oxidative damage to proteins and lipids. The AKR1C3 SNP rs10508293, located in intron 4 of the gene, is of unknown functional significance. NOS2 produces nitric oxide, which is an important signaling molecule. However, nitric oxide can also increase oxidative stress by reacting with superoxide to produce the potent oxidant, peroxynitrite. The NOS2 polymorphism rs1060826 relevant to this study is located in exon 22. It does not change the amino acid sequence in the protein. MPO is found in neutrophils and macrophages where it produces hypochlorous acid. This enzyme can also activate xenobiotics and its products can cause protein oxidation. For our analysis of the MPO gene, we chose the -764A/G SNP (rs2243828) as a proxy for the -463G/A SNP (rs2333227). The latter polymorphism affects the transcription rate of the gene. Importantly, it has been linked to survival rates for breast cancer patients treated with cyclophosphamide. The -764A/G SNP was used due to technical difficulty genotyping the -463G/A SNP. There is 100% genotype concordance between these two SNPs. We found that patients with at least 1 copy of the AKR1C3 T allele had better overall and progression-free survival than those with the CC genotype. We also found that patients who were homozygous for the NOS2 G allele had better overall and progression-free survival than those with the AA genotype. In complete agreement with the study of breast cancer patients, the MPO GG genotype was associated with worse overall and progression-free survival, as compared to the AA genotype. Our additional tests for polymorphisms associated with hematologic toxicity identified the -212A/G SNP (rs1883112) in NCF4. This gene encodes p40-phox, a regulatory subunit of NAD(P)H oxidase. The NCF4 GG genotype has previously been linked to a two-fold reduction in the risk of anthracycline-induced cardiotoxicity among Non-Hodgkin lymphoma patients. We now report a similar reduction in hematologic toxicity for patients homozygous for the G allele, as compared to those with the AA genotype (hazard ratio, 0.48; 95% confidence interval, 0.19–0.95; unadjusted P = 0.038). This is the first report linking polymorphisms in AKR1C3, NOS2, MPO and NCF4 with outcome in aggressive lymphomas. Further work is needed to elucidate the mechanism(s) by which the SNP genotypes would influence treatment outcome. Overall Survival Progression-Free Survival Gene Genotype N Adjusted HR 95% CI P value* Adjusted HR 95% CI P value* AKR1C3 CC 25 1.0 (ref) 1.0 (ref) CT 75 0.40 0.23-0.72 0.0021 0.36 0.21-0.64 0.00042 TT 237 0.45 0.27-0.74 0.0018 0.48 0.29-0.78 0.0028 NOS2 AA 32 1.0 (ref) 1.0 (ref) AG 16 0.54 0.24-1.23 0.14 0.52 0.24-1.13 0.098 GG 284 0.52 0.32-0.86 0.01 0.55 0.35-0.87 0.011 MPO AA 205 1.0 (ref) 1.0 (ref) AG 113 1.31 0.93-1.84 0.12 1.17 0.86-1.61 0.32 GG 22 2.54 1.50-4.30 0.0005 1.96 1.18-3.24 0.0093 * P values not adjusted for multiple comparisons. Disclosures: No relevant conflicts of interest to declare.
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