mda-7/IL-24: multifunctional cancer-specific apoptosis-inducing cytokine

Abstract

"Differentiation therapy" provides a unique and potentially effective, less toxic treatment paradigm for cancer. Moreover, combining "differentiation therapy" with molecular approaches presents an unparalleled opportunity to identify and clone genes mediating cancer growth control, differentiation, senescence, and programmed cell death (apoptosis). Subtraction hybridization applied to human melanoma cells induced to terminally differentiate by treatment with fibroblast interferon (IFN-beta) plus mezerein (MEZ) permitted cloning of melanoma differentiation associated (mda) genes. Founded on its novel properties, one particular mda gene, mda-7, now classified as a member of the interleukin (IL)-10 gene family (IL-24) because of conserved structure, chromosomal location, and cytokine-like properties has become the focus of attention of multiple laboratories. When administered by transfection or adenovirus-transduction into a spectrum of tumor cell types, melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) induces apoptosis, whereas no toxicity is apparent in normal cells. mda-7/IL-24 displays potent "bystander antitumor" activity and also has the capacity to enhance radiation lethality, to induce immune-regulatory activities, and to inhibit tumor angiogenesis. Based on these remarkable attributes and effective antitumor therapy in animal models, this cytokine has taken the important step of entering the clinic. In a Phase I clinical trial, intratumoral injections of adenovirus-administered mda-7/IL-24 (Ad.mda-7) was safe, elicited tumor-regulatory and immune-activating processes, and provided clinically significant activity. This review highlights our current understanding of the diverse activities and properties of this novel cytokine, with potential to become a prominent gene therapy for cancer.

Fig. 1

Schematic of the differentiation induction subtraction hybridization (DISH) approach. This procedure has been used to identify and clone genes displaying differential expression as a function of induction of irreversible growth arrest, terminal differentiation, and loss of tumorigenic potential in HO-1 human melanoma cells. Temporally spaced libraries are constructed from actively growing HO-1 cells and from HO-1 cells treated with combination of IFN-β plus MEZ. The actively growing HO-1 cDNA library is then subtracted from the IFN-β plus MEZ-treated HO-1 library resulting in a subtracted cDNA library enriched for differentially expressed genes that associate with a multitude of processes, some of which are indicated in this figure. Further details of the subtraction approach and its application with reverse Northern blotting of cDNAs and high throughput cDNA microarrays can be found in Jiang and Fisher (1993) and Huang et al. (1999a, (reproduced, by permission of the publisher, from Fisher et al., 2003).

Fig. 2

Schematic representation of the MDA-7/IL-24 polypeptide showing various predicted and established protein motifs (reproduced in modified form, by permission of the publisher, from Sauane et al., 2003b).

Fig. 3

Expression of mda-7/IL-24 message in the human immune system. Human multiple tissue Northern blot consisting of poly(A)+ mRNA from different tissues shows tissue-specific expression of mda-7/IL-24. The mRNAs immobilized on the blot are from spleen (1), thymus (2), prostate (3), testis (4), ovary (5), small intestine (6), colon (7), and peripheral blood leukocytes (8) (reproduced, by permission of the publisher, from Huang et al., 2001).

Fig. 4

MDA-7/IL-24-related molecules: alignment of mouse (FISP), rat (c49a/MOB-5), and human protein sequences showing identical (*) conserved (:) and similar (.) amino acid residues (reproduced, by permission of the publisher, from Sauane et al., 2003b).

Fig. 5

Reduction in mda-7/IL-24 mRNA expression as a consequence of human melanoma progression. Quantitative RT-PCR-based analysis of mda-7/IL-24 mRNA expression versus GAPDH expression in independent normal melanocytes, primary melanoma, radial or early vertical growth phase (RGP and VGP, respectively), and metastatic melanoma cell cultures and patient-derived samples. Results are expressed as the ratio of mda-7/IL-24 mRNA to GAPDH mRNA. These data indicate progressive reduction or complete loss of mda-7/IL-24 expression in association with melanoma progression.

Fig. 6

Inhibition of the p38 MAPK pathway protects FO-1 melanoma cells from Ad.mda-7-mediated cell death. (A) FO-1 cells were infected with either Ad.vec or Ad.mda-7 (100 pfu/cell) and treated with 1 μM SB203580 or infected with Ad.p38DN (an adenovirus expressing a dominant-negative p38 mutant gene) (100 pfu/cell). Cell viability was measured by MTT assay after 4 days. Cell viability of Ad.vec-treated cells was regarded as 1. *Significant differences from Ad.mda-7 (P<0.0001). (B) FO-1 cells were infected with either Ad.vec or Ad.mda-7 (100 pfu/cell) and treated with 1 μM SB203580 for 3 days. DNA was isolated from the cells and fragmentation was analyzed as described in Sarkar et al. (2002b). (C) FO-1 cells were infected with either Ad.vec or with Ad.mda-7 (100 pfu/cell) and treated with 1 μM SB203580 or infected with Ad.p38DN (100 pfu/cell). Percentage of apoptotic cells at days 1 and 3 after infection in each group were plotted (reproduced, by permission of the publisher, from Sarkar et al., 2002b).

Fig. 7

Proposed model for Ad.mda-7-induced apoptosis in prostate cancer cell lines. Following Ad.mda-7 infection, MDA-7 protein affects mitochondria directly or indirectly, causing alterations in mitochondrial function (decrease in Δψ_m and MPT) and ROS production. The reductions in Δψ_m and MPT are caspase-independent because they are not inhibited by the general caspase inhibitor z-VAD.fmk. Moreover, MPT can be blocked by inhibitors of MPT, such as CsA and BA, and can be promoted by activators of MPT, such as PK11195, a PBzR agonist. ROS inhibitors (NAC and Tiron) block Ad.mda-7-induced apoptosis, whereas ROS producers (As₂2O₃ and NSC656240) enhance apoptosis only in the context of prostate cancer cells. Abbreviations: Δψ_m, mitochondrial transmembrane potential; MPT, mitochondrial permeability transition; ROS, reactive oxygen species; z-VAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; CsA, cyclosporin A; BA, bongkrekic acid; PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; PBzR, peripheral benzodiazepine receptors; As₂O₃, arsenic trioxide; NSC656240, dithiophene (reproduced, by permission of the publisher, from Lebedeva et al., 2003c).

Fig. 8

The combination of Ad.mda-7 plus AS K-ras PS ODN or AS K-ras plasmids synergistically inhibits colony formation in mutant K-ras MIA PaCa-2 pancreatic carcinoma cells. (Upper) Effect of Ad.mda-7 plus AS K-ras PS ODN on MIA PaCa-2 colony formation. Cells were infected with 100 pfu/cell of Ad.vec or Ad.mda-7, treated with 0.5 μM AS K-ras PS ODN plus 10 μl of Lipofectamine, reseeded at a density of 400 cells per plate, and fixed and stained with Giemsa after 3 weeks. (Lower) Effect of Ad.mda-7 plus AS K-ras plasmid transfection on MIA PaCa-2 G418-resistant colony formation. Cells were infected with 100 pfu/cell of Ad.vec or Ad.mda-7, transfected with 10 μg of plasmid (either control pcDNA3.1 lacking insert or the pcDNA3.1 vector containing a 346-nt AS K-ras fragment), reseeded at a density of 2.5×10⁵ cells per plate, and selected in 400 μg/mL G418, and G418-resistant colonies were fixed and stained with Giemsa after 3 weeks (reproduced, by permission of the publisher, from Su et al., 2001).

Fig. 9

Comparative mechanism of mda-7/IL-24 action after infection of various cell lines with Ad.vec, Ad.mda-7, and Ad.SP-mda-7 (adenovirus expressing an mda-7/IL-24 cDNA lacking the signal peptide). (A) mda-7/IL-24 inhibits C8161 Matrigel invasiveness without altering C8161 viability. C8161 cells were infected with 100 pfu/cell of Ad.vec, Ad.mda-7, or Ad.SP-mda-7. After 24 hr, 1 × 10⁶ cells were allowed to invade for 48 hr through transwell inserts (8-μm pores) coated with Matrigel. The cells that invaded through the Matrigel-coated inserts were stained, counted, and photographed under a light microscope at ×20 magnification. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in parallel to ascertain whether the inhibition of invasion was associated with a decrease in growth or viability of infected C8161 cells, bars, ±SD. Direct cell counts were performed on all surviving, attached cells in the lower chamber to quantitate the relative efficiency of invasiveness. (B) “Bystander” suppression of anchorage-independent growth of DU-145 cells after adenovirus infection of P69 cells. P69 cells were seeded at 2 × 10⁵/6-cm plate, infected 24 hr later with 25 pfu/cell of Ad.vec, Ad.mda-7, or Ad.SP-mda-7 and overlaid with 1 × 10⁵ DU-145 cells suspended in 0.4% agar. Fourteen days later, with agar medium feeding every 4 days, the number of anchorage-independent DU-145 colonies >2 mm was enumerated microscopically. Average number of colonies ±SD from triplicate plates. Qualitatively similar results were obtained in 2 additional studies. pfu, plaque-forming unit; IL, interleukin (reproduced, by permission of the publisher, from Sauane et al., 2004b).

Fig. 10

Overview of the signaling pathways associated with Ad.mda-7 and MDA-7/IL-24 activity in cancer cells and in the immune system. Abbreviations: P, phosphorylation; PHA, phytohemagglutinin; LPS, lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon-γ; GM-CSF, granulocyte macrophage-colony stimulating factor; VEGF; vascular endothelial growth factor; TGF-β, transforming growth factor-β; PI3K/PKB, phosphatidylinositol 3-kinase/protein kinase B; FAK, focal adhesion kinase; MMP, matrix metalloproteinase; PKR, double-stranded RNA-dependent protein kinase R; MAPK, mitogen-activated protein kinase; eIF2α, eukaryotic translation initiation factor-2α; Tyk2, tyrosine kinase-2; STAT, signal transducer and activator of transcription; GADD, growth-arrest and DNA-damage inducible; Hsp, heat shock protein; Pp2A, protein phosphatase-2A; iNOS, inducible nitric oxide synthase (reproduced, by permission of the publisher, from Lebedeva et al., 2005a).

Fig. 11

Spread of mda-7/IL-24 RNA, DNA, and protein and biological effects (apoptosis) 24 hr after intratumoral injection. (A) Schematic representation of serial sections of tumor. (B) Decay of INGN 241 (Ad.mda-7) vector at the injection site. Immunohistochemical staining of different tumor sections and the median numbers of DNA and RNA copies determined by PCR and RT-PCR, respectively, are shown for each section. (C) Spread of MDA-7/IL-24 protein and biological effect (apoptosis) at the injection site. Protein expression correlates with apoptosis. Serial sections from each tumor were evaluated for MDA-7/IL-24 expression and TUNEL reactivity using immunohistochemistry. (D) Data from TUNEL assay and immunohistochemistry are plotted to indicate signals compared to distance from injection site (reproduced, by permission of the publisher, from Lebedeva et al., 2005a).

Fig. 12

Model illustrating the possible molecular basis of mda-7/IL-24 cancer cell-mediated apoptosis. The effects of known physiological and ectopic overexpression of mda-7/IL-24 are shown on left and right sides of the figure, respectively. Normally, mda-7/IL-24 binds to cognate receptors and activates STAT-1 and -3 transcription factors to mediate pathways affecting cell growth. Because mda-7/IL-24 mRNA and protein are normally seen in subpopulations of immune cells and melanocytes, effects are likely initiated in these cell types but might also affect neighboring non-producing cells because the protein is secreted. When normally or ectopically overexpressed, current findings indicate localization to the ER/Golgi compartments, whether or not the protein contains a secretory signal. Accumulation of MDA-7/IL-24 protein in this compartment triggers apoptosis that could apparently involve induction of pathways described currently as ER stress. However, MDA-7/IL-24 additionally acts indirectly on mitochondria to generate reactive oxygen species. A combination of pathways triggered by mda-7/IL-24 results in transformed cell-specific apoptosis. IL, interleukin; ER, endoplasmic reticulum; STAT, signal transducers and activators of transcription; ROS, reactive oxygen species (reproduced, by permission of the publisher, from Sauane et al., 2004b).