Yonghua Yang

Role of Acetylation and Extracellular Location of Heat Shock Protein 90α in Tumor Cell Invasion

Heat shock protein (hsp) 90 is an ATP-dependent molecular chaperone that maintains the active conformation of client oncoproteins in cancer cells. An isoform, hsp90alpha, promotes extracellular maturation of matrix metalloproteinase (MMP)-2, involved in tumor invasion and metastasis. Knockdown of histone deacetylase (HDAC) 6, which deacetylates lysine residues in hsp90, induces reversible hyperacetylation and attenuates ATP binding and chaperone function of hsp90. Here, using mass spectrometry, we identified seven lysine residues in hsp90alpha that are hyperacetylated after treatment of eukaryotic cells with a pan-HDAC inhibitor that also inhibits HDAC6. Depending on the specific lysine residue in the middle domain involved, although acetylation affects ATP, cochaperone, and client protein binding to hsp90alpha, acetylation of all seven lysines increased the binding of hsp90alpha to 17-allyl-amino-demethoxy geldanamycin. Notably, after treatment with the pan-HDAC inhibitor panobinostat (LBH589), the extracellular hsp90alpha was hyperacetylated and it bound to MMP-2, which was associated with increased in vitro tumor cell invasiveness. Treatment with antiacetylated hsp90alpha antibody inhibited in vitro invasion by tumor cells. Thus, reversible hyperacetylation modulates the intracellular and extracellular chaperone function of hsp90, and targeting extracellular hyperacetylated hsp90alpha may undermine tumor invasion and metastasis.

HDAC6 inhibition enhances 17-AAG–mediated abrogation of hsp90 chaperone function in human leukemia cells

Histone deacetylase 6 (HDAC6) is a heat shock protein 90 (hsp90) deacetylase. Treatment with pan-HDAC inhibitors or depletion of HDAC6 by siRNA induces hyperacetylation and inhibits ATP binding and chaperone function of hsp90. Treatment with 17-allylamino-demothoxy geldanamycin (17-AAG) also inhibits ATP binding and chaperone function of hsp90, resulting in polyubiquitylation and proteasomal degradation of hsp90 client proteins. In this study, we determined the effect of hsp90 hyperacetylation on the anti-hsp90 and antileukemia activity of 17-AAG. Hyperacetylation of hsp90 increased its binding to 17-AAG, as well as enhanced 17-AAG-mediated attenuation of ATP and the cochaperone p23 binding to hsp90. Notably, treatment with 17-AAG alone also reduced HDAC6 binding to hsp90 and induced hyperacetylation of hsp90. This promoted the proteasomal degradation of HDAC6. Cotreatment with 17-AAG and siRNA to HDAC6 induced more inhibition of hsp90 chaperone function and depletion of BCR-ABL and c-Raf than treatment with either agent alone. In addition, cotreatment with 17-AAG and tubacin augmented the loss of survival of K562 cells and viability of primary acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) samples. These findings demonstrate that HDAC6 is an hsp90 client protein and hyperacetylation of hsp90 augments the anti-hsp90 and antileukemia effects of 17-AAG.

Hydroxamic Acid Analogue Histone Deacetylase Inhibitors Attenuate Estrogen Receptor-α Levels and Transcriptional Activity: A Result of Hyperacetylation and Inhibition of Chaperone Function of Heat Shock Protein 90

Purpose: The molecular chaperone heat shock protein (hsp)-90 maintains estrogen receptor (ER)-α in an active conformation, allowing it to bind 17β-estradiol (E2) and transactivate genes, including progesterone receptor (PR)-β and the class IIB histone deacetylase HDAC6. By inhibiting HDAC6, the hydroxamic acid analogue pan-HDAC inhibitors (HA-HDI; e.g., LAQ824, LBH589, and vorinostat) induce hyperacetylation of the HDAC6 substrates α-tubulin and hsp90. Hyperacetylation of hsp90 inhibits its chaperone function, thereby depleting hsp90 client proteins. Here, we determined the effect of HA-HDIs on the levels and activity of ERα, as well as on the survival of ERα-expressing, estrogen-responsive human breast cancer MCF-7 and BT-474 cells. Experimental Design: Following exposure to HA-HDIs, hsp90 binding, polyubiquitylation levels, and transcriptional activity of ERα, as well as apoptosis and loss of survival, were determined in MCF-7 and BT-474 cells. Results: Treatment with HA-HDI induced hsp90 hyperacetylation, decreased its binding to ERα, and increased polyubiquitylation and depletion of ERα levels. HA-HDI treatment abrogated E2-induced estrogen response element-luciferase expression and attenuated PRβ and HDAC6 levels. Exposure to HA-HDI also depleted p-Akt, Akt, c-Raf, and phospho-extracellular signal–regulated kinase-1/2 levels, inhibited growth, and sensitized ERα-positive breast cancer cells to tamoxifen. Conclusions: These findings show that treatment with HA-HDI abrogates ERα levels and activity and could sensitize ERα-positive breast cancers to E2 depletion or ERα antagonists.

Molecular and biologic characterization and drug sensitivity of pan-histone deacetylase inhibitor–resistant acute myeloid leukemia cells

Hydroxamic acid analog pan-histone deacetylase (HDAC) inhibitors (HA-HDIs) have shown preclinical and clinical activity against human acute leukemia. Here we describe HA-HDI-resistant human acute myeloid leukemia (AML) HL-60 (HL-60/LR) cells that are resistant to LAQ824, vorinostat, LBH589, and sodium butyrate. HL-60/LR cells show increased expression of HDACs 1, 2, and 4 but lack HDAC6 expression, with concomitant hyperacetylation of heat shock protein 90 (hsp90). Treatment with HA-HDI failed to further augment hsp90 acetylation, or increase the levels of p21 or reactive oxygen species (ROSs), in HL-60/LR versus HL-60 cells. Although cross-resistant to antileukemia agents (eg, cytarabine, etoposide, and TRAIL), HL-60/LR cells are collaterally sensitive to the hsp90 inhibitor 17-AAG. Treatment with 17-AAG did not induce hsp70 or deplete the hsp90 client proteins AKT and c-Raf. HL-60/LR versus HL-60 cells display a higher growth fraction and shorter doubling time, along with a shorter interval to generation of leukemia and survival in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus, resistance of AML cells to HA-HDIs is associated with loss of HDAC6, hyperacetylation of hsp90, aggressive leukemia phenotype, and collateral sensitivity to 17-AAG. These findings suggest that an hsp90 inhibitor-based antileukemia therapy may override de novo or acquired resistance of AML cells to HA-HDIs.

Panobinostat treatment depletes EZH2 and DNMT1 levels and enhances decitabine mediated de-repression of JunB and loss of survival of human acute leukemia cells

The PRC2 complex protein EZH2 is a histone methyltransferase that is known to bind and recruit DNMT1 to the DNA to modulate DNA methylation. Here, we determined that the pan-HDAC inhibitor panobinostat (LBH589) treatment depletes DNMT1 and EZH2 protein levels, disrupts the interaction of DNMT1 with EZH2, as well as de-represses JunB in human acute leukemia cells. Similar to treatment with the hsp90 inhibitor 17-DMAG, treatment with panobinostat also inhibited the chaperone association of heat shock protein 90 with DNMT1 and EZH2, which promoted the proteasomal degradation of DNMT1 and EZH2. Unlike treatment with the DNA methyltransferase inhibitor decitabine, which demethylates JunB promoter DNA, panobinostat treatment mediated chromatin alterations in the JunB promoter. Combined treatment with panobinostat and decitabine caused greater attenuation of DNMT1 and EZH2 levels than either agent alone, which was accompanied by more JunB de-repression and loss of clonogenic survival of K562 cells. Co-treatment with panobinostat and decitabine also caused more loss of viability of primary AML but not normal CD34+ bone marrow progenitor cells. Collectively, these findings indicate that co-treatment with panobinostat and decitabine targets multiple epigenetic mechanisms to de-repress JunB and exerts antileukemia activity against human acute myeloid leukemia cells.

Treatment with Panobinostat Induces Glucose-Regulated Protein 78 Acetylation and Endoplasmic Reticulum Stress in Breast Cancer Cells

Increased levels of misfolded polypeptides in the endoplasmic reticulum (ER) triggers the dissociation of glucose-regulated protein 78 (GRP78) from the three transmembrane ER-stress mediators, i.e., protein kinase RNA-like ER kinase (PERK), activating transcription factor-6 (ATF6), and inositol-requiring enzyme 1α, which results in the adaptive unfolded protein response (UPR). In the present studies, we determined that histone deacetylase-6 (HDAC6) binds and deacetylates GRP78. Following treatment with the pan-histone deacetylase inhibitor panobinostat (Novartis Pharmaceuticals), or knockdown of HDAC6 by short hairpin RNA, GRP78 is acetylated in 11 lysine residues, which dissociates GRP78 from PERK. This is associated with the activation of a lethal UPR in human breast cancer cells. Coimmunoprecipitation studies showed that binding of HDAC6 to GRP78 requires the second catalytic and COOH-terminal BUZ domains of HDAC6. Treatment with panobinostat increased the levels of phosphorylated-eukaryotic translation initiation factor (p-eIF2α), ATF4, and CAAT/enhancer binding protein homologous protein (CHOP). Panobinostat treatment also increased the proapoptotic BIK, BIM, BAX, and BAK levels, as well as increased the activity of caspase-7. Knockdown of GRP78 sensitized MCF-7 cells to bortezomib and panobinostat-induced UPR and cell death. These findings indicate that enforced acetylation and decreased binding of GRP78 to PERK is mechanistically linked to panobinostat-induced UPR and cell death of breast cancer cells. Mol Cancer Ther; 9(4); 942–52. ©2010 AACR.

Cotreatment with Vorinostat Enhances Activity of MK-0457 (VX-680) against Acute and Chronic Myelogenous Leukemia Cells

Purpose: We determined the effects of vorinostat (suberoylanalide hydroxamic acid) and/or MK-0457 (VX-680), an Aurora kinase inhibitor on the cultured human (HL-60, OCI-AML3, and K562) and primary acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML), as well as on the murine pro-B BaF3 cells with ectopic expression of the unmutated and mutant forms of Bcr-Abl. Experimental Design: Following exposure to MK-0457 and/or vorinostat, apoptosis, loss of viability, as well as activity and levels of Aurora kinase and Bcr-Abl proteins were determined. Results: Treatment with MK-0457 decreased the phosphorylation of Aurora kinase substrates including serine (S)10 on histone H3 and survivin, and led to aberrant mitosis, DNA endoreduplication as well as apoptosis of the cultured human acute leukemia HL-60, OCI-AML3, and K562 cells. Combined treatment with vorinostat and MK-0457 resulted in greater attenuation of Aurora and Bcr-Abl (in K562) kinase activity and levels as well as synergistically induced apoptosis of OCI-AML3, HL-60, and K562 cells. MK-0457 plus vorinostat also induced synergistic apoptosis of BaF3 cells with ectopic overexpression of wild-type or mutant Bcr-Abl. Finally, cotreatment with MK-0457 and vorinostat induced more loss of viability of primary AML and imatinib-refractory CML than treatment with either agent alone, but exhibited minimal toxicity to normal CD34+ progenitor cells. Conclusions: Combined in vitro treatment with MK-0457 and vorinostat is highly active against cultured and primary leukemia cells. These findings merit in vivo testing of the combination against human AML and CML cells, especially against imatinib mesylate–resistant Bcr-AblT315I–expressing CML Cells.

Cotreatment with BCL-2 antagonist sensitizes cutaneous T-cell lymphoma to lethal action of HDAC7-Nur77-based mechanism

Pan-histone deacetylase inhibitors, for example, vorinostat and panobinostat (LBH589; Novartis Pharmaceuticals, East Hanover, NJ), have shown clinical efficacy against advanced cutaneous T-cell lymphoma (CTCL). However, the molecular basis of this activity remains unclear. HDAC7, a class IIA histone deacetylase (HDAC), is overexpressed in thymocytes, where it represses expression of the proapoptotic nuclear orphan receptor Nur77. Here, we demonstrate that treatment with panobinostat rapidly inhibits the in vitro and intracellular activity, as well as the mRNA and protein levels of HDAC7, and induces expression and translocation of Nur77 to the mitochondria. There, Nur77 converts death resistance protein Bcl-2 into a killer protein, promoting cell death of cultured and patient-derived human CTCL cells. Treatment with panobinostat improved survival of athymic nude mice implanted with human CTCL cells. Ectopic expression of Nur77 induced apoptosis and sensitized HH cells to panobinostat, whereas combined knockdown of Nur77 and its family member Nor1 was necessary to inhibit panobinostat-induced apoptosis of CTCL cells. Cotreatment with the Bcl-2/Bcl-x(L) antagonist ABT-737 decreased resistance and synergistically induced apoptosis of human CTCL cells. These findings mechanistically implicate HDAC7 and Nur77 in sensitizing human CTCL cells to panobinostat as well as suggest that cotreatment with an anti-Bcl-2 agent would augment the anti-CTCL activity of panobinostat.

Co-treatment with heat shock protein 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG) and vorinostat: a highly active combination against human Mantle Cell Lymphoma (MCL) cells

Heat shock protein (hsp) 90 inhibitors promote proteasomal degradation of pro-growth and pro- survival hsp90 client proteins, including CDK4, c-RAF and AKT, and induce apoptosis of human lymphoma cells. The pan-histone deacetylase inhibitor vorinostat has also been shown to induce growth arrest and apoptosis of lymphoma cells. Here, we determined the effects of the more soluble, orally bio-available, geldanamycin analogue 17-NN-dimethyl ethylenediamine geldanamycin (DMAG, Kosan Biosciences Inc) and/or vorinostat in cultured and primary human MCL cells. While vorinostat induced accumulation in the G1 phase, treatment with DMAG arrested MCL cells in the G2/M phase of the cell cycle. Both agents dose-dependently induced apoptosis of MCL cells. Vorinostat also induced hyperacetylation of hsp90 and disrupted the association of hsp90 with its co-chaperones p23 and cdc37, as well as with its client proteins CDK4 and c-RAF. Treatment of MCL cells with vorinostat or 17-DMAG was associated with the induction of p21 and p27, as well as with depletion of c-Myc, c-RAF, AKT and CDK4. Compared to treatment with either agent alone, co-treatment with DMAG and vorinostat markedly attenuated the levels of cyclin D1 and CDK4, as well as of c-Myc, c-RAF and AKT. Combined treatment with DMAG and vorinostat synergistically induced apoptosis of the cultured MCL cells, as well as induced more apoptosis of primary MCL cells than either agent alone. Therefore, these findings support the rationale to determine the in vivo efficacy of co- treatment with vorinostat and DMAG against human MCL cells.

Abstract B21: Targeting autophagy induced by pan‐HDAC inhibitor panobinostat and promoted by acetylated hsp70: A novel therapy for breast cancer

Among the hallmarks of cancer is the stress phenotype, which is collectively induced by hypoxia, deprivation of nutrients, acidosis and increased reactive oxygen species, and exhibited as metabolic and proteinmisfolding/denaturing (proteotoxic) stress. This results in the constitutive activation of the heat shock response with elevated levels of heat shock proteins (hsp), and induction of autophagy, for avoiding cell death. Autophagy is a conserved catabolic pathway in which cytoplasmic macromolecules and organelles are sequestered in autophagosomes, which fuse with lysosomes for protein degradation and amino acid recycling. Elevated levels of hsp70 and hsp90 promote proper protein folding and inhibit both the intrinsic and extrinsic pathways of apoptosis. In our present studies, we have determined in vitro that the stress induced by nutrient withdrawal or treatment with pan‐HDAC inhibitor (HDI) panobinostat (PS) (Novartis Pharmaceuticals) resulted in hyperacetylation of hsp70, which induced autophagy in the cultured breast cancer MB‐231 and MCF‐7 cells. The initial steps of autophagy include vesicle nucleation, vesicle expansion to form the phagophore, edges of which fuse to create the autophagosome. This process is triggered by a lipid kinase (PI3KC3) signaling complex, consisting of Vps34, Vps15, and autophagy‐associated genes (ATG) 6 and ATG14, which mediate vesicle nucleation. We determined that hyperacetylated hsp70 significantly enhanced the stability and activity of VPS34, which led to binding of VPS34 to Beclin1 (ATG6). Hyperacetylated hsp70 also recruited the PHD domain containing E3 ligase for sumoylation, KAP1, and induced the sumoylation of VPS34 by SUMO‐1. This was documented by confocal immunostaining with fluorescence‐conjugated anti‐VPS34 and anti‐SUMO‐1 antibodies, as well as by biochemical determination of the in vitro and in vivo sumoylation of VPS34. PS treatment also induced LC3II accumulation and formation of authophagosomes, the latter determined morphologically by electron microscopy. PS‐induced, KAP‐1 dependent sumoylation of VPS34 and downstream autophagosome formation were abrogated by siRNA to hsp70. Also, VPS34 expression was attenuated and the processing of LC3 was inhibited in the cells from the hsp70 knockout as compared to the control mice. Importantly, we also determined that, as compared to treatment with each agent alone, co‐treatment with PS and the inhibitor of autophagy 3‐ methl‐adenosine (3‐MA) or chloroquine significantly enhanced cell death of breast cancer MB‐231 and SUM159T cells. Furthermore, we determined the in vivo role of hyperacetylated hsp70 in autophagy. In cohorts of mice, following the growth of the orthotopically‐implanted MB‐231 xenografts in the mammary fat pads of NOD/SCID mice to a size of 100, 200, 500 and 1000 cu mm, tumor cell‐lysates were obtained. Western blot analyses demonstrated size‐dependent increase in the intracellular levels of hyperacetylated hsp70, Beclin1 and LC3II. Collectively, these findings suggest that stress phenotype associated heat shock response and increase in acetylated hsp70 promotes autophagy. Our findings also demonstrate that in established breast cancers, autophagy can be selectively targeted by cotreatment with the pan‐HDAC inhibitor and an inhibitor of autophagy to eliminate breast cancer cells. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B21.