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The present studies focused on determining whether the autophagy-inducing drug OSU-03012 (AR-12) could enhance the toxicity of recombinant adenoviral delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) in glioblastoma multiforme (GBM) cells. The toxicity of a recombinant adenovirus to express MDA-7/IL-24 (Ad.mda-7) was enhanced by OSU-03012 in a diverse panel of primary human GBM cells. The enhanced toxicity correlated with reduced ERK1/2 phosphorylation and expression of MCL-1 and BCL-XL, and was blocked by molecular activation of ERK1/2 and by inhibition of the intrinsic, but not the extrinsic, apoptosis pathway. Both OSU-03012 and expression of MDA-7/IL-24 increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) that correlated with increased levels of autophagy and expression of dominant negative PERK blocked autophagy induction and tumor cell death. Knockdown of ATG5 or Beclin1 suppressed OSU-03012 enhanced MDA-7/IL-24-induced autophagy and blocked the lethal interaction between the two agents. Ad.mda-7-infected GBM cells secreted MDA-7/IL-24 into the growth media and this conditioned media induced expression of MDA-7/IL-24 in uninfected GBM cells. OSU-03012 interacted with conditioned media to kill GBM cells and knockdown of MDA-7/IL-24 in these cells suppressed tumor cell killing. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.
In the United States, glioblastoma multiforme (GBM) is diagnosed in ~20,000 patients per annum. High-grade tumors such as anaplastic astrocytoma and GBM account for the majority of astrocytic tumors.1 Even under optimal conditions, in which essentially all of the tumor can be surgically removed and the patients are maximally treated with radiation and chemotherapy, the mean survival of this disease is only extended from 2 to 3 months to 1 year.1 These realities emphasize the need to develop therapies against this devastating and consistently fatal disease.
The mda-7 gene (recently renamed Interleukin 24, IL-24) was isolated from human melanoma cells induced to undergo terminal differentiation by treatment with fibroblast interferon and mezerein.2 The RNA and protein expression of MDA-7/IL-24 is decreased in advanced melanomas, with nearly undetectable levels in metastatic disease.2–4 This novel cytokine is a member of the interleukin-10 (IL-10) gene family.5–13 Enforced expression of MDA-7/IL-24, by use of a recombinant adenovirus Ad.mda-7, inhibits the growth and kills a broad spectrum of cancer cells, without exerting any harmful effects in normal human epithelial or fibroblast cells.9–15 In view of its potent cancer-specific apoptosis-inducing ability and tumor growth-suppressing properties in human tumor xenograft animal models, mda-7/IL-24 was evaluated in a Phase I clinical trial in patients with advanced cancers.10,11,13,16–18 This study indicated that Ad.mda-7 injected intra-tumorally was safe and with repeated injection, manifested noteworthy clinical activity.
The apoptotic pathways by which Ad.mda-7 leads to cell death in tumor cells are not fully understood, however, current data suggests an intrinsic complexity and a contribution of proteins central for the onset of growth inhibition and apoptosis, including BCL-XL BCL-2 and BAX.9–15 In human melanoma cell lines, but not in normal melanocytes, Ad.mda-7 infection induces a significant decrease in both BCL-2 and BCL-XL levels, with only a modest upregulation of BAX and BAX expression.19 This data supports the hypothesis that Ad.mda-7 increases the ratio of pro-apoptotic to anti-apoptotic proteins in cancer cells, thereby facilitating induction of apoptosis.9–15,19 The ability of Ad.mda-7 to promote apoptosis in DU-145 prostate cancer cells, which does not produce BAX, indicates that MDA-7/IL-24 can initiate apoptosis in tumor cells through a BAX-independent pathway.9–13 In prostate cancer cells, over-expression of either BCL-2 or BCL-XL protects cells from Ad.mda-7-induced toxicity in a cell type-dependent fashion.20 In a single ovarian cancer cell line, MDA-7/IL24 was reported to kill via the extrinsic apoptosis pathway.21 Accordingly, MDA-7/IL-24 toxicity appears to occur by multiple distinct pathways in different cell types, but in all of these studies cell killing is associates with a profound induction of mitochondrial dysfunction.22
More recently, MDA-7/IL-24 toxicity has been coupled with alterations in endoplasmic reticulum stress signaling.23 In these studies; MDA-7/IL-24 physically associates with BiP/GRP78 and nullifies the protective actions of this ER-chaperone protein. In addition to virus-administered mda-7/IL-24, delivery of this cytokine as a bacterially expressed GST fusion protein, GST-MDA-7,24 retains cancer-specific killing, selective ER localization and induces similar signal transduction changes in cancer cells.23 We have found that a high concentration of GST-MDA-7 kills primary human glioma cells and does so in a PERK-dependent fashion that is dependent on elevated levels of autophagy and mitochondrial dysfunction.26–28
The ability of MDA-7/IL-24 to modulate cell-signaling processes in transformed cells has been examined by several laboratories.14–32 Prior work by our groups has shown, using bacterially synthesized GST-MDA-7 protein,24 that in the 0.25–2.0 nM concentration range GST-MDA-7 primarily produces growth arrest with slight cell killing, whereas at ~20-fold greater concentrations, this cytokine causes profound growth arrest and tumor cell death.24,27,28 Our laboratories have also demonstrated that Ad.mda-7 kills melanoma cells in part by promoting p38 MAPK-dependent activation of the growth arrest and DNA damage inducible genes, including GADD153, GADD45 and GADD34.30 In primary GBM cells, however, we noted p38 MAPK signaling provided a protective signal.28 Other groups have argued that inhibition of PI3K signaling, but not ERK1/2 signaling, modestly promotes Ad.mda-7 lethality in breast and lung cancer cells.31,32
The present studies were designed to evaluate the relative merit of enhancing autophagy in parallel with expression of MDA-7/IL-24, using the novel drug that is derived from Celecoxib, OSU-03012 (AR-12), and that is presently entering phase I trials.33,34 OSU-03012, unlike the parent drug, does not inhibit COX2 but has an order of magnitude greater toxicity towards tumor cells. Our prior studies have demonstrated that OSU-03012, like MDA-7/IL-24, also kills primary human GBM cells in a PERK-dependent fashion. However, unlike MDA-7/IL-24, OSU-03012 lethality was dependent on AIF release from the mitochondrion and not on cytochrome c and caspases. The present studies have discovered that Ad.mda-7 induced cell killing is enhanced by OSU-03012 that does not correlate with enhanced additional PERK phosphorylation, but that is nonetheless dependent on increased levels of toxic autophagy and mitochondrial dysfunction. OSU-03012 also enhances the potency of secreted MDA-7/IL-24 acting via a ‘bystander’ effect on uninfected tumor cells.35,36
In primary human GBM cells, infection with a recombinant adenovirus to express MDA-7/IL-24 (Ad.mda-7) caused cell killing that was enhanced by treatment with the drug OSU-03012 (AR-12) (Fig. 1A). Similar survival data were obtained in colony formation assays (e.g., Fig. 1B). Although OSU-03012 has been shown at higher concentrations than those used herein to inhibit PDK-1, which would predict enhanced drug toxicity in GBM cells lacking PTEN, we discovered that GBM14 cells that lack PTEN function exhibited a similar sensitivity to OSU-03012 and to the OSU-03012 + Ad.mda-7 combination as did GBM12 cells that express wild type PTEN.
Treatment of Ad.mda-7 infected cells with OSU-03012 facilitated MDA-7/IL-24-induced reductions in BCL-XL and MCL-1 expression and further decreased ERK1/2 phosphorylation (Fig. 1C). Overexpression of BCL-XL, or inhibition of components in the intrinsic apoptosis pathway, reduced Ad.mda-7 or OSU-03012 toxicity as single agents and abolished the interaction between these agents (Fig. 1D). Inhibition of the extrinsic pathway, as judged by overexpression of the caspase 8 inhibitor c-FLIP-s, did not alter Ad.mda-7 or OSU-03012 lethality.
As ERK1/2 was inactivated to a greater extent in cells treated with OSU-03012 + Ad.mda-7 we determined whether maintenance of ERK1/2 activity altered OSU-03012 + Ad.mda-7 toxicity. Expression of constitutively activated MEK1 EE maintained ERK1/2 phosphorylation and acted to partially sustain expression of BCL-XL and MCL-1 (Fig. 2A). Overexpression of MCL-1 also protected GBM cells from OSU-03012 + Ad.mda-7 lethality (Fig. 2B). Collectively our data demonstrates that OSU-03012 enhances Ad.mda-7 lethality by facilitating the MDA-7/IL-24-induced lowering of mitochondrial protective protein expression.
Prior studies with OSU-03012 and with GST-MDA-7 in primary human GBM and renal carcinoma cells had demonstrated that both agents activated PKR-like endoplasmic reticulum kinase (PERK) and both induced a toxic form of autophagy.27,29,33,34 Infection of primary human GBM cells with Ad.mda-7 also activated PERK, however treatment of GBM cells expressing MDA-7/IL-24 with OSU-03012 did not cause additional PERK activation, and actually reduced PERK phosphorylation (Fig. 3A). However, OSU-03012 + Ad.mda-7 treatment did increase the processing/expression of LC3-II over and above that induced by treatment of cells with the individual agents; this is indicative that the drug combination causes elevated levels of autophagy. Expression of dominant negative PERK suppressed the lethal interaction between OSU-03012 + Ad.mda-7 (Fig. 3B). In agreement with these findings OSU-03012 + Ad.mda-7 exposure caused an additive increase in the vesicularization of a transfected LC3-GFP construct, an effect that was blocked by expression of dominant negative PERK (Fig. 3C).
We next investigated the role of autophagy in the lethal interaction between OSU-03012 and Ad.mda-7. Knockdown of ATG5 or Beclin1 expression inhibited Ad.mda-7- or OSU-03012-induced autophagy (data not shown). Knockdown of ATG5 or Beclin1 expression profoundly reduced the toxicity of Ad.mda-7, OSU-03012 or combined treatment with both agents in GBM cells (Fig. 3D). Collectively our findings in Figures 2 and and33 demonstrate that ER stress/autophagy signaling induced by combined Ad.mda-7 and OSU-03012 treatment plays a key role in the toxic interaction between these agents in primary human GBM cells.
One probable reason for our prior findings demonstrating that MDA-7/IL-24 is very effective in killing invasive GBM cells in vivo is that MDA-7/IL-24 is a secreted cytokine.35,36 As a secreted protein MDA-7/IL-24 has the potential to act on uninfected GBM cells, thereby causing tumor cell death in a larger number of tumor cells. Conditioned media from Ad.mda-7 infected GBM cells induced expression of MDA-7/IL-24 in uninfected GBM cells and enhanced the toxicity of ionizing radiation (Fig. 4A and B). Transfection of uninfected GBM cells with an siRNA to knock down MDA-7/IL-24 expression prevented conditioned media from Ad.mda-7 infected GBM cells from increasing MDA-7/IL-24 expression, as well as enhancing the toxicity of OSU-03012 or of ionizing radiation (Fig. 4B). Treatment of GBM cells with OSU-03012, 24 h following conditioned media transfer, generated a greater cytotoxic response than simultaneous exposure to conditioned media and OSU-03012 (Fig. 4C cf B). Expression of dominant negative PERK partially reduced the toxic ‘bystander’ effect of conditioned media containing MDA-7/IL-24 (Fig. 4D).
Previous studies have noted that GST-MDA-7 or Ad.mda-7 reduces proliferation and causes tumor cell-specific killing of malignant glioma cells via induction of a toxic form of autophagy. We have also previously shown that OSU-03012 (AR-12) kills GBM cells through induction of autophagy. The studies in this manuscript were designed to determine whether OSU-03012 and Ad.mda-7 could cooperate in killing GBM cells and if so; what mechanism(s) were involved in tumor cell killing.
Infection of GBM cells with a dose of Ad.mda-7 virus particles that caused modest levels of toxicity ~48 h after exposure correlated with activation of the JNK1/2 and p38 MAPK pathways and well as of PERK. In parallel this treatment suppressed ERK1/2 and to a lesser extent AKT signaling. Multiple studies using a number of toxic stimuli document that prolonged JNK1-3 and/or p38 MAPK activation in a wide variety of cell types can trigger cell death.37–43 It is also well established that the balance between the readouts of ERK1/2 and JNK1-3 signaling can represent a general key homeostatic mechanism that regulates cell survival versus cell death processes.37–43 Prior studies in primary human GBM cells using GST-MDA-7 have demonstrated that inhibition of ERK1/2 signaling represents a key pro-apoptotic signal generated by GST-MDA-7 exposure. In prior studies OSU-03012 at low concentrations and as a single agent did not significantly modulate the activities of any of the most widely examined signal transduction pathways, e.g., ERK1/2, JNK1/2, p38 MAPK or AKT.38,39 We found that OSU-03012 facilitated MDA-7IL-24-induced suppression of ERK1/2 signaling which in turn resulted in lower expression of the mitochondrial protective proteins MCL-1 and BCL-XL.
Infection of cells with Ad.mda-7 decreased the expression of BCL-XL and MCL-1, which we have previously demonstrated with GST-MDA-7 was due to ER stress signaling via PERK-eIF2α.27,28,43 Lower MCL-1 and BCL-XL expression not only facilitated mitochondrial dysfunction and release of cytochrome c into the cytosol, but as we have recently published, enhanced the induction of autophagy by untethering Beclin1.43–45 Freed Beclin1 in turn can interact with Vps34 to promote autophagy.44 We have also shown that MDA-7/IL-24-induced JNK pathway signaling mediated activation of the pro-apoptotic proteins BAX and BAK; OSU-03012 did not further increase JNK pathway signaling. Thus the MDA-7/IL-24-induced ratio change of pro- to anti-apoptotic proteins is exacerbated by inhibiting protective signaling pathways, leading to greater levels of tumor cell death.
Prior studies have demonstrated that GST-MDA-7 lethality or OSU-03012 lethality as single agents in GBM cells required the induction of a toxic form of autophagy and that this process was dependent on PERK signaling.27,28 A priori we hypothesized that if there was a less than additive lethal interaction between Ad.mda-7 and OSU-03012 our data would tend to argue that both agents were acting on a single molecule, e.g., PERK to promote autophagy and cell death. Alternatively, we considered that if Ad.mda-7 and OSU-03012 caused a greater than additive amount of cell killing then both agents had unique modes of action. Although OSU-03012 did not further enhance MDA-7/IL-24-induced PERK activation, both agents caused an additive increase in LC3-II levels and in the vesicularization of a transfected LC3-GFP construct. Inhibition of autophagy, either at the level of PERK or by knockdown of ATG5/Beclin1, blocked the lethal interaction between Ad.mda-7 and OSU-03012. BiP/GRP78 has been defined as a key target for MDA-7/IL-24, with MDA-7/IL-24 binding to this chaperone resulting in its release from PERK, and activation of PERK.23 The precise molecular target for OSU-03012 is at present unclear. OSU-03012 was originally proposed to be an inhibitor of PDK-1, however, we have previously noted that in the concentration range of drug used in our studies AKT phosphorylation did not change.33,34 As OSU-03012 was shown to enhance HSP70 and decrease HSP90 expression, it is possible that a key mode of action for this drug may be to alter chaperone function, which will result in plieotropic drug effects on many different proteins including the ability to stabilize lysosomes and mitochondria. Further studies will be required to unravel the biological consequences and molecular basis of OSU-03012 actions on cancer cells.
GBM was one of the first malignancies considered to be treatable through viral delivery of genetic-based therapeutics.45 A problem of efficacy, however, exists for all gene therapy approaches, and one that is intensified by the highly invasive and diffuse nature of GBM compared to other tumor cell types. This problem highlights the need for development of a toxic ‘bystander’ effect in tumor cells that have not been infected by virus during the primary infection process. By the rules of simple mass-action, i.e., the total number of non-transformed cells within and around a GBM tumor compared to the total number of transformed cells in a tumor to the total number of virus particles being infused, it is not possible for all tumor cells in a highly invasive tumor cell type such as GBM to be infected by a non-replicative, and in all likelihood even a conditionally replicative, adenovirus. Furthermore, many prior studies in GBM using gene therapeutic vectors have often expressed intracellular proteins that are not normally expressed or secreted, which will frequently result in only those cells that have been virally infected being subjected to the actions of the therapeutic agent. The expression of MDA-7/IL-24 overcomes the limitation associated with lack of a ‘bystander’ effect following gene therapeutic intervention in the majority of previous studies.35,36 We found that MDA-7/IL-24 is secreted from infected GBM cells and media containing secreted MDA-7/IL-24 induced apoptosis in uninfected GBM cells and promoted the toxicity of either OSU-03012 or ionizing radiation.
In conclusion, the data in this manuscript demonstrates that MDA-7/IL-24 interacts with OSU-03012 to enhance killing of primary human GBM cells in a greater than additive manner. Our data also indicates that the use of two (or more) agents that increase autophagy will facilitate GBM cell apoptosis. Since both MDA-7/IL-24 and OSU-03012 are currently undergoing evaluation in the clinic for patients with diverse cancers, future studies combining these agents, assuming no or limited toxicity will be evident, offers potential for developing improved therapies for GBM and possibly other cancers.
Phospho-/total-ERK1/2, Phospho-/total-JNK1-3, Phospho (S473)-/total-AKT, Phospho-/total-p38 MAPK, antibodies were purchased from both Cell Signaling Technologies (Worcester, MA) and from Santa Cruz Biotechnology (Santa Cruz, CA). Trypsin-EDTA, DMEM and RPMI medium, and penicillin-streptomycin were purchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island, NY). Dr. C.D. James, (UCSF) very generously originally supplied primary human GBM cells (GBM6, GBM12, GBM14) and information on the genetic background of such cells. Dr. S. Spiegel (VCU) supplied the plasmid to express LC3-GFP. Other reagents were of the highest quality commercially available.27,28
All GBM lines were cultured at 37°C 5% (v/v CO2) in vitro using RPMI supplemented with 5% (v/v) fetal calf serum and 10% (v/v) Non-essential amino acids. For short-term cell killing assays and immunoblotting, cells were plated at a density of 3 × 103 per cm2 and were treated with the various drugs, as indicated. In vitro small molecule inhibitor treatments were from a 100 mM stock solution of each drug and the maximal concentration of Vehicle (DMSO) in media was 0.02% (v/v). For adenoviral infection, cells were infected 12 h after plating and the expression of the recombinant viral transgene allowed to occur for 12 h prior to any additional experimental procedure. Cells were not cultured in reduced serum media during any study.27,28
Cells were treated with various viral multiplicities of infection, as indicated in the Figure legends. For SDS PAGE and immunoblotting, cells were lysed in either a non-denaturing lysis buffer, and prepared for immunoprecipitation as described in ref.34 or in whole-cell lysis buffer (0.5 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 0.02% bromophenol blue), and the samples were boiled for 30 min. After immunoprecipitation, samples were boiled in whole cell lysis buffer. The boiled samples were loaded onto 10–14% SDS-PAGE and electrophoresis was run overnight. Proteins were electrophoretically transferred onto 0.22 μm nitrocellulose, and immunoblotted with indicated primary antibodies against the different proteins.27,28
We generated and purchased previously noted recombinant adenoviruses to express constitutively activated and dominant negative AKT and MEK1 proteins, dominant negative caspase 9, XIAP, c-FLIP-s, CRM A and BCL-XL (Vector Biolabs, Philadelphia, PA). Cells were infected with these adenoviruses at an approximate m.o.i. of 50. Cells were incubated for 24 h to ensure adequate expression of transduced gene products prior to drug exposures.
Cells were harvested by trypsinization with Trypsin/EDTA for ~10 min at 37°C. As some apoptotic cells detached from the culture substratum into the medium, these cells were also collected by centrifugation of the medium at 1,500 rpm for 5 min. The pooled cell pellets were resuspended and mixed with trypan blue dye. Trypan blue staining, in which blue dye incorporating cells were scored as being dead, was performed by counting of cells using a light microscope and a hemacytometer. Five hundred cells from randomly chosen fields were counted and the number of dead cells was counted and expressed as a percentage of the total number of cells counted. For confirmatory purposes the extent of apoptosis was evaluated by assessing Hoechst and TUNEL stained cytospin slides under fluorescent light microscopy and scoring the number of cells exhibiting the “classic” morphological features of apoptosis and necrosis. For each condition, 10 randomly selected fields per slide were evaluated, encompassing at least 1,500 cells. Alternatively, the Annexin V/propidium iodide assay was carried out to determine cell viability as per the manufacturer’s instructions (BD PharMingen) using a Becton Dickinson FACScan flow cytometer (Mansfield, MA).27,28
Plasmid DNA (0.5 μg/total plasmid transfected) was diluted into 50 μl of RPMI growth media that lacked supplementation with FBS or with penicillin-streptomycin. Lipofectamine 2000 reagent (1 μl) (Invitrogen, Carlsbad, CA) was diluted into 50 μl growth media that lacked supplementation with FBS or with penicillin-streptomycin. The two solutions were then mixed together and incubated at room temperature for 30 min. The total mix was added to each well (4-well glass slide or 12-well plate) containing 200 μl growth media that lacked supplementation with FBS or with penicillin-streptomycin. The cells were incubated for 4 h at 37°C, after which time the media was replaced with RPMI growth media containing 5% (v/v) FBS and 1x pen-strep.
Where indicated, 12 h after transfection LC3-GFP transfected cells were infected with either Ad.cmv or Ad.mda-7 and cultured for 24 h. Cells were then stained with Lysotracker Red Dye (Invitrogen) at the indicated time points for 20 min. Lysotracker Red Dye stained cells were visualized immediately after staining on a Zeiss Axiovert 200 microscope using the rhodamine filter. LC3-GFP transfected cells were visualized at the indicated time points on the Zeiss Axiovert 200 microscope using the FITC filter.
Comparison of the effects of various treatments was performed using one way analysis of variance and a two tailed Student’s t-test. Differences with a p-value of <0.05 were considered statistically significant. Experiments shown are the means of multiple individual points from multiple experiments (±SEM).
Support for the present study was provided; to P.D. from PHS grants (P01-CA104177, R01-CA108325, R01-DK52825), The Jim Valvano “V” foundation, and Department of Defense Award (DAMD17-03-1-0262); to S.G. from PHS grants (R01-CA63753; R01-CA77141) and a Leukemia Society of America grant 6405-97; to P.B.F. from PHS grants (P01-CA104177, R01-CA097318; R01-CA134721; P01-NS031492), The Samuel Waxman Cancer Research Foundation (SWCRF); to D.T.C. from PHS grant (P01-CA104177). P.D. is The Universal Inc., Professor in Signal Transduction Research. D.S. is a Harrison Scholar in Cancer Research in the VCU Massey Cancer Center, VCU, School of Medicine. P.B.F. holds the Thelma Newmeyer Corman Chair in Cancer Research at the VCU Massey Cancer Center, VCU, School of Medicine a SWCRF Investigator.