In various organisms, high intracellular manganese provides protection against oxidative damage through unknown pathways. Herein we use a genetic approach in S. cerevisiae to analyze factors that promote manganese as an anti-oxidant in cells lacking Cu/Zn superoxide dismutase (sod1Δ). Unlike certain bacterial systems [1], oxygen resistance in yeast correlates with high intracellular manganese without a lowering of iron. This manganese for anti-oxidant protection is provided by the Nramp transporters Smf1p and Smf2p, with Smf1p playing a major role. In fact, loss of manganese transport by Smf1p together with loss of the Pmr1p manganese pump is lethal to sod1Δ cells in spite of normal manganese SOD2 activity. Manganese-phosphate complexes are excellent superoxide dimustase mimics in vitro [2], yet through genetic disruption of phosphate transport and storage, we observed no requirement for phosphate in manganese suppression of oxidative damage. If anything, elevated phosphate correlated with profound oxidative stress in sod1Δ mutants. The efficacy of manganese as an anti-oxidant was drastically reduced in cells that hyper-accumulate phosphate without effects on MnSOD activity. Non-SOD manganese can provide a critical backup for Cu/Zn SOD1, but only under appropriate physiologic conditions.

The interaction between tumor cells and macrophages is crucial in promoting tumor invasion and metastasis. In this study, we examined a novel mechanism of intercellular communication, namely membranous actin-based tunneling nanotubes (TNTs), that occurs between macrophages and tumor cells in the promotion of macrophage-dependent tumor cell invasion. The presence of heterotypic TNTs between macrophages and tumor cells induced invasive tumor cell morphology, which was dependent on EGF-EGFR signaling. Furthermore, reduction of a protein involved in TNT formation, M-Sec (TNFAIP2), in macrophages inhibited tumor cell elongation, blocked the ability of tumor cells to invade in 3D and reduced macrophage-dependent long-distance tumor cell streaming in vitro. Using an in vivo zebrafish model that recreates macrophagemediated tumor cell invasion, we observed TNT-mediated macrophage-dependent tumor cell invasion, distant metastatic foci and areas of metastatic spread. Overall, our studies support a role for TNTs as a novel means of interaction between tumor cells and macrophages that leads to tumor progression and metastasis.

During metastasis to distant sites, tumor cells migrate to blood vessels. In vivo, breast tumor cells utilize a specialized mode of migration known as streaming where a linear assembly of tumor cells migrate directionally towards blood vessels on fibronectin-collagen I-containing ECM fibers in response to chemotactic signals. We have successfully reconstructed tumor cell streaming in vitro by co-plating tumors cells, macrophages, and endothelial cells on 2.5 um thick ECM-coated micro-patterned substrates. We found that tumor cells and macrophages, when plated together on the micro-patterned substrates, do not demonstrate sustained directional migration in only one direction (sustained directionality) but show random bi-directional walking. Sustained directionality of tumor cells as seen in vivo was established in vitro when beads coated with human umbilical vein endothelial cells (HUVEC) were placed at one end of the micro-patterned “ECM fibers” within the assay. We demonstrated that these endothelial cells supply the HGF required for the chemotactic gradient responsible for sustained directionality. Using this in vitro reconstituted streaming system, we found that directional streaming is dependent on, and most effectively blocked by, inhibiting the HGF/C-Met signaling pathway between endothelial cells and tumor cells. Key observations made with the in vitro reconstituted system implicating C-Met signaling were confirmed in vivo in mammary tumors using the in vivo invasion assay and intravital multiphoton imaging of tumor cell streaming. These results establish HGF/C-Met as a central organizing signal in blood vessel directed tumor cell migration in vivo and highlight a promising role for C-Met inhibitors in blocking tumor cell streaming and metastasis in vivo and for use in human trials.

Much of what is currently understood about the cell biology of metals involves their interactions with proteins. By comparison, little is known about metal interactions with intracellular inorganic compounds such as phosphate. Here we examined the role of phosphate in metal metabolism in vivo by genetically perturbing the phosphate content of Saccharomyces cerevisiae cells. Yeast pho80 mutants cannot sense phosphate and have lost control of phosphate uptake, storage and metabolism. We report here that pho80 mutants specifically elevate cytosolic and non-vacuolar phosphate and this in turn causes a wide range of metal homeostasis defects. Intracellular levels of the hard metal cations sodium and calcium increase dramatically, and cells become susceptible to toxicity from the transition metals manganese, cobalt, zinc and copper. Disruptions in phosphate control also elicit an iron starvation response, as pho80 mutants were seen to up-regulate iron transport genes. The iron responsive transcription factor Aft1p appears activated in cells with high phosphate in spite of normal intracellular iron levels. The high phosphate of pho80 mutants can be lowered by mutating Pho4p, the transcription factor for phosphate uptake and storage genes. Such lowering of phosphate by pho4 mutations reversed the high calcium and sodium of pho80 mutants and prevented the iron starvation response. However, pho4 mutations only partially reversed toxicity from heavy metals, representing a novel outcome of phosphate dysregulation. Overall these studies underscore the importance of maintaining a charge balance in the cell; a disruption in phosphate metabolism can dramatically impact on metal homeostasis.