OPA1 Regulates Lipid Metabolism and Cold-Induced Browning of White Adipose Tissue in Mice.

Mitochondria play a vital role in white adipose tissue (WAT) homeostasis including adipogenesis, fatty acid synthesis, and lipolysis. We recently reported that the mitochondrial fusion protein optic atrophy 1 (OPA1) is required for induction of fatty acid oxidation and thermogenic activation in brown adipocytes. In the current study we investigated the role of OPA1 in WAT function in vivo. We generated mice with constitutive or inducible knockout of OPA1 selectively in adipocytes. Studies were conducted under baseline conditions, at thermoneutrality, following high-fat feeding or during cold exposure. OPA1 deficiency reduced mitochondrial respiratory capacity in white adipocytes, impaired lipolytic signaling, repressed expression of de novo lipogenesis and triglyceride synthesis pathways, and promoted adipose tissue senescence and inflammation. Reduced WAT mass was associated with hepatic triglycerides accumulation and glucose intolerance. Moreover, mice deficient for OPA1 in adipocytes had impaired adaptive thermogenesis and reduced cold-induced browning of subcutaneous WAT and were completely resistant to diet-induced obesity. In conclusion, OPA1 expression and function in adipocytes are essential for adipose tissue expansion, lipid biosynthesis, and fatty acid mobilization of WAT and brown adipocytes and for thermogenic activation of brown and beige adipocytes.

AP-2α-Mediated Activation of E2F and EZH2 Drives Melanoma Metastasis.

In melanoma metastasis, the role of the AP-2α transcription factor, which is encoded by TFAP2A, is controversial as some findings have suggested tumor suppressor activity while other studies have shown high TFAP2A expression in node-positive melanoma associated with poor prognosis. Here we demonstrate that AP-2α facilitates melanoma metastasis through transcriptional activation of genes within the E2F pathway including EZH2. A BioID screen found that AP-2α interacts with members of the nucleosome remodeling and deacetylase (NuRD) complex. Loss of AP-2α removed activating chromatin marks in the promoters of EZH2 and other E2F target genes through activation of the NuRD repression complex. In melanoma cells, treatment with tazemetostat, an FDA-approved and highly specific EZH2 inhibitor, substantially reduced anchorage-independent colony formation and demonstrated heritable antimetastatic effects, which were dependent on AP-2α. Single-cell RNA sequencing analysis of a metastatic melanoma mouse model revealed hyperexpansion of Tfap2a High/E2F-activated cell populations in transformed melanoma relative to progenitor melanocyte stem cells. These findings demonstrate that melanoma metastasis is driven by the AP-2α/EZH2 pathway and suggest that AP-2α expression can be used as a biomarker to predict responsiveness to EZH2 inhibitors for the treatment of advanced melanomas. SIGNIFICANCE: AP-2α drives melanoma metastasis by upregulating E2F pathway genes including EZH2 through inhibition of the NuRD repression complex, serving as a biomarker to predict responsiveness to EZH2 inhibitors.

AP-2α Regulates S-Phase and Is a Marker for Sensitivity to PI3K Inhibitor Buparlisib in Colon Cancer.

Activating protein 2 alpha (AP-2α; encoded by TFAP2A) functions as a tumor suppressor and influences response to therapy in several cancer types. We aimed to characterize regulation of the transcriptome by AP-2α in colon cancer. CRISPR-Cas9 and short hairpin RNA were used to eliminate TFAP2A expression in HCT116 and a panel of colon cancer cell lines. AP-2α target genes were identified with RNA sequencing and chromatin immunoprecipitation sequencing. Effects on cell cycle were characterized in cells synchronized with aphidicolin and analyzed by FACS and Premo FUCCI. Effects on invasion and tumorigenesis were determined by invasion assay, growth of xenografts, and phosphorylated histone H3 (PHH3). Knockout of TFAP2A induced significant alterations in the transcriptome including repression of TGM2, identified as a primary gene target of AP-2α. Loss of AP-2α delayed progression through S-phase into G2-M and decreased phosphorylation of AKT, effects that were mediated through regulation of TGM2. Buparlisib (BKM120) repressed in vitro invasiveness of HCT116 and a panel of colon cancer cell lines; however, loss of AP-2α induced resistance to buparlisib. Similarly, buparlisib repressed PHH3 and growth of tumor xenografts and increased overall survival of tumor-bearing mice, whereas, loss of AP-2α induced resistance to the effect of PI3K inhibition. Loss of AP-2α in colon cancer leads to prolonged S-phase through altered activation of AKT leading to resistance to the PI3K inhibitor, Buparlisib. The findings demonstrate an important role for AP-2α in regulating progression through the cell cycle and indicates that AP-2α is a marker for response to PI3K inhibitors. IMPLICATIONS: AP-2α regulated cell cycle through the PI3K cascade and activation of AKT mediated through TGM2. AP-2α induced sensitivity to Buparlisib/BKM120, indicating that AP-2α is a biomarker predictive of response to PI3K inhibitors.

AP-2γ Is Required for Maintenance of Multipotent Mammary Stem Cells.

Mammary gland ductal morphogenesis depends on the differentiation of mammary stem cells (MaSCs) into basal and luminal lineages. The AP-2γ transcription factor, encoded by Tfap2c, has a central role in mammary gland development but its effect in mammary lineages and specifically MaSCs is largely unknown. Here, we utilized an inducible, conditional knockout of Tfap2c to elucidate the role of AP-2γ in maintenance and differentiation of MaSCs. Loss of AP-2γ in the basal epithelium profoundly altered the transcriptomes and decreased the number of cells within several clusters of mammary epithelial cells, including adult MaSCs and luminal progenitors. AP-2γ regulated the expression of genes known to be required for mammary development, including Cebpb, Nfkbia, and Rspo1. As a result, AP-2γ-deficient mice exhibited repressed mammary gland ductal outgrowth and inhibition of regenerative capacity. The findings demonstrate that AP-2γ can regulate development of mammary gland structures potentially regulating maintenance and differentiation of multipotent MaSCs.

Nitrogen cycling microbiomes are structured by plant mycorrhizal associations with consequences for nitrogen oxide fluxes in forests.

Volatile nitrogen oxides (N2 O, NO, NO2 , HONO, …) can negatively impact climate, air quality, and human health. Using soils collected from temperate forests across the eastern United States, we show microbial communities involved in nitrogen (N) cycling are structured, in large part, by the composition of overstory trees, leading to predictable N-cycling syndromes, with consequences for emissions of volatile nitrogen oxides to air. Trees associating with arbuscular mycorrhizal (AM) fungi promote soil microbial communities with higher N-cycle potential and activity, relative to microbial communities in soils dominated by trees associating with ectomycorrhizal (ECM) fungi. Metagenomic analysis and gene expression studies reveal a 5 and 3.5 times greater estimated N-cycle gene and transcript copy numbers, respectively, in AM relative to ECM soil. Furthermore, we observe a 60% linear decrease in volatile reactive nitrogen gas flux (NOy  ≡ NO, NO2 , HONO) as ECM tree abundance increases. Compared to oxic conditions, gas flux potential of N2 O and NO increase significantly under anoxic conditions for AM soil (30- and 120-fold increase), but not ECM soil-likely owing to small concentrations of available substrate ( NO 3 - ) in ECM soil. Linear mixed effects modeling shows that ECM tree abundance, microbial process rates, and geographic location are primarily responsible for variation in peak potential NOy flux. Given that nearly all tree species associate with either AM or ECM fungi, our results indicate that the consequences of tree species shifts associated with global change may have predictable consequences for soil N cycling.

TFAP2C regulates carbonic anhydrase XII in human breast cancer.

The expression of carbonic anhydrase XII (CA12) is associated with the expression of estrogen receptor alpha (ERα) in breast cancer and is linked to a good prognosis with a lower risk of metastasis. Transcription Factor Activator Protein 2γ (TFAP2C, AP-2γ) governs luminal breast cancer phenotype through direct and indirect regulation of ERα and ERα-associated genes, GATA3, FOXA1, EGFR, CDH1, DSP, KRT7, FBP1, MYB, RET, KRT8, MUC1, and ERBB2-genes which are responsible for the luminal signature in breast cancer. Herein, utilizing chromatin immunoprecipitation and direct sequencing (ChIP-seq), we show that CA12 is regulated by AP-2γ through binding with its promoter region in luminal breast cancer cell lines and indirectly through a distal estrogen-responsive region in ERα-positive cell lines by upregulation of ERα. CA12 is transcriptionally silenced in basal breast cancer cell lines through histone deacetylation and CpG methylation of the promoter region and can be re-activated with Trichostatin A (histone deacetylase inhibitor) and/or 5-aza-dC (an inhibitor of DNA methylation). Strong concordance in co-expression of CA12 and ESR1 (R2 = 0.1128, p = 0486) and TFAP2C (R2 = 0.1823, p = 0.0105) was found using a panel of primary breast tumor samples (n = 35), supporting a synergetic role of AP-2γ and ERα in activation of CA12. Our results highlight the essential role of AP-2γ in maintaining the luminal breast cancer phenotype and provide evidence that epigenetic mechanisms silence luminal gene expression in the basal phenotype. Additional studies to decipher mechanisms that drive epigenetic silencing of AP-2γ target genes are a critical area for further research.

Fine Sediment Removal Influences Biogeochemical Processes in a Gravel-bottomed Stream.

The transport and processing of nutrients and organic matter in streams are important functions that influence the condition of watersheds and downstream ecosystems. In this study, we investigated the effects of streambed sediment removal on biogeochemical cycling in Fawn River, a gravel-bottomed river in Indiana, U.S.A. We measured stream metabolism as well as nitrogen (N) and phosphorus (P) retention in both restored and unrestored reaches of Fawn River to examine how sediment removal affected multiple biogeochemical functions at the reach scale. We also assessed the properties of restored and unrestored streambed sediments to elucidate potential mechanisms driving observed reach-scale differences. We found that sediment removal led to lower rates of primary productivity and ecosystem respiration in the restored reach, likely due to macrophyte removal and potentially due to changes to sediment organic matter quality. We found minimal differences in N and P retention, suggesting that these processes are controlled at larger spatial or temporal scales than were examined in this study. Denitrification enzyme activity was lower in sediments from the restored reach compared to the unrestored reach, suggesting that restoration may have decreased N removal. Our results indicate that most near-term changes in biogeochemical function following restoration could be attributed to macrophyte removal, although effects from sediment removal may emerge over longer timescales.

Microbial mechanisms and ecosystem flux estimation for aerobic NOy emissions from deciduous forest soils.

Reactive nitrogen oxides (NOy; NOy = NO + NO2 + HONO) decrease air quality and impact radiative forcing, yet the factors responsible for their emission from nonpoint sources (i.e., soils) remain poorly understood. We investigated the factors that control the production of aerobic NOy in forest soils using molecular techniques, process-based assays, and inhibitor experiments. We subsequently used these data to identify hotspots for gas emissions across forests of the eastern United States. Here, we show that nitrogen oxide soil emissions are mediated by microbial community structure (e.g., ammonium oxidizer abundances), soil chemical characteristics (pH and C:N), and nitrogen (N) transformation rates (net nitrification). We find that, while nitrification rates are controlled primarily by chemoautotrophic ammonia-oxidizing archaea (AOA), the production of NOy is mediated in large part by chemoautotrophic ammonia-oxidizing bacteria (AOB). Variation in nitrification rates and nitrogen oxide emissions tracked variation in forest communities, as stands dominated by arbuscular mycorrhizal (AM) trees had greater N transformation rates and NOy fluxes than stands dominated by ectomycorrhizal (ECM) trees. Given mapped distributions of AM and ECM trees from 78,000 forest inventory plots, we estimate that broadleaf forests of the Midwest and the eastern United States as well as the Mississippi River corridor may be considered hotspots of biogenic NOy emissions. Together, our results greatly improve our understanding of NOy fluxes from forests, which should lead to improved predictions about the atmospheric consequences of tree species shifts owing to land management and climate change.

Inhibiting the SUMO Pathway Represses the Cancer Stem Cell Population in Breast and Colorectal Carcinomas.

Many solid cancers have an expanded CD44+/hi/CD24-/low cancer stem cell (CSC) population, which are relatively chemoresistant and drive recurrence and metastasis. Achieving a more durable response requires the development of therapies that specifically target CSCs. Recent evidence indicated that inhibiting the SUMO pathway repressed tumor growth and invasiveness, although the mechanism has yet to be clarified. Here, we demonstrate that inhibition of the SUMO pathway repressed MMP14 and CD44 with a concomitant reduction in cell invasiveness and functional loss of CSCs in basal breast cancer. Similar effects were demonstrated with a panel of E1 and E3 SUMO inhibitors. Identical results were obtained in a colorectal cancer cell line and primary colon cancer cells. In both breast and colon cancer, SUMO-unconjugated TFAP2A mediated the effects of SUMO inhibition. These data support the development of SUMO inhibitors as an approach to specifically target the CSC population in breast and colorectal cancer.

Unanticipated Geochemical and Microbial Community Structure under Seasonal Ice Cover in a Dilute, Dimictic Arctic Lake.

Despite most lakes in the Arctic being perennially or seasonally frozen for at least 40% of the year, little is known about microbial communities and nutrient cycling under ice cover. We assessed the vertical microbial community distribution and geochemical composition in early spring under ice in a seasonally ice-covered lake in southwest Greenland using amplicon-based sequencing that targeted 16S rRNA genes and using a combination of field and laboratory aqueous geochemical methods. Microbial communities changed consistently with changes in geochemistry. Composition of the abundant members responded strongly to redox conditions, shifting downward from a predominantly heterotrophic aerobic community in the suboxic waters to a heterotrophic anaerobic community in the anoxic waters. Operational taxonomic units (OTUs) of Sporichthyaceae, Comamonadaceae, and the SAR11 Clade had higher relative abundances above the oxycline and OTUs within the genus Methylobacter, the phylum Lentisphaerae, and purple sulfur bacteria (PSB) below the oxycline. Notably, a 13-fold increase in sulfide at the oxycline was reflected in an increase and change in community composition of potential sulfur oxidizers. Purple non-sulfur bacteria were present above the oxycline and green sulfur bacteria and PSB coexisted below the oxycline, however, PSB were most abundant. For the first time we show the importance of PSB as potential sulfur oxidizers in an Arctic dimictic lake.

Combined Flux Chamber and Genomics Approach Links Nitrous Acid Emissions to Ammonia Oxidizing Bacteria and Archaea in Urban and Agricultural Soil.

Nitrous acid (HONO) is a photochemical source of hydroxyl radical and nitric oxide in the atmosphere that stems from abiotic and biogenic processes, including the activity of ammonia-oxidizing soil microbes. HONO fluxes were measured from agricultural and urban soil in mesocosm studies aimed at characterizing biogenic sources and linking them to indigenous microbial consortia. Fluxes of HONO from agricultural and urban soil were suppressed by addition of a nitrification inhibitor and enhanced by amendment with ammonium (NH4(+)), with peaks at 19 and 8 ng m(-2) s(-1), respectively. In addition, both agricultural and urban soils were observed to convert (15)NH4(+) to HO(15)NO. Genomic surveys of soil samples revealed that 1.5-6% of total expressed 16S rRNA sequences detected belonged to known ammonia oxidizing bacteria and archaea. Peak fluxes of HONO were directly related to the abundance of ammonia-oxidizer sequences, which in turn depended on soil pH. Peak HONO fluxes under fertilized conditions are comparable in magnitude to fluxes reported during field campaigns. The results suggest that biogenic HONO emissions will be important in soil environments that exhibit high nitrification rates (e.g., agricultural soil) although the widespread occurrence of ammonia oxidizers implies that biogenic HONO emissions are also possible in the urban and remote environment.

A novel animal model for locally advanced breast cancer.

BACKGROUND: Locally advanced breast cancer (LABC) poses complex management issues due to failure of response to chemotherapy and progression to local complications such as skin erosion, superinfection, and lymphedema. Most cell line and animal models are not adequate to study LABC. METHODS: A patient-derived xenograft (IOWA-1T) from a patient with LABC was characterized for expression profile, short tandem repeat profile, oncogenic mutations, xenograft growth, and response to therapy. RESULTS: Short tandem repeat profile authenticated the cell line as derived from a human woman. The primary tumor and derived xenografts were weakly estrogen receptor alpha positive (<5%), progesterone receptor negative, and HER2 nonamplified. Expression array profile compared to MCF-7 and BT-549 cell lines indicate that IOWA-1T was more closely related to basal breast cancer. IOWA-1T harbors a homozygous R248Q mutation of the TP53 gene; in vitro invasion assay was comparable to BT-549 and greater than MCF-7. IOWA-1T xenografts developed palpable tumors in 9.6 ± 1.6 days, compared to 49 ± 13 days for parallel experiments with BT-20 cells (p < 0.002). Tumor xenografts became locally advanced, growing to >2 cm in 21.6 ± 2 days, characterized by skin erosion necessitating euthanasia. The SUMO inhibitor anacardic acid inhibited the outgrowth of IOWA-1T xenografts, while doxorubicin had no effect on tumorigenesis. CONCLUSIONS: IOWA-1T is a novel cell line with an expression pattern consistent with basal breast cancer. Xenografts recapitulated LABC and provide a novel model for testing therapeutic drugs that may be effective in cases resistant to conventional chemotherapy.

A synthesis of methane emissions from 71 northern, temperate, and subtropical wetlands.

Wetlands are the largest natural source of atmospheric methane. Here, we assess controls on methane flux using a database of approximately 19 000 instantaneous measurements from 71 wetland sites located across subtropical, temperate, and northern high latitude regions. Our analyses confirm general controls on wetland methane emissions from soil temperature, water table, and vegetation, but also show that these relationships are modified depending on wetland type (bog, fen, or swamp), region (subarctic to temperate), and disturbance. Fen methane flux was more sensitive to vegetation and less sensitive to temperature than bog or swamp fluxes. The optimal water table for methane flux was consistently below the peat surface in bogs, close to the peat surface in poor fens, and above the peat surface in rich fens. However, the largest flux in bogs occurred when dry 30-day averaged antecedent conditions were followed by wet conditions, while in fens and swamps, the largest flux occurred when both 30-day averaged antecedent and current conditions were wet. Drained wetlands exhibited distinct characteristics, e.g. the absence of large flux following wet and warm conditions, suggesting that the same functional relationships between methane flux and environmental conditions cannot be used across pristine and disturbed wetlands. Together, our results suggest that water table and temperature are dominant controls on methane flux in pristine bogs and swamps, while other processes, such as vascular transport in pristine fens, have the potential to partially override the effect of these controls in other wetland types. Because wetland types vary in methane emissions and have distinct controls, these ecosystems need to be considered separately to yield reliable estimates of global wetland methane release.