Pubertal exposure to dietary advanced glycation end products disrupts ductal morphogenesis and induces atypical hyperplasia in the mammary gland.

BACKGROUND: Advanced glycation end products (AGEs) are reactive metabolites intrinsically linked with modern dietary patterns. Processed foods, and those high in sugar, protein and fat, often contain high levels of AGEs. Increased AGE levels are associated with increased breast cancer risk, however their significance has been largely overlooked due to a lack of direct cause-and-effect relationship. METHODS: To address this knowledge gap, FVB/n mice were fed regular, low AGE, and high AGE diets from 3 weeks of age and mammary glands harvested during puberty (7 weeks) or adulthood (12 weeks and 7 months) to determine the effects upon mammary gland development. At endpoint mammary glands were harvested and assessed histologically (n ≥ 4). Immunohistochemistry and immunofluorescence were used to assess cellular proliferation and stromal fibroblast and macrophage recruitment. The Kruskal-Wallis test were used to compare continuous outcomes among groups. Mammary epithelial cell migration and invasion in response to AGE-mediated fibroblast activation was determined in two-compartment co-culture models. In vitro experiments were performed in triplicate. The nonparametric Wilcoxon rank sum test was used to compare differences between groups. RESULTS: Histological analysis revealed the high AGE diet delayed ductal elongation, increased primary branching, as well as increased terminal end bud number and size. The high AGE diet also led to increased recruitment and proliferation of stromal cells to abnormal structures that persisted into adulthood. Atypical hyperplasia was observed in the high AGE fed mice. Ex vivo fibroblasts from mice fed dietary-AGEs retain an activated phenotype and promoted epithelial migration and invasion of non-transformed immortalized and tumor-derived mammary epithelial cells. Mechanistically, we found that the receptor for AGE (RAGE) is required for AGE-mediated increases in epithelial cell migration and invasion. CONCLUSIONS: We observed a disruption in mammary gland development when mice were fed a diet high in AGEs. Further, both epithelial and stromal cell populations were impacted by the high AGE diet in the mammary gland. Educational, interventional, and pharmacological strategies to reduce AGEs associated with diet may be viewed as novel disease preventive and/or therapeutic initiatives during puberty.

Impaired glucose metabolism underlies articular cartilage degeneration in osteoarthritis.

Osteoarthritis (OA) is the leading joint disease characterized by cartilage destruction and loss of mobility. Accumulating evidence indicates that the incidence and severity of OA increases with diabetes, implicating systemic glucose metabolism in joint health. However, a definitive link between cellular metabolism in articular cartilage and OA pathogenesis is not yet established. Here, we report that in mice surgically induced to develop knee OA through destabilization of medial meniscus (DMM), expression of the main glucose transporter Glut1 is notably reduced in joint cartilage. Inducible deletion of Glut1 specifically in the Prg4-expressing articular cartilage accelerates cartilage loss in DMM-induced OA. Conversely, forced expression of Glut1 protects against cartilage destruction following DMM. Moreover, in mice with type I diabetes, both Glut1 expression and the rate of glycolysis are diminished in the articular cartilage, and the diabetic mice exhibit more severe cartilage destruction than their nondiabetic counterparts following DMM. The results provide proof of concept that boosting glucose metabolism in articular chondrocytes may ameliorate cartilage degeneration in OA.

Chronic Engineered Nanoparticle Additions Alter Insect Emergence and Result in Metal Flux from Aquatic Ecosystems into Riparian Food Webs.

Freshwater ecosystems are exposed to engineered nanoparticles (NPs) through discharge from wastewater and agricultural runoff. We conducted a 9-month mesocosm experiment to examine the combined effects of chronic NP additions on insect emergence and insect-mediated contaminant flux to riparian spiders. Two NPs (copper, gold, plus controls) were crossed by two levels of nutrients in 18 outdoor mesocosms open to natural insect and spider colonization. We collected adult insects and two riparian spider genera, Tetragnatha and Dolomedes, for 1 week on a monthly basis. We estimated a significant decrease in cumulative insect emergence of 19% and 24% after exposure to copper and gold NPs, irrespective of nutrient level. NP treatments led to elevated copper and gold tissue concentrations in adult insects, which resulted in terrestrial fluxes of metals. These metal fluxes were associated with increased gold and copper tissue concentrations for both spider genera. We also observed about 25% fewer spiders in the NP mesocosms, likely due to reduced insect emergence and/or NP toxicity. These results demonstrate the transfer of NPs from aquatic to terrestrial ecosystems via emergence of aquatic insects and predation by riparian spiders, as well as significant reductions in insect and spider abundance in response to NP additions.

Preventing ovariectomy-induced weight gain decreases tumor burden in rodent models of obesity and postmenopausal breast cancer.

BACKGROUND: Obesity and adult weight gain are linked to increased breast cancer risk and poorer clinical outcomes in postmenopausal women, particularly for hormone-dependent tumors. Menopause is a time when significant weight gain occurs in many women, and clinical and preclinical studies have identified menopause (or ovariectomy) as a period of vulnerability for breast cancer development and promotion. METHODS: We hypothesized that preventing weight gain after ovariectomy (OVX) may be sufficient to prevent the formation of new tumors and decrease growth of existing mammary tumors. We tested this hypothesis in a rat model of obesity and carcinogen-induced postmenopausal mammary cancer and validated our findings in a murine xenograft model with implanted human tumors. RESULTS: In both models, preventing weight gain after OVX significantly decreased obesity-associated tumor development and growth. Importantly, we did not induce weight loss in these animals, but simply prevented weight gain. In both lean and obese rats, preventing weight gain reduced visceral fat accumulation and associated insulin resistance. Similarly, the intervention decreased circulating tumor-promoting growth factors and inflammatory cytokines (i.e., BDNF, TNFα, FGF-2), with greater effects in obese compared to lean rats. In obese rats, preventing weight gain decreased adipocyte size, adipose tissue macrophage infiltration, reduced expression of the tumor-promoting growth factor FGF-1 in mammary adipose, and reduced phosphorylated FGFR indicating reduced FGF signaling in tumors. CONCLUSIONS: Together, these findings suggest that the underlying mechanisms associated with the anti-tumor effects of weight maintenance are multi-factorial, and that weight maintenance during the peri-/postmenopausal period may be a viable strategy for reducing obesity-associated breast cancer risk and progression in women.

Advances in Analyzing the Breast Cancer Lipidome and Its Relevance to Disease Progression and Treatment.

Abnormal lipid metabolism is common in breast cancer with the three main subtypes, hormone receptor (HR) positive, human epidermal growth factor 2 (HER2) positive, and triple negative, showing common and distinct lipid dependencies. A growing body of studies identify altered lipid metabolism as impacting breast cancer cell growth and survival, plasticity, drug resistance, and metastasis. Lipids are a class of nonpolar or polar (amphipathic) biomolecules that can be produced in cells via de novo synthesis or acquired from the microenvironment. The three main functions of cellular lipids are as essential components of membranes, signaling molecules, and nutrient storage. The use of mass spectrometry-based lipidomics to analyze the global cellular lipidome has become more prevalent in breast cancer research. In this review, we discuss current lipidomic methodologies, highlight recent breast cancer lipidomic studies and how these findings connect to disease progression and therapeutic development, and the potential use of lipidomics as a diagnostic tool in breast cancer. A better understanding of the breast cancer lipidome and how it changes during drug resistance and tumor progression will allow informed development of diagnostics and novel targeted therapies.

Succession, regression and loss: does evidence of saltwater exposure explain recent changes in the tree communities of North Carolina's Coastal Plain?

BACKGROUND AND AIMS: Coastal plant communities globally are highly vulnerable to future sea-level rise and storm damage, but the extent to which these habitats are affected by the various environmental perturbations associated with chronic salinization remains unclear. In this study, we examine the relationship between North Carolina wetland tree community composition and basal area change and indicators of salinization such as soil salt ion content and elevation. METHODS: We surveyed 34 forest plots in forested, freshwater wetlands across the Albemarle-Pamlico Peninsula. A subset of our study sites had been sampled during the previous decade as part of the Carolina Vegetation Survey, enabling us to investigate the environmental effects on current community structure, and community change over time. KEY RESULTS: Multi-variate (ordination) analysis and linear regression models of tree community composition revealed that elevation and soil salt content were correlated with changes in total site tree basal area. Shifts in tree community composition were, however, only weakly correlated with indicators of salinization, specifically elevation, soil sulphate and sodium, but not chloride. While the majority of plots experienced gains in basal area over the past decade, consistent with secondary succession, sites with high soil salt content or low elevation experienced basal area (biomass) loss during the same period. CONCLUSIONS: The key factors associated with chronic saltwater intrusion (soil ion content) likely explain recent changes in tree biomass, and potential shifts in community composition in low-elevation sites along the North Carolina coast. Not only is it probable that other coastal forest ecosystems worldwide will experience similar stressors and shifts in community biomass and structure as sea levels rise, but the ability of these habitats to deliver key ecosystem services like carbon sequestration and flood defence will be compromised as a result.

Copper and Gold Nanoparticles Increase Nutrient Excretion Rates of Primary Consumers.

Freshwater ecosystems are exposed to engineered nanoparticles through municipal and industrial wastewater-effluent discharges and agricultural nonpoint source runoff. Because previous work has shown that engineered nanoparticles from these sources can accumulate in freshwater algal assemblages, we hypothesized that nanoparticles may affect the biology of primary consumers by altering the processing of two critical nutrients associated with growth and survivorship, nitrogen and phosphorus. We tested this hypothesis by measuring the excretion rates of nitrogen and phosphorus of Physella acuta, a ubiquitous pulmonate snail that grazes heavily on periphyton, exposed to either copper or gold engineered nanoparticles for 6 months in an outdoor wetland mesocosm experiment. Chronic nanoparticle exposure doubled nutrient excretion when compared to the control. Gold nanoparticles increased nitrogen and phosphorus excretion rates more than copper nanoparticles, but overall, both nanoparticles led to higher consumer excretion, despite contrasting particle stability and physiochemical properties. Snails in mesocosms enriched with nitrogen and phosphorus had overall higher excretion rates than ones in ambient (no nutrients added) mesocosms. Stimulation patterns were different between nitrogen and phosphorus excretion, which could have implications for the resulting nutrient ratio in the water column. These results suggest that low concentrations of engineered nanoparticles could alter the metabolism of consumers and increase consumer-mediated nutrient recycling rates, potentially intensifying eutrophication in aquatic systems, for example, the increased persistence of algal blooms as observed in our mesocosm experiment.

Differential Reactivity of Copper- and Gold-Based Nanomaterials Controls Their Seasonal Biogeochemical Cycling and Fate in a Freshwater Wetland Mesocosm.

Reliable predictions of the environmental fate and risk of engineered nanomaterials (ENMs) require a better understanding of ENM reactivity in complex, biologically active systems for chronic low-concentration exposure scenarios. Here, simulated freshwater wetland mesocosms were dosed with ENMs to assess how their reactivity and seasonal changes in environmental parameters influence ENM fate in aquatic systems. Copper-based ENMs (Kocide), known to dissolve in water, and gold nanoparticles (AuNPs), stable against dissolution in the absence of specific ligands, were added weekly to mesocosm waters for 9 months. Metal accumulation and speciation changes in the different environmental compartments were assessed over time. Copper from Kocide rapidly dissolved likely associating with organic matter in the water column, transported to terrestrial soils and deeper sediment where it became associated with organic or sulfide phases. In contrast, Au accumulated on/in the macrophytes where it oxidized and transferred over time to surficial sediment. A dynamic seasonal accumulation and metal redox cycling were found between the macrophyte and the surficial sediment for AuNPs. These results demonstrate the need for experimental quantification of how the biological and chemical complexity of the environment, combined with their seasonal variations, drive the fate of metastable ENMs.

Liver X receptor-α activation enhances cholesterol secretion in lactating mammary epithelium.

Liver X receptors (LXRs) are ligand-dependent transcription factors activated by cholesterol metabolites. These receptors induce a suite of target genes required for de novo synthesis of triglycerides and cholesterol transport in many tissues. Two different isoforms, LXRα and LXRß, have been well characterized in liver, adipocytes, macrophages, and intestinal epithelium among others, but their contribution to cholesterol and fatty acid efflux in the lactating mammary epithelium is poorly understood. We hypothesize that LXR regulates lipogenesis during milk fat production in lactation. Global mRNA analysis of mouse mammary epithelial cells (MECs) revealed multiple LXR/RXR targets upregulated sharply early in lactation compared with midpregnancy. LXRα is the primary isoform, and its protein levels increase throughout lactation in MECs. The LXR agonist GW3965 markedly induced several genes involved in cholesterol transport and lipogenesis and enhanced cytoplasmic lipid droplet accumulation in the HC11 MEC cell line. Importantly, in vivo pharmacological activation of LXR increased the milk cholesterol percentage and induced sterol regulatory element-binding protein 1c (Srebp1c) and ATP-binding cassette transporter a7 (Abca7) expression in MECs. Cumulatively, our findings identify LXRα as an important regulator of cholesterol incorporation into the milk through key nodes of de novo lipogenesis, suggesting a potential therapeutic target in women with difficulty initiating lactation.

Metformin inhibits stromal aromatase expression and tumor progression in a rodent model of postmenopausal breast cancer.

BACKGROUND: Obesity and type II diabetes are linked to increased breast cancer risk in postmenopausal women. Patients treated with the antidiabetic drug metformin for diabetes or metabolic syndrome have reduced breast cancer risk, a greater pathologic complete response to neoadjuvant therapy, and improved breast cancer survival. We hypothesized that metformin may be especially effective when targeted to the menopausal transition, as this is a lifecycle window when weight gain and metabolic syndrome increase, and is also when the risk for obesity-related breast cancer increases. METHODS: Here, we used an 1-methyl-1-nitrosourea (MNU)-induced mammary tumor rat model of estrogen receptor (ER)-positive postmenopausal breast cancer to evaluate the long-term effects of metformin administration on metabolic and tumor endpoints. In this model, ovariectomy (OVX) induces rapid weight gain, and an impaired whole-body response to excess calories contributes to increased tumor glucose uptake and increased tumor proliferation. Metformin treatment was initiated in tumor-bearing animals immediately prior to OVX and maintained for the duration of the study. RESULTS: Metformin decreased the size of existing mammary tumors and inhibited new tumor formation without changing body weight or adiposity. Decreased lipid accumulation in the livers of metformin-treated animals supports the ability of metformin to improve overall metabolic health. We also found a decrease in the number of aromatase-positive, CD68-positive macrophages within the tumor microenvironment, suggesting that metformin targets the immune microenvironment in addition to improving whole-body metabolism. CONCLUSIONS: These findings suggest that peri-menopause/menopause represents a unique window of time during which metformin may be highly effective in women with established, or at high risk for developing, breast cancer.

Engineered nanoparticles interact with nutrients to intensify eutrophication in a wetland ecosystem experiment.

Despite the rapid rise in diversity and quantities of engineered nanomaterials produced, the impacts of these emerging contaminants on the structure and function of ecosystems have received little attention from ecologists. Moreover, little is known about how manufactured nanomaterials may interact with nutrient pollution in altering ecosystem productivity, despite the recognition that eutrophication is the primary water quality issue in freshwater ecosystems worldwide. In this study, we asked two main questions: (1) To what extent do manufactured nanoparticles affect the biomass and productivity of primary producers in wetland ecosystems? (2) How are these impacts mediated by nutrient pollution? To address these questions, we examined the impacts of a citrate-coated gold nanoparticle (AuNPs) and of a commercial pesticide containing Cu(OH)2 nanoparticles (CuNPs) on aquatic primary producers under both ambient and enriched nutrient conditions. Wetland mesocosms were exposed repeatedly with low concentrations of nanoparticles and nutrients over the course of a 9-month experiment in an effort to replicate realistic field exposure scenarios. In the absence of nutrient enrichment, there were no persistent effects of AuNPs or CuNPs on primary producers or ecosystem productivity. However, when combined with nutrient enrichment, both NPs intensified eutrophication. When either of these NPs were added in combination with nutrients, algal blooms persisted for >50 d longer than in the nutrient-only treatment. In the AuNP treatment, this shift from clear waters to turbid waters led to large declines in both macrophyte growth and rates of ecosystem gross primary productivity (average reduction of 52% ± 6% and 92% ± 5%, respectively) during the summer. Our results suggest that nutrient status greatly influences the ecosystem-scale impact of two emerging contaminants and that synthetic chemicals may be playing an under-appreciated role in the global trends of increasing eutrophication. We provide evidence here that chronic exposure to Au and Cu(OH)2 nanoparticles at low concentrations can intensify eutrophication of wetlands and promote the occurrence of algal blooms.

Size-Based Differential Transport, Uptake, and Mass Distribution of Ceria (CeO2) Nanoparticles in Wetland Mesocosms.

Trace metals associated with nanoparticles are known to possess reactivities that are different from their larger-size counterparts. However, the relative importance of small relative to large particles for the overall distribution and biouptake of these metals is not as well studied in complex environmental systems. Here, we have examined differences in the long term fate and transport of ceria (CeO2) nanoparticles of two different sizes (3.8 vs 185 nm), dosed weekly to freshwater wetland mesocosms over 9 months. While the majority of CeO2 particles were detected in soils and sediments at the end of nine months, there were significant differences observed in fate, distribution, and transport mechanisms between the two materials. Small nanoparticles were removed from the water column primarily through heteroaggregation with suspended solids and plants, while large nanoparticles were removed primarily by sedimentation. A greater fraction of small particles remained in the upper floc layers of sediment relative to the large particles (31% vs 7%). Cerium from the small particles were also significantly more bioavailable to aquatic plants (2% vs 0.5%), snails (44 vs 2.6 ng), and insects (8 vs 0.07 µg). Small CeO2 particles were also significantly reduced from Ce(IV) to Ce(III), while aquatic sediments were a sink for untransformed large nanoparticles. These results demonstrate that trace metals originating from nanoscale materials have much greater potential than their larger counterparts to distribute throughout multiple compartments of a complex aquatic ecosystem and contribute to the overall bioavailable pool of the metal for biouptake and trophic transfer.

Functional traits of the understory plant community of a pyrogenic longleaf pine forest across environmental gradients.

Understanding and predicting the response of plant communities to environmental changes and disturbances such as fire requires an understanding of the functional traits present in the system, including within and across species variability, and their dynamics over time. These data are difficult to obtain as few studies provide comprehensive information for more than a few traits or species, rarely cover more than a single growing season, and usually present only summary statistics of trait values. As part of a larger study seeking to understand the dynamics of plant communities in response to different prescribed fire regimes, we measured the functional traits of the understory plant communities located in over 140 permanent plots spanning strong gradients in soil moisture in a pyrogenic longleaf pine forest in North Carolina, USA, over a four-year period from 2011 and 2014. We present over 120,000 individual trait measurements from over 130 plant species representing 91 genera from 47 families. We include data on the following 18 traits: specific leaf area, leaf dry matter content, leaf area, leaf length, leaf width, leaf perimeter, plant height, leaf nitrogen, leaf carbon, leaf carbon to nitrogen ratio, water use efficiency, time to ignition, maximum flame height, maximum burn temperature, mass-specific burn time, mass-specific smolder time, branching architecture, and the ratio of leaf matter consumed by fire. We also include information on locations, soil moisture, relative elevation, soil bulk density, and fire histories for each site.

The glucose transporter GLUT1 is required for ErbB2-induced mammary tumorigenesis.

BACKGROUND: Altered tumor cell metabolism is an emerging hallmark of cancer; however, the precise role for glucose in tumor initiation is not known. GLUT1 (SLC2A1) is expressed in breast cancer cells and is likely responsible for avid glucose uptake observed in established tumors. We have shown that GLUT1 was necessary for xenograft tumor formation from primary mammary cells transformed with the polyomavirus middle-T antigen but that it was not necessary for growth after tumors had formed in vivo, suggesting a differential requirement for glucose depending on the stage of tumorigenesis. METHODS: To determine whether GLUT1 is required early during mammary tumorigenesis, we crossed MMTV-NIC mice, which express activated HER2/NEU/ERBB2 and Cre recombinase, to Slc2a1 Flox/Flox (GLUT1Flox/Flox) mice to generate NIC-GLUT1+/+, NIC-GLUT1Flox/+, and NIC-GLUT1Flox/Flox mice. In addition, we evaluated effects of glucose restriction or GLUT1 inhibition on transformation in MCF10A-ERBB2 breast epithelial cells in three-dimensional culture. Finally, we utilized global gene expression profiling data of primary human breast tumors to determine the relationship between SLC2A1 and stage of tumorigenesis. RESULTS: All of the NIC-GLUT1+/+ mice developed tumors in less than 200 days. In contrast, only 1 NIC-GLUT1Flox/Flox mouse and 1 NIC-GLUT1Flox/+ mouse developed mammary tumors, even after 18 months. Mammary gland development was not disrupted in NIC mice lacking GLUT1; however, epithelial content of mature glands was reduced compared to NIC-GLUT1Flox/+ mice. In MCF10A-ERBB2 cells, glucose restriction or GLUT1 inhibition blocked transformation induced by activated ERBB2 through reduced cell proliferation. In human breast cancers, SLC2A1 was higher in ductal carcinoma in situ compared to the normal breast, but lower in invasive versus in situ lesions, suggesting the requirement for GLUT1 decreases as tumors progress. CONCLUSIONS: This study demonstrates a strict requirement for GLUT1 in the early stages of mammary tumorigenesis in vitro and in vivo. While metabolic adaptation has emerged as a hallmark of cancer, our data indicate that early tumor cells rely heavily on glucose and highlight the potential for glucose restriction as a breast cancer preventive strategy.

Inhibiting tyrosine phosphorylation of protein kinase Cδ (PKCδ) protects the salivary gland from radiation damage.

Radiation therapy for head and neck cancer can result in extensive damage to normal adjacent tissues such as the salivary gland and oral mucosa. We have shown previously that tyrosine phosphorylation at Tyr-64 and Tyr-155 activates PKCδ in response to apoptotic stimuli by facilitating its nuclear import. Here we have identified the tyrosine kinases that mediate activation of PKCδ in apoptotic cells and have explored the use of tyrosine kinase inhibitors for suppression of irradiation-induced apoptosis. We identify the damage-inducible kinase, c-Abl, as the PKCδ Tyr-155 kinase and c-Src as the Tyr-64 kinase. Depletion of c-Abl or c-Src with shRNA decreased irradiation- and etoposide-induced apoptosis, suggesting that inhibitors of these kinases may be useful therapeutically. Pretreatment with dasatinib, a broad spectrum tyrosine kinase inhibitor, blocked phosphorylation of PKCδ at both Tyr-64 and Tyr-155. Expression of "gate-keeper" mutants of c-Abl or c-Src that are active in the presence of dasatinib restored phosphorylation of PKCδ at Tyr-155 and Tyr-64, respectively. Imatinib, a c-Abl-selective inhibitor, also specifically blocked PKCδ Tyr-155 phosphorylation. Dasatinib and imatinib both blocked binding of PKCδ to importin-α and nuclear import, demonstrating that tyrosine kinase inhibitors can inhibit nuclear accumulation of PKCδ. Likewise, pretreatment with dasatinib also suppressed etoposide and radiation induced apoptosis in vitro. In vivo, pre-treatment of mice with dasatinib blocked radiation-induced apoptosis in the salivary gland by >60%. These data suggest that tyrosine kinase inhibitors may be useful prophylactically for protection of nontumor tissues in patients undergoing radiotherapy of the head and neck.

Thyroid hormone responsive protein Spot14 enhances catalysis of fatty acid synthase in lactating mammary epithelium.

Thyroid hormone responsive protein Spot 14 has been consistently associated with de novo fatty acid synthesis activity in multiple tissues, including the lactating mammary gland, which synthesizes large quantities of medium chain fatty acids (MCFAs) exclusively via FASN. However, the molecular function of Spot14 remains undefined during lactation. Spot14-null mice produce milk deficient in total triglyceride and de novo MCFA that does not sustain optimal neonatal growth. The lactation defect was rescued by provision of a high fat diet to the lactating dam. Transgenic mice overexpressing Spot14 in mammary epithelium produced total milk fat equivalent to controls, but with significantly greater MCFA. Spot14-null dams have no diminution of metabolic gene expression, enzyme protein levels, or intermediate metabolites that accounts for impaired de novo MCFA. When [(13)C] fatty acid products were quantified in vitro using crude cytosolic lysates, native FASN activity was 1.6-fold greater in control relative to Spot14-null lysates, and add back of Spot14 partially restored activity. Recombinant FASN catalysis increased 1.4-fold and C = 14:0 yield was enhanced 4-fold in vitro following addition of Spot14. These findings implicate Spot14 as a direct protein enhancer of FASN catalysis in the mammary gland during lactation when maximal MCFA production is needed.

Modulation of tumor fatty acids, through overexpression or loss of thyroid hormone responsive protein spot 14 is associated with altered growth and metastasis.

INTRODUCTION: Spot14 (S14), encoded by the THRSP gene, regulates de novo fatty acid synthesis in the liver, adipose, and lactating mammary gland. We recently showed that S14 stimulated fatty acid synthase (FASN) activity in vitro, and increased the synthesis of fatty acids in mammary epithelial cells in vivo. Elevated de novo fatty acid synthesis is a distinguishing feature of many solid tumors compared with adjacent normal tissue. This characteristic is thought to be acquired during tumor progression, as rapidly proliferating cells have a heightened requirement for membrane phospholipids. Further, overexpression of FASN is sufficient to stimulate cell proliferation. While many studies have focused on the FASN enzyme in cancer biology, few studies have addressed the roles of proteins that modify FASN activity, such as S14. METHODS: Tumor fatty acids were modulated using two mouse models, mouse mammary tumor virus (MMTV)-neu mice overexpressing S14 and MMTV-polyomavirus middle T antigen (PyMT) mice lacking S14, and associations between elevated or impaired fatty acid synthesis on tumor latency, growth, metastasis, and signaling pathways were investigated. We evaluated S14-dependent gene expression profiles in mouse tumors by microarray and used publicly available microarray datasets of human breast tumors. RESULTS: S14 overexpression in the MMTV-Neu transgenic model is associated with elevated medium-chain fatty acids, increased proliferation and a shorter tumor latency, but reduced tumor metastasis compared to controls. Loss of S14 in the MMTV-PyMT model decreased FASN activity and the synthesis of medium-chain fatty acids but did not alter tumor latency. Impaired fatty acid synthesis was associated with reduced solid tumor cell proliferation, the formation of cystic lesions in some animals, and decreased phosphorylation of Src and protein kinase B (Akt). Analysis of gene expression in these mouse and human tumors revealed a relationship between S14 status and the expression of genes associated with luminal epithelial differentiation. CONCLUSIONS: This study demonstrates a potential role for S14 in regulating mammary tumor growth and fatty acid synthesis in vivo. Furthermore, these results suggest that modulating the amount of medium chain fatty acids, by changing the levels of S14, has the potential to impact malignant mammary tumor phenotypes.

The insulin receptor plays an important role in secretory differentiation in the mammary gland.

Insulin is known to be an important regulator of milk secretion in the lactating mammary gland. Here we examine the role of insulin signaling in mammary development in pregnancy using a mouse with a floxed insulin receptor (IR) crossed with a mouse expressing Cre specifically in the mammary gland. In the mammary glands of these IR(fl/fl) Cre(+) mice, expression of IR is significantly diminished throughout development. Glands from these mice had 50% fewer alveoli at midpregnancy; casein and lipid droplets were diminished by 60 and 75%, respectively, indicating a role for IR both in alveolar development and differentiation. In an acinar preparation from mammary epithelial cells (MEC) isolated from pregnant mice, insulin stimulated lumen formation, mammary cell size, acinar size, acinar casein content, and the formation of lipid droplets with a Km of ∼1.7 nM. IGF-I and IGF-II had no effect at concentrations below 50 nM, and a function blocking antibody to the IGF type 1 receptor did not alter the response to insulin. We conclude that insulin interacting with IR is essential for mammary differentiation during murine pregnancy. Using array analysis, we then examined the expression of genes up- or downregulated >1.5-fold in the IR(fl/fl) Cre(+) MECs, finding significant downregulation of differentiation specific genes and upregulation of cell cycle and extracellular matrix genes. We conclude that insulin fosters differentiation and may inhibit cell proliferation in the mammary gland of the midpregnant mouse.

Lactation and neonatal nutrition: defining and refining the critical questions.

This paper resulted from a conference entitled "Lactation and Milk: Defining and refining the critical questions" held at the University of Colorado School of Medicine from January 18-20, 2012. The mission of the conference was to identify unresolved questions and set future goals for research into human milk composition, mammary development and lactation. We first outline the unanswered questions regarding the composition of human milk (Section I) and the mechanisms by which milk components affect neonatal development, growth and health and recommend models for future research. Emerging questions about how milk components affect cognitive development and behavioral phenotype of the offspring are presented in Section II. In Section III we outline the important unanswered questions about regulation of mammary gland development, the heritability of defects, the effects of maternal nutrition, disease, metabolic status, and therapeutic drugs upon the subsequent lactation. Questions surrounding breastfeeding practice are also highlighted. In Section IV we describe the specific nutritional challenges faced by three different populations, namely preterm infants, infants born to obese mothers who may or may not have gestational diabetes, and infants born to undernourished mothers. The recognition that multidisciplinary training is critical to advancing the field led us to formulate specific training recommendations in Section V. Our recommendations for research emphasis are summarized in Section VI. In sum, we present a roadmap for multidisciplinary research into all aspects of human lactation, milk and its role in infant nutrition for the next decade and beyond.

Clinical characterization of the mutational landscape of 24,639 real-world samples from patients with myeloid malignancies.

Myeloid neoplasms represent a broad spectrum of hematological disorders for which somatic mutation status in key driver genes is important for diagnosis, prognosis and treatment. Here we summarize the findings of a targeted, next generation sequencing laboratory developed test in 24,639 clinical myeloid samples. Data were analyzed comprehensively and as part of individual cohorts specific to acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Overall, 48,015 variants were detected, and variants were found in all 50 genes in the panel. The mean number of mutations per patient was 1.95. Mutation number increased with age (Spearman's rank correlation coefficient, ρ = 0.29, P < 0.0001) and was higher in patients with AML than MDS or MPN (Student's t-test, P < 0.0001). TET2 was the most common mutation detected (19.1% of samples; 4,695/24,639) including 7.7% (1,908/24,639) with multi-hit TET2 mutations. Mutation frequency was correlated between patients with cytopenias and MDS (Spearman's, ρ = 0.97, P < 2.2×10-16) with the MDS diagnostic gene SF3B1 being the only notable outlier. This large retrospective study shows the utility of NGS testing to inform clinical decisions during routine clinical care and highlights the mutational landscape of a broad population of myeloid patients.