Pharmacological inhibition of MDA-9/Syntenin blocks breast cancer metastasis through suppression of IL-1β

Melanoma differentiation associated gene-9 (MDA-9), Syntenin-1, or syndecan binding protein is a differentially regulated prometastatic gene with elevated expression in advanced stages of melanoma. MDA-9/Syntenin expression positively associates with advanced disease stage in multiple histologically distinct cancers and negatively correlates with patient survival and response to chemotherapy. MDA-9/Syntenin is a highly conserved PDZ-domain scaffold protein, robustly expressed in a spectrum of diverse cancer cell lines and clinical samples. PDZ domains interact with a number of proteins, many of which are critical regulators of signaling cascades in cancer. Knockdown of MDA-9/Syntenin decreases cancer cell metastasis, sensitizing these cells to radiation. Genetic silencing of MDA-9/Syntenin or treatment with a pharmacological inhibitor of the PDZ1 domain, PDZ1i, also activates the immune system to kill cancer cells. Additionally, suppression of MDA-9/Syntenin deregulates myeloid-derived suppressor cell differentiation via the STAT3/interleukin (IL)-1β pathway, which concomitantly promotes activation of cytotoxic T lymphocytes. Biologically, PDZ1i treatment decreases metastatic nodule formation in the lungs, resulting in significantly fewer invasive cancer cells. In summary, our observations indicate that MDA-9/Syntenin provides a direct therapeutic target for mitigating aggressive breast cancer and a small-molecule inhibitor, PDZ1i, provides a promising reagent for inhibiting advanced breast cancer pathogenesis.

Breast cancer remains the second leading cause of death among women in the United States (1). Prognosis for early-stage disease is favorable, whereas late-stage disease with tumor cell spread beyond the primary site (i.e., metastasis) frequently heralds poorer outcomes (1). Therapy of metastatic disease usually involves systemic chemotherapy combined with radiation, providing mostly palliative options to reduce metastatic outgrowth (2). Multiple unique and distinct biological steps and an interplay between transformed and nontransformed cells highlight complexities of the metastatic process, which habitually thwarts clinical intervention. In principle, targeting these processes independently or collectively could culminate in effective antimetastatic therapies.Melanoma differentiation-associated gene-9 (mda-9), also known as Syntenin-1 or syndecan binding protein (SDCBP), was cloned in our laboratory using subtraction hybridization from terminal differentiating metastasis-derived human melanoma cells treated with interferon (IFN)-β and the protein kinase C activator, mezerein (3, 4) (designated as mda-9/Syntenin). Preferential elevated expression of mda-9/Syntenin is evident in histologically distinct tumors and contributes to several steps in the metastatic process (5). These include tumor cell invasion and migration (6, 7), induction of angiogenesis through secretion of proangiogenic factors (8–10), enhancement of epithelial–mesenchymal transition (EMT) (11, 12), regulation of the expression of integrins affecting cell-adhesion processes (13), exosome biogenesis and exosome-mediated signaling in cell–cell communication (14), and recently immune-modulation suppressing host-immune surveillance (15). Cancer cell-independent functions of MDA-9/Syntenin also contribute to metastatic progression by regulating immunosuppressive cell infiltration in the metastatic niche (16). Based on its relevance to the invasive and metastatic phenotype of cancers, MDA-9/Syntenin represents a prospective target for rational design of antimetastatic drugs.Differential expression of MDA-9/Syntenin in cancer versus adjacent normal tissue is often a predictor of poor clinical outcomes (8). A relationship exists between MDA-9/Syntenin (SDCBP) and breast cancer in rat mammary tumors (genomic localization) (17) and in metastasis and clinical situations in human triple negative and other human breast cancers (11, 15, 18). MDA-9/Syntenin plays a pivotal role in EMT induction that includes initiation of Smad-dependent EMT through interaction with TGF-βR1, disrupting receptor internalization (11). Physical interaction between MDA-9/Syntenin and TGF-β activates small GTPases, Rho A, and CDC 42 (12). In addition, MDA-9/Syntenin enhances primary tumor growth and lung metastasis through immune evasion by up-regulating PD-L1 (program death ligand 1) through STAT3 activation, causing T cell apoptosis (15). In breast cancer, MDA-9/Syntenin affects tumor cell proliferation in estrogen receptor-negative breast cancer, causing cells to bypass the G1/S checkpoint promoting S-phase entry (19). MDA-9/Syntenin is also considered a potential antigen in breast cancer (20). These observations endorse MDA-9/Syntenin as a prospective target for the therapy of breast cancer metastasis.Disturbing MDA-9/Syntenin protein:protein interactions is viewed as a viable strategy to disrupt key downstream signaling pathways regulating cancer cell invasion and metastasis (reviewed in ref. 5). Fragment-based drug discovery guided by NMR identified a first in-class interaction inhibitor of the PDZ1 domain of MDA-9/Syntenin, PDZ1i (21), displaying efficacy against glioblastoma multiforme, neuroblastoma, and prostate cancer (5, 13, 22, 23). PDZ1i suppresses cancer cell-autonomous and nonautonomous functions of MDA-9/Syntenin, culminating in strong antiinvasive and antimetastatic properties in vitro and in vivo, without inducing overt cytostatic or toxic effects in normal or most cancer cells. Informed by the crystal structure of MDA-9/Syntenin, peptide-based inhibitory molecules have been developed and validated in cell-based assays (24). Additionally, a genetic approach using adenovirus-mediated delivery of shmda-9 has shown efficacy in xenografted human melanoma (8) and prostate cancer (25) in nude mice. These investigations confirm MDA-9/Syntenin as a viable target for suppressing both primary and metastatic tumor growth and support further evaluation of PDZ1i on breast cancer metastasis.Evidence from both experimental models and clinical studies show a relationship between abundance of tumor-infiltrating immune cells and metastasis (26). To create a permissive environment in a secondary site, disseminated tumor cells employ multiple strategies, including reducing host immune surveillance (27). In several mouse tumor models, myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid cells with immunosuppressive properties, are expanded in the blood, lymph nodes, and spleen (28). They help shape the microenvironment and metastatic niches by regulating both innate and adaptive immunity (29). In breast cancer mouse models, MDSCs accumulate in the lungs prior to metastatic spread (30) and promote immune suppression by producing reactive oxygen species and arginase (Arg-1) (31). Not surprisingly, various chemotherapeutic agents, such as gemcitabine (32), 5-flurouracil (33), and docetaxel (34) decrease MDSC accumulation in the tumors’ stroma, thereby enhancing antitumor immune responses (29). Similar soluble factors are operational in primary tumors and metastases, including granulocyte-macrophage colony-stimulating factor, interleukins (e.g., IL-6, IL-1β), and vascular endothelial growth factor (VEGF), causing MDSC infiltration. Tumor cells in the metastatic niche that produce various cytokines or growth factors also regulate this process.We have now explored a potential role of MDA-9/Syntenin in breast cancer progression with specific emphasis on a relevant interleukin, IL-1β, representing an important inflammatory cytokine mediating cancer pathogenesis and tumor progression (35). Inflammation regulates fundamental pathways that are causative of the cancer phenotype, including proliferation, survival, and migration (36). IL-1β regulates tumor initiation/progression, angiogenesis, Th17 cell differentiation, and expansion of MDSCs (35). Additionally, IL-1β controls macrophage recruitment and invasion, and metastasis of cancer cells (35). Based on these seminal roles in orchestrating the neoplastic process, IL-1β represents a potential therapeutic target and its regulation deserves further analysis. We now confirm that MDA-9/Syntenin, which can be obstructed by the small-molecule inhibitor PDZ1i, regulates IL-1β, thereby directly controlling breast cancer pathogenesis.