Chaperone Protein Hsp90 Research Articles

Mechanisms of Protein Homeostasis Regulation in Cancer Development

The proteome of eukaryotic cells represents a complex system. Its components are exposed to various intrinsic and extrinsic stresses. Therefore, the function of the cellular proteome is dependent on the existence of compensatory mechanisms balancing the inner protein homeostasis - proteostasis. These mechanisms involve the network of molecular chaperones and transcriptional program regulating their expression. The process of cancerogenesis is accompanied by significant changes in the intracellular milieu of cancer cells - temperature, pH, availability of nutrients. On the one hand, these changes represent a consequence of the deregulated growth of the tumor tissue; on the other hand, they can be a source of selection pressure, which allows the emergence of resistant and aggressive tumor cell populations. Description of the proteostatic apparatus components and the mechanism of their involvement in the tumor tissue development is provided in this review article.This review focuses on the description of two causally linked groups of proteostatic events; their mutual coordination is crucial to the process of tumor cell and by extension the entire tumor tissue response to environmental and internal stress factors. The first group of these processes is represented by the "executory" role of molecular chaperones from HSP70, HSP90 and so-called small molecular chaperone protein families. These proteins are involved in maintaining stability of cellular proteins essential for proliferation, apoptosis, senescence, migration and phenotypic plasticity of tumor cells. The second group of the described processes comprises the posttranslational control of the "systemic" role of the transcription factor HSF1 in regulating the gene expression of molecular chaperones and other genes specifically regulated by this transcription factor in the tumor and stromal cells.Key words: molecular chaperones - heat-shock factor 1 - cancer - protein homeostasisThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 15. 5. 2016Accepted: 25. 5. 2016.

Anticancer Effects of Targeting Hsp70 in Tumor Stromal Cells.

The stress-induced chaperone protein Hsp70 enables the initiation and progression of many cancers, making it an appealing therapeutic target for development. Here, we show that cancer cells resistant to Hsp70 inhibitors in vitro remain sensitive to them in vivo, revealing the pathogenic significance of Hsp70 in tumor stromal cells rather than tumor cells as widely presumed. Using transgenic mouse models of cancer, we found that expression of Hsp70 in host stromal cells was essential to support tumor growth. Furthermore, genetic ablation or pharmacologic inhibition of Hsp70 suppressed tumor infiltration by macrophages needed to enable tumor growth. Overall, our results illustrate how Hsp70 inhibitors mediate the anticancer effects by targeting both tumor cells and tumor stromal cells, with implications for the broad use of these inhibitors as tools to ablate tumor-associated macrophages that enable malignant progression. Cancer Res; 76(20); 5926-32. ©2016 AACR.

Acidosis Acts through HSP90 in a PHD/VHL-Independent Manner to Promote HIF Function and Stem Cell Maintenance in Glioma.

Hypoxia is a common feature of solid tumors, which controls multiple aspects of cancer progression. One important function of hypoxia and the hypoxia-inducible factors (HIF) is the maintenance of cancer stem-like cells (CSC), a population of tumor cells that possess stem cell-like properties and drives tumor growth. Among the changes promoted by hypoxia is a metabolic shift resulting in acidification of the tumor microenvironment. Here, we show that glioma hypoxia and acidosis functionally cooperate in inducing HIF transcription factors and CSC maintenance. We found that these effects did not involve the classical PHD/VHL pathway for HIF upregulation, but instead involved the stress-induced chaperone protein HSP90. Genetic or pharmacologic inactivation of HSP90 inhibited the increase in HIF levels and abolished the self-renewal and tumorigenic properties of CSCs induced by acidosis. In clinical specimens of glioma, HSP90 was upregulated in the hypoxic niche and was correlated with a CSC phenotype. Our findings highlight the role of tumor acidification within the hypoxic niche in the regulation of HIF and CSC function through HSP90, with implications for therapeutic strategies to target CSC in gliomas and other hypoxic tumors. Cancer Res; 76(19); 5845-56. ©2016 AACR.

CHIP mediates down-regulation of nucleobindin-1 in preosteoblast cell line models

Nucleobindin-1 (NUCB1), also known as Calnuc, is a highly conserved, multifunctional protein widely expressed in tissues and cells. It contains two EF-hand motifs which have been shown to play a crucial role in binding Ca2+ ions. In this study, we applied comparative two-dimensional gel electrophoresis to characterize differentially expressed proteins in HA-CHIP over-expressed and endogenous CHIP depleted MC3T3-E1 stable cell lines, identifying NUCB1 as a novel CHIP/Stub1 targeted protein. NUCB1 interacts with and is down-regulated by CHIP by both proteasomal dependent and independent pathways, suggesting that CHIP-mediated down-regulation of nucleobindin-1 might play a role in osteoblast differentiation. The chaperone protein Hsp70 was found to be important for CHIP and NUCB1 interaction as well as CHIP-mediated NUCB1 down-regulation. Our findings provide new insights into understanding the stability regulation of NUCB1.

HSP70 Inhibition Limits FAK-Dependent Invasion and Enhances the Response to Melanoma Treatment with BRAF Inhibitors.

The stress-inducible chaperone protein HSP70 (HSPA1) is implicated in melanoma development, and HSP70 inhibitors exert tumor-specific cytotoxic activity in cancer. In this study, we documented that a significant proportion of melanoma tumors express high levels of HSP70, particularly at advanced stages, and that phospho-FAK (PTK2) and BRAF are HSP70 client proteins. Treatment of melanoma cells with HSP70 inhibitors decreased levels of phospho-FAK along with impaired migration, invasion, and metastasis in vitro and in vivo Moreover, the HSP70 inhibitor PET-16 reduced levels of mutant BRAF, synergized with the BRAF inhibitor PLX4032 in vitro, and enhanced the durability of response to BRAF inhibition in vivo Collectively, these findings provide strong support for HSP70 inhibition as a therapeutic strategy in melanoma, especially as an adjuvant approach for overcoming the resistance to BRAF inhibitors frequently observed in melanoma patients. Cancer Res; 76(9); 2720-30. ©2016 AACR.

Sex- and corticotropin-releasing factor receptor 2- dependent actions of urocortin 1 during inflammation

We investigated whether corticotropin-releasing factor receptor 2 (CRF2) and its high-affinity agonist urocortin 1 (Ucn1) mediate sex-specific signaling and immune responses. Intrarectal trinitrobenzene sulfonic acid was used to induce experimental colitis in wild-type, CRF2 knockout (CRF2KO), and heterozygous (CRF2Ht) mice of both sexes. Changes in plasma extravasation, organ weight, survival, immune cell numbers, inflammatory cytokines, and the MAPK signaling pathway were assessed. Stored intestinal biopsies from patients with Crohn's disease (CD) and age- and sex-matched individuals without inflammatory bowel disease (IBD) were examined by immunofluorescence and confocal microscopy to characterize Ucn1 and CRF receptor expression. CRF2Ht mice of both sexes showed decreased survival during colitis compared with other genotypes. Ucn1 improved survival in male mice alone. Ucn1 restored colon length and spleen and adrenal weight and decreased colonic TNF-α, IL-6, and IL-1β levels in male CRF2Ht mice alone. CRF2Ht mice of both sexes showed decreased phosphorylation of MAPK p38 and heat shock protein 27 (Hsp27) levels. Ucn1 restored p-Hsp27 levels in male CRF2Ht mice alone. Expression of the chaperone protein Hsp90 decreased during colitis, except in male CRF2Ht mice. Taken together, our data indicate that sex shows significant interaction with genotype and Ucn1 during colitis. Human duodenal and colonic biopsies revealed that sex-specific differences exist in levels of CRF receptors and Ucn1 expression in patients with CD compared with the matched non-IBD subjects. To conclude, Ucn1 mediates sex-specific immune and cellular signaling responses via CRF2, emphasizing the need for inclusion of females in preclinical studies.

Sex‐ and Corticotropin‐releasing Factor Receptor 2‐dependent Actions of Urocortin 1 During Colitis

We investigated whether corticotropin‐releasing factor receptor 2 (CRF2R) and its high affinity agonist urocortin 1 (Ucn1) mediate sex‐specific signaling and immune responses using an in vivo model of inflammatory bowel disease (IBD). Male and female wild‐type (WT) and CRF2R heterozygous (CRF2R+/−) mice, immunoassays, flow cytometry, and microscopy, were used to examine sex‐specific responses. CRF2R+/− mice of both sexes were more susceptible to colitis‐induced mortality than their WT littermates. Ucn1 rescued male CRF2R+/− mice from colitis‐induced mortality, whereas it increased mortality in female CRF2R+/− mice. Ucn1 restored colon length, spleen and adrenal mass, and decreased colonic TNF‐α and IL‐1β levels to baseline levels in male CRF2+/− mice alone. Treatment with monoclonal anti‐TNF‐a ameliorated colitis‐induced inflammation in both sexes, suggesting that Ucn1 actions are upstream of TNF‐α and gender‐specific. CRF2R+/− mice of both sexes showed decreased phosphorylation of MAPK p38 and cytoprotective heat shock protein 27 (Hsp27) levels. Ucn1 restored p‐Hsp27 levels in male CRF2R+/− mice alone. Baseline expression of the chaperone protein Hsp90 was reduced in male CRF2R+/− mice to start with and did not change during colitis or after Ucn1 treatment, suggesting that CRF2R interaction with Hsp90 is critical. Expression levels and subcellular localization of CRF2R and Ucn1 in pediatric IBD patients was dysregulated and gender‐specific. To conclude, CRF2R is a key determinant of the observed sex‐specific immune and cellular signaling responses. We established that Ucn1 mediates sex‐specific responses during IBD via CRF2R, emphasizing the need for inclusion of females in preclinical studies.Support or Funding InformationNIH/NIDDK R01‐080787 to AB

Structural Dynamics of Hsp90 Resolved by a Novel Multi-Pair FRET Approach

Three-dimensional models of proteins at atomic resolution are an essential prerequisite for mechanistic insight into protein function. However, in solution the protein exists as a dynamic equilibrium of global and local conformations. Therefore, a comprehensive understanding of protein function requires knowledge on the structure - dynamics relationship. We developed a novel hybrid approach based on single molecule Forster Resonance Energy Transfer [1, 2] that integrates hundreds of time resolved intramolecular distances together with x-ray structure information and MD simulations. Simultaneous determination of fluctuations and time resolved anisotropies enables a new level of accuracy and confidence. We demonstrate the power of our method by determining the dynamic structure of the molecular chaperone and heat shock protein Hsp90. Our resulting mean structure for the closed state of Hsp90 resembles the corresponding x-ray structure of yeast Hsp90 [3] to 2.5 A. Beyond that, we resolved the previously unknown open structure of this multi-domain protein. Surprisingly, the open state shows much larger fluctuations on relevant timescales than the closed state. Finally, we show how this method can be applied to quantify inter-domain dynamics and dynamics of small elements. We anticipate that this dynamic structure determination will solve many long standing questions regarding flexible parts in large proteins and protein-protein interactions.[1] Muschielok A. et al.; Nature Methods 2008; 5, 965-971.[2] Kalinin S. et al.; Nature Methods 2012; 9(12):1218-1225.[3] Ali M.M. et al.; Nature 2006, 440(7087):1013-1017.

The importance of being chaperoned: HSP90 and necroptosis

Necroptosis is a form of regulated cell death implicated in a range of inflammatory diseases. In this issue of Cell Chemical Biology, Li et al. (2016) describe a novel inhibitor of necroptosis that targets the chaperone protein HSP90.

Stromal cell-derived factor 2 is critical for Hsp90-dependent eNOS activation.

Endothelial nitric oxide synthase (eNOS) catalyzes the conversion of l-arginine and molecular oxygen into l-citrulline and nitric oxide (NO), a gaseous second messenger that influences cardiovascular physiology and disease. Several mechanisms regulate eNOS activity and function, including phosphorylation at Ser and Thr residues and protein-protein interactions. Combining a tandem affinity purification approach and mass spectrometry, we identified stromal cell-derived factor 2 (SDF2) as a component of the eNOS macromolecular complex in endothelial cells. SDF2 knockdown impaired agonist-stimulated NO synthesis and decreased the phosphorylation of eNOS at Ser(1177), a key event required for maximal activation of eNOS. Conversely, SDF2 overexpression dose-dependently increased NO synthesis through a mechanism involving Akt and calcium (induced with ionomycin), which increased the phosphorylation of Ser(1177) in eNOS. NO synthesis by iNOS (inducible NOS) and nNOS (neuronal NOS) was also enhanced upon SDF2 overexpression. We found that SDF2 was a client protein of the chaperone protein Hsp90, interacting preferentially with the M domain of Hsp90, which is the same domain that binds to eNOS. In endothelial cells exposed to vascular endothelial growth factor (VEGF), SDF2 was required for the binding of Hsp90 and calmodulin to eNOS, resulting in eNOS phosphorylation and activation. Thus, our data describe a function for SDF2 as a component of the Hsp90-eNOS complex that is critical for signal transduction in endothelial cells.

Adenomatous polyposis coli mutants dominantly activate Hsf1-dependent cell stress pathways through inhibition of microtubule dynamics.

Cancer cells up-regulate cell stress pathways, including the protein chaperone Hsp90. Increases in Hsp90 are believed "buffer" mutant protein activities necessary for cancer phenotypes. Activation of the cell stress pathway also alters the transcriptional landscape of cells in ways that are critical for cancer progression. However, it is unclear when and how the cell stress pathway is de-regulated during cancer progression. Here we report that mutations in adenomatous polyposis coli (APC) found in colorectal cancer activate cell stress pathways in mouse intestinal crypt cells, prior to loss of heterozygosity at APC or to the appearance of canonical intestinal cancer markers. Hsp90 levels are elevated in normal APC heterozygote crypt cells and further elevated in non-cancer cells adjacent to dysplasias, suggesting that the Hsp90 stress pathway marks the "cancer-field" effect. Expression of mutant APC in normal human epithelial cells is sufficient to activate a cell stress pathway via perturbations in microtubule dynamics. Inhibition of microtubule dynamics is sufficient to activate an Hsf1-dependent increase in gene transcription and protein levels. We suggest that the early activation of this Hsf1 dependent cell stress pathway by mono-allelic mutations in APC can affect cell programming in a way that contributes to cancer onset.

Particulate cytoplasmic structures with high concentration of ubiquitin-proteasome accumulate in myeloid neoplasms.

BackgroundIncreased plasma levels of proteasome have been associated with various neoplasms, especially myeloid malignancies. Little is known of the cellular origin and release mechanisms of such proteasome. We recently identified and characterized a novel particulate cytoplasmic structure (PaCS) showing selective accumulation of ubiquitin-proteasome system (UPS) components. PaCSs have been reported in some epithelial neoplasms and in two genetic disorders characterized by hematopoietic cell dysplasia and increased risk of leukemia. However, no information is available about PaCSs in hematopoietic neoplasms.MethodsPaCSs were investigated by ultrastructural, immunogold, and immunofluorescence analysis of bone marrow (BM) biopsies and peripheral blood (PB) cell preparations of 33 consecutive, untreated, or relapsed patients affected by different hematopoietic neoplasms. BM and PB samples from individuals with non-neoplastic BM or healthy donors were studied as controls. Granulocytes and platelet proteasome content was measured by immunoblotting and plasma proteasome levels by ELISA.ResultsPaCSs with typical, selective immunoreactivity for polyubiquitinated proteins and proteasome were widespread in granulocytic cells, megakaryocytes, and platelets of patients with myeloproliferative neoplasms (MPN). In acute myeloid leukemia and myelodysplastic syndromes (MDS), PaCSs were only occasionally detected in blast cells and were found consistently in cells showing granulocytic and megakaryocytic maturation. Conversely, PaCSs were poorly represented or absent in non-neoplastic hematopoietic tissue or lymphoid neoplasms. In MPN granulocytes and platelets, the presence of PaCSs was associated with increased amounts of proteasome in cell lysates. PaCSs were often localized in cytoplasmic blebs generating PaCSs-filled plasma membrane vesicles observable in the BM intercellular space. In MPN and MDS, accumulation of PaCSs was associated with significant increase in plasma proteasome. Immunogold analysis showed that PaCSs of myeloid neoplasia selectively concentrated the chaperone proteins Hsp40, Hsp70, and Hsp90. ConclusionsPaCSs accumulate in cells of myeloid neoplasms in a lineage- and maturation-restricted manner; in particular, they are widespread in granulocytic and megakaryocytic lineages of MPN patients. PaCSs development was associated with excess accumulation of polyubiquitinated proteins, proteasome, and chaperone molecules, indicating impairment of the UPS-dependent protein homeostasis and a possible link with Hsp90-related leukemogenesis. A mechanism of PaCSs discharge by leukemic cells could contribute to increased plasma proteasome of MPN and MDS.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0169-6) contains supplementary material, which is available to authorized users.

RAN translation at CGG repeats induces ubiquitin proteasome system impairment in models of fragile X-associated tremor ataxia syndrome.

Fragile X-associated tremor ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a CGG trinucleotide repeat expansion in the 5' UTR of the Fragile X gene, FMR1. FXTAS is thought to arise primarily from an RNA gain-of-function toxicity mechanism. However, recent studies demonstrate that the repeat also elicits production of a toxic polyglycine protein, FMRpolyG, via repeat-associated non-AUG (RAN)-initiated translation. Pathologically, FXTAS is characterized by ubiquitin-positive intranuclear neuronal inclusions, raising the possibility that failure of protein quality control pathways could contribute to disease pathogenesis. To test this hypothesis, we used Drosophila- and cell-based models of CGG-repeat-associated toxicity. In Drosophila, ubiquitin proteasome system (UPS) impairment led to enhancement of CGG-repeat-induced degeneration, whereas overexpression of the chaperone protein HSP70 suppressed this toxicity. In transfected mammalian cells, CGG repeat expression triggered accumulation of a UPS reporter in a length-dependent fashion. To delineate the contributions from CGG repeats as RNA from RAN translation-associated toxicity, we enhanced or impaired the production of FMRpolyG in these models. Driving expression of FMRpolyG enhanced induction of UPS impairment in cell models, while prevention of RAN translation attenuated UPS impairment in cells and suppressed the genetic interaction with UPS manipulation in Drosophila. Taken together, these findings suggest that CGG repeats induce UPS impairment at least in part through activation of RAN translation.

The Chaperone Protein Hsp90 is Upregulated in Human Pulmonary Hypertension

INTRODUCTIONHuman and animal pulmonary arterial hypertension (PAH) is characterized by a selective activation of several transcription factors including STAT3, NFAT and HIF‐1α, which regulates pulmonary artery smooth muscle cells (PASMC) proliferation and apoptosis. The activation mechanisms of these factors in PAH remain elusive. Hsp90 is a major molecular chaperone that is plays a pivotal role in assisting correct folding and functionality of its client proteins. The Hsp90 client proteins include a wide variety of transcription factors like NFAT, STAT3 and HIF‐1α. Recent evidences have shown that miR‐223 may regulates HsP90 expression in cancer, which share many similarities with PAH. We thus hypothesized that the miR‐223/Hsp90 axis is aberrantly expressed in PAH patients, contributing to PAH‐PASMC phenotype.METHODS and RESULTSUsing immunoblot, we showed that Hsp90 is upregulated in lungs, and PASMC of PAH patients compared to non‐PAH donors. This upregulation in Hsp90 is associated with a significant downregulation of miR223 in PAH; upregulation of HIF‐1α, NFAT and STAT3 activation measured by immunoblot and nuclear translocation assay. In primary cultured PASMC from 3 PAH and 3 control patients, we demonstrated that miR‐223 upregulation using mimic or Hsp90 molecular inhibition using siRNAs significantly reduces PAH‐PASMC proliferation and resistance to apoptosis. Finally, using monocrotaline‐induced PAH, we showed that miR223 nebulization decreases total pulmonary resistance by decreasing distal PA wall thickness.CONCLUSIONThe miR223/Hsp90 may represent a new signal hub in PAH, accounting for the activation of several transcription factors contributing to PAH‐PASMC phenotype and thus vascular remolding seen in PAH.

Development of a Nonradioactive Steroid Receptor Ligand Binding Assay Using Fluorescence Polarization for Studying Hsp90‐Mediated Chaperone Protein Activity

While glucocorticoid receptor (GR) ligand binding has historically been measured using radiolabeled agonists, such as [3H]dexamethasone, new methods have sought to use fluorescently conjugated ligands, such as dexamethasone‐fluorescein (Dex‐Fl), as more sustainable alternatives. The molecular chaperone protein hsp90 has been shown to be essential for facilitating GR ligand binding, and structurally unrelated hsp90 inhibitors are currently in various phases of clinical investigation. The purpose of our study was to develop a fluorescence polarization (FP)‐based assay for measuring GR binding activity suitable for use in undergraduate research environments, and then apply this method to evaluating the extent to which specific agonists and antagonists of the GR and hsp90 chaperone machinery affect ligand binding. We constructed a versatile FP assay with comparable results to the previously used radiation‐based method. Interestingly, GR agonists dexamethasone and cortisol produced equivalent Dex‐Fl competition to the GR antagonist RU486, and did so in a concentration‐dependent manner. Data from hsp90 and hsp70 inhibitors showed mixed results, with hsp90 antagonists radicicol, NVP‐AUY922, and BIIB012 having limited effects, while the hsp70 antagonist novobiocin showed a potentiation of FP‐detectable steroid binding. Using the described FP method to evaluate steroid binding can be applied to broader studies examining clinically relevant GR and hsp90‐based drug therapies.

Nucleotide Dependency of Endogenous Hsp90 Chaperone Protein Machinery and Its Association with Baculovirally Expressed Glucocorticoid Receptor

Hsp90 and hsp70 are essential eukaryotic chaperone proteins required for facilitating functional activity of mammalian transcription factors, including the glucocorticoid receptor (GR). Both chaperones exist in ATP‐ and ADP‐bound states, and understanding the relationship between nucleotide‐bound forms of hsp90 and hsp70, their affiliated cochaperones, and GR activation has significant pharmacological implications. The goal of this study was to investigate the extent to which hsp90 and hsp70 nucleotide interactions affect production of a GR‐hsp90 heterocomplex capable of steroid binding activity. Reticulocyte lysate (RL) containing high endogenous concentrations of hsp90 and hsp70 was incubated in the presence or absence of an ATP regenerating system and fractionated by size exclusion chromatography (SEC). ATP incubation did not appear to alter total global protein elution, as detected by A280 spectrophotometry. However, SDS‐PAGE and western blot analysis identified nucleotide‐associated alterations in hsp90 SEC elution profiles. In order to further compare nucleotide dependency and chaperone effects, full‐length GR, produced in baculovirally infected insect cells, was immunoadsorbed, stripped of endogenous chaperones, and reassembled into a heterocomplex with hsp90 ± ATP. SDS‐PAGE and western blotting showed hsp90 derived from RL−ATP samples were tightly bound to the GR, while hsp90 from RL+ATP failed to stay bound to the GR. The difference in SEC elution profiles and co‐immunoadsorption imply a notable role of nucleotide alterations in the hsp90 chaperone machinery and functional interactions with the GR.

Tetratricopeptide repeat domain 9A negatively regulates estrogen receptor alpha activity.

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.

Bone Marrow Adipocyte-Derived Free Fatty Acids Induce Gene Signature Linking Transcription with Metabolic Changes That Contribute to Survival of Acute Monocytic Leukemia Cells

Adipocytes are the prevalent stromal cell type in adult bone marrows (BM). With increasing age, BM stroma-resident mesenchymal stem cells (MSCs), increase their capacity to differentiate into adipocytes, which leads to the progressive accumulation of fat in the BM space. It is conceivable that the increased BM adipocyte content promotes leukemogenesis and negatively affects responsiveness to chemotherapy. We previously reported that free fatty acids (FFAs) promote the metabolic shift from pyruvate oxidation to fatty acid oxidation (FAO), which causes uncoupling of mitochondrial oxidative phosphorylation and promotes leukemia cell survival (Samudio, J Clin Invest. 2010). We further demonstrated the prominent antiapoptotic effects of BM-derived adipocytes co-cultured with cells from acute monocytic leukemia (AMoL), a poor-prognosis subtype of AML (Tabe ASH. 2013). Proteomic analysis with isobaric tags for relative and absolute quantification (iTRAQ) showed upregulation of protein folding pathways which increases the expression of antiapoptotic chaperone proteins HSP70 and HSP90, of integrin-mediated cell adhesion and migration pathways and downregulation of oxidative phosphorylation along with repression of cytochrome c.Metacore gene ontology (GO) analysis identified NF-kB, c-Jun, SP1, AP-1, and HMG as the potent relevant transcription factors that closely interact with and activate chaperone proteins, chromatin, and gene transcription.In this study, we characterized a gene signature linking transcription with metabolic changes that contribute to AMoL cell survival under conditions mimicking aging BM with prevalent adipocytes. We confirmed the antiapoptotic role of FFAs produced by the primary BM MSC-derived adipocytes via pharmacologic inhibition of FAO by etomoxir (EX), which inhibits fatty acid entry into the mitochondria. EX (50mM) treatment reversed the prosurvival effects of adipocytes on serum-starved U937 monoblast cells (% Annexin V, -/+ EX: mono-culture, 30.1±9.0 / 31.5±4.6; co-culture with adipocytes, 8.9±2.1 / 29.6±9.2; P=0.02).To assess the molecular links between metabolic pathways and gene expression triggered by BM adipocytes, we performed RNA-seq transcriptome analysis with a next generation sequencer system HiSeq1500 (Illumina) using TopHat software for alignment and Cufflinks software for identifying differential gene expression. RNA-Seq detected upregulation of 21 genes in U937 cells after co-culture with BM-derived adipocytes (false discovery rate, <0.05). Specifically, 10 of these upregulated genes were significantly decreased by EX treatment, including transcription factor–activating PPARg2 promoter KLF9, co-chaperone immunophilin protein FKBP5, chemokine CXCL12 receptor CXCR4, receptor tyrosine kinase FLT3, PI3K negative regulator PIK3IP1, and immunosuppressive transcriptional regulator TSC22D3. GO pathway analysis further revealed that co-culture with adipocytes induced upregulation of antioxidant thioredoxin peroxidase activity, which was reversed by EX. Together with the iTRAQ GO results, the antioxidative chaperone proteins might play critical roles in regulation of FAO with repression of oxidative phosphorylation in AMoL cells in adipocytes abundant BM.DNA array (GeneSQUARE) analysis and quantitative RT-PCR detected upregulation of fatty acid binding protein 4 (FABP4), scavenger receptor CD36, nuclear receptor PPARG, and antiapoptotic Bcl-2 in U937 cells co-cultured with adipocytes. It is known that FFAs, the ligand of nuclear receptor PPARγ, activate PPARγ and promote FFA uptake through transcriptional induction of CD36 and FABP4 in monocytic cells. EX treatment, which blocks the entry of fatty acids into the mitochondria, induced prominent elevation of FABP4 in U937 cells co-cultured with adipocytes. These results indicate that the FABP4-mediated internalization and ligation of fatty acids to PPARγ facilitates transcriptional activation.From the transcriptome analysis and the mitochondrial uncoupling metabolic changes, we conclude that the survival of AMoL cells depends on the cooperative interactions between lipid metabolism and transcriptional activation of factors associated with chaperones, chemokines, and integrins. Strategies targeting FAO warrant further exploration in patients with monocytic leukemia, which is highly dependent on altered lipid metabolism. DisclosuresNo relevant conflicts of interest to declare.

The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70.

Protein-damaging stress stimulates cell destruction through apoptosis; however, non-lethal proteotoxic stress induces an adaptive response leading to the increased synthesis of heat shock proteins, which inhibit apoptosis. In this study, we sought to determine the mechanism responsible for the accumulation of the BH3-only protein NOXA in heat-stressed cells and its prevention by the heat shock protein HSP70. Analysis of transcript levels by RT-qPCR revealed that miR-23a levels decreased in heat-stressed cells and that this was correlated with an increased abundance of NOXA mRNA, which contains a miR-23a binding site in its 3′ untranslated region. Cells overexpressing HSP70 had higher levels of miR-23a, maintained these levels after heat shock and accumulated lower levels of NOXA mRNA and protein. The enhanced abundance of mir-23a in these HSP70-expressing cells is primarily due to its increased stability although higher levels of pri/pre-miR-23a expression, nuclear export and maturation were also contributing factors. Stable overexpression of miR-23a in the acute lymphoblastic T-cell line PEER resulted in reduced basal and heat-induced levels of NOXA mRNA and significantly inhibited heat-induced apoptosis. Additionally, stable overexpression of an shRNA targeting miR-23a in U937 lymphoma cells produced stable knockdown of miR-23a and resulted in increased NOXA mRNA and an increased sensitivity to heat-induced apoptosis. These results demonstrate the novel finding that hyperthermia affects the abundance of a microRNA that targets the expression of a pro-apoptotic protein and that HSP70 protects cells from heat-induced apoptosis by regulating the abundance of this microRNA. We speculate that the inhibition of miRNA transcription in heat-stressed cells could represent a general mechanism for apoptosis induction that is regulated by the molecular chaperone protein HSP70. Furthermore, we propose that HSP70 could be beneficial to tumor cells by helping to maintain the expression of oncogenic miRNAs under conditions of cellular stress.

Abstract 1612: Gene expression and proteomic analysis to identify predictive biomarkers of response in the ENCHANT-1 Trial (NCT01677455), a Phase 2 Proof of Concept study evaluating first-line ganetespib monotherapy in women with metastatic HER2 positive or triple negat

Abstract Background: The chaperone protein HSP90 is required for the stabilization and activation of many client proteins critical to breast cancer growth and aggressiveness, such as HER2, HIF1-α, EGFR, ER, PI3K, AKT, P53 and VEGFR. Ganetespib, a novel triazolone inhibitor of HSP90, has shown activity in breast cancer in both preclinical models (HER2+, ER+/PR+ and TNBC), and in early clinical trials (patients with HER2+ disease, as well as TNBC). Ganetespib has been well tolerated in clinical trials with a favorable safety profile. The ENCHANT-1 trial was designed to further evaluate ganetespib single agent activity in metastatic breast cancer (mBC) and identify potential predictive biomarkers. Methods: The ENCHANT-1 trial is an international, first-line phase 2 study in mBC patients: Cohort A, HER2+ (up to n=35) and Cohort B, TNBC (up to n=35). Patients with previously untreated metastatic disease are eligible for treatment with ganetespib at 150 mg/m2 twice weekly on 3 out of 4 wks. Primary endpoint: ORR assessed using RECIST1.1 criteria. Key secondary endpoints include early metabolic effects as assessed by PET/CT at week 3. Tissue samples were collected from all pts at baseline prior to initiation of treatment; fresh biopsies at C1D18 and end of treatment were optional. Gene expression will be evaluated using RNAseq to survey mutations and gene expression levels in breast cancer genes. A reverse phase protein microarray assay will be used to map the activated protein signaling architecture of laser capture micro-dissected tumor cells from collected tissue samples to evaluate the level of phosphorylation/activation of HSP90 clients and coordinated signaling pathways. Analysis of ∼150 signaling proteins is planned. Results and Conclusions: The study was initiated in 27 centers globally. At the time of submission, a total of 35 patients were enrolled; TNBC (n= 30) and HER2+ (n=5). In an interim analysis, of the evaluable 4 patients in HER2-positive cohort, 2 had objective response (OR), and 2 stable disease (SD). In the TNBC cohort, of the evaluable 10 patients, 2 had OR, 3 SD and 5 PD. Full molecular analysis to identify biomarkers of response is underway and will be reported at the meeting. Citation Format: Emanuel F. Petricoin, Julia Wulfkuhle, Tamas Hickish, Iman El-Hariry, Vienna Reichert, Vojislav M. Vukovic, David A. Cameron, Ahmad Awada, Neil Spector. Gene expression and proteomic analysis to identify predictive biomarkers of response in the ENCHANT-1 Trial (NCT01677455), a Phase 2 Proof of Concept study evaluating first-line ganetespib monotherapy in women with metastatic HER2 positive or triple negat [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1612. doi:10.1158/1538-7445.AM2014-1612