MDA-7/IL-24, NOVEL ANTICANCER CYTOKINE: TRANSLATION FROM THE LABORATORY TO THE CLINIC [FROM BENCH TO BEDSIDE] I.Gst.

Agung Raka Mahasadu1, Senior Clerkship in Faculty of Medicine, Udayana University, ABSTRACT The novel cytokine melanoma differentiation associated gene-7 (MDA-7) was identified by subtractive hybridization in the mid-1990s as a protein whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to nontransformed cells. Since its identification sixteen years ago, MDA-7 has been the object of intense focus because of its unique biological properties. Firstly, mda-7 is a secreted protein having cytokine-like properties and belonging to the IL-10 cytokine family. Based on this consideration, MDA-7 was renamed IL-24. Secondly, if delivered by means of an adenoviral vector, MDA-7 induces selective apoptosis in cancer cells of diverse origin, while sparing their normal cellular counterparts. As such, mda-7 has become a novel tool for cancer gene therapy. Multiple studies have demonstrated that expression of MDA-7/IL-24 in a wide variety of tumor cell types, but not in corresponding equivalent non-transformed cells, causes their growth arrest and ultimately cell death. In addition, MDA-7/IL-24 has been noted to be a radiosensitizing cytokine, which in part is due to the generation of reactive oxygen species (ROS) and ceramide that cause endoplasmic reticulum stress. Phase I clinical trial data has shown that a recombinant adenovirus expressing MDA-7/IL-24 (Ad.mda-7 (INGN-241)) was safe and had measurable tumoricidal effects in over 40% of patients, which strongly argues that MDA-7/IL-24 may have significant therapeutic value. The present review examines the biological properties of mda-7, the signaling pathways that contribute to its unique cancer-specific apoptosisinducing properties and its potential therapeutic applications. Introduction Two important hallmarks of neoplastic transformation are aberrant growth and abnormal differentiation. Thus, induction of growth arrest and differentiation is a potential therapeutic intervention for cancer. Several chemical agents that modulate intracellular signal transduction pathways have differentiation-inducing properties in cancers of diverse tissue types. It is hypothesized that cancer cells retain the appropriate genetic information necessary for terminal cell differentiation, but these

genes are either not expressed or they are expressed at sub-threshold levels that preclude biological activity. A well-established model for studying differentiation therapy in cancer is human melanoma. Several in vitro cultures of malignant human melanoma cells cease proliferating and display a differentiated phenotype, analogous to normal melanocytes. To determine the mechanism of terminal cell differentiation, multiple molecular approaches are being used to define the complete spectrum of gene expression changes associated withand potentially mediatingdifferentiation. Classical subtraction hybridization procedures and improved modificationsof this scheme. These approaches have uncovered a diverse spectrum of melanoma differentiation associated MDA genes, both known and unknown, which are upregulated during terminal differentiation. A novel MDA gene, MDA-7, was identified with a subtraction hybridization protocol using temporally-spaced cDNA libraries isolated from terminally differentiated human melanoma cells treated with sinterferon and the protein kinase C activator mezerein. MDA-7, a cytokine-like molecule (also known as IL-24) that has ubiquitous cancer growth-suppressor properties. The purpose of this review is to provide a brief chronicle of the discovery, current understanding of the mode of action, and results of clinical studies of mda7/IL-24, suggesting that this novel cytokine may indeed be the long sought, proverbial magical bullet for cancer and its potential therapeutic applications. What Is MDA-7?: The Discovery of MDA-7/IL-24 and Its Biologic Effects The MDA-7 gene is selectively up-regulated during terminal differentiation of human melanoma cells. The MDA-7/IL-24 cDNA is 1718-bp in length with an open reading frame encoding a polypeptide of 206 amino acids with a predicted size of 23.8 kDa. The predicted reading frame is flanked by 5-and 3-untranslated sequences of 274 bp and 823 bp, respectively.The 3-UTR contains three consensus elements (AUUUA) involved in mRNA stability and three polyadenylation signals (AAUAAA). The

MDA-7/IL-24 gene is composed of seven exons and six introns and is located in chromosome 1q32 within an interleukin (IL)-10 related gene cluster containing four genes (IL-10, IL-19, IL-20, and mda-7) in linear order spanning 195 kb of genomic DNA. MDA-7/IL-24 displays at least two distinct biological functions. At low concentration, MDA-7/IL-24 functions predominantly as a cytokine. However, when overexpressed at supra-physiological levels using a replication-incompetent adenoviral vector, MDA-7/1L-24 shows cancer cell specific growth inhibitory properties without negatively affecting normal cells. These two important functional attributes of MDA-7/1L-24 have stimulated growing interest in understanding the mechanism of action of this interesting molecule. The MDA-7/IL-24 protein contains a consensus signal sequence and a proteolytic cleavage site permitting secretion from cells. Expression of MDA-7/IL-24 protein in normal cells is restricted to cells of the immune system and melanocytes. MDA-7/IL-24 expression is decreased during pathologic progression of melanocytes to melanomas as demonstrated by both RT-PCR and immunohistochemistry. This correlation between the loss of gene expression and tumor invasion suggests that mda-7/IL-24 might function as a tumor suppressor. Further studies in vitro indicated that the functional antitumor attributes of MDA-7/IL-24 were dependent on MDA7/IL-24 expression level. When present at low physiological levels, mda-7/IL-24 exhibited specific immunostimulatory properties. Other published studies over the last 15 years have demonstrated that enforced expression of MDA-7/IL-24, either by transfection of tumor cells with a plasmid containing the cDNA for MDA-7/IL-24 or by use of a recombinant adenovirus to deliver the gene, Ad.mda-7, rapidly inhibits the growth of a broad-spectrum of cancer cells, resulting in tumor cell death within 2448 h. When expressed, MDA-7/IL-24 is secreted from cells, as would be expected for a cytokine. Of considerable note, when MDA-7/IL-24 was over-expressed in nontransformed cells little change was observed in either cell growth or cell viability.

Molecular Pathways Involved in MDA-7/IL-24 Activity

Expanding studies in diverse systems reveal that the signal transduction pathways mediating Ad.MDA-7-induced apoptosis are versatile and different signaling pathways are activated in different cell lines (summarized in Fig. 2).These studies underscore the complexity of action of this novel cytokine.  IL-20/IL-22 receptor independence. Within the IL-10 family, IL-19, IL-20 and IL-24 exhibit

substantial sharing of receptor complexes; all three are capable of signaling through IL-20R1/IL-20R2, and both IL-20 and IL-24 can also use IL-22R1/IL-20R2. Nevertheless, the biological activities of these three cytokines appear quite distinct; since only mda-7/IL-24 induces tumor-specific apoptosis, and this effect can be receptor-independent. Initial studies using mammalian cell synthesized MDA-7/IL-24 protein; a protein that is a dimer and glycosylated, demonstrated that purified MDA-7/IL-24 interacted with two type II cytokine hetero-dimeric receptor complexes: IL-20R1/IL-20R2 (type 1 IL-20R) and IL-22R1/IL-20R2 (type 2 IL20R). In one of the first of these studies, nontransformed BHK cells stably transfected with IL-20 and IL-22 receptors were treated with MDA-7/IL-24; at low pM concentrations of MDA-7/IL-24 (<100 pM) growth was promoted whereas at higher concentrations (>100 pM) it inhibited cell proliferation. In cells transfected transfected to express IL-20 receptor complexes, MDA-7/IL-24 activated multiple STAT transcription factors. However, in ovarian carcinoma cells, which express endogenous IL-20 receptor complexes, it was noted that MDA-7/IL-24 at low nM concentrations promoted growth inhibition without altering STAT transcription factor phosphorylation or function. Treatment with tyrosine kinase specific inhibitors or a JAK-selective inhibitor does not alter Ad.MDA-7-induced apoptosis in diverse cancer cell lines. In addition, there is no correlation between the pattern of expression of IL-20R1, IL-20R2, and IL-22R

mRNA and susceptibility to Ad.MDA-7-induced cell death in different cell lines. Therefore, signaling events leading to Ad.MDA-7-induced apoptosis might be tyrosine kinase independent and can be distinguished from MDA-7/IL-24 cytokine function-related properties mediated by the IL-20/IL-22 receptor complexes that require JAK/STAT kinase activity.

 Mitochondrial pathway and reactive oxygen species (ROS) production.  ER-stress and its link to mitochondria. MDA-7/IL-24 has also been shown to kill cancer cells by causing endoplasmic reticulum (ER) stress. ER stress may be caused by misfolded protein accumulation followed by the activation of a highly conserved unfolded protein response (UPR). These events lead to apoptosis through the induction of GADD genes. The hypothesis of a role for MDA-7/IL-24-induced ER stress and cancer cell apoptosis is supported by the fact that MDA-7/IL-24 induces GADD genes and further activates p38MAPK in the context of transformed cells.

 Multiple signaling pathways regulate MDA-7/IL-24-induced apoptosis in cancer cells. Initial studies in human melanoma, subsequently confirmed in malignant glioma and prostate carcinomas, confirm a role of p38 mitogen-activated protein kinase (MAPK) and induction of the growth arrest and DNA damage inducible gene (GADD) family, including GADD34, GADD45, and GADD153, in selective induction of apoptosis. The GADD gene family consists of GADD34, GADD45a, GADD45h, GADD45g, and GADD153, which encode highly acidic proteins. Overexpression of each GADD gene causes growth inhibition and/or apoptosis

and combined overexpression of the GADD genes leads to synergistic or cooperative antiproliferative effects.

 MDA-7/IL-24 can induce apoptosis by an intracellular process in a JAK/STAT independent manner. The canonical mechanism by which diverse cytokines, including members of the IL10 gene family, are proposed to mediate bioactivity is by binding to defined cell surface receptors and activating JAK/STAT signaling pathways. In the case of mda7/IL-24, JAK/STAT activation is induced after binding of MDA-7/IL-24 protein to the IL-20R1/IL-22R1 and IL-20R2/IL-22R1 receptors. Immune properties of MDA-7/IL-24 Attacking Cancer on Many Fronts: MDA-7/IL-24 Inhibits Tumor Angiogenesis and Expression Radiosensitizes Cancer Cells

Translation of mda-7/IL-24 into the clinic: A good beginning! Concluding Perspective on MDA-7/IL-24: A Gene With A Potentially Bright Future