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Cancer Research 66, 11869, December 15, 2006. doi: 10.1158/0008-5472.CAN-06-1887
© 2006 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

N-Glycosylation of MDA-7/IL-24 Is Dispensable for Tumor Cell–Specific Apoptosis and "Bystander" Antitumor Activity

Moira Sauane1, Pankaj Gupta1, Irina V. Lebedeva1, Zao-zhong Su1, Devanand Sarkar1,2, Aaron Randolph4, Kristoffer Valerie4, Rahul V. Gopalkrishnan1,2 and Paul B. Fisher1,2,3

Departments of 1 Urology, 2 Pathology, and 3 Neurosurgery, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, New York; and 4 Department of Radiation Oncology and Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia

Requests for reprints: Paul B. Fisher, Departments of Pathology and Urology, Columbia University Medical Center, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032. Phone: 212-305-3642; Fax: 212-305-8177; E-mail: pbf1{at}columbia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Biochemical and genetic mutation–based analyses confirm that the MDA-7/IL-24 protein can induce transformed cell–specific apoptosis through a mechanism involving endoplasmic reticulum (ER) stress–associated pathways. Covalent modifications by N-linked glycans in the ER contribute to the conformational maturation and biological functions of many proteins. Because MDA-7/IL-24 is a glycosylated protein, we investigated the role of glycosylation in mediating the specific biological and "bystander" antitumor activities of this cytokine. An adenovirus vector expressing a nonsecreted and nonglycosylated version of MDA-7/IL-24 protein was generated via deletion of its signal peptide and point mutations of its three N-glycosylated sites. In this study, we showed that this intracellular nonglycosylated protein was as effective as wild-type MDA-7/IL-24 protein in inducing apoptosis in multiple tumor cell lines. Both constructs (a) displayed transformed cell specificity and localization to the ER compartment, (b) mediated apoptosis through JAK/STAT-independent and p38MAPK-dependent pathways, (c) induced sustained ER stress as evidenced by expression of ER stress markers (BiP/GRP78, GRP94, XBP-1, and eIF2{alpha}), and (d) generated proteins that physically interacted with BiP/GRP78. Additionally, an expression construct containing the mda-7/IL-24 signal peptide linked to the mutated nonglycosylated mda-7/IL-24 gene retained the ability to induce bystander antitumor activity. These studies reveal that MDA-7/IL-24 glycosylation is not mandatory for inducing cell death or bystander activities in different cancer cells, providing new insights into the mechanism by which MDA-7/IL-24 induces apoptosis and ER stress. (Cancer Res 2006; 66(24): 11869-77)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A novel tumor cell–specific apoptosis-inducing gene, melanoma differentiation-associated gene 7 (mda-7), was identified by subtraction hybridization from human melanoma cells induced to growth arrest and terminally differentiate by treatment with fibroblast IFN and mezerein (1, 2). Initial studies confirmed that the expression of this gene correlated with the induction of irreversible growth arrest, cancer reversion, and terminal differentiation in human melanoma cells (1, 2). Additional investigations by our group and others have confirmed its gene therapeutic potential in other human cancers, including malignant glioma and breast, prostate, and ovary cancer–derived cells (3–7). Several independent studies showed that a majority of human cancer–derived cell lines, including prostate, breast, cervical, lung, fibrosarcoma, colorectal, melanoma, and glioblastoma, undergo apoptosis when exposed to mda-7/IL-24 (3–7). In contrast, no significant growth-inhibitory effect occurred when this gene was transduced into normal human breast or prostate epithelial, endothelial, melanocyte, astrocyte, or fibroblast cells (3–7). This property of mda-7/IL-24 suggested translational potential for the gene-based therapy of multiple cancers. Moreover, based on preclinical cell culture and animal modeling studies, successful phase I trials have now been done and a phase II clinical trial is in preparation (6–9). MDA-7/IL-24 has been delivered to cells, tumor xenografts, and patient tumors in clinical trials via a nonreplicating adenovirus (Ad.mda-7). These studies are contributing significantly to our understanding of the underlying basis of mda-7/IL-24 activity. Elucidation of the mechanistic basis of the selective antitumor action of this novel cytokine will provide valuable insights ensuring safe use, improving efficacy, identifying potential pharmacologic adjuvants or substitutes including small molecule mimetics, and possibly uncovering important additional information for developing enhanced therapeutic applications (4).

Structural and motif sequence homology, in addition to functional conservation and chromosomal localization, indicated that mda-7 belongs to the IL-10 gene family of cytokines and has therefore been designated IL-24 (2, 4–7, 10–14). We previously showed that mda-7/IL-24's cancer cell–specific activity could occur through mechanisms independent of binding to its currently recognized cognate receptors and might even occur independent of receptor function (15). Follow-up studies assessed whether the potent proapoptotic activity observed with Ad.mda-7 was due to intracellular or secreted MDA-7/IL-24 protein (16). We confirmed that Ad.mda-7 infection of cancer cells promoted endoplasmic reticulum (ER) stress and showed that mda-7/IL-24-mediated apoptosis could be triggered through an intracellular mechanism (confirmed by deletion of the signal peptide of the mda-7/IL-24 sequence) and occurred efficiently in the absence of protein secretion (16). A potential intracellular mode of killing was further confirmed using a bacterially expressed and purified GST-MDA-7 fusion protein (17).

Most secreted proteins of eukaryotic cells enter the secretory pathway through the translocation channel at the membrane of the ER (18). The lumen of the ER is the site where translocated proteins assume their secondary structure and where the assembly of oligomeric complexes occurs. It is also where cotranslational and posttranslational modifications occur. Once proteins acquire their fully folded native conformation, they can proceed in the secretory pathway. Many of the proteins that fold in the ER are covalently modified by the cotranslational addition of N-linked glycans that contribute not only to their conformational maturation but also to their multiple biological functions (19, 20). In fact, glycans provide polar surface groups, thus enhancing the solubility and preventing the aggregation of the polypeptide, on one hand, and enabling the nascent glycoproteins to interact with a number of ER-resident chaperones, on the other (18, 19).

The mRNA encoding mda-7/IL-24 is ~2 kb in length, generating a predicted protein of 23.8 kDa belonging to the four-helix bundle family of cytokine molecules (2, 12). The open reading frame encodes a molecule that is 206 amino acids in length, which is a precursor form of the ultimate cleaved, posttranslationally processed and secreted mature product. There are three consensus asparagine glycosylation residues at amino acids 85, 99, and 126, which are N-glycosylated, resulting in a mature secreted product showing multiple bands on denaturing protein gel electrophoresis. This is likely due to partial and complete sugar modification on the available N-glycosylation sites (5, 14).

The present study was designed to assess the relevance of MDA-7/IL-24 glycosylation and secretion in mediating cancer-selective cell-killing and "bystander" antitumor activity. Our results indicate that MDA-7/IL-24 glycosylation is not mandatory for cell death or bystander antitumor activity in cancer cells. Furthermore, we document that mda-7/IL-24 lacking the signal peptide (SP–mda-7) and missing both the signal peptide and the three N-glycosylation sites (SP–gly–.mda-7) are indistinguishable from wild-type MDA-7/IL-24 protein in specifically up-regulating BiP/GRP78, GRP94, XBP-1, and phosphorylation of eIF2{alpha}, and they induce the same signal transduction pathways.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, adenoviruses, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assays, fluorescence-activated cell sorting analysis, and cell counting. DU-145, U231, T47D, A549, HBL-100, HO-1, and C8161 cells were obtained from the American Type Culture Collection (Rockville, MD). HO-1 cells were originally obtained from Dr. B. Giovanella (Stehlin Foundation, Houston, TX). FM516-SV immortalized normal human melanocytes (referred to as FM-516) have been previously described (21). The human fibrosarcoma 2fTGH and its derivative cell lines were provided by Dr. G. Stark (Cleveland Clinic, Cleveland, OH; ref. 16), and an immortalized normal human prostate epithelial cell line (P69; ref. 22) was provided by Dr. J. Ware (MCV, Richmond, VA). Primary human fetal astrocytes were established as previously described and used between passage 3 and 6 (23). The culture and maintenance of cells, and construction, propagation, and utilization of adenoviruses have been previously described (16). For the present studies, three mda-7/IL-24 cDNAs were engineered in replication-incompetent adenoviruses, Ad.mda-7 (encoding a full-length mda-7/IL-24 cDNA), Ad.SP–mda-7 (encoding a full-length mda-7/IL-24 cDNA lacking the sequence encoding the signal peptide), and Ad.SP–gly–.mda-7 (encoding an mda-7/IL-24 cDNA in which all three N-glycosylation sites were mutated and the signal peptide encoding sequence was deleted; Fig. 1A ). The SP–gly–.mda-7 mutant was generated using a PCR mutagenesis strategy wherein positions 85, 99, and 126 of the MDA-7/IL-24 peptide (SwissProt accession no. Q13007) were mutated from asparagine to glutamine residues so as to prevent N-glycosylation of the mutated protein. The triple-mutated MDA-7/IL-24 protein, deleted for the signal peptide was designated SP–gly–.mda-7. The mutated cDNA was cloned into a nonreplicating adenoviral vector, Ad.SP–gly–.mda-7. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, fluorescence-activated cell sorting analysis, and cell counts were done by standard protocols as previously described (16).


Figure 1
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  		Figure 1. Comparative growth inhibition, apoptosis induction, and MDA-7/IL-24 expression in cells infected with Ad.vec, Ad.mda-7, Ad.SP–mda-7, and Ad.SP–gly–.mda-7. A, expression vectors and adenoviruses encoding wild-type and mutant MDA-7/IL-24. Schematic representation of gene constructs incorporated into the pREP4 expression construct or used to generate recombinant adenoviruses. The full-length mda-7/IL-24 construct encodes a 206 amino acid protein with a 48 amino acid signal peptide and three N-glycosylation sites at amino acid positions 85, 99, and 126 in the protein. The first three constructs were used to make both pREP4 expression vectors and adenoviruses, and the fourth construct was cloned into a pREP4 expression vector. B, growth inhibition in different tumor cell lines. Cells were infected with 100 pfu/cell of Ad.vec, Ad.mda-7, Ad.SP–mda-7, or Ad.SP–gly–.mda-7 and cell viability was determined by the MTT proliferation assay 5 days after infection. Top, numbers represent a ratio of specific treatments indicated versus untreated cells; columns, average of three independent experiments; bars, ±SD. Bottom, apoptosis induction in cancer cell lines: cells were treated as described in above and the percentage of the cells displaying hypodiploidy (Ao), a measure of apoptosis, was determined 24 hours later by fluorescence-activated cell sorting analysis using the CellQuest software (Becton Dickinson, Mountain View, CA) as described in (ref. 15). C, MDA-7/IL-24 protein and mda-7/IL-24 mRNA expression in DU-145 cells. Protein lysates were collected from uninfected (control) DU-145 cells and after infection with Ad.vec, Ad.mda-7, Ad.SP–mda-7, or Ad.SP–gly–.mda-7. Samples (50 µg) were run on 12% SDS-PAGE, transferred to a nitrocellulose membrane and stained with rabbit anti-mda-7/IL-24 antibody as described in Materials and Methods. Total RNA was prepared and the expressions of mda-7/IL-24 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs were determined by Northern blotting analysis. D, protein glycosylation. Protein extracts from Ad.mda-7, Ad.SP–mda-7, or Ad.SP–gly–.mda-7–infected cells were untreated or treated with glycopeptidase F (glyco F) and then evaluated by Western blot using anti-MDA-7/IL-24 antibody.

 
Coimmunoprecipitation of BiP/GRP78 with MDA-7/IL-24. Cells were infected with Ad.vec. or Ad.mda-7 or transfected with Flag-tagged MDA-7/IL-24 and Myc-tagged BiP/GRP78. After 48 hours, cells were rinsed with ice-cold PBS and lysed in 1 mL of immunoprecipitation buffer containing 25 mmol/L of Tris-Cl (pH 8.0), 137 mmol/L of NaCl, 2.5 mmol/L of KCl, 1% Triton X-100, and protease inhibitor cocktail (Roche, Nutley, NJ). The samples were centrifuged at 4°C for 10 minutes at 13,000 rpm and the supernatants were incubated with 10 µL of 50% protein agarose at 4°C for 1 hour to eliminate nonspecific interactions. Samples were centrifuged and mixed with anti-BiP/GRP78 or 9E10 anti-Myc monoclonal antibodies (1:200 dilution; Sigma, St. Louis, MO) and rotated overnight at 4°C. Immunocomplexes were precipitated with 25 µL of 50% protein A agarose for 2 hours. The immunoprecipitates were washed very gently with the immunoprecipitation buffer thrice, resuspended in 50 µL of 10 mmol/L Tris-Cl (pH 8.0) and 1 mmol/L EDTA, and resolved by SDS-PAGE. Western blot analysis was done as previously described (15–17) using the following primary antibodies at 1:1,000 dilutions: anti-MDA-7/IL-24, anti-Flag M2, anti-BiP/GRP78, and anti-Myc. Secondary antibodies specific for heavy chains of IgG were used as the light chain of IgG interfered with the detection of MDA-7/IL-24 because of its similar size.

Northern blotting analyses. Fifteen micrograms of total RNA was denatured, electrophoresed in a 1.2% agarose gel with 3% formaldehyde, and transferred onto a nylon membrane. The blots were probed with an {alpha}-32P[dCTP] full-length human mda-7/IL-24 cDNA probe, then stripped, and reprobed with an {alpha}-32P[dCTP] human gapdh probe. Following hybridization, the filters were washed and exposed for autoradiography.

Western blot analyses. Cell lines were grown on 10 cm plates and protein extracts were prepared with radioimmunoprecipitation assay buffer containing a cocktail of protease inhibitors. Fifty micrograms of protein were applied to a 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with polyclonal antibodies to MDA-7/IL-24, EF1{alpha}, BiP/GRP78, calnexin, calreticulin, GRP94, XBP-1, phospho-JNK, phospho-p38MAPK and total PKR, JNK, and p38 antibodies.

Bystander tumor cell growth inhibition assay. Normal immortal P69 prostate epithelial cells were seeded at 2 x 105/6 cm dish and transfected with 30 µg of the indicated expression plasmid. The expression plasmids included pREP4 (control vector plasmid), mda-7 (full-length mda-7/IL-24), SP–.mda-7 (mda-7/IL-24 missing the signal peptide), SP–.gly–.mda-7 (N-glycosylation mutant mda-7/IL-24 gene lacking the signal peptide), or gly–.mda-7 (N-glycosylation mutant of mda-7/IL-24 containing the signal peptide; Fig. 1A). After 24 hours, cells were washed five times with PBS and overlaid with 6 mL of 0.4% Nobel agar containing 1 x 105 DU-145 cells. Following 14 days of incubation, during which overlay cells were re-fed every 4 days, macroscopic colonies ≥2 mm were scored. Colonies were enumerated from triplicate plates and values expressed as an average ±SD.

Immunofluorescence. Cells were seeded onto chamber slides (Falcon; BD Biosciences, San Jose, CA) and maintained in DMEM with 10% fetal bovine calf serum, 24 hours postinfection, cells were fixed with 2% paraformaldehyde, permeabilized by 0.1% Triton X-100, and then incubated with primary antibodies: rabbit anti-MDA-7/IL-24, GM130 (BD PharMingen, San Diego, CA), LAMP1/2 (Santa Cruz Biotechnology, Santa Cruz, CA), calreticulin (BD PharMingen), and Mitrotrack marker (Molecular Probes, Eugene OR). Controls were incubated with only the secondary antibodies under the same experimental conditions. FITC-conjugated donkey anti-mouse IgG or anti-rabbit IgG (Molecular Probes) were used for visualization on a Zeiss LSM 510 fluorescence microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparative growth inhibition and apoptosis induction in cancer cells infected with Ad.SP–mda-7, Ad.SP–gly–.mda-7 and Ad.mda-7. Initial studies determined if infection with an adenovirus expressing a nonglycosylated and nonsecreted version of MDA-7/IL-24 protein (Ad.SP–gly–.mda-7) produced growth suppression (loss of viability) and apoptosis in tumor cells in a manner analogous to that observed using a full-length MDA-7/IL-24 protein (Ad.mda-7) or a nonsecreted form of the full-length MDA-7/IL-24 protein (Ad.SP–mda-7; Fig. 1A and B; ref. 16). Parallel experiments were done with normal primary human fetal astrocytes, FM-516, P69, and HBL-100 to substantiate potential differential susceptibility to these viruses. These experiments confirmed that infection with Ad.SP–gly–.mda-7, Ad.SP–mda-7, or Ad.mda-7 induced equivalent growth suppression and decreases in viability in susceptible tumor cell lines (C8161, DU-145, U231, and T47D), without affecting the viability of comparable normal cells (Fig. 1B, top).

We next determined the mechanism by which Ad.SP–gly–.mda-7 induced a reduction in growth and decreased survival in cancer cells. For this evaluation, we also included Ad.mda-7 and Ad.SP–mda-7. Infection with all three viruses induced a similar increase in the proportion of tumor cells undergoing apoptosis as reflected by an increase in the percentage of cells with a sub-G0/G1 (A0) DNA content (Fig. 1B, bottom). In contrast, no significant change in the percentage of apoptotic cells was found following infection of primary human fetal astrocytes, FM-516, P69, and HBL-100 with any of the viruses (Fig. 1B, bottom).

Based on its structure, i.e., absence of a signal peptide, infection with Ad.SP–gly–.mda-7 is predicted to produce an N-glycosylated mutated MDA-7/IL-24 protein in target cells without promoting secretion. Experiments were done to confirm this property and to determine the relative levels of mda-7/IL-24 mRNA and MDA-7/IL-24 protein produced following infection with Ad.SP–gly–.mda-7, Ad.SP–mda-7, and Ad.mda-7. Northern blot analysis confirmed that comparable levels of MDA-7/IL-24 mRNA were produced following infection with Ad.SP–gly–.mda-7, Ad.SP–mda-7, and Ad.mda-7 (Fig. 1C, bottom right). Additionally, DU-145 cells were infected with the three viruses and the levels of MDA-7/IL-24 proteins in the supernatants and pellets quantified by Western blotting 24 hours postinfection (Fig. 1C). Intracellular protein was observed in extracts of DU-145 cells infected with Ad.SP–gly–.mda-7, Ad.SP–mda-7, or Ad.mda-7. In contrast, secreted MDA-7/IL-24 protein was only detected in the supernatants from Ad.mda-7-infected cell lines at 24, 48, and 72 hours postinfection (Fig. 1C; data not shown). Intracellular MDA-7/IL-24 proteins expressed by Ad.SP–gly–.mda-7 and Ad.SP–mda-7 differed from the wild-type Ad.mda-7 expressed protein. In Ad.SP–gly–.mda-7- and Ad.SP–mda-7-infected cells, a single protein of 17 or 23 kDa, respectively, was detected, whereas a series of higher molecular weight species were apparent in Ad.mda-7-infected cells, most likely representing posttranslationally processed forms of the MDA-7/IL-24 protein. As predicted, treatment of cell extracts with the enzyme N-glycosidase F, which removes N-linked oligosaccharides, reduced the molecular mass of the 32, 30, 27, and 23 kDa MDA-7/IL-24 proteins to ~17 kDa (Fig. 1D). Treatment with N-glycosidase F reduced the molecular mass of the MDA-7/IL-24 proteins expressed by Ad.SP–mda-7 from 23 to 17 kDa. This 17 kDa species seems to represent the completely deglycosylated form of the MDA-7/IL-24 protein, which does not change following treatment with N-glycosidase F (Fig. 1D).

Comparative activation of signal transduction pathways in cells infected with Ad.SP–mda-7, Ad.mda-7, and Ad.SP–gly–.mda-7. Activation of p38MAPK and up-regulation of PKR, in particular, cancer cells upon Ad.mda-7 infection has been shown to be essential in mediating mda-7/IL-24-induced apoptosis in specific tumor cells (24–26). Based on this consideration, we determined if p38MAPK or PKR activation might also play a role in mda-7/IL-24-induced killing in Ad.SP–gly–.mda-7-infected cells. DU-145 cells were uninfected or infected with the different viruses and analyzed by SDS-PAGE followed by Western blotting with anti-phospho-JNK, anti-JNK, anti-phospho-p38MAPK, anti-p38MAPK, and anti-PKR antibodies. Treatment with Ad.SP–gly–.mda-7, Ad.SP–mda-7, or Ad.mda-7 promoted p38MAPK phosphorylation in DU-145 cells, whereas it did not affect total p38MAPK (Fig. 2A ). Moreover, the specific p38MAPK inhibitor, SB203580, partially blocked the killing effect of Ad.SP–gly–.mda-7, indicating that p38MAPK is a contributing signaling pathway regulating cell growth and viability (Fig. 2B). Infection of DU-145 cells with the three different adenoviruses expressing mda-7/IL-24 did not alter the total levels or phosphorylation of JNK, whereas PKR expression was enhanced. Further studies are being done to define the relevance of PKR activation by the different viruses expressing variant forms of MDA-7/IL-24. These results document that all three variant mda-7/IL-24-expressing viruses similarly modulate defined and parallel signal transduction pathways in DU-145 cells, i.e., activation and up-regulation of p38MAPK and PKR, respectively. Additionally, as documented previously for Ad.mda-7, the Ad.SP–gly–.mda-7 virus was capable of decreasing viability in cells deficient in JAK/STAT signaling, further demonstrating the functional equivalence of these viruses (Fig. 3C ).


Figure 2
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  		Figure 2. p38MAPK and PKR activation following MDA-7/IL-24 expression in DU-145 cells. A, activation of the p38MAPK and PKR pathway was determined by Western blotting using total and phosphospecific antibodies 24 hours postinfection with 100 pfu/cell of the different viruses. Additionally, the levels of total and phospho-JNK were determined by Western blotting after viral infection. B, effect of p38MAPK inhibitor on mda-7/IL-24-induced killing in prostate cancer cell lines. Cells were incubated in the absence or presence of SB203580 (5 µmol/L) after infection with 100 pfu/cell of Ad.vec, Ad.SP–gly–.mda-7, or Ad.mda-7. Cell viability was determined by MTT assay 6 days after infection. MTT absorbance of untreated control cells was set to 1 to determine the relative number of viable cells. Columns, average of three independent experiments; bars, ± SD. C, apoptotic activity after Ad.vector, Ad.SP–gly–.mda-7, Ad.SP–mda-7, or Ad.mda-7 infection in JAK/STAT-deficient cell lines. The cell lines indicated were infected with 150 pfu/cell of Ad.vector, Ad.SP–gly–.mda-7, Ad.SP–mda-7, or Ad.mda-7. Cells were analyzed for cell viability by MTT assay 5 days after infection. MTT absorbance of untreated control cells was set to 1 to determine the relative number of viable cells. Results shown are an average of three independent experiments.

 

Figure 3
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  		Figure 3. Localization of the mutated N-glycosylation-deficient MDA-7/IL-24 protein after infection with Ad.SP–gly–.mda-7. A, MDA-7/IL-24 protein localization was analyzed by indirect immunofluorescence after infection of DU-145 FM-516, C8161, or HO-1 cells with 100 pfu/cell of Ad.SP–gly–.mda-7 or Ad.vec. Forty-eight hours postinfection, cells were fixed and MDA-7/IL-24 protein was detected by indirect immunofluorescence using anti-MDA-7/IL-24 antibody. Images of ER were obtained using anticalreticulin, as described in Materials and Methods. Images of the different compartments and MDA-7/IL-24 were merged. B, MDA-7/IL-24 regulates the levels of specific chaperone protein expression. Cells were infected with Ad.vec, Ad.mda-7, or Ad.SP–gly–.mda-7 and protein changes in BiP/GRP78, calnexin, calreticulin, GRP94, XBP-1, total eIF2{alpha}, and p-eIF2{alpha} were evaluated using Western blot analyses. C, MDA-7/IL-24 and SP–.gly–.MDA-7/IL-24 proteins bind to BiP/GRP78. Coimmunoprecipitation of MDA-7/IL-24 protein with BiP/GRP78 protein. DU-145 cells were infected with 100 pfu/cell of Ad.vec, Ad.mda-7 or Ad. SP–.gly–mda-7 and immunoprecipitation analysis was done 48 hours later using BiP/GRP78 antibodies.

 
Localization of N-glycosylation mutant MDA-7/IL-24 protein to the ER/Golgi compartments. We previously reported that MDA-7/IL-24 localizes in the ER subcellular compartment after infection with Ad.mda-7 or Ad.SP–mda-7 (16, 17). Experiments were done to determine if retention of the N-glycosylation sites contribute to this subcellular localization. DU-145, FM-516 (normal SV40-immortalized human melanocyte), C8161 (metastatic melanoma), and HO-1 (metastatic melanoma) cells were infected with Ad.SP–gly–.mda-7 and subcellular localization of MDA-7/IL-24 protein was determined. The ER was specifically labeled with anticalreticulin antibody (red) and MDA-7/IL-24 protein was recognized by green immunofluorescence with a specific anti–MDA-7/IL-24 antibody. Because these signal-peptideless mutants of MDA-7/IL-24 protein do not contain an export signal, they are predicted to remain in the cytosol. However, confocal overlapping imaging results show that MDA-7/IL-24 protein (yellow staining) localizes primarily in the ER (Fig. 3A), indicating that a significant fraction of this protein is able to enter the ER and that proteins derived from wild-type and mutant virus seem to have overlapping patterns of localization within the cell. The possible mechanism of this localization might be (a) the presence of cryptic internalization signals, the identity of which are currently unknown, that become active in the absence of the actual signal peptide and/or (b) adenovirus infection producing relatively large amounts of protein that even in the absence of a specific targeting sequence possesses the ability to cross membranes and accumulate in the ER/Golgi due to charge and/or tertiary structure.

Regulation of ER chaperone protein levels after Ad.mda-7 or Ad.SP–gly–.mda-7 infection. Misfolded proteins are retained in the ER and induce an unfolded protein response (UPR) pathway (27). Calnexin and calreticulin are molecular chaperones of the ER that bind to newly synthesized glycoproteins through a lectin site specific for monoglucosylated oligosaccharides, whereas BiP/GRP78 and GRP94 depend on the presence of unfolded hydrophobic regions in proteins (27). To determine if chaperone proteins were activated after Ad.mda-7 or Ad.SP–gly–.mda-7 infection, we measured the steady-state levels of specific proteins (BiP/GRP78, calnexin, calreticulin, and GRP94) whose up-regulation frequently correlates with UPR. Additionally, we determined the phosphorylation of eIF2{alpha}, a key downstream event of the UPR that mediates inhibition of protein translation. In addition, we measured the levels of XBP-1 (X-box DNA-binding protein, a UPR-specific b-ZIP transcription factor; ref. 28) whose up-regulation and splicing stimulates the transcription of specific chaperones. Because the binding of misfolded proteins to calreticulin depends on the presence of monoglucosylated N-linked glycans, the analysis of this chaperone represents an indirect indicator of whether glycosylation is essential for mda-7/IL-24-induced apoptosis. Enhanced XBP-1, BiP/GRP78, and GRP94 protein levels were apparent at 1 or 2 days after infection with Ad.mda-7 or Ad.SP–gly–.mda-7, indicating selective modulation of specific chaperone proteins (Fig. 3B). In addition, Ad.mda-7 and Ad.SP–gly–.mda-7 induced equivalent phosphorylation of eIF2{alpha}.

Previous studies have documented that MDA-7/IL-24 directly interacts with the ER resident chaperone BiP/GRP78 (24). Because full-length MDA-7/IL-24, as well as SP–gly–MDA-7/IL-24 protein both localize in the ER upon infection with adenovirus, we investigated whether SP–gly–MDA-7/IL-24 also interacts with BiP/GRP78, as previously described for full-length MDA-7/IL-24 protein. This was the case because both MDA-7/IL-24 and SP–gly–MDA-7/IL-24 proteins coimmunoprecipitated with BiP/GRP78, demonstrating a physical interaction between these two molecules (Fig. 3C).

The bystander antitumor activity of MDA-7/IL-24 occurs independent of N-glycosylation. A prominent component of mda-7/IL-24 antitumor activity involves its' ability to induce a profound bystander antitumor effect. Previous studies have documented that the mechanism of the "antitumor bystander" effect partially differs from the mechanism of tumor cell–specific apoptotic effect mediated by Ad.mda-7 or GST-MDA-7/IL-24 in that bystander activity is dependent on the presence of canonical IL-20/IL-22 receptor complexes on target tumor cells, whereas intracellular killing is receptor-independent (29). Based on these considerations, we investigated whether N-glycosylation of MDA-7/IL-24 protein was mandatory for this activity. To determine if the nonglycosylated mutant form of the MDA-7/IL-24 protein could provoke a bystander effect on nonexpressing cells, a dual normal/tumor cell culture agar overlay diffusion protocol was employed (16, 29). For this assay, normal immortal human prostate epithelial (P69) cells (22), which are resistant to killing by MDA-7/IL-24, although serving as a source of production of this cytokine (16), were transfected with various expression plasmids followed by overlaying with agar containing susceptible DU-145 cells or resistant A549 lung carcinoma cells (Fig. 4 ). Using this strategy, transfection of P69 cells with full-length mda-7/IL-24 or an expression construct in which the signal peptide of mda-7/IL-24 was incorporated in the gly–.mda-7/IL-24 construct (gly–.mda-7; Fig. 1A) resulted in a reduction in both the number and size of DU-145 colonies growing in agar (and data not shown). In contrast, transfection with a control expression vector (not containing an insert) or expression vectors that produce full-length SP–MDA-7/IL-24 or SP–gly–.MDA-7/IL-24 protein that are not secreted did not alter the growth of DU-145 cells in the overlay medium. In the case of A549 cells, which do not contain a complete set of IL-20/IL-22 receptors necessary for bystander antitumor activity (29), transfection with the various expression constructs had no effect on DU-145 growth in suspension culture (Fig. 4B). These results confirm that N-glycosylation of the MDA-7/IL-24 protein is not required for direct bystander antitumor activity.


Figure 4
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  		Figure 4. Bystander suppression of anchorage-independent growth of DU-145 cells following adenovirus infection of P69 cells. Immortal normal P69 cells were seeded in 10 cm plates at a density of 4 x 106 cells per plate. The next day, the cells were transfected with various constructs as indicated by Lipofectamine, following the manufacturer's protocol (Invitrogen, Lipofectomune 2000 Reagent). After 24 hours, the transfected cells were trypsinized and re-seeded into 6 cm plates at a density of 5 x 105 cells per plate. The next day, the cells were overlaid with 8 mL of complete DMEM plus 0.3% agar suspended with DU-145 or A549 cells at a density of 1 x 105 cell per plate. After 6 to 8 days of incubation with an overlay of DMEM plus 0.3% agar every 2 to 3 days, colonies >2 mm in size were scored. Columns, averages of at least three plates; bars, SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A recent phase I clinical trial indicates that mda-7/IL-24, administered by adenovirus, is safe and induces significant clinical responses in patients with solid carcinomas and melanomas (6–9). These observations confirm the cancer therapeutic properties of this intriguing cytokine. They also mandate detailed studies to define the precise mechanism of action of mda-7/IL-24, and in particular, how this gene differentially affects cancer versus normal cells. This information is mandatory for developing approaches to more efficiently and effectively use this molecule in cancer gene therapy.

In the present study, we investigated the role of posttranslational modification of MDA-7/IL-24, specifically N-glycosylation, in mediating cancer-selective killing and bystander antitumor properties. In eukaryotic cells, a wide variety of secretory and membrane proteins have one or more N-linked glycans in their outer domains that contribute not only to their conformational maturation but also to their multiple biological functions (18–20). Although it is established that MDA-7/IL-24 has three N-linked glycosylation sites and exists as variably sized protein products, presumably resulting from posttranslational modifications, the functional significance of these modifications in mediating various biological and biochemical properties of this cytokine are unknown. Using a series of viruses and expression vectors (Fig. 1A), we now show that N-glycosylation is not required to elicit specific biological and biochemical effects in tumor cells. We confirm that a mutant MDA-7/IL-24 protein lacking N-glycosylation retains the same localization in the ER/Golgi compartment, stimulates the same signal transduction pathways (i.e., p38MAPK and PKR), and enhances the expression of the same chaperone proteins (i.e., BiP/GRP78, GRP94 and XBP-1) as does the wild-type MDA-7/IL-24 protein. Moreover, when secretion of the mutant N-glycosylated MDA-7/IL-24 protein occurs by adding an mda-7/IL-24 signal peptide to the gly–.mda-7 cDNA (gly–.mda-7), a similar antitumor bystander activity is observed as with the secreted unmodified MDA-7/IL-24 protein (mda-7). These data raise the intriguing question as to what the actual role of N-glycosylation is in mediating MDA-7/IL-24 action? Perhaps these modifications are necessary to induce a subset of MDA-7/IL-24 functions, such as angiogenesis inhibition and/or immunomodulation. In particular, N-glycosylation of MDA-7/IL-24 may play a role in regulating molecular stability, in vivo activity, and/or immune regulatory properties. Further studies are required to address these questions.

A unique observation for a cytokine is that intracellular MDA-7/IL-24 protein is active in inducing transformed cell–specific apoptosis in a STAT1- and STAT3-independent manner (16). Additionally, multiple studies now indicate that cancer-selective apoptosis induction by MDA-7/IL-24 can involve changes in multiple signal transduction pathways and ER stress may play a pivotal role in this process (15–17, 24–26). In the present study, we document up-regulation of both p38MAPK and PKR, but not JNK, in DU-145 cells by both full-length and mutated N-glycosylation deficient MDA-7/IL-24. This observation supports a similar mode of intracellular signal transduction pathway changes leading to apoptosis induction by these two versions of MDA-7/IL-24.

A highly conserved UPR signal transduction pathway is activated by ER stress caused by misfolded protein accumulation (30–32). The UPR is characterized by the coordinated activation of multiple signal transduction pathways that lead to the suppression of the initiation step of protein synthesis, and trigger the expression of genes encoding ER chaperones, enzymes, and structural components of the ER (31). Prolonged activation of this pathway ultimately leads to apoptosis. The UPR can be triggered by unfolding proteins in the lumen of the ER, resulting in de novo synthesis of ER proteins (such as the "glucose-regulated proteins" BIP/GRP-78 and GRP94) that assist in protein folding. We now show that both N-glycosylated and unglycosylated MDA-7/IL-24 proteins are equally effective in inducing elevated levels of BiP/GRP78 and GRP94 in DU-145 cells. In contrast, no up-regulation of calnexin or calreticulin, which is dependent on the presence of both monoglucosylated N-linked glycans and unfolded regions on nascent glycoproteins, were observed in DU-145 cells infected with viruses expressing wild-type or N-glycosylated mutant MDA-7/IL-24 protein. Additionally, both wild-type and N-glycosylated mutant MDA-7/IL-24 enhanced XBP-1 expression and phosphorylation of eIF2{alpha}, providing a further link between the activity of MDA-7/IL-24 and induction of UPR and ER stress responses in cancer cells. These results indicate that specific ER stress responses, potentially mediated by up-regulation of BiP/GRP78 and GRP94, are elicited by MDA-7/IL-24, and similar changes occur when the protein is unglycosylated or glycosylated.

Several additional lines of evidence support the proposal that ER stress, generated by an UPR, is a major factor in eliciting tumor-specific apoptosis by MDA-7/IL-24. Ad.mda-7 infection in cancer cells induces the growth arrest and DNA damage inducible gene family, classically associated with the stress response, including the ER stress pathways (25). The induction of growth arrest and DNA damage genes and further upstream events such as activation of p38MAPK and PKR are promoted by mda-7/IL-24 in a transformed cell–specific manner and induction of these pathways now seem to occur independent of the glycosylation of MDA-7/IL-24. Additionally, treatment with Ad.mda-7 also specifically activates the p44/42MAPK pathway and produces an up-regulation of the inositol 1,4,5-trisphosphate receptor in H1299 cells (33). The inositol 1,4,5-trisphosphate receptor is an intracellular calcium–release channel implicated in apoptosis and localized in the ER. Earlier reports identified putative conserved functional HSP70-like chaperone (BiP/GRP78)–binding sites in both the helix C and F motifs of MDA-7/IL-24 (34). Recent studies from our laboratory show that mutation(s) of these sites in MDA-7/IL-24 prevent this cytokine from inducing cancer cell–specific apoptosis (24). Additionally, a microarray study indicated that mda-7/IL-24 is able to induce the expression of ER stress response genes, including BiP/GRP78 (35). Overall, these findings and our present study, suggest a series of events mediated by MDA-7/IL-24 that promote apoptosis, including up-regulation of specific signal transduction pathways and gene products involved in the ER stress response (Fig. 5 ).


Figure 5
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  		Figure 5. Proposed pathway of action of full-length and mutated versions of mda-7/IL-24 in inducing apoptosis in cancer cells. Schematic of the role of ER stress and activation of UPR by wild-type, signal peptide minus and signal peptide minus plus N-glycosylation mutated MDA-7/IL-24 in inducing apoptosis in DU-145 cells. Under normal conditions, IRE1 and PERK are bound to and inactivate BiP/GRP78. The accumulation of unfolded proteins leads to the release of BiP/GRP78, which is recruited to facilitate folding, and subsequent activation of IRE1 and PERK. As a result, the UPR is initiated, which involves both transcriptional activation of the ER stress-response genes and overall translational repression. IRE1, inositol-requiring enzyme 1; BiP/GRP78, glucose-regulated protein 78; PERK, PKR-like ER kinase; BAP, BiP-associated protein; ERdj3, stress-inducible ER DnaJ homologue; ROS, reactive oxygen species; GADDs, growth arrest and DNA damage inducible genes.

 
As recently emphasized, a significant component of mda-7/IL-24's therapeutic efficacy in vivo involves its ability to induce a potent bystander antitumor effect (8, 9, 36). Current studies support the hypothesis that this activity is mediated by a functional set of cell surface receptors consisting of IL-20R1/IL-20R2 and/or IL-22R1/IL-20R2 (29). Similarly, induction of an antiangiogenic effect by MDA-7/IL-24 also requires these receptors on endothelial cells in the tumor vasculature (37–39). The present study uncovers an interesting and potentially important phenomenon in which the lack of N-glycosylation of the MDA-7/IL-24 protein is not mandatory for antitumor bystander activity, although further studies are required to determine if this is also the case for its antiangiogenic or immunomodulatory activities.

In summary, the present studies document that N-glycosylation is not obligatory to induce a number of changes promoted by normally N-glycosylated MDA-7/IL-24, including cancer-selective growth suppression and apoptosis-induction, localization in the ER/Golgi compartment, up-regulation of p38MAPK and PKR, elevation in XBP-1, BiP/GRP78, and GRP94 chaperone protein levels, phosphorylation of eIF2{alpha}, and induction of bystander antitumor activity. These results provide additional support for the importance of ER stress and the UPR in mediating apoptosis-induction by MDA-7/IL-24 protein in cancer cells. Because a number of agents that synergize with MDA-7/IL-24 in inducing apoptosis in tumor cells, including radiation and chemotherapy, promote ER stress, the present studies suggest that combining MDA-7/IL-24 with agents promoting UPR may provide a means of enhancing the therapeutic applications of this gene for cancer therapy.


   Acknowledgments
 
Grant support: NIH/National Cancer Institute grants R01 CA097318, R01 CA098172, P01 CA104177, the Samuel Waxman Cancer Research Foundation, and the Chernow Endowment (P.B. Fisher), W81XWH-04-1-0433 (M. Sauane), and DAMD17-03-1-0290 (D. Sarkar).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Confocal studies were done at the Optical Microscopy Facility, a core facility of the Herbert Irving Comprehensive Cancer Center.


   Footnotes
 
Note: P.B. Fisher is a Michael and Stella Chernow Urological Cancer Research Scientist, and a Samuel Waxman Cancer Research Foundation Investigator.

Received 5/23/06. Revised 9/10/06. Accepted 10/10/06.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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