Elucidation of Genomic Structure, Biological Properties, and Functional Status of Canine MDA-7 and Its Receptors

Abstract

Human melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a tumor suppressor gene with interleukin properties. Ectopic expression of MDA-7 protein causes growth suppression and induces apoptosis in a wide variety of cancer cells. In this study, we identified a canine ortholog of the human mda-7 gene, and elucidated its genomic structue and biological properties. Canine mda-7 was mapped to chromosome 7, located in a cluster of IL-10 family members. Canine MDA-7 was found to be endogenously expressed in cultured normal canine epidermal keratinocytes (NCEKs). When compared to human mda-7 mRNA, canine mda-7 mRNA had a very short 3’ untranslated region. Pre-mRNA transcribed from the canine MDA-7 locus was alternatively spliced (Exon skipping and use of alternative 5’ donor sites) to yield five splice variants in cultured NCEKs. Canine mda-7 splice variant1 (sv1) was the predominant splice variant expressed in NCEKs, while sv2 and sv5 were expressed at intermediate levels and sv3 and sv4 at the lowest level. These splice variants encode four isoforms of canine MDA-7 protein, which have similar amino acid sequences at their amino terminus. Canine MDA-7 is constitutively expressed in NCEKs and its expression was induced in PBMCs after lipopolysaccharide (LPS) stimulation. Similarly, expression of canine mda-7 mRNA was increased in NCEKs after LPS stimulation. Canine MDA-7 mRNA was not expressed in most canine tumor cells and tumor samples. However, one cancer cell line, canine mammary tumor-12 (CMT12) expressed canine MDA-7 at very high levels. When ectopically expressed from a plasmid vector, it suppressed the growth of canine and human tumor cells. Canine MDA-7 treated cancer cells accumulated in G2/M phase and underwent apoptosis. Canine MDA-7 has cytotoxic effects on cancer cells but not on normal canine fibroblasts (NCFs). In addition, canine MDA-7 protein isoforms also have growth inhibitory effects on cancer cells. Canine MDA-7 has a 28 amino acid long signal peptide sequence, and a possible cleavage site between the 28th and 29th amino acid. Canine MDA-7 was determined to be actively secreted and could bind to and signal through human MDA-7 receptors (IL-20R1/IL-20R2 and IL-22R1/IL-20R2). Secreted canine MDA-7 showed bystander antitumor activity against human tumor cells. In this study, we identified and showed that canine IL-20R1 and IL-22R1 subunits were expressed in NCEKs, canine tissues and tumor cells. A truncated mRNA sequence encoding for the extracellular domain of canine IL-20R2 subunit was also identified. The truncated canine IL-20R2 subunit does not have a transmembrane or intracellular domain and thus, it cannot translocate into the cell membrane to make functional canine MDA-7 receptor. Canine MDA-7 did not show bystander antitumor activity against canine tumor cells. This is either due to a lack of expression of functional MDA-7 receptors or due to expession of a truncated canine IL-20R2 subunit, which can block the activity of canine IL-24. In summary, we have shown that canine MDA-7 is indeed an ortholog of MDA-7. It has strikingly similarities in amino acid sequence, genomic and protein structure to human MDA-7. However, the canine MDA-7 receptor system appears to be defective, due to the absence of a full-length IL-20R2 subunit, leading to a failure of the mechanism responsible for the bystander antitumor effect. Although the canine MDA-7 has the potential to be used for cancer gene therapy to treat canine cancers, however, the lack of a bystander effect in dogs may limit its usefulness.