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[Cancer Research 66, 8182-8191, August 15, 2006]
© 2006 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

BiP/GRP78 Is an Intracellular Target for MDA-7/IL-24 Induction of Cancer-Specific Apoptosis

Pankaj Gupta1, Mark R. Walter4, Zao-zhong Su1, Irina V. Lebedeva1, Luni Emdad1, Aaron Randolph5, Kristoffer Valerie5, Devanand Sarkar1 and Paul B. Fisher1,2,3

Departments of 1 Pathology, 2 Neurosurgery, and 3 Urology, Herbert Irving Comprehensive Cancer Center, College of Physicians and Surgeons, Columbia University Medical Center, New York, New York; 4 Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama; and 5 Department of Radiation Oncology and Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia

Requests for reprints: Paul B. Fisher, Departments of Pathology and Urology, College of Physicians and Surgeons, Columbia University Medical Center, BB-1501, 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
 
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a unique member of the IL-10 gene family that induces cancer-selective growth suppression and apoptosis in a wide spectrum of human cancers in cell culture and animal models. Additionally, recent clinical trials confirm safety and document significant clinical activity of mda-7/IL-24 in patients with diverse solid cancers and melanomas. Despite intensive study the molecular basis of tumor-cell selectivity of mda-7/IL-24 is not well characterized. Using deletion analysis, a specific mutant of MDA-7/IL-24, M4, consisting of amino acids 104 to 206, is described that retains the cancer-specific growth-suppressive and apoptosis-inducing properties of the full-length protein. Employing rationally designed mutational analysis, we show that MDA-7/IL-24 and M4 physically interact with BiP/GRP78 through their C and F helices, localize in the endoplasmic reticulum, and activate p38 MAPK and GADD gene expression, culminating in cancer-selective apoptosis. These studies provide novel mechanistic insights into the discriminating antitumor activity of MDA-7/IL-24 by elucidating BiP/GRP78 as a defined intracellular target of action and present an unparalleled opportunity to develop improved therapeutic versions of this cancer-specific apoptosis-inducing cytokine. (Cancer Res 2006; 66(16): 8182-91)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is an intriguing multifunctional gene product that exhibits considerable potential as a gene therapy for cancer (reviewed in refs. 1–4). When administered by means of a replication-incompetent adenovirus (Ad.mda-7), growth suppression and apoptosis are induced in a broad spectrum of tumor cells both in vitro and in vivo in human tumor xenograft models, whereas no harmful effects are observed in normal cells (1, 2). In a phase I clinical trial involving intratumoral injection of Ad.mda-7 into advanced carcinomas and melanomas, this novel cytokine was found to be safe and showed profound tumor-specific apoptosis induction and significant clinical activity (1–6). Based on these provocative findings, further clinical trials are ongoing using mda-7/IL-24 for melanoma and other human cancers.

Studies in melanoma cells establish that Ad.mda-7 alters the ratio of proapoptotic to antiapoptotic proteins culminating in apoptosis, effects not observed in normal or immortal human melanocytes (7). Experiments investigating the mechanism underlying this differential apoptotic effect showed that Ad.mda-7 induced a family of growth arrest and DNA damage inducible (GADD) genes, GADD153, GADD45{alpha}, and GADD34, through p38 mitogen-activated protein kinase (MAPK) activation in melanoma cells but not in normal immortal melanocytes (8). Activation of the GADDs following infection with Ad.mda-7 has also been shown to occur selectively in human malignant glioma and prostate and ovarian carcinomas versus normal primary astrocytes, prostate epithelial cells, and mesothelial cells (9).6 These findings suggest that induction of these key molecules is essential for mda-7/IL-24-mediated apoptosis induction in specific cancer cells.

MDA-7/IL-24 is a 206-amino-acid residue protein with an NH2-terminal 48-amino-acid signal peptide resulting in a 158-amino-acid secreted cytokine, which is variably glycosylated (10, 11). Sequence analysis reveals that mda-7/IL-24 is a member of the class II cytokine family that includes IL-10, IL-19, IL-20, IL-22, IL-26, and IFN-{gamma} (12). In these contexts, mda-7/IL-24 is expected to adopt an {alpha}-helical structure (six {alpha}-helices labeled A-F) similar to the crystal structure of IL-10 (12, 13). Like other class II cytokines, MDA-7/IL-24 binds to cell-surface receptors (IL-20R1/IL-20R2 or IL-22R1/IL-20R2 heterodimers; refs. 14, 15) and activates the Janus-activated kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway (12, 14, 15). Consistent with its role as a cytokine, exogenously added MDA-7/IL-24 has been shown to induce apoptosis in cancer cells, which is dependent on the presence of its cognate cell-surface receptors (16, 17). However, using a series of tyrosine kinase inhibitors, a JAK-selective inhibitor, and cells defective in specific JAK/STAT signaling pathways, we showed that tyrosine kinase activation was not required for Ad.mda-7-induced apoptosis, suggesting that mda-7/IL-24 cancer–specific apoptotic activity was JAK/STAT independent (18). Further support for lack of a canonical cytokine mechanism of inducing cancer-specific apoptosis comes from studies using an adenovirus expressing a nonsecreted version of mda-7/IL-24 devoid of the signal peptide, Ad.SP-mda-7 (19). Ad.SP-mda-7 displayed comparable apoptosis-inducing activity as full-length Ad.mda-7 (19). These findings illustrate that mda-7/IL-24-mediated apoptosis can be triggered through an undefined intracellular mode of action as well as via secretion or by a combination of both processes (2, 16, 19).

Several lines of evidence suggest that mda-7/IL-24 intracellular-mediated apoptosis may involve endoplasmic reticulum signaling. First, localization studies employing Ad.mda-7 and Ad.SP-mda-7 indicate that the endoplasmic reticulum/Golgi compartment is a primary site of localization of MDA-7/IL-24 (19). Second, Ad.mda-7 induces GADD gene expression that is classically associated with "endoplasmic reticulum stress" responses (8, 9). Third, Ad.mda-7 infection of H1299 non–small-cell lung carcinoma cells leads to up-regulation of inositol triphosphate receptor, an endoplasmic reticulum–localized intracellular calcium release channel implicated in apoptosis (20).

Previous studies have documented that the class II cytokine IFN-{gamma} directly interacts with the endoplasmic reticulum–resident chaperone BiP/GRP78 (21). Based on amino acid sequence homology among the class II cytokines, mda-7/IL-24 also contains one or more putative BiP/GRP78 binding sites that could affect endoplasmic reticulum signaling responses. Because BiP/GRP78 is involved in binding unfolded polypeptides to promote folding into a three-dimensional structure, we reasoned it might be possible to identify a deletion mutant of mda-7/IL-24 that would activate endoplasmic reticulum signaling in a similar manner as wild-type mda-7/IL-24. Our studies now confirm the importance of the endoplasmic reticulum chaperone protein BiP/GRP78 as an intracellular target for MDA-7/IL-24 to subsequently activate the downstream targets p38 MAPK and GADD in mediating apoptosis selectively in cancer cells. We also identify a truncated version of MDA-7/IL-24, M4, consisting of amino acids 104 to 206 of the full-length protein, which retains BiP/GRP78 binding, localizes in the endoplasmic reticulum, and induces biochemical changes promoting growth suppression and apoptosis uniquely in tumor cells. The present studies provide additional support for a noncanonical intracellular mode of apoptosis induction by the IL-10 family member mda-7/IL-24 and suggest that a small-molecule mimetic of mda-7/IL-24 activity may be developed that selectively induces apoptosis in cancer cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection assays. HeLa, DU-145, and T47D cells were grown in DMEM supplemented with 10% FCS at 37°C in a 5% CO2 incubator. SV40 T antigen–immortalized normal human prostate epithelial cells P69 were grown in serum-free medium containing epidermal growth factor (22). Cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the protocol of the manufacturer and were incubated for 24 to 48 hours before further experimental manipulation was done as outlined in specific figure legends.

Construction of MDA-7/IL-24 mutants. Serial NH2-terminal deletion mutants of MDA-7/IL-24 (M1 to M6) were generated by PCR using a common antisense primer and corresponding sense primers (Supplementary Table). M1 (amino acids 48-206) was devoid of the signal peptide. In M2 (amino acids 63-206), the {alpha}-helical domain A was disrupted in the middle. In M3 (amino acids 80-206), the {alpha}-helical domain B was disrupted in the middle. M4 (amino acids 104-206) contained the C, D, E, and F {alpha}-helical domains. M5 (amino acids 131-206) contained only the D, E, and F {alpha}-helical domains, whereas M6 (amino acids 159-206) contained the E and F {alpha}-helical domains. A Kozak sequence including the start codon (GCCACCATG) was added in front of the mutants for better expression. Mutants were cloned into the HindIII and BamHI sites of the vector pREP4 (Invitrogen), which contains a hygromycin resistance gene selection marker. Additional mutations were made in deletion mutant M4 (M4A-M4G) by PCR using the primers as described in Supplementary Table (Fig. 3A). In these mutants, either helix C or F or both were deleted or mutated. Residues TLLEFYLK in {alpha}-helix C were mutated to residues AGDATAGA whereas residues KALGEVD in {alpha}-helix F were mutated to residues GAHGAVA by PCR-based mutagenesis. A similar approach was employed to make mutations in the helices C and F of full-length MDA-7/IL-24. In mutant MDA7(C), TLLEFYLK residues in {alpha}-helix C were mutated to residues TLAGSRLG. The construct was generated by adding a restriction site XbaI in the middle by PCR (Supplementary Table). The 5' and 3' DNA fragments were amplified and the fragments were ligated into HindIII and BamHI sites of pREP4. In mutant MDA7 (C/F), the same mutations were made in {alpha}-helix C and KALGEVD residues in {alpha}-helix F were mutated to residues GAHGAVA. For making the mutant MDA7 (C/F), mutant MDA7 (C) was used as a template. However, MDA7 (C/F) contained residues 1 to 199 of full-length wild-type MDA7/IL-24 due to absence of a restriction endonuclease site at the end. The construct M4A served as a control for MDA7 (C/F) as removal of the last seven residues had no effect on the activity of M4 (Fig. 3E). Various MDA7 deletion mutants, M4 mutants and MDA7 (C) and MDA7 (C/F), were also cloned in HindIII and BamHI sites of the vector pCMV3X Flag vector (Sigma, St. Louis, MO) containing three Flag tags at the NH2 terminus. The authenticity of all the constructs was confirmed by sequence analysis. Myc-tagged BiP construct was a kind gift from Prof. Ron Prywes (Columbia University, New York, NY; ref. 23).


Figure 3
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  		Figure 3. Mutation analysis of helix C and helix F of M4 and MDA-7/IL-24 confirms the importance of these regions in mediating cancer-selective growth inhibitory activity. A, schematic representation of mutations generated in M4 targeting the C and F helices. Regions were either deleted or mutated and the resultant constructs were cloned in the vector pREP4. Mutations were constructed as described in Materials and Methods. B, HeLa, DU-145, and P69 cells were transfected with different deletion constructs of M4 and colony formation assays were done as described in Materials and Methods. C, schematic of mutations at helices C and F of full-length MDA-7/IL-24. D, HeLa and P69 cells were transfected with MDA-7/IL-24, M4, and mutant constructs of MDA-7/IL-24, and colony formation assay was done in the presence of hygromycin. B and D, columns, mean of three independent experiments; bars, SD.

 
Construction of recombinant adenoviruses. The construction of Ad.mda-7 (replication-incompetent adenovirus expressing mda-7/IL-24) was previously described (24) and similar strategies were employed to construct Ad.M4 (24). Adenovirus infection was done as previously described (24, 25). The empty adenoviral vector (Ad.vec) was used as a control.

Cell viability assays. Cells were seeded in 96-well tissue culture plates (1.5 x 103 per well) and, the next day, infected with Ad.vec, Ad.mda-7, and Ad.M4 at multiple plaque-forming units (pfu) for different time points as described in Results. Cell viability was assayed by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described (7) and the data were presented as a ratio between optical densities of treated cells to Ad.vec-infected cells.

Colony formation assays. Cells were transfected with 20 µg of DNA, plated in 60-mm dishes at 1 x 105 for cancer cells or 2 x 105 for normal cells the next day, and selected for colony-forming ability in the presence of hygromycin (400 µg/mL for HeLa, 300 µg/mL for DU-145, and 200 µg/mL for the other cell lines). To determine the effect of adenovirus transduction, cells were infected with 10, 50, or 100 pfu/cell with Ad.vec, Ad.mda-7, and Ad.M4. The next day, 200 to 500 cells were seeded to determine colony-forming ability. In both cases, after 2 weeks of incubation, colonies were fixed, stained with 5% Giemsa solution, and colonies of >50 cells were enumerated (24).

RNA isolation and Northern blot analysis. Total RNA was extracted from the cells by using Qiagen RNeasy kit according to the protocol of the manufacturer and Northern blotting was done as previously described (24). The membranes were hybridized with 32P-labeled cDNA probes that include full-length mda-7/IL-24, GADD34, GADD153, and GAPDH (24).

Western blot analysis. Total cell lysate preparation and Western blotting were done as described (7). The primary antibodies were anti–MDA-7/IL-24 (rabbit polyclonal; 1:5,000) and anti–phospho p38 MAPK and anti–p38 MAPK (Cell Signaling Technology, Inc., Danvers, MA; rabbit polyclonal; 1:1,000). Horseradish peroxidase–conjugated antirabbit secondary antibody (1:10,000) was used. Blots were visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

Annexin V binding assays. Cells were seeded in six-well plates (5 x 105 per well) and, the next day, cells were infected with 100 pfu/cell of different adenoviruses for 24 hours. Annexin V binding assay was done by flow cytometry as described (26).

Coimmunoprecipitation of BiP/GRP78 with MDA-7/IL-24 and its mutants. Cells were infected with Ad.vec, Ad.M4, or Ad.mda-7 or transfected with Flag-tagged MDA7 or M4 and Myc-tagged BiP. After 48 hours, cells were rinsed with ice-cold PBS and lysed in 1 mL of immunoprecipitation buffer containing 25 mmol/L Tris-Cl (pH 8.0), 137 mmol/L NaCl, 2.5 mmol/L 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 A 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 (Sigma; 1:200 dilution) 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 described (7) 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 chain of immunoglobulin G (IgG) were used as the light chain of IgG interfered with detection of MDA-7/IL-24 because of similar size.

Immunofluorescence analysis. Cells (1 x 105) were grown on two chamber slides (BD Falcon Biosciences, Bedford, MA) and, the next day, were infected with 50 pfu/cell of Ad.mda-7 or Ad.M4. After 24 hours, cells were fixed in 4% paraformaldehyde in PBS for 30 minutes and permeabilized by 0.1% Triton X-100 in PBS for 10 minutes. Cells were rinsed in PBS, blocked in 5% bovine serum albumin in PBS for 2 hours, and then incubated with anti-MDA-7/IL-24 and anti-calregulin antibodies overnight (1:500 dilution of both antibodies). Cells were washed 3 x 5 minutes each in PBS and incubated with anti-rabbit-FITC and anti-mouse-rhodamine secondary antibodies (Molecular Probes, Carlsbad, CA) for 2 hours. Cells were washed 3 x 5 minutes each in PBS. Slides were mounted and cells were visualized using a Zeiss LSM 510 fluorescence microscope and a 100x objective. For localization studies, cells were transfected with Flag-tagged DNA constructs and, after 24 hours, immunostaining was done as described earlier using mouse anti-Flag M2 (Sigma) and rabbit anti-BiP/GRP78 primary antibodies overnight. Anti-mouse-FITC and anti-rabbit-rhodamine secondary antibodies were used to detect Flag-tagged proteins and BiP/GRP78, respectively.

Tumorigenicity studies. Tumorigenecity studies were done using T47D human breast carcinoma cell lines in athymic nude mice exactly as described (27, 28). Tumors were established on both flanks of the animals and intratumoral injections (1 x 108 pfu) of different adenoviruses were given only to tumors on the left side. The injections were given thrice a week for the first week and then twice a week for 2 more weeks to a total of seven injections.

Statistical analysis. Statistical analysis of the results was done using the Analysis ToolPack provided by Microsoft Excel. A two-sample Student's t test, assuming unequal variances, was used to determine the equality of the means of two samples. The confidence level {alpha} was 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mapping functional regions of mda-7/IL-24 that mediate cancer-specific growth suppression. To confirm that mda-7/IL-24 induces apoptosis through an intracellular receptor-independent mechanism, a series of mda-7/IL-24 deletion mutants (M1-M6) were constructed guided by secondary structure predictions of MDA-7/IL-24 defined by amino acid sequence and structural homology with IL-10 (Fig. 1A ; ref. 29). In the first mutant (M1), the signal peptide that directs secretion of mda-7/IL-24 is deleted. In M2, the signal peptide and residues before {alpha}-helix A are deleted. Mutants M3 to M6 correspond to peptides that contain putative MDA-7/IL-24 {alpha}-helices B, C, D, E, and F (M3); C, D, E, and F (M4); D, E, and F (M5); and E and F (M6). This strategy was adopted to define fragments of MDA-7/IL-24 that might be biologically active even if they cannot adopt a completely folded three-dimensional structure or be secreted into the culture medium to bind cell-surface receptors.


Figure 1
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  		Figure 1. Identifying the regions of functional activity of MDA-7/IL-24. A, schematic representation of NH2-terminal deletion mutations generated in the mda-7/IL-24 gene. Fragments were cloned in the expression vector pREP4. B, effect of various deletion mutants on colony formation in cancer and normal cells. HeLa, DU-145, and P69 cells were transfected with different deletion constructs of mda-7/IL-24 and, the next day, cells were subcultured and selected for colony formation ability in the presence of hygromycin for 2 weeks. Columns, mean of three independent experiments; bars, SD. C, expression of Flag-tagged deletion constructs of MDA-7/IL-24 protein after transient transfection into HeLa cells using anti-Flag antibody.

 
Mutants M1 to M6 were transiently expressed in cancer (HeLa and DU-145) and normal (P69) cell lines and their ability to suppress cell growth was evaluated (Fig. 1B). None of these mutants suppressed growth of SV40 T antigen–immortalized normal human prostate epithelial (P69) cells (Fig. 1B). As predicted from previous studies (19), M1, which is devoid of the signal peptide amino acids 1 to 47, significantly inhibited growth in HeLa and DU-145 cells (Fig. 1B). Deletion of residues from 1 to 62 and 1 to 79 of the full-length mda-7/IL-24 gene in constructs M2 and M3, respectively, resulted in molecules that were devoid of growth-suppressive activity (Fig. 1B). Surprisingly, deletion of residues 1 to 103 (M4, corresponding to {alpha}-helices C, D, E, and F) retained the cancer-specific growth suppression activities of full-length MDA-7/IL-24 (Fig. 1B). M4 also inhibited colony formation in LNCaP (prostate carcinoma) and T47D (breast carcinoma) cells (data not shown), but not in P69 (Fig. 1B) or FM516-SV (an SV40 T antigen–immortalized normal human melanocyte cell line) cells.6 Further deletion of residues 1 to 130 (M5) or 1 to 158 (M6) rendered the molecule inactive in cancer-specific cell growth suppression activity.

To characterize the expression levels of the mutants, M1 to M6 were subcloned into a pCMV3x Flag vector. The Flag-tagged M1 to M6 were expressed in HeLa, DU-145, and P69 cells and the expression was analyzed by Western blotting using an anti-Flag antibody. Functional protein was synthesized for full-length MDA-7/IL-24, M1, M2, M3, and M4, but not for M5 and M6 constructs (Fig. 1C).

Ad.M4 induces cancer cell–specific apoptosis. To analyze the biological function of M4 and to ensure efficient delivery to all cells, we constructed a replication-incompetent type 5 adenovirus expressing M4, Ad.M4. HeLa, DU-145, and P69 cells were infected with 50 pfu/cell of Ad.vec (adenovirus lacking a gene insert), Ad.mda-7, or Ad.M4 and, 24 hours later, the expression levels of mda-7/IL-24 and M4 mRNA and proteins were analyzed by Northern blot (Fig. 2A, left , and data not shown) and Western blot (Fig. 2A, right, and Supplementary data) analyses, respectively. Both of these adenoviruses generated authentic mRNA and protein. Ad.M4 produced a single protein of ~15 kDa whereas Ad.mda-7 generated multiple bands because of glycosylation, ranging in size from ~20 to ~30 kDa (Fig. 2A, right, and Supplementary data). Similar results were obtained in normal primary human fetal astrocytes or FM516-SV cells (data not shown).


Figure 2
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  		Figure 2. M4 exhibits similar biological properties and activities as full-length MDA-7/IL-24. A, HeLa cells were infected with Ad.mda-7 or Ad.M4 at 100 pfu/cell and mda-7/IL-24 and M4 mRNA and protein were analyzed by Northern blot (left) and Western blot (right) analyses 48 hours postinfection. B, top, the indicated cell type was seeded in 96-well plates and infected with the indicated pfu of Ad.vec, Ad.mda-7, or Ad.M4. After 5 days, cell viability was assessed by MTT assay and plotted as the ratio to Ad.vec treatment. Bottom, the indicated cells were infected with the indicated plaque-forming units of adenovirus and colony formation assays were done as described in Materials and Methods. C, the indicated cells were seeded in six-well plates at a density of 2 x 105 per well and, the next day, were infected with 100 pfu/cell of Ad.vec, Ad.mda-7, or Ad.M4. After 24 hours, Annexin V binding was analyzed by flow cytometry. B and C, columns, mean of three independent experiments; bars, SD. D, DU-145 (top) and P69 (bottom) cells were infected with 100 pfu/cell of Ad.M4 or Ad.mda-7. After 24 hours, cells were fixed and MDA-7/IL-24 and M4 protein was detected by indirect immunofluorescence using anti-mda-7/IL-24 rabbit polyclonal antibodies. Colocalization was determined by using antibodies against the endoplasmic reticulum marker protein, calregulin. Images of MDA-7/IL-24 and calregulin were merged.

 
The effect of Ad.M4 and Ad.mda-7 on the survival of cancer and normal cells was evaluated. HeLa, DU-145, T47D, and P69 cells were infected with 100 pfu/cell of Ad.vec or 10, 25, 50, or 100 pfu/cell of Ad.M4 or Ad.mda-7, and cell viability was monitored after 5 days. These experiments confirmed a dose-dependent decrease in cell viability in DU-145, HeLa, and T47D cells following infection with Ad.M4 or Ad.mda-7 (Fig. 2B). In contrast, no discernible effect was evident on the viability of P69 cells even after infection with 100 pfu/cell of Ad.M4 or Ad.mda-7 (Fig. 2B). Definitive decreases in cancer cell viability were evident with as little as 10 pfu/cell of Ad.M4 or Ad.mda-7 in DU-145 and HeLa cells (Fig. 2B, top). Inhibition of long-term viability was documented using clonal survival assays (Fig. 2B, bottom). In these experiments, Ad.M4 and Ad.mda-7 infection resulted in a profound decrease in survival of DU-145 and HeLa cells, even when cells were infected with 10 pfu/cell of virus. In contrast, even with 100 pfu/cell of Ad.M4 or Ad.mda-7, no significant effect was apparent on colony formation in P69 cells. These studies confirm similar restricted antiproliferative and antisurvival effects of Ad.M4 versus Ad.mda-7 in cancer cells, with no toxic effects in normal cells.

Annexin V staining, which monitors early apoptotic changes, was determined by fluorescence-activated cell sorting analysis in P69, DU-145, HeLa, and T47D cells 24 hours after infection with 100 pfu/cell of Ad.vec, Ad.M4, or Ad.mda-7 (Fig. 2C). Whereas significant increases in apoptotic cells were observed in DU-145, HeLa, and T47D cells following Ad.M4 and Ad.mda-7 infection, no such change was evident in P69 cells (Fig. 2C) and in primary human fetal astrocytes and FM516-SV cells (data not shown). These findings confirm that M4 retains the same cancer-specific growth-suppressive and apoptosis-inducing properties as the full-length molecule. Furthermore, M4 does not contain a signal sequence and is not secreted from cells. This data provides additional support for a novel intracellular mode of cancer cell–specific killing by mda-7/IL-24.

M4, like mda-7/IL-24, localizes in the endoplasmic reticulum. Because MDA-7/IL-24 localizes in the endoplasmic reticulum in both normal and cancer cells (18), we next determined if M4 displays similar localization. DU-145 and P69 cells were infected with Ad.M4 or Ad.mda-7 and subcellular localization of M4 and MDA-7/IL-24 was determined by immunofluorescence analysis (Fig. 2D). Immunofluorescence detection was standardized at different time points to avoid ambiguous changes in localization that might occur as a result of loss of internal membrane integrity due to apoptotic events induced by M4 or MDA-7/IL-24 in DU-145 cells. Like full-length MDA-7/IL-24 protein, M4 was localized in the endoplasmic reticulum in both cancer and normal cells, as evidenced by overlapping localization of these proteins with the endoplasmic reticulum–resident protein calregulin (Fig. 2D).

Hydrophobic residues in the C and F helices of the M4 and MDA-7/IL-24 proteins are required for biological activity. Studies by Vandenbroeck et al. (21) identified a conserved DnaK/BiP/GRP78 binding site in all IL-10 family members, including mda-7/IL-24, which may be necessary to assist in the folding of these molecules. The conserved DnaK/BiP/GRP78 binding site is located on helix C and consists of the eight-residue sequence TLLEFYLK in MDA-7/IL-24. In the three-dimensional structure of IL-10 and IFN-{gamma}, the helix C DnaK/BiP/GRP78 binding site is positioned next to a second highly conserved amino acid sequence (KALGEVD in MDA-7/IL-24) located in helix F. In contrast to the conserved segment in helix C, the conserved sequence in helix F has not been shown to interact with DnaK/BiP/GRP78. Because MDA-7/IL-24 and M4 both localize to the endoplasmic reticulum on expression in cells, we investigated a potential role of these conserved residue segments, located in helices C and F, in mediating killing by M4 and MDA-7/IL-24.

To explore the role of conserved residues in helices C (TLLEFYLK) and F (KALGEVD) of MDA-7/IL-24 in inducing cancer cell–specific killing, a second set of mutants were made (M4A-M4G; Fig. 3A ). The last seven residues of MDA-7/IL-24 were deleted in M4A, resulting in the mutant containing residues 104 to 199. This construct M4A was kept as a control due to lack of restriction site at the COOH terminus. M4B (residues 119-206) corresponds to a deletion of helix C. M4C is the same length as M4 (104-206), but helix C residues TLLEFYLK were mutated to AGDATAGA. In M4D (residues 104-187), the entire F helix was deleted. In M4E, conserved residues in helix F (KALGEVD) were mutated to GAHGAVA. M4F (residues 119-187) is a double deletion mutant in which both MDA-7/IL-24 helices C and F were removed. Finally, M4G is a double mutation construct where both the conserved residues in helices C and F were mutated as previously described for mutants M4C and M4E (Fig. 3A).

The various constructs were evaluated for functional activity using colony formation assays in HeLa, DU-145, and P69 cells. Following transfection, mda-7/IL-24, M4, and M4A reduced colony formation to an equivalent degree in both HeLa and DU-145 cells, without significantly altering colony formation in normal P69 cells (Fig. 3B). Mutations or deletions of either the C or F helices, M4B, M4C, M4D, and M4E, modestly reduced colony formation in HeLa and DU-145 cells as compared with transfection with mda-7/IL-24, M4, or M4A. The M4F and M4G mutants, which contain deletions or mutations in both C and F helices, were devoid of (HeLa) or displayed minimal (DU-145) colony inhibitory activity (Fig. 3B). None of these additional mutants affected colony formation in P69 cells (Fig. 3B). These data confirm the importance of the C and F helices of the MDA-7/IL-24 protein in mediating cancer-specific activity of the M4 deletion mutant of mda-7/IL-24. When either site was mutated or deleted, there was a disruption of the activity, and functionality was essentially extinguished when both sites were deleted or mutated.

To confirm the previous observations, mutations were generated in the conserved regions of helices C and F in full-length MDA-7/IL-24. In MDA7 (C), helix C of MDA-7/IL-24 was mutated from TLLEFYLK to TLAGSRLG. In mutant MDA7 (C/F), {alpha}-helix C residues TLLEFYLK were mutated to residues TLAGSRLG and {alpha}-helix F residues KALGEVD were mutated to residues GAHGAVA (Fig. 3C). The mutation introduced in MDA7 (C) is different than other helix C mutations made in M4 due to difficulties in generating the construct, whereas mutations in the conserved region of helix F were identical to those used in the M4 constructs.

The effect of these mutations on clonal colony formation in cancer and normal cell lines was evaluated (Fig. 3D). As predicted, mutations in helix C disrupted the functional activity of MDA-7/IL-24 and mutations in both the C and F (C/F) helices further abrogated the cancer-specific inhibitory activity of MDA-7/IL-24 (Fig. 3D). Moreover, colonies were morphologically smaller than colonies formed in control vector-transfected cells, suggesting retention of some growth-modulating activity.6 No effect was observed in P69 cells with either MDA-7/IL-24 mutant (Fig. 3D). These studies confirm that both the C and F helices of the MDA-7/IL-24 protein are crucial for maintaining optimum mda-7/IL-24 cancer–specific growth-suppressive activity.

Endoplasmic reticulum chaperone BiP/GRP78 interacts with MDA-7/IL-24 and M4. We next examined whether the conserved residues in helices C and F of MDA-7/IL-24 interact with BiP/GRP78 as previously described for IFN-{gamma}. Infection with Ad.mda-7 or Ad.M4 followed by immunoprecipitation using anti-BiP/GRP78 antibody and immunoblotting with anti-MDA-7/IL-24 antibody confirmed a physical interaction between these molecules (Fig. 4A ).


Figure 4
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  		Figure 4. Full-length MDA-7/IL-24 and M4 proteins bind to BiP/GRP78. A, coimmunoprecipitation of MDA-7/IL-24 and M4 with endogenous BiP/GRP78. HeLa cells were infected with 100 pfu/cell of Ad.mda-7, Ad.M4, or Ad.vec and immunoprecipitation analysis was done 48 hours later using BiP/GRP78 antibodies. B, left, coimmunoprecipitation of Flag-tagged MDA-7/IL-24 or M4 with BiP/GRP78. Flag-tagged MDA-7/IL-24 or M4 and Myc-tagged BiP/GRP78 were cotransfected into HeLa cells. Forty-eight hours posttransfection, BiP/GRP78 was immunoprecipitated using anti-Myc monoclonal antibody. Samples were washed gently and separated on 12% SDS-PAGE and probed with anti-Flag M2 antibody. B, left, top, coimmunoprecipitation of MDA-7/IL-24 and M4 with BiP/GRP78 is confirmed; bottom, expression and immunoprecipitation profile of Myc-tagged BiP/GRP78 using anti-Myc antibody. Right, the MDA-7/IL-24 deletion mutants M1, M2, and M3 bind to BiP/GRP78. Samples, as above in B (left), were immunoprecipitated using anti-Myc monoclonal antibody and immunoblotting was done using anti-Flag M2 antibody. C, expression of the indicated Flag-tagged constructs was confirmed by Western blot analysis using anti-Flag M2 antibody. D, coimmunoprecipitation experiments were done using the mutants described in (C) and probed with anti-Flag M2 antibody.

 
To further explore the interaction between BiP/GRP78 and MDA-7/IL-24 or M4, we transiently transfected Flag-tagged MDA-7/IL-24 or M4 and Myc-tagged BiP/GRP78 in HeLa cells and did coimmunoprecipitation studies. The tagged constructs were used because probing with anti-BiP/GRP78 antibody generated high background. Immunoprecipitation was done using 9E10 anti-Myc monoclonal antibody and immunoblotting was done with anti-FLAG M2 and anti-Myc antibodies, the latter for monitoring the efficiency of immunoprecipitation. MDA-7/IL-24 and M4 coimmunoprecipitated with BiP/GRP78 showing a physical interaction between these two molecules (Fig. 4B, left). Coimmunoprecipitation of MDA-7/IL-24 and M4 with BiP/GRP78 was also observed when polyclonal anti-BiP/GRP78 antibody was used for immunoprecipitation (data not shown). Similarly, cotransfection of HeLa cells with Flag-tagged M1, M2, or M3 and Myc-tagged BiP/GRP78 showed interaction of these MDA-7/IL-24 mutants with BiP/GRP78 (Fig. 4B, right). Experiments were also done in a reverse direction (i.e., immunoprecipitation was done using anti-Flag antibody and the membrane was probed with the anti-Myc antibody; data not shown). These experiments also confirmed BiP/GRP78 interaction with MDA-7/IL-24 and M4, as well as M1, M2, and M3, only when both molecules were simultaneously transfected into HeLa cells (data not shown).

To investigate further the putative roles of the C and F helices of MDA-7/IL-24 and M4 in mediating interaction with BiP/GRP78, Flag-tagged C and F helix mutants of MDA-7/IL-24 and M4 were constructed. Figure 4C confirms expression of the Flag-tagged wild-type MDA-7/IL-24 and M4 as well as the C plus F (C/F) mutants of MDA-7/IL-24 and M4 by Western blot using anti-Flag antibody. These Flag-tagged constructs and Myc-tagged BiP/GRP78 were transfected into HeLa (Fig. 4D) and P69 cells (data not shown), immunoprecipitation was done using anti-Myc antibody, and the membrane was probed with anti-Flag and anti-Myc antibodies (Fig. 4D). The C/F helix mutants of MDA-7/IL-24 and M4 lost their ability to bind to BiP/GRP78 (Fig. 4D). Similar results were obtained using normal P69 cells (data not shown). These studies confirm that BiP/GRP78 interacts with MDA-7/IL-24 and M4 through the conserved residues in helices C and/or F and mutation of these residues prevents binding and abrogates apoptosis induction by MDA-7/IL-24 and M4. The observation that MDA-7/IL-24:BiP/GRP78 interactions occur in both normal and cancer cells indicates that although this interaction is necessary for apoptosis induction, it does not confer cancer cell specificity.

The interaction of MDA-7/IL-24 with BiP/GRP78 was further confirmed by immunofluorescence studies. Flag-tagged MDA-7/IL-24, M1, M2, M3, and M4 were transfected into HeLa cells and double immunofluorescence studies were done using anti-Flag and anti-BiP/GRP78 antibodies. MDA-7/IL-24 and its deletion mutants, which interacted with BiP/GRP78 in coimmunoprecipitation assays, also localized in the endoplasmic reticulum with BiP/GRP78 (Fig. 5A ). In total, these findings suggest that although BiP/GRP78 interaction is necessary, it is not the only event that determines apoptosis induction by MDA-7/IL-24.


Figure 5
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  		Figure 5. Full-length MDA-7/IL-24 protein and proteins encoded by the M1, M2, M3, and M4 mutants colocalize with BiP/GRP78 in the endoplasmic reticulum. A, HeLa cells were transiently transfected with Flag-tagged full-length MDA-7/IL-24 or the indicated deletion mutants of MDA-7/IL-24. Twenty-four hours posttransfection, cells were fixed and MDA-7/IL-24 protein was detected by indirect immunofluorescence using anti-Flag M2 antibody. Colocalization was determined by using anti-BiP/GRP78 antibody. Images of BiP/GRP78 and MDA-7/IL-24 were merged. B, HeLa cells were transfected with the indicated constructs and, 24 hours posttransfection, the expression levels of phosphorylated and total p38 MAPK were analyzed by Western blot analysis. C, HeLa cells were transfected with the indicated constructs and, 24 hours later, the expression levels of GADD34, GADD153, and GAPDH genes were analyzed by Northern blotting. D, proposed model of the molecular mechanism of mda-7/IL-24-induced apoptosis. MDA-7/IL-24 protein (gray diamond) delivered by Ad.mda-7 localizes in the endoplasmic reticulum where it interacts with BiP/GRP78 (or other chaperones), which might result in activation of a yet unidentified molecule (X) and generation of endoplasmic reticulum stress that involves activation of p38 MAPK and induction of GADD family genes, culminating in apoptosis. Additionally, interaction of MDA-7/IL-24 with BiP/GRP78 might squelch the interaction of BiP/GRP78 with other cancer cell–specific molecule(s), necessary for proliferation, inducing their degradation with resultant apoptosis. The secreted MDA-7/IL-24 interacts with its cognate receptors on the cell surface, which activates a signaling cascade, resulting in apoptosis. However, whether this signaling cascade also involves endoplasmic reticulum stress remains to be determined.

 
M4 induces activation of p38 MAPK and GADD gene family expression. Activation of p38 MAPK followed by induction of GADD family genes plays a critical role in Ad.mda-7-induced apoptosis in multiple cell types (8, 9). Based on these considerations, we tested whether MDA-7/IL-24 and its mutants, including M1, M2, M3, M4, and various point or deletion mutations in M4, retain the ability to induce p38 MAPK phosphorylation and GADD gene expression. As shown in Fig. 5B and C, full-length MDA-7/IL-24, M1, and M4 proteins retained the ability to maximally promote p38 MAPK phosphorylation and induce downstream targets of p38 MAPK such as GADD34 and GADD153 mRNAs. In contrast, M2, M3, M5, and M6 did not induce p38 MAPK phosphorylation or GADD gene expression. Similarly, reduced phosphorylation of p38 MAPK and induction of GADD34 and GADD153 mRNAs were apparent in helix C or helix C plus F (C/F) mutants of full-length MDA-7/IL-24, MDA7 (C) and MDA7 (C/F), and in the M4 mutants, M4C, M4E, M4F, and M4G (Fig. 5B and C). Although the mechanism by which MDA-7/IL-24 and M4 induce p38 MAPK phosphorylation remains to be determined, the present study identifies a relevant downstream target gene family that is activated after BiP/GRP78 binding and is critical for MDA-7/IL-24 and M4 to induce apoptosis selectively in cancer cells (Fig. 5 D).

M4 retains antitumor properties in vivo in a human tumor nude mouse xenograft model. We next determined tumor suppressor function of Ad.mda-7, Ad.M4, and Ad.Sp-mda-7 in vivo. For the tumor studies, we employed a scheme used previously (27, 28), in which tumors were established on both sides of an animal and the therapeutic agent was applied to one side of the animal and its effects on the injected and noninjected tumor sites were determined over time. This approach provides insight into "antitumor bystander" activity, which is an inherent property of mda-7/IL-24 that significantly increases its therapeutic utility (16, 27). Intratumoral injection of Ad.M4 and Ad.Sp-mda-7 in established T47D human breast cancer xenografts in nude mice significantly inhibited tumor growth only on the left side (injected site), but not on the uninjected right side, when compared with that of control (untreated) or Ad.vec-injected animals (Fig. 6 ). However, injection of Ad.mda-7 completely eradicated tumors on the left side and markedly inhibited the growth of the tumors on the right side. These findings indicate that although Ad.M4 and Ad.Sp-mda-7 significantly inhibited tumor growth because of the lack of secretory ability, they did not show any antitumor bystander activity.


Figure 6
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  		Figure 6. Ad.M4 displays potent antitumor activity in vivo in an experimental human breast tumor xenograft nude mouse model. Subcutaneous tumors were established in both flanks of athymic nude mice using T47D human breast carcinoma cells and intratumoral injections of the indicated adenovirus were given only to tumors on the left side. Top and bottom, tumor volume on the left and right side, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we sought to define the mechanism by which MDA-7/IL-24, an IL-10 family cytokine, selectively induces apoptosis in cancer cells without interactions with its cell-surface receptors (IL-20R1/IL-20R2 or IL-22R1/IL-20R2) and without JAK/STAT activation. To address this question, a series of MDA-7/IL-24 deletion mutants were constructed and evaluated for growth-suppressing and apoptosis-inducing activity in cancer and normal cells. This analysis revealed the MDA-7/IL-24 deletion mutant containing amino acids 104 to 206 (M4) exhibits apoptosis-inducing activities indistinguishable from the full-length protein by activating p38 MAPK and promoting GADD34 and GADD153 gene expression. M4 also inhibits tumor grown in vivo although it lacks the bystander activity because it cannot be secreted.

An essential requirement for MDA-7/IL-24-, M1-, and M4-mediated cancer cell apoptosis is an interaction with the endoplasmic reticulum chaperone BiP/GRP78. Disrupting interactions of MDA-7/IL-24 or M4 with BiP/GRP78 by mutating the conserved BiP/GRP78 binding site in helices C and F prevented cancer cell apoptosis and the activation of p38 MAPK and the GADD genes. These results suggest that MDA-7/IL-24 binding to the chaperone BiP/GRP78 in a cancer cell–specific context may induce endoplasmic reticulum stress signals, and ultimately apoptosis, by activating the GADD genes through p38 MAPK (Fig. 5D). An important question, which is unresolved, is why MDA-7/IL-24 does not induce apoptosis in normal cells although it is localized in the endoplasmic reticulum and interacts with BiP/GRP78 in these cells. Cancer is often associated with mutations in protein kinases such as Ras or Raf. Mutated BRAF (V600EBRAF), detected in ~7% of cancers, has constitutive kinase activity stimulating cancer cell proliferation and survival (30). V600EBRAF is a Hsp90 client protein requiring Hsp90 for its folding and stability, and disruption of Hsp90 binding by anticancer drugs induces degradation of V600EBRAF but not of wild-type BRAF (31). A possibility is that BiP/GRP78 interacts with similar cancer cell–specific molecules that are required for cell proliferation and MDA-7/IL-24 triggers apoptosis by squelching away BiP/GRP78, thus inducing degradation of these molecules. This possibility is currently being investigated.

Unexpectedly, MDA-7/IL-24 mutants M2 and M3, which do not induce apoptosis, have intact BiP/GRP78 binding sites in helices C and F. Thus, M2 and M3 mutants bind BiP/GRP78 and localize to the endoplasmic reticulum, but these mutants do not induce similar p38 MAPK phosphorylation or GADD gene expression. These results suggest BiP/GRP78 binding is essential, but not sufficient, for MDA-7/IL-24-mediated cancer cell apoptosis. The results are consistent with the primary role of BiP/GRP78 of assisting in the proper folding of a variety of secreted proteins, including other members of the IL-10 family, which contain conserved BiP/GRP78 binding sites in their sequences (21). What is hard to reconcile is the observation that inactive M2 and M3 mutants both contain residues 104 to 206 of MDA-7/IL-24 (M4), which is able to selectively induce apoptosis in cancer cells. Considering that these inactive constructs interact with BiP/GRP78, it is clear that in addition to stabilization by chaperones, a higher level of regulation may be operational to control the activity of MDA-7/IL-24. This regulation might be mediated by the interaction of MDA-7/IL-24 with as yet unidentified protein(s) (Protein X), in addition to BiP/GRP78 to which M1 and M4, but not M2 or M3, interact thereby activating downstream signaling cascades, such as p38 MAPK phosphorylation and subsequent GADD gene induction (Fig. 5D).

At least some insight into the connection between MDA-7/IL-24:BiP/GRP78 interactions and cancer cell apoptosis can be obtained from recent studies on the activation mechanisms of the endoplasmic reticulum stress response (32). In particular, the activation of the membrane-associated transcription factor ATF6, which induces several endoplasmic reticulum stress response genes, is controlled by a competition between the luminal domain of ATF6 and unfolded proteins in the endoplasmic reticulum for BiP/GRP78 (23). Under normal conditions, ATF6 is kept in a sequestered inactive form by interactions with BiP/GRP78. However, dissociation of BiP/GRP78 from ATF6 activates the transcription factor, which results in the induction of several endoplasmic reticulum stress response genes (33). Thus, in cancer cells, the expression of MDA-7/IL-24, M1, and M4 may compete for BiP/GRP78 leading to ATF6 activation, and possibly other signaling molecules that regulate cancer cell apoptosis. In contrast, the additional MDA-7/IL-24 residues found in the inactive M2 and M3 mutants may shield or prevent high-affinity interactions with BiP/GRP78 and possibly other endoplasmic reticulum proteins that regulate apoptosis (Fig. 5D). The identification of such proteins is currently being pursued. Our studies also show that the endoplasmic reticulum is extremely sensitive to the type of protein/peptide required to induce BiP/GRP78-mediated cancer cell apoptosis.

In summary, MDA-7/IL-24 is an {alpha}-helical cytokine that has tremendous potential as a gene therapy for cancer (1–4). In this study, we have identified a peptide of MDA-7/IL-24 (M4, residues 104-206) that mimics the biological properties of the full-length protein including in vivo tumor suppression properties. The experimental evidence confirms that interactions with the endoplasmic reticulum chaperone BiP/GRP78 are critical for the ability of MDA-7/IL-24 or M4 to induce cancer cell apoptosis. This data provides an explanation for how virally expressed MDA-7/IL-24 induces apoptosis without the need for cell-surface receptor interactions or the JAK/STAT signaling pathway (18). It also provides a possible explanation for why MDA-7/IL-24 is able to kill diverse types of cancer cells. Finally, the effectiveness of MDA-7/IL-24 in selectively inducing apoptosis in cancer cells, but not normal cells (24, 34), is consistent with cancer cells already being under significant metabolic stress. Thus, MDA-7/IL-24 peptides may lead to new therapeutics that selectively target and kill cancer cells based on their increased level of stress compared with normal cells.


   Acknowledgments
 
Grant support: NIH grants CA097318, CA098712, P01 CA104177, and AI47300; University of Alabama at Birmingham Cancer Center Internal Award; the Samuel Waxman Cancer Research Foundation; and the Chernow Endowment.

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.

We thank Nichollaq Vozhilla for help with the animal tumor modeling studies and Dr. Ron Prywes for providing valuable reagents.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

P.B. Fisher is the Michael and Stella Chernow Urological Cancer Research Scientist and a Samuel Waxman Cancer Research Foundation Investigator.

6 Unpublished data. Back

Received 2/14/06. Revised 5/ 5/06. Accepted 6/ 9/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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