Mena deficiency delays tumor progression and decreases metastasis in polyoma middle-T transgenic mouse mammary tumors

Introduction The actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development. Methods To investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice. Results Mena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching. Conclusions Deficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena deficiency during development causes defects in invasive processes involved in mammary gland development. These findings suggest that functional intervention targeting Mena in breast cancer patients may provide a valuable treatment option to delay tumor progression and decrease invasion and metastatic spread leading to an improved prognostic outcome.National Cancer Institute (U.S.). Integrative Cancer Biology Program (grant U54 CA112967)Virginia and D.K. Ludwig Fund for Cancer Researc

Response to nitrogen addition reveals metabolic and ecological strategies of soil bacteria

The nitrogen (N) cycle represents one of the most well-studied systems, yet the taxonomic diversity of the organisms that contribute to it is mostly unknown, or linked to poorly characterized microbial groups. While new information has allowed functional groups to be refined, they still rely on a priori knowledge of enzymes involved and the assumption of functional conservation, with little connection to the role the transformations, plays for specific organisms. Here, we use soil microcosms to test the impact of N deposition on prokaryotic communities. By combining chemical, genomic and transcriptomic analysis, we are able to identify and link changes in community structure to specific organisms catalysing given chemical reactions. Urea deposition led to a decrease in prokaryotic richness, and a shift in community composition. This was driven by replacement of stable native populations, which utilize energy from N-linked redox reactions for physiological maintenance, with fast responding populations that use this energy for growth. This model can be used to predict response to N disturbances and allows us to identify putative life strategies of different functional and taxonomic groups, thus providing insights into how they persist in ecosystems by niche differentiation

Competition and community succession link N transformation and greenhouse gas emissions in urine patches

Nitrous oxide (N₂O) is a strong greenhouse gas produced by biotic/abiotic processes directly linked to both fungal and prokaryotic communities that produce, consume or create conditions leading to its emission. In soils exposed to nitrogen (N) in the form of urea, an ecological succession is triggered resulting in a dynamic turnover of microbial populations. However, knowledge of the mechanisms controlling this succession and the repercussions for N₂O emissions remain incomplete. Here, we monitored N₂O production and fungal/prokaryotic community changes (via 16S and 18S amplicon sequencing) in soil microcosms exposed to urea. Contributions of microbes to emissions were determined using biological inhibitors. Results confirmed that urea leads to shifts in microbial community assemblages by selecting for certain microbial groups (fast growers) as dictated through life history strategies. Urea reduced overall community diversity by conferring dominance to specific groups at different stages in the succession. The diversity lost under urea was recovered with inhibitor addition through the removal of groups that were actively growing under urea indicating that species replacement is mediated in part by competition. Results also identified fungi as significant contributors to N₂O emissions, and demonstrate that dominant fungal populations are consistently replaced at different stages of the succession. These successions were affected by addition of inhibitors which resulted in strong decreases in N₂O emissions, suggesting that fungal contributions to N₂O emissions are larger than that of prokaryotes

Fungal and bacterial contributions to codenitrification emissions of N₂O and N₂ following urea deposition to soil

Grazed pastures contribute significantly to anthropogenic emissions of N₂O but the respective contributions of archaea, bacteria and fungi to codenitrification in such systems is unresolved. This study examined the relative contributions of bacteria and fungi to rates of denitrification and codenitrification under a simulated ruminant urine event. It was hypothesised that fungi would be primarily responsible for both codenitrification and total N₂O and N₂ emissions. The effects of bacterial (streptomycin), fungal (cycloheximide), and combined inhibitor treatments were measured in a laboratory mesocosm experiment, on soil that had received ¹⁵N labelled urea. Soil inorganic-N concentrations, N₂O and N₂ gas fluxes were measured over 51 days. On Days 42 and 51, when nitrification was actively proceeding in the positive control, the inhibitor treatments inhibited nitrification as evidenced by increased soil NH₄ + -N concentrations and decreased soil NO₂ ⁻ -N and NO 3 ⁻ -N concentrations. Codenitrification was observed to contribute to total fluxes of both N₂O (≥ 33%) and N 2 (≥ 3%) in urine-amended grassland soils. Cycloheximide inhibition decreased NH₄ ⁺ –¹⁵N enrichment and reduced N₂O fluxes while reducing the contribution of codenitrification to total N₂O fluxes by ≥ 66 and ≥ 42%, respectively. Thus, given archaea do not respond to significant urea deposition, it is proposed that fungi, not bacteria, dominated total N₂O fluxes, and the codenitrification N₂O fluxes, from a simulated urine amended pasture soil

Urea treatment decouples intrinsic pH control over N₂O emissions in soils

Soil N₂O emission potential is commonly investigated under idealized denitrifying conditions (e.g. nitrate-N supplied and anaerobic soil), with pH commonly identified as a major determinant of N₂O emission potential. However, under urine patch conditions in grazed pastures soils a more complex series of abiotic and biotic factors may influence emissions due to the complex N transformations that occur following urea hydrolysis. These transformations may decouple native and/or expected controls of N₂O emissions encountered under classic denitrifying conditions. Here, we tracked O₂, CO₂, NO, N₂O and N₂ emissions from urine amended soils (i.e. simulating a urine patch) to determine putative controls of N₂O emissions within 13 different pasture soils from northern (Ireland) and southern hemispheres (New Zealand). Incubations were performed under aerobic conditions±artificial urine (13.3 mg N vial¯¹) equivalent to field ruminant urine deposition rates of 1000 kg N ha¯¹. Results revealed that pH was not an important regulator of the emission ratio (N₂O /(NO + N₂O + N₂)) in urine amended soils. Within urine affected soils, a new set of variables emerged as regulators of N₂O emissions, likely due to the unique environment created within this system. We show that urine results in decoupling of the initial soil pH control of the emission ratio allowing other regulators such as nitrite to dominate. In addition, we observed that the emission ratio of N₂O increased linearly with the rate of N- gas loss (NO + N₂O + N₂ μmol N h¯¹), O₂ consumption was positively associated with ammonia oxidising bacteria (AOB) and that the production of NO and N₂O were also enhanced under urine conditions