Neuroblastoma is a frequently lethal childhood tumor in which MYC gene deregulation, commonly as MYCN amplification, portends poor outcome. Identifying the requisite biopathways downstream of MYC may provide therapeutic opportunities. We used transcriptome analyses to show that MYCN-amplified neuroblastomas have coordinately deregulated myriad polyamine enzymes (including ODC1, SRM, SMS, AMD1, OAZ2, and SMOX) to enhance polyamine biosynthesis. High-risk tumors without MYCN amplification also overexpress ODC1, the ratelimiting enzyme in polyamine biosynthesis, when compared with lower-risk tumors, suggesting that this pathway may be pivotal. Indeed, elevated ODC1 (independent of MYCN amplification) was associated with reduced survival in a large independent neuroblastoma cohort. As polyamines are essential for cell survival and linked to cancer progression, we studied polyamine antagonism to test for metabolic dependence on this pathway in neuroblastoma. The Odc inhibitor A-difluoromethylornithine (DFMO) inhibited neuroblast proliferation in vitro and suppressed oncogenesis in vivo. DFMO treatment of neuroblastoma-prone genetically engineered mice (TH-MYCN) extended tumor latency and survival in homozygous mice and prevented oncogenesis in hemizygous mice. In the latter, transient Odc ablation permanently prevented tumor onset consistent with a time-limited window for embryonal tumor initiation. Importantly, we show that DFMO augments antitumor efficacy of conventional cytotoxics in vivo. This work implicates polyamine biosynthesis as an arbiter of MYCN oncogenesis and shows initial efficacy for polyamine depletion strategies in neuroblastoma, a strategy that may have utility for this and other MYC-driven embryonal tumors. [Cancer Res 2008;68(23):9735-45]

Correcting T cell immunosuppression may unleash powerful antitumor responses, however, knowledge about the mechanisms and modifiers that may be targeted to improve therapy remains incomplete. Here we report that polyamine elevation in cancer, a common metabolic aberration in aggressive lesions, contributes significantly to tumor immunosuppression and that a polyamine depletion strategy can exert antitumor effects that may also promote immunity. A polyamine-blocking therapy (PBT) that combines the well-characterized ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) with AMXT1501, a novel inhibitor of the polyamine transport system, blocked tumor growth in immunocompetent mice but not in athymic nude mice lacking T cells. PBT had little effect on the proliferation of epithelial tumor cells but it increased the number of apoptotic cells. Analysis of CD45+ tumor immune infiltrates revealed that PBT decreased levels of Gr-1+CD11b+ myeloid suppressor cells and increased CD3+ T cells. Strikingly, in a model of neoadjuvant therapy, mice administered PBT one week before surgical resection of engrafted mammary tumors exhibited resistance to subsequent tumor re-challenge. Collectively, our results indicate that therapies targeting polyamine metabolism do not act exclusively as anti-proliferative agents, but also act strongly to prevent immune escape by the tumor. PBT may offer a general approach to heighten immune responses in cancer.

Coagulation proteases and the generation of thrombin are increased in breast tumor epithelial and stromal cells. Since thrombin can modify tumor cell behavior directly through the activation of protease-activated receptors (PARs) or indirectly by generating fibrin matrices, the effect of dabigatran etexilate, a direct thrombin inhibitor, on breast cancer development was evaluated. Dabigatran inhibited invasiveness of MDA-MB-231 breast carcinoma cells across Matrigel-coated membranes at concentrations that had no effect on the proliferation index of cultured tumor cells. In vivo evaluation of invasiveness of MDA-MB-231 cells in tracheal xenotransplants in nude mice orally administered dabigatran etexilate twice daily at a dose of 45 mg/kg over 4 weeks demonstrated less invasion of tumor cells through the tracheal wall compared to vehicle-treated mice. To evaluate the effect of dabigatran on the development of metastatic foci, 4T1 tumor cells were injected orthotopically in the mammary fat pads of syngeneic Balb/c mice. Dabigatran etexilate treatment exhibited evidence of antitumor activity with a 50% reduction in tumor volume at 4 weeks following orthotopic injection of 4T1 cells in syngeneic Balb/c mice with no weight loss in treated mice. Dabigatran etexilate reduced both 4T1 tumor cells in the blood and liver micrometastases by 50-60%. These results suggest that oral administration of the direct thrombin inhibitor, dabigatran etexilate, inhibits both invasion and metastasis of malignant breast tumors, suggesting that it may be beneficial in not only preventing thrombotic events in cancer patients, but also as adjunct therapy to treat malignant tumors.