Specific Aims:Malignant Mesothelioma remains one of the “die-hard” tumors for which there remains a lack of effective treatment. Unresponsiveness to most of the current therapies may, in part, be explained by the resistance to apoptosis. Disruption of histone acetyltransferase (HAT) and histone deacetylase(HDAC) function has been associated with the development of cancer and resistance to apopstosis[cohen hy]. A potential inhibitor of HDAC in cancer has provided a rationale for inhibiting HDAC activities to achieve therapeutic effects against tumors. Histone Deacetylase Inhibitors (HDACi) have shown considerable promise against a variety of cancers including mesothelioma. Although histones are thought to be primary targets of HATs and HDACs, non-histone proteins, including transcriptional factors, nuclear import proteins, signal transduction molecules and DNA repair enzymes can also be regulated by acetylation and deacetylation [xang]. Accordingly, HDACi may mediate tumor cell death via effects on non-histone proteins, in addition to their effects on histones. As a consequence, it is still unclear exactly how HDAC inhibitors target non-histone proteins and affect the mitochondria. Given range of response these HDACi can elicit, and apparent lack of toxicity to normal cells, the use of such inhibitors in combination with other anti-cancer agents, may prove to be therapeutically beneficial. However, lack of mechanism of HDACi function on mitochondria in mesothelioma becomes an important problem, because without it, developing a successful treatment against mesothelioma is unlikely.

The goal of this proposal is to understand the underlying mechanisms responsible for inhibition of tumor growth by HDACi, in order to develop effective intreventions. The primary objective her is to define the molecular mechanisms by which HDACi alter the mitochondrial membrane potential in mesothelioma cells. The Central Hypothesis is that HDACi attenuate Bcl-xL expression through transcriptional regulation and decrease AKT activity through post-translational modification. Our hypothesis is formulated based on the findings reported by other groups that Malignant Mesothelioma expresses high Akt kinase activity [garland, LL], elevated Bcl-xL expression [soini] that cooperates to maintain mitochondrial membrane potential and promotes tumor survival. Our rationale is that once the molecular mechanism by which HDACi alter the mitochondrial membrane potential is determined, this knowledge will potentially lead to improved therapeutic approaches in treatment of mesothelioma.

Specific Aim #1: Define the mechanism of HDACi mediated reduction in Bcl-xL transcription. HDACi increases expression of TEL by selectively modifying (via acetylation) transcriptional repressor that competes against other ETS transcriptional factors at the level of Bcl-xL promoter to decrease its transcriptional activity.

Specific Aim #2: Determine the mechanism of AKT kinase-dependent HDAC inhibitor-mediated modification of the mitochondrial membrane potential. Downstream targets of the AKT kinase, including hexokinase and BH3 only members of the Bcl-2 family proteins, required to regulate the mitochondrial membrane potential are selectively modified in response to the HDAC inhibitors.

The proposed work is potentially innovative, the mechanism of HDACi targeting mitochondria to disrupt tumor survival and growth via upregulation and modification of transcriptional repressor (TEL) leading to decreased pro-apoptotic (Bcl-xL) protein transcription has previously been not addressed in Malignant Mesothelioma. This mechanism of action will contribute to the development of improved therapeutics.

Background and Significance

Significance

Targeting mitochondria has recently emerged as therapeutic strategy in cancer research. Bcl-2 and Bcl-xL were discovered to bind to the outer mitochondrial membrane and function to block cell death [XXXX]. In addition to Bcl-2 family proteins, highly-activated Akt kinase was discovered to promote tumor survival by maintaining the mitochondrial membrane potential [xxx]. Many pro-apoptotic Bcl-2 family proteins are targets for Akt kinase[xxxx]. In addition, the activated Akt kinase phosphorylates hexokinase that translocates to mitochondria and bind to the outer mitochondrial membrane, voltage-dependent anion channel and blocks the out-flow of cytochrome c[xxx]. Such findings suggest that targeting of mitochondria by blocking the Bcl-xL expression and Akt kinase will lead to the development of significant cell apoptosis.

HDAC inhibitors (HDACi) are investigated as anti-cancer agents in clinical trials [ververis][XXXX][XXXX]. Nevertheless, mechanisms underlying the anti-proliferative effects remain to be defined. Altough these inhibitors have been shown to activate the transcription of a defined set of genes through chromatin remodeling [XXXX], increasing evidence suggests that modifications of the epigenetic histone code may not represent a primary cause for HDAC inhibitor-mediated growth inhibiton and apoptosis in cancer cells [XXXX][XXXX][XXXX]. To date, at least two distinct histone acetylation independent mechanisms have been described for action of HDAC inhibitors on cellular targets. First, because certain HDAC members can mediate the deacetylation of non-histone proteins, their inhibiton interferes with signaling processes in which these proteins are involved, independent of the activity of HDACi in histone acetylation. Example, HDAC3 regulates NF-kB signaling in nucleus by deacetylating the Rel-A subunit [XXXX]. Second, HDACs 1 and 6 have been shown to form complexes with protein phosphatases 1 (PP1) [XXXX][XXXX], of which HDACi could facilitate the dephosphorylation of Akt and other signaling kinases[xxx]x\[xxx].

Our contribution here is expected to be detailed understanding of how HDAC inhibitors affect the mitochondrial membrane potential and induce apoptosis in mesothelioma. This contribution is significant because it is expected to provide the knowledge needed to develop clinical strategies to treat mesothelioma successfully.

Background

Over-expressed Bcl-xL maintains mitochondrial membrane potential

Mitochondria plays an important role in the intrinsic apoptosis pathway. The key regulators of MOMP (Mitochondrial Outer Membrane Potential), members of the Bcl-2 Family Proteins [XXXX] are divided into pro-apoptotic (promote MOMP loss) and anti-apoptotic (prevent MOMP loss). The anti-apoptotic protein Bcl-xL is prevalent in many solid tumors, including lung cancer and mesothelioma[XXXX], and contributes to cell survival and drug resistance. Since cell survival and death is determined by the competitive action of death agonists and antagonists, and the balance between expression of pro- and anti- apoptosis genes, any approach to alter the balance in favour of cell

apoptosis will be of therapeutic benefit. RNA interference targeting Bcl-xL and BH3 mimetic 2-methoxy antimycin A3 induced apoptosis in mesothelioma following decreased mitochondrial membrane potential [XXXX].

Bcl-xL transcriptional regulation with ETS family gene

Analysis of human Bcl-xL promoter reveals that the promoter has nine potential Ets-binding sties(EBS)[habens]. Ets1, and Ets2, which in turn activate Bcl-xL to promote cell survival[XXXX]. The Ets family of transcription factors consists of more than 20 members[seth a]. Most Ets family proteins are nuclear targets for activation of many signaling pathways, including MAPK signaling pathway and different post-translational modifications including phosphorylation, glycosylation, acetylation, ubiquitination and sumoylation[charlet]. Among these Ets family transcriptional factors, Ets-1, Ets-2 and PU.1[XXXX] were discovered to have a positive regulation of Bcl-xL expression at the promoter level. In contrast, the transcriptional repression memeber of the Ets family, TEL has been reported to decrease Bcl-xL expression[XXXX]. As a member of Ets family protein, TEL preferentially binds to the sequence T(G/T)(A/C)GGAAGT[XXXX] and functions as a transcriptional repressor via interactions with the mSin3A, N-CoR, and SMRT corepressors[XXXX], as well as with histone deacetylase 3[XXXX]. TEL is negatively regulated by phosphorylation and sumoylation[roukens, maki]. Once phosphorylated and sumolyated, TEL is removed from the promoter and thus has abrogated transcriptional repression. TEL is then exported to the cytoplasm through the CRM1 mediated nuclear export system[XXXX]. Although there is currently no direct evidence to support the conclusion that TEL becomes acetylated following HDACi treatment, another member of the Ets family genes, Er81 was reported to be acetylated[XXXX]. Since ubiquitination, sumoylation and acetylation can all occur on lysine residues[XXXX], TEL may potentially undergo a cascade of acetylation that serves to modulate its functions.

Akt maintains the mitochondrial membrane potential through Hexokinase and BH3 only proteins

Akt elevates mitochondrial hexokinase(mtHK) association and activity at mitochondria and requires active mtHK to inhibit apoptosis[XXXX]. Mammalian cells have four HK isoforms, of which HKI and HKII associate with the Outer Mitochondrial Membrane (OMM). An amino-terminal hydrophobic domain found only in HKI and HKII mediates this interaction. HKI, possibly HKII, bind to the voltage-dependent anion channel (VDAC)[XXXX]. HKII has been shown to inhibit the mitochondrial binding of BAX[XXXX]. Akt was shown to maintain mitochondrial potential and prevent the release of apoptotic factors by regulating HKs to antagonize BAX and BAK activity.

Akt is also expected to directly or indirectly phosphorylate targets that affects the interactions between hexokinase and its mitochondrial receptor VDAC. Although Akt phosphorylation motifs are found in HKII, it has not yet been established whether Akt directly phosphorylates mitochondrial hexokinases or whether such modifications impact hexokinase - mitochondria interactions. Akt is also expected to indirectly influnace this interaction through Akt - mediated changes in the activity of downstream effectors capable of modifying hexokinase or VDAC in a manner that influences their interaction. Such demonstration suggests that GSK3β, which is phosphorylated and thus inhibited by Akt, will disrupt mitochondrial hexokinase association via phosphorylation of a putative hexokinase docking site on

VDAC[xxx]. This suggests at least one mechanism whereby Akt, through direct phosphorylation and inactivation of GSK3β, will augment mitochondrial hexokinase interaction and promote cell survival.

Akt has been shown to negatively regulate the activity of several pro-apoptotic members of the Bcl-2 family. It has also been reported that Akt phosphorylates and directly inhibits the activity of the BH3-only protiens, BAD and BimEL, as well as FoxO3a transcription factor. FoxO3a has been shown to induce the expression of the BH3-only Bcl-2 family member BIM and PUMA [XXXX].Dephosphorylated Bad following attenuated Akt activity could displace "activator" only BH3 only proteins such as Bid, Bim or PUMA from Bcl-xL and in turn activate Bax and Bak[XXXX].

Interrupting the connection between HDACs and PP1 attenuated Akt phosphorylation.

HDACi has been found to mediate tumor cell death via multiple possible effects on non-histone proteins. Their inhibition interferes with signaling processes in which these proteins are involved independently of the direct activity of HDAC inhibitors in transcriptional activation. Studies have confirmed the existence of a cellular HDAC1-PP1 complex and showed that this complex was disrupted by treatment of cells with TSA (trichostatin A) [XXXX]. TSA was used to demonstrate that HDACi facilitate dephosphorylation of Akt by altering the dynamics of HDAC-PP1 complexes [XXXX]. HDACi selectively target HDACs 1 and 6 to disrupt the respective HDAC-PP1 complexes, resulting in increased association of PP1 and Akt[XXXX]. This PP1 faciliated kinase dephosphorylation underscores the diverse functions of HDAC inhibitors in mediating anti-neoplastic activities at different cellular levels. PP1 is regulated by its interaction with a variety of protein subunits that target the catalytic subunit (PP1c) to specific subcellular compartments and determine its localization, activity and substrate selectivity[XXXX]. In addition to the phosphatase of Akt, PP1α (major subunti of PP1c) is also a BAD phosphatase that dephosphorylates BAD on Ser-112, 136 prior to inducing apoptosis [XXXX]. In addition to decreasing Bcl-xL or Bcl-2 at transcriptional level, HDACi mediate dephosphorylation of Akt and BAD by disrupting HDAC-PP1 complexes, thereby lowering the apoptotic threshold of treated cells.

Research Startegy

Aim #1: Define the mechanism of HDAC inhibitor-mediated reduction of Bcl-xL transcription

The objective is to determine the mechanism by which HDACi reduce Bcl-xL expression at the transcriptional level in mesothelioma. To attain the objective of this section, we will test the working hypothesis that HDACi increase the expression of TEL transcriptional repressor which competes against other ETS transcriptional factors at the level of Bcl-xL promoter to decrease its transcriptional activity.

Experimental Plan

Identification of TEL binding sites on Bcl-xL reporter promoter.

To more definitely evaluate the role of HDACi in attanuated Bcl-xL expression through TEL induction, we will perform a deletion mutation analysis of the Bcl-xL promoter sequence utilizing transient transfection techniques. We will transfect mesothelioma cell line H2542 with the vector construct

containing either entire promoter sequence or ETS binding deletion mutants utilizing a liposomal protocol and record comparative Bcl-xL promoter (luciferase) activity in the presence and absence of HDACi SAHA (Suberoyl anilide hydroxamic acid).

Electrophoresis mobility shift assay (EMSA) will be performed to monitor the interaction between the ETS transcription factor TEL and ETS binding sites identified on Bcl-xL promoter using dsDNA probe covering the binding site. The EMSA will be carried out as described previously[XXX]. The association bewteen the ETS family members and ETS binding sites will be quantitatively presented as the amount of radiation signal of retarded shift band. In addition to using mutant dsDNA probe, we will use "super-shift EMSA" by adding anti-TEL and control antibody into the reaction complex to further confirm the binding capacity.

Define the role of Acetylation of TEL in down-regulation of Bcl-xL promoter activity.

His-tagged TEL stably transfected cells will be exposed to SAHA or DMSO control. His-tagged TEL proteins will be precipitated from the cell lysates using Dynabeads magnetic beads. The precipitated His-TEL protein will be processed for mass spectrometric analysis to identify the exact location of acetylation. Based on the results, a series of mutations of TEL will be generated by site directed mutagenesis in His-TEL replacing lysine amino acid with arginine (blocking acetylation) and explore if acetylated TEL is retained in the nucleus compared to the non-acetylated one. His-tagged wild type TEL and acetylation mutants will be transfected into H2542 mesothelioma cell lines and treated with SAHA, 24 hours later cells will be fixed using formaldehyde and immunofluorescence technique using a FITC-labeled anti-His monoclonal anti-body will be used to detect the translocation of TEL protein. We will also examine whether acetylated TEL has a stronger Bcl-xl promoter binding capacity using ChIP assay technique. Briefly, H2542 cells transfected with His-tagged TEL and acetylation mutants will be treated with SAHA, 24 hours later cells will be lysed and protein-DNA is crosslinked with formaldehyde and sonicated to generate smaller protein-DNA fragments ( 50 - 200 bp), cell lysate is immunoprecipitated using His - tagged antibody to pull down protein-DNA complex, reverse cross-linked and DNA is eluted using phenol-chloroform extraction and quantitative PCR is done with primers specific for Bcl-xL. A super-shift EMSA assay will be performed along with ChIP assay on the above samples, and the Bcl-xL expression will be measured by western blotting.

Study the proteins involved in TEL based transcriptional repression at Bcl-xL promoter level.

In order to determine possible involvement of TEL in repression complex on the Bcl-xL promoter, His-tagged TEL will be precipitaed from the His-TEL cDNA transfected H2542 cell line. Western blot will be used to detect whether mSin3a, N-CoR and HDAC3/9 are present in the precipiated complex. Binding of these repressors to the Bcl-xL promoter will be determined via ChIP assay with anti-mSIN3a, N-CoR and HDAC3/9 antibodies. Repression of Bcl-xL transcription will be measured by promoter reporter analysis and real-time PCR, in the presence of control siRNA or siRNA against mSin3a, N-CoR or HDAC3/9.

Aim #2: Determine the mechanism of Akt kinase dependent HDACi mediated modification of the mitochondrial membrane potential.

The objective here is to determine the mechanism of Akt kinase dependent HDACi mediated reduction of the mitochondrial membrane potential. We will test the hypothesis that downstream targets of the Akt kinase, including protein phosphatases (PP1), Hexokinase, Mcl-1 and BH3 only proteins, Bad and Bim requred to regulate the mitochondrial membrane potential are selectively modified in response to HDACi.

Experimental Plan

HDACi induces dissociation of hexokinase from mitochondria through activation of GSK-3β by attenuating Akt kinase activity.

We will determine the phosphorylation status of Hexokinase I and II by immunoprecipitating Hexokinase and analyze by western blotting with monoclonal antibody against RXRXXS/T motif phosphorylated by Akt kinases in H2542 upon treatment with HDACi[XXX]. We will also explore physical interaction between Akt and HK I and II on SAHA treatment by immunoprecipitation and western blotting. Briefly H2542 cells treated SAHA or vehicle control for 24 hours will be lysed and proteins immunoprecipitated with anti-Akt antibody and western blotting with anti-Hexokinase, or vice versa. Confocal microscopy technique will be used to show co-localization of Hexokinase and Akt under the above treatments.

Alternatively, we will determine whether attenuating Akt kinase activity by SAHA leads to GSK-3β activation which leads to VDAC phosphorylation and dissociation from Hexokinase. We will test to determine if SAHA increased GSK-3β activity in H2542 cell line. GSK-3β activity will be measured with a kinase kit. PI3K inhibitor LY2940002 and commercially available Akt kinase inhibitors will be used as positive controls and the GSK-3β inhibitor will be used as a negative control. To evaluate whether SAHA promotes activation of GSK-3β resulting in disruption of Hexokinase binding to VDAC, H2542 cells will be treated with various doses of SAHA overnight with or without Akt inhibitor, GSK-3β inhibitor or both. Cells will be harvested and mitochondria and cytosolic fractions will be isolated. VDAC protein will be immunoprecipitated from the mitochondrial fraction using anti-VDAC antibody and precipiate will be blotted with anti-phosphothreonine antibody. HK I and II will be blotted both in the cytoplasmic fraction and mitochondiral fraction under the above treatment conditions and compared to determine the protein translocation and localization.

Identify the impact of dephophorylated Bad on mitochondrial membrane potential

To evaluate the impact of dehphosphorylated Bad on mitochondrial membrane potential, we will identify serine residues in Bad protein that dephosphorylated after SAHA treatment. H2542 cells will be treated with SAHA or solvent control overnight. The phosphorylated Bad protein will then be detected by westernblot using three antibodies specific to three regulatory serines (S112, S116 and S155) [XXX]. Once these phosphorylated serines are determined we will create a series of mutants to creat non-phosphorylatable Bad cDNA by replacing one, two or three serine with alanine through site-directed mutagenesis. In order to determine whether dephophorylated Bad induced by SAHA neutralzed the Bcl-

xL function, wild type His-tagged Bad cDNA and non-phosphorylatable mutant cDNA vectors will be transfected into H2542 and will be treated with SAHA or solvent control. The interaction between Bcl-xL and Bad will be measure by immunoprecipitation-westernblotting. The mitochondrial membrane potential will be determined using JC-1 staining and apoptosis will be measured using TUNEL assay.

Characterizing the impact of Bim on Mesothelioma cells after SAHA treatmentIn order to determine the role of phosphorylation of Bim in response to SAHA, phopshorylation of

Bim will be measured by IP - Western Blotting. Briefly, Bim and Bim-EL will be immunoprecipitated from H2542 cells exposed to various doses of SAHA. Phopshorylated Bim/Bim-EL will be measured by anti-phosphoserine antibody. We will measure Bim association with Akt, p38, JNK or Erk kinase with antibodies specific for each. Once we have determined which of these kinases in addition to Akt are primarily responsible for phosphorylation of Bim and Bim-EL, small molecular inhibitor of targeting that specific kinase will be added to the H2542 cells and the phopshorylation of Bim will be compared to control. In order to determine whether or not phopsphorylation of Bim contributes to HDACi induced decrease in MOMP, phosphorylation specific Bim mutants will be created and transfected into H2542 cell line and IC50 to SAHA will be evaluated, Mitochondrial membrane potential will be measured by JC-1 staining.

Anticipated ResultsWe expect up-regulation of TEL expression by SAHA will decrease Bcl-xL transcriptional activity

by binding to one or more ETS binding site in the Bcl-xL promoter. This finding will strongly support the hypothesis that TEL transcriptional repressor competes against other ETS transcriptional factors at the level of Bcl-xL promoter. Further, we expect to find that acetylated TEL has enhanced Bcl-xL promoter binding capacity relative to unmodified TEL.

We expect that relocation of hexokinase from the mitochondria to the cytosol will contribute to the alteration of mitochondrial membrane potential. Additionally, we anticipate that the dephosphorylation of Bad following SAHA treatment would serve to free other BH3 only proteins like Bid, Bim and PUMA, from Bcl-xL to activate Bak and Bax, which inturn will decrease the mitochondrial membrane potential. Also, we expect to find phoshorylation of VDAC channel after SAHA treatment. The phosphorylation of VDAC could let hexokinase disassociate from mitochondria.

Potential problems and proposed solutionsIt is possible that TEL might have additional mechanisms involved in transcriptional silencing.

There are multiple transcriptional repressors in the ETS family which potentially bind to the Bcl-xL promoter. In this case, we will study whether Bcl-6, Net and ERF could synergistically repress Bcl-xL transcription in addition to TEL.

There is a possibility that PPI is not the main target of SAHA for the dephosphorylating proteins. In that unlikely event, we will focus on other kinases which could potentially affect Akt and its downstream targets. We will also focus on PP2 whcih is not associated with HDAC but would dephosphorylate Bad and GSK-3β. It is also possible that highly expressed Bcl-xL alone would maintain the mitochondrial membrane potential despite no Akt activity. In that case, we would consider including additional mesothelioma cells line with low or no Bcl-xL.

Future work

We will study the role of soluble factors like cytokines on BNIP family proteins in the mitochondria membrane potential change after SAHA treatment. HDACi potentially up-regulate BNIP family proteins through activating FOXO transcriptional factor and by inducing inflammatory cytokines that results in induction of autophagy. we will evaluate BAGs/Hsp70 as molecular switch between autophagy and apoptosis.