Srilatha Nalluri

Targeting levels or oligomerization of nucleophosmin 1 induces differentiation and loss of survival of human AML cells with mutant NPM1

Nucleophosmin 1 (NPM1) is an oligomeric, nucleolar phosphoprotein that functions as a molecular chaperone for both proteins and nucleic acids. NPM1 is mutated in approximately one-third of patients with AML. The mutant NPM1c+ contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal, which causes NPM1c+ to be localized in the cytoplasm. Here, we determined the effects of targeting NPM1 in cultured and primary AML cells. Treatment with siRNA to NPM1 induced p53 and p21, decreased the percentage of cells in S-phase of the cell cycle, as well as induced differentiation of the AML OCI-AML3 cells that express both NPMc+ and unmutated NPM1. Notably, knockdown of NPM1 by shRNA abolished lethal AML phenotype induced by OCI-AML3 cells in NOD/SCID mice. Knockdown of NPM1 also sensitized OCI-AML3 to all-trans retinoic acid (ATRA) and cytarabine. Inhibition of NPM1 oligomerization by NSC348884 induced apoptosis and sensitized OCI-AML3 and primary AML cells expressing NPM1c+ to ATRA. This effect was significantly less in AML cells coexpressing FLT3-ITD, or in AML or normal CD34+ progenitor cells expressing wild-type NPM1. Thus, attenuating levels or oligomerization of NPM1 selectively induces apoptosis and sensitizes NPM1c+ expressing AML cells to treatment with ATRA and cytarabine.

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.

Metabolomic Profiling Reveals a Role for Androgen in Activating Amino Acid Metabolism and Methylation in Prostate Cancer Cells

Prostate cancer is the second leading cause of cancer related death in American men. Development and progression of clinically localized prostate cancer is highly dependent on androgen signaling. Metastatic tumors are initially responsive to anti-androgen therapy, however become resistant to this regimen upon progression. Genomic and proteomic studies have implicated a role for androgen in regulating metabolic processes in prostate cancer. However, there have been no metabolomic profiling studies conducted thus far that have examined androgen-regulated biochemical processes in prostate cancer. Here, we have used unbiased metabolomic profiling coupled with enrichment-based bioprocess mapping to obtain insights into the biochemical alterations mediated by androgen in prostate cancer cell lines. Our findings indicate that androgen exposure results in elevation of amino acid metabolism and alteration of methylation potential in prostate cancer cells. Further, metabolic phenotyping studies confirm higher flux through pathways associated with amino acid metabolism in prostate cancer cells treated with androgen. These findings provide insight into the potential biochemical processes regulated by androgen signaling in prostate cancer. Clinically, if validated, these pathways could be exploited to develop therapeutic strategies that supplement current androgen ablative treatments while the observed androgen-regulated metabolic signatures could be employed as biomarkers that presage the development of castrate-resistant prostate cancer.

Role of CAAT/Enhancer Binding Protein Homologous Protein in Panobinostat-Mediated Potentiation of Bortezomib-Induced Lethal Endoplasmic Reticulum Stress in Mantle Cell Lymphoma Cells

Purpose: Bortezomib induces unfolded protein response (UPR) and endoplasmic reticulum stress, as well as exhibits clinical activity in patients with relapsed and refractory mantle cell lymphoma (MCL). Here, we determined the molecular basis of the improved in vitro and in vivo activity of the combination of the pan-histone deacetylase inhibitor panobinostat and bortezomib against human, cultured, and primary MCL cells. Experimental Design: Immunoblot analyses, reverse transcription-PCR, and immunofluorescent and electron microscopy were used to determine the effects of panobinostat on bortezomib-induced aggresome formation and endoplasmic reticulum stress in MCL cells. Results: Treatment with panobinostat induced heat shock protein 90 acetylation; depleted the levels of heat shock protein 90 client proteins, cyclin-dependent kinase 4, c-RAF, and AKT; and abrogated bortezomib-induced aggresome formation in MCL cells. Panobinostat also induced lethal UPR, associated with induction of CAAT/enhancer binding protein homologous protein (CHOP). Conversely, knockdown of CHOP attenuated panobinostat-induced cell death of MCL cells. Compared with each agent alone, cotreatment with panobinostat increased bortezomib-induced expression of CHOP and NOXA, as well as increased bortezomib-induced UPR and apoptosis of cultured and primary MCL cells. Cotreatment with panobinostat also increased bortezomib-mediated in vivo tumor growth inhibition and improved survival of mice bearing human Z138C MCL cell xenograft. Conclusion: These findings suggest that increased UPR and induction of CHOP are involved in enhanced anti-MCL activity of the combination of panobinostat and bortezomib. Clin Cancer Res; 16(19); 4742–54. ©2010 AACR.

TIMP-1 Inhibits Apoptosis in Lung Adenocarcinoma Cells via Interaction with Bcl-2

Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target.

Heat shock protein 90 inhibition depletes TrkA levels and signaling in human acute leukemia cells

Nerve growth factor (NGF) induces autophosphorylation and downstream progrowth and prosurvival signaling from the receptor tyrosine kinase TrkA. Overexpression or activating mutation of TrkA has been described in human acute myeloid leukemia cells. In the present study, we show the chaperone association of TrkA with heat shock protein 90 (hsp90) and the inhibitory effect of the hsp90 inhibitor, 17-DMAG, on TrkA levels and signaling in cultured and primary myeloid leukemia cells. Treatment with 17-DMAG disrupted the binding of TrkA with hsp90 and the cochaperone cdc37, resulting in polyubiquitylation, proteasomal degradation, and depletion of TrkA. Exposure to 17-DMAG inhibited NGF-induced p-TrkA, p-AKT, and p-ERK1/2 levels, as well as induced apoptosis of K562, 32D cells with ectopic expression of wild-type TrkA or the constitutively active mutant ΔTrkA, and of primary myeloid leukemia cells. Additionally, 17-DMAG treatment inhibited NGF-induced neurite formation in the rat pheochromocytoma PC-12 cells. Cotreatment with 17-DMAG and K-252a, an inhibitor of TrkA-mediated signaling, induced synergistic loss of viability of cultured and primary myeloid leukemia cells. These findings show that TrkA is an hsp90 client protein, and inhibition of hsp90 depletes TrkA and its progrowth and prosurvival signaling in myeloid leukemia cells. These findings also support further evaluation of the combined activity of an hsp90 inhibitor and TrkA antagonist against myeloid leukemia cells. Mol Cancer Ther; 9(8); 2232–42. ©2010 AACR.

Abstract 5294: Increased angiogenesis resulting from TIMP-1 inhibition of TSP-1 is mediated by PI3kinase and FAK interactions

Tissue Inhibitor of Matrix Metalloprotease -1 (TIMP-1), one of the four natural inhibitors of matrix metalloproteinases was initially identified for its Erythroid Potentiating Activity. Since then, it has been shown to independently induce cell proliferation and inhibit apoptosis in a number of cell types. TIMP-1 has also been shown to promote, but also more often to inhibit angiogenesis. Previously, we have shown that TIMP-1 overexpression in a lung adenocarcinoma cell line (H2009) resulted in more aggressive and vascular tumors in nude mice and increased capillary network formation and tumorigenicity in vitro. An angiogenesis pathway-specific gene array had identified a 3-fold reduction in thrombospondin 1 (TSP-1) levels in the TIMP-1 overexpressing clone HB-1. TSP-1 siRNA clone exhibited a similar increase in in vitro angiogenesis assays. In the present study, we have sought to identify the signaling pathway involved whereby TIMP-1 inhibits TSP-1. To this end, we have used specific inhibitors to inhibit TIMP1 initiated signaling pathways. MAP kinase, PI3/Akt inhibitors were used, either singly or in combination with Focal Adhesion kinase (FAK) inhibitor. Our results indicate a synergistic interaction between PI3/Akt pathway and FAK signaling, which restores TSP-1 levels by approximately 3-fold relative to control. Gelatin zymography of serum-free conditioned media (SFCM) revealed a reduction in proMMP-9 level in the HB-1 clone versus H2009. Additionally MMP9 specific ELISA reveals a two-fold reduction in MMP9 levels. This is in tune with the role of TSP-1 as an inhibitor of MMP-9 activation such that decreased TSP-1 level results in increased activation of MMP-9 with a concomitant reduction in the extracellular level of proMMP-9. Thus our findings define interaction of TIMP-1 via PI3kinase and FAK signaling. resulting in TSP-1 decrease and subsequent downstream promotion of angiogenesis with increased tumor kinetics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5294. doi:1538-7445.AM2012-5294

Abstract 4955: TIMP-1 inhibits apoptosis in lung adenocarcinoma cells via Bcl-2 interaction

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tissue inhibitors of matrix metalloproteinases have multifunctional properties, orchestrating diverse biological activities. Previous studies revealed a novel function of TIMP-1 as an inhibitor of apoptosis in mammalian cells. However, the mechanisms by which TIMP-1 exerts its anti-apoptotic effect are not completely understood. The antiapoptotic effect has been correlated with higher levels of Bcl-Xl. Our earlier studies in a CNS model of metastasis, using HB-1, a TIMP-1 overexpressing lung adenocarcinoma cell line (H2009), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in apoptosis in our model. Utilizing an apoptosis -specific gene array, we found that TIMP-1 overexpression results in 3-fold increased expression of Bcl-2. H2009 and the TIMP1 over-expressing clone - HB1 were treated with staurosporine to induce apoptosis, followed by analysis for poly ADP-ribose polymerase (PARP) cleavage. HB1 showed marked reduction in PARP cleavage in comparison to the controls. Treatment of HB1 cells with ABT737, an inhibitor of Bcl-2, restored apoptosis resulting in PARP cleavage in this clone. To further confirm this mechanism, we induced transient inhibition of Bcl-2 with siRNA, which also restored PARP cleavage in the HB-1 clone. Thus both chemical and biological inhibition resulted in increased apoptosis. Additionally, immunoprecipitation of Bcl-2 from cell lysate co-immunoprecipitated TIMP-1 suggesting a direct interaction between TIMP-1 and Bcl-2. These investigations identify a relationship between TIMP-1 and Bcl-2 in apoptosis inhibition, contributing to aggressive tumor kinetics, utilizing non-MMP dependent mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4955. doi:1538-7445.AM2012-4955

Abstract 4860: Pan-HDAC inhibitor treatment sensitizes breast cancer cells to autophagy inhibitor 2-phenylethynesulfonamide: Molecular mechanism

The stress phenotype of transformed cells is associated with increased levels of heat shock proteins (hsp) and autophagy to avoid 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. The molecular cascade initiating autophagosome formation is triggered by a lipid kinase (PI3KC3) signaling complex (Vps34, Vps15, ATG6 and ATG14) that mediates phagophore nucleation. We have previously reported that treatment with panobinostat (PS), a pan-histone deacetylase (HDAC) inhibitor induced hyperacetylation of hsp70 and hsp90. In the present study we demonstrate that treatment of MB231 cells with PS increased the association of hyperacetylated hsp70 with the PI3KC3 VPS34, ATG6 (Beclin-1) and KAP1 (an E3 ligase for sumoylation). PS treatment also induced the levels of ATG7, ATG5 and LC3-II (a phosphatidylethanolamine conjugated ATG8) in MB-231 cells. Treatment with 2-phenylethynesulfonamide (PES), a small molecule inhibitor of hsp70 function resulted in the abrogation of PS-induced interaction of hsp70 with hsp40, VPS34 and (the lysosome-associated membrane protein A (LAMP-2A), a protein involved in chaperone mediated autophagy. Consequently, as compared to treatment with each agent alone, co-treatment with PS and PES resulted in significantly more cell death in MB-231 and SUM159PT cells (p Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4860.

Abstract 640: SIRT2 deacetylates peroxiredoxin and increases sensitivity to oxidative stress in human breast cancer cells

SIRT2, the mammalian ortholog of yeast Hst2, is a predominantly cytosolic and nuclear member of sirtuins, which is the class III, NAD+ dependent, histone deacetylase (HDAC) family. Sirtuins play a key role in modulating cellular stress resistance, longevity and cancer transformation. SIRT2 is known to deacetylate FOXO1 and FOXO3, thereby regulating FOXO-regulated stress resistance. In the cytosol, SIRT2 co-localizes with microtubules and deacetylates α-tubulin at the lysine-40, indicating a role in proper cytokinensis. Here, we determined other biologically important SIRT2 targets and their functions. Knockdown and overexpression of SIRT2 in HEK293 cells significantly altered the acetylation status of many proteins, including a group of proteins ranging in molecular weight from 23-50 kDa. In cells with knockdown of SIRT2, differential in-gel electrophoresis of cytosolic extract of cells followed by mass spectrometric analysis identified several significantly hyper-acetylated proteins, notably peroxiredoxin 1. Peroxiredoxins are ubiquitous family of evolutionarily conserved, thiol-dependent peroxidases, which catalyze the reduction of hydrogen peroxide. SIRT2 co-immunoprecipitated with peroxiredoxin 1, and ectopic overexpression of SIRT2 attenuated acetylation of peroxiredoxin 1 in HEK 293 cells. While acetylation activates, reduced acetylation is known to inhibit the activity of peroxiredoxins. Ectopic overexpression of SIRT2, but not the catalytically dead SIRT2 mutant, markedly increased reactive oxygen species (ROS) levels as well as increased the sensitivity of the human breast cancer MCF7 and MDA MB231 cells to oxidant stress induced by hydrogen peroxide. SIRT2 overexpression in breast cancer cells also increased their sensitivity to ROS generating agents such as arsenic trioxide and menadione. These findings demonstrate that SIRT2 plays an important role in regulating oxidative stress in cells by modulating the activity of peroxiredoxin 1. The findings also highlight the possibility that increased SIRT2 levels and activity can be therapeutically exploited for augmenting the antitumor effects of therapeutic agents that induce cancer cell death by increasing intracellular ROS levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 640.