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.

To study the effects of de novo induction of ornithine decarboxylase (ODC) activity in adult, quiescent skin, we generated transgenic mice in which the suprabasal expression of an inducible form of the ODC protein fused to a modified estrogen receptor ligand-binding domain (ODCER) is driven by an involucrin promoter. After topical treatment with the inducing agent 4-hydroxytamoxifen (4OHT), ODC activity and putrescine levels were dramatically increased in the epidermis but not in the dermis of transgenic mice. 4OHT treatment stimulated both proliferation as measured by bromodeoxyuridine incorporation in basal epidermal cells and differentiation shown by increased expression of differentiation markers. Furthermore, induction of ODC activity did not rescue primary epidermal keratinocyte cultures isolated from ODCER2 mice from a calcium-triggered DNA synthesis block, as measured by [3H]thymidine incorporation. In vivo induction of epidermal ODC enzyme activity significantly stimulated the vascularization of ODCER transgenic skin. Increased expression of interleukin-1beta and keratin 6, markers of keratinocyte activation seen in wound healing, was also observed in 4OHT-treated transgenic skin. These results suggest that de novo suprabasal induction of ODC activity in adult mouse skin activates keratinocytes and stimulates vascularization in the dermal layer in a manner similar to skin undergoing wound healing.

Cancer is often associated with an increased risk of thrombotic events which are exacerbated by treatment with chemotherapeutics such as cyclosphosphamide (CP). Evidence suggests that thrombin can stimulate tumor progression via formation of fibrin and activation of protease-activated receptors (PARs) and platelets. We examined the effect of co-treatment with CP and dabigatran etexilate, a direct inhibitor of thrombin, using the murine orthotopic 4T1 tumor model. Mice receiving co-treatment with both low dose CP and dabigatran etexilate had significantly smaller mammary tumors and fewer lung metastases than mice treated with CP or dabigratran etexilate alone. Co-treatment with dabigatran etexilate and low dose CP also significantly decreased the number of arginase C Gr-1 C CD11b C myeloid derived suppressor cells as well as levels of TGF-b in spleens from tumor bearing mice. 4T1 tumors express procoagulant tissue factor (TF) and spontaneously release TF C microparticles which are potent procoagulant factors that promote thrombin generation. Treatment with dabigatran etexilate alone prevented tumor-induced increases in circulating TF C microparticles and also decreased the numbers of tumor-induced activated platelets by 40%. These results show that co-treatment with dabigatran etexilate and CP synergistically inhibits growth and metastasis of mammary tumors, suggesting that oral administration of the thrombin inhibitor dabigatran etexilate may be beneficial in not only preventing thrombotic events in cancer patients but also in treating malignant tumors themselves.

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The levels of polyamines are elevated in neoplastic lesions as compared with normal tissues, and cancer cells tend to manifest a robust dependence on these compounds for proliferation and survival. We have recently demonstrated that a novel approach to polyamine depletion suppresses tumor growth in a T cell-dependent manner, highlighting a poorly appreciated role of polyamines as strong modulators of antitumor immune responses.

Elevated expression of ornithine decarboxylase (ODC) and increased synthesis of polyamines are hallmarks of epithelial tumorigenesis. The skin and tumors of K6/ODC and ODC/ Ras transgenic mice, in which overexpression of ODC has been targeted to hair follicles, were found to exhibit intrinsically high histone acetyltransferase (HAT) activity. We identified Tip60 as a candidate enzyme for contributing significantly to this abnormally high HAT activity. Compared with normal littermate controls, the levels of Tip60 protein and an alternative splice variant Tip53 were found to be greater in K6/ODC mouse skin. Furthermore, skin tumors that spontaneously develop in ODC/Ras bigenic mice typically have substantially more Tip60 protein than adjacent non-tumorbearing skin and exhibit a unique pattern of Tip60 size variants and chemically modified protein isoforms. Steadystate Tip60 and Tip53 mRNA levels were not affected in ODC-overexpressing skin and tumors, implying novel posttranscriptional regulation by polyamines. Given the diverse roles of Tip60, the overabundance of Tip60 protein is predicted to have biological consequences. Compared with normal littermate skin, we detected altered association of Tip60 with E2F1 and a subset of newly identified Tip60-interacting transcription factors in ODC transgenic mouse skin and tumors. E2F1 was shown to be bound in greater amounts to up-regulated target genes in ODC-overexpressing skin. Thus, up-regulation of Tip60 protein, influencing the expression of Tip60-regulated genes, could play a contributing role in polyaminemediated tumor promotion. (Cancer Res 2006; 66(16): 8116-22)