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PDF file - 5906K, Supplemental Table I - Inhibition of mammary carcinogenesis in Wistar-Furth rats by mda-7/IL-24 Supplemental Table II - Microarray analysis of differential gene expression in FE1.2 + IL-24S compared to FE1.2 + IL-24AS cells. Supplemental Figure 1: Serum starvation induces GAS3 expression in both FE1.2 and FE1.3 cells. Supplemental Figure 2: GAS3 downregulation decreases the growth of cells in soft agar. Supplemental Figure 3: STAT3 inhibition reduced attachment of attaches cells to culture plates. Supplemental Figure 4: GAS3 expression reduced attachment of confluent cells to culture plates. Supplemental Figure 5: Binding of GAS3 to β1 integrin using immunoprecipitation assay. Supplemental Figure 6: GAS3 expression inhibits attachment to and proliferation on fibronectin coated culture plates. Supplemental Figure 7: GAS3 expression increases attachment to laminin coated culture plates. Supplemental Figure 8: GAS3 expression by immunohistochemistry.

Melanoma differentiation-associated gene (MDA)-7)/interleukin (IL)-24, a member of the IL-10 family of cytokines, inhibits growth of various human cancer cells, yet the underlying mechanism is largely unknown. Here, we report that mda-7/IL-24 efficiently suppresses the development of rat mammary tumors in vivo. Microarray analysis for genes differentially expressed in rat mammary tumor cells overexpressing MDA-7/IL-24 compared with those that do not express this cytokine identified growth arrest-specific gene-3 (gas3) as a target for mda-7/IL-24. Upregulation of gas3 by mda-7/IL-24 was STAT3 dependent. Induction of gas3 inhibited attachment and proliferation of tumor cells in vitro and in vivo by inhibiting the interaction of β1 integrin with fibronectin. A mutated GAS3, which is unable to bind β1 integrin, was also unable to inhibit fibronectin-mediated attachment and cell growth both in adherent and suspension cultures, suggesting that GAS3 exerts its effects through interaction with and regulation of β1 integrin. Thus, mda-7/IL-24 inhibits breast cancer growth, at least in part, through upregulation of GAS3 and disruption of β1 integrin function. Importantly, the expression of the mda-7/IL-24 receptor, IL-20R1, is highly correlated with GAS3 expression in human breast cancer (P = 1.02 × 10−9), and the incidence of metastases is significantly reduced in patients with HER2+ breast cancer expressing high-levels of IL-20R1. Together, our results identify a novel MDA-7/IL-24-GAS3-β1integrin–fibronectin signaling pathway that suppresses breast cancer growth and can be targeted for therapy. Mol Cancer Res; 11(6); 593–603. ©2013 AACR.