Overexpression of transforming growth factor B (TGF-B) is frequently associated with metastasis and poor prognosis, and TGF-B antagonism has been shown to prevent metastasis in preclinical models with surprisingly little toxicity. Here, we have used the transplantable 4T1 model of metastatic breast cancer to address underlying mechanisms. We showed that efficacy of the anti-TGF-B antibody 1D11 in suppressing metastasis was dependent on a synergistic combination of effects on both the tumor parenchyma and microenvironment. The main outcome was a highly significant enhancement of the CD8+ T-cell-mediated antitumor immune response, but effects on the innate immune response and on angiogenesis also contributed to efficacy. Treatment with 1D11 increased infiltration of natural killer cells and T cells at the metastatic site, and enhanced expression of coactivators (NKG2D) and cytotoxic effectors (perforin and granzyme B) on CD8+ T cells. On the tumor cells, increased expression of an NKG2D ligand (Rae1;) and of a death receptor (TNFRSF1A) contributed to enhanced immune cell-mediated recognition and lysis. The data suggest that elevated TGF-B expression in the tumor microenvironment modulates a complex web of intercellular interactions that aggregately promote metastasis and progression. TGF-B antibodies reverse this effect, and the absence of a major effect of TGF-B antagonism on any one cell compartment may be critical for a good therapeutic window and the avoidance of autoimmune complications. [Cancer Res 2008;68(10):3835-43]

Tumor-associated myeloid cells regulate tumor growth and metastasis, and their accumulation is a negative prognostic factor for breast cancer. Here we find calcium/calmodulin-dependent kinase kinase (CaMKK2) to be highly expressed within intratumoral myeloid cells in mouse models of breast cancer, and demonstrate that its inhibition within myeloid cells suppresses tumor growth by increasing intratumoral accumulation of effector CD8 + T cells and immune-stimulatory myeloid subsets. Tumor-associated macrophages (TAMs) isolated from Camkk2 −/− mice expressed higher levels of chemokines involved in the recruitment of effector T cells compared to WT. Similarly, in vitro generated Camkk2 −/− macrophages recruit more T cells, and have a reduced capability to suppress T cell proliferation, compared to WT. Treatment with CaMKK2 inhibitors blocks tumor growth in a CD8 + T cell-dependent manner, and facilitates a favorable reprogramming of the immune cell microenvironment. These data, credential CaMKK2 as a myeloid-selective checkpoint, the inhibition of which may have utility in the immunotherapy of breast cancer.

Background: Folylpolyglutamates are in the cytosol and mitochondria and the enzyme that makes these compounds, folylpolyglutamate synthetase (FPGS) is in both compartments. Results: Folylpolyglutamates cannot traverse mitochondrial membranes in either direction. Conclusion: Subcellular isoforms of FPGS are required to establish and maintain subcellular folate compartmentalization and function. Significance: Mitochondrial folates are a separate metabolic pool not in equilibrium with cytosol.

Oomycetes in the genus Phytophthora are among the most damaging plant pathogens worldwide. Two important species are Phytophthora cinnamomi, which causes root rot in thousands of native and agricultural plants, and Phytophthora agathidicida, which causes kauri dieback disease in New Zealand. As is the case for other Phytophthora species, management options for these two pathogens are limited. Here, we have screened over 100 compounds for their anti-oomycete activity, as a potential first step toward identifying new control strategies. Our screening identified eight compounds that showed activity against both Phytophthora species. These included five antibiotics, two copper compounds and a quaternary ammonium cation. These compounds were tested for their inhibitory action against three stages of the Phytophthora life cycle: mycelial growth, zoospore germination, and zoospore motility. The inhibitory effects of the compounds were broadly similar between the two Phytophthora species, but their effectiveness varied widely among life cycle stages. Mycelial growth was most successfully inhibited by the antibiotics chlortetracycline and paromomycin, and the quaternary ammonium salt benzethonium chloride. Copper chloride and copper sulfate were most effective at inhibiting zoospore germination and motility, whereas the five antibiotics showed relatively poor zoospore inhibition. Benzethonium chloride was identified as a promising antimicrobial, as it is effective across all three life cycle stages. While further testing is required to determine their efficacy and potential phytotoxicity in planta, we have provided new data on those agents that are, and those that are not, effective against P. agathidicida and P. cinnamomi. Additionally, we present here the first published protocol for producing zoospores from P. agathidicida, which will aid in the further study of this emerging pathogen.