Susan K. Gilmour

Use of dabigatran etexilate to reduce breast cancer progression.

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.

Tip60 protein isoforms and altered function in skin and tumors that overexpress ornithine decarboxylase.

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-tumor-bearing skin and exhibit a unique pattern of Tip60 size variants and chemically modified protein isoforms. Steady-state 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 polyamine-mediated tumor promotion. (

Elevated Ornithine Decarboxylase Levels Activate Ataxia Telangiectasia Mutated–DNA Damage Signaling in Normal Keratinocytes

We examined the effect of increased expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on cell survival in primary cultures of keratinocytes isolated from the skin of K6/ODC transgenic mice (Ker/ODC) and their normal littermates (Ker/Norm). Although elevated levels of ODC and polyamines stimulate proliferation of keratinocytes, Ker/ODC undergo apoptotic cell death within days of primary culture unlike Ker/Norm that continue to proliferate. Phosphorylation of ataxia telangiectasia mutated (ATM) and its substrate p53 are significantly induced both in Ker/ODC and in K6/ODC transgenic skin. Chromatin immunoprecipitation analyses show that the increased level of p53 in Ker/ODC is accompanied by increased recruitment of p53 to the Bax proximal promoter. ATM activation is polyamine dependent because alpha-difluoromethylornithine, a specific inhibitor of ODC activity, blocks its phosphorylation. Ker/ODC also displays increased generation of H(2)O(2), acrolein-lysine conjugates, and protein oxidation products as well as polyamine-dependent DNA damage, as measured by the comet assay and the expression of the phosphorylated form of the histone variant gamma H2AX. Both reactive oxygen species generation and apoptotic cell death of Ker/ODC may, at least in part, be due to induction of a polyamine catabolic pathway that generates both H(2)O(2) and cytotoxic aldehydes, because spermine oxidase (SMO) levels are induced in Ker/ODC. In addition, treatment with MDL 72,527, an inhibitor of SMO, blocks the production of H(2)O(2) and increases the survival of Ker/ODC. These results show a novel activation of the ATM-DNA damage signaling pathway in response to increased ODC activity in nontumorigenic keratinocytes.

A novel polyamine blockade therapy activates an anti-tumor immune response

Most tumors maintain elevated levels of polyamines to support their growth and survival. This study explores the anti-tumor effect of polyamine starvation via both inhibiting polyamine biosynthesis and blocking the upregulated import of polyamines into the tumor. We demonstrate that polyamine blockade therapy (PBT) co-treatment with both DFMO and a novel polyamine transport inhibitor, Trimer PTI, significantly inhibits tumor growth more than treatment with DFMO or the Trimer PTI alone. The anti-tumor effect of PBT was lost in mice where CD4+ and CD8+ T cells were antibody depleted, implying that PBT stimulates an anti-tumor immune effect that is T-cell dependent. The PBT anti-tumor effect was accompanied by an increase in granzyme B+, IFN-γ+ CD8+ T-cells and a decrease in immunosuppressive tumor infiltrating cells including Gr-1+CD11b+ myeloid derived suppressor cells (MDSCs), CD4+CD25+ Tregs, and CD206+F4/80+ M2 macrophages. Stimulation with tumor-specific peptides elicited elevated antigen-specific IFN-γ secretion in splenocytes from PBT-treated mice, indicating that PBT treatment stimulates the activation of T-cells in a tumor-specific manner. These data show that combined treatment with both DFMO and the Trimer PTI not only deprives polyamine-addicted tumor cells of polyamines, but also relieves polyamine-mediated immunosuppression in the tumor microenvironment, thus allowing the activation of tumoricidal T-cells.

Thrombin inhibition and cisplatin block tumor progression in ovarian cancer by alleviating the immunosuppressive microenvironment

Cancer is often associated with an increased risk of thrombotic complications which can be aggravated by treatment with chemotherapeutics such as cisplatin. Multiple lines of evidence suggest that thrombin activity promotes tumor growth and metastasis. We examined the effect of co-treatment with dabigatran etexilate, a direct thrombin inhibitor, and cisplatin using the murine ID8 ovarian cancer model. Mice receiving co-treatment with both dabigatran etexilate and low dose cisplatin had significantly smaller tumors, developed less ascites and had lower levels of circulating activated platelets and tissue factor (TF) positive microparticles than those treated with dabigatran etexilate or cisplatin alone. Co-treatment with dabigatran etexilate and cisplatin significantly decreased the number of Gr1+/CD11b+ myeloid derived suppresser cells and CD11b+/CD11c+ dendritic cells in the ascites of ID8 tumor-bearing mice. Co-treatment also significantly reduced levels of pro-tumorigenic cytokines including TGF-β, VEGF, IL-6, IL-10, and MCP-1 in the ascites while increasing IFN-γ production by CD8+ effector T cells in the tumor ascites. These results demonstrate that co-treatment with dabigatran etexilate significantly augments the anti-tumor activity of cisplatin in ovarian tumor progression by alleviating the immunosuppressive microenvironment, suggesting that thrombin may be a potential therapeutic target for treatment of ovarian cancer.

A Novel Polyamine-Targeted Therapy for BRAF Mutant Melanoma Tumors

Mutant serine/threonine protein kinase B-Raf (BRAF) protein is expressed in over half of all melanoma tumors. Although BRAF inhibitors (BRAFi) elicit rapid anti-tumor responses in the majority of patients with mutant BRAF melanoma, the tumors inevitably relapse after a short time. We hypothesized that polyamines are essential for tumor survival in mutant BRAF melanomas. These tumors rely on both polyamine biosynthesis and an upregulated polyamine transport system (PTS) to maintain their high intracellular polyamine levels. We evaluated the effect of a novel arylpolyamine (AP) compound that is cytotoxic upon cellular entry via the increased PTS activity of melanoma cells with different BRAF mutational status. Mutant BRAF melanoma cells demonstrated greater PTS activity and increased sensitivity to AP compared to wild type BRAF (BRAFWT) melanoma cells. Treatment with an inhibitor of polyamine biosynthesis, α-difluoromethylornithine (DFMO), further upregulated PTS activity in mutant BRAF cells and increased their sensitivity to AP. Furthermore, viability assays of 3D spheroid cultures of mutant BRAF melanoma cells demonstrated greater resistance to the BRAFi, PLX4720, compared to 2D monolayer cultures. However, co-treatment with AP restored the sensitivity of melanoma spheroids to PLX4720. These data indicate that mutant BRAF melanoma cells are more dependent on the PTS compared to BRAFWT melanoma cells, resulting in greater sensitivity to the PTS-targeted cytotoxic AP compound.