Sherry Niessen

Monoacylglycerol Lipase Regulates a Fatty Acid Network that Promotes Cancer Pathogenesis

Tumor cells display progressive changes in metabolism that correlate with malignancy, including development of a lipogenic phenotype. How stored fats are liberated and remodeled to support cancer pathogenesis, however, remains unknown. Here, we show that the enzyme monoacylglycerol lipase (MAGL) is highly expressed in aggressive human cancer cells and primary tumors, where it regulates a fatty acid network enriched in oncogenic signaling lipids that promotes migration, invasion, survival, and in vivo tumor growth. Overexpression of MAGL in nonaggressive cancer cells recapitulates this fatty acid network and increases their pathogenicity-phenotypes that are reversed by an MAGL inhibitor. Impairments in MAGL-dependent tumor growth are rescued by a high-fat diet, indicating that exogenous sources of fatty acids can contribute to malignancy in cancers lacking MAGL activity. Together, these findings reveal how cancer cells can co-opt a lipolytic enzyme to translate their lipogenic state into an array of protumorigenic signals. PAPERFLICK:

The CREB Coactivator TORC2 Functions as a Calcium- and cAMP-Sensitive Coincidence Detector

Elevations in circulating glucose and gut hormones during feeding promote pancreatic islet cell viability in part via the calcium- and cAMP-dependent activation of the transcription factor CREB. Here, we describe a signaling module that mediates the synergistic effects of these pathways on cellular gene expression by stimulating the dephosphorylation and nuclear entry of TORC2, a CREB coactivator. This module consists of the calcium-regulated phosphatase calcineurin and the Ser/Thr kinase SIK2, both of which associate with TORC2. Under resting conditions, TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins. Triggering of the calcium and cAMP second messenger pathways by glucose and gut hormones disrupts TORC2:14-3-3 complexes via complementary effects on TORC2 dephosphorylation; calcium influx increases calcineurin activity, whereas cAMP inhibits SIK2 kinase activity. Our results illustrate how a phosphatase/kinase module connects two signaling pathways in response to nutrient and hormonal cues.

TRB3 Links the E3 Ubiquitin Ligase COP1 to Lipid Metabolism

During fasting, increased concentrations of circulating catecholamines promote the mobilization of lipid stores from adipose tissue in part by phosphorylating and inactivating acetyl–coenzyme A carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis. Here, we describe a parallel pathway, in which the pseudokinase Tribbles 3 (TRB3), whose abundance is increased during fasting, stimulates lipolysis by triggering the degradation of ACC in adipose tissue. TRB3 promoted ACC ubiquitination through an association with the E3 ubiquitin ligase constitutive photomorphogenic protein 1 (COP1). Indeed, adipocytes deficient in TRB3 accumulated larger amounts of ACC protein than did wild-type cells. Because transgenic mice expressing TRB3 in adipose tissue are protected from diet-induced obesity due to enhanced fatty acid oxidation, these results demonstrate how phosphorylation and ubiquitination pathways converge on a key regulator of lipid metabolism to maintain energy homeostasis.

A streamlined platform for high-content functional proteomics of primary human specimens

Achieving information content of satisfactory breadth and depth remains a formidable challenge for proteomics. This problem is particularly relevant to the study of primary human specimens, such as tumor biopsies, which are heterogeneous and of finite quantity. Here we present a functional proteomics strategy that unites the activity-based protein profiling and multidimensional protein identification technologies (ABPP-MudPIT) for the streamlined analysis of human samples. This convergent platform involves a rapid initial phase, in which enzyme activity signatures are generated for functional classification of samples, followed by in-depth analysis of representative members from each class. Using this two-tiered approach, we identified more than 50 enzyme activities in human breast tumors, nearly a third of which represent previously uncharacterized proteins. Comparison with cDNA microarrays revealed enzymes whose activity, but not mRNA expression, depicted tumor class, underscoring the power of ABPP-MudPIT for the discovery of new markers of human disease that may evade detection by other molecular profiling methods.

Polo-like kinase 4 kinase activity limits centrosome overduplication by autoregulating its own stability

Plk4 phosphorylates itself in trans to prevent accumulation and self-limit kinase activity, which may be important for regulating centriole duplication.

Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening

Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.

Click-generated triazole ureas as ultrapotent in vivo–active serine hydrolase inhibitors

Serine hydrolases (SHs) are a diverse enzyme class representing > 1% of all human proteins. The biological functions for most SHs remain poorly characterized due to a lack of selective inhibitors to probe their activity in living systems. Here, we show that a substantial number of SHs can be irreversibly inactivated by 1,2,3-triazole ureas, which exhibit negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the SH superfamily, including peptidases (acyl-peptide hydrolase or APEH), lipases (platelet-activating factor acetylhyrolase-2 or PAFAH2), and uncharacterized hydrolases (α, β-hydrolase 11 or ABHD11), with exceptional potency in cells (sub-nM) and mice (< 1 mg/kg). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T-cells. These data designate 1,2,3-triazole ureas as a pharmacologically privileged chemotype for SH inhibition that shows broad activity across the SH class coupled with tunable selectivity for individual enzymes.

A Hormone-Dependent Module Regulating Energy Balance

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.

Monoacylglycerol Lipase Exerts Dual Control over Endocannabinoid and Fatty Acid Pathways to Support Prostate Cancer

Cancer cells couple heightened lipogenesis with lipolysis to produce fatty acid networks that support malignancy. Monoacylglycerol lipase (MAGL) plays a principal role in this process by converting monoglycerides, including the endocannabinoid 2-arachidonoylglycerol (2-AG), to free fatty acids. Here, we show that MAGL is elevated in androgen-independent versus androgen-dependent human prostate cancer cell lines, and that pharmacological or RNA-interference disruption of this enzyme impairs prostate cancer aggressiveness. These effects were partially reversed by treatment with fatty acids or a cannabinoid receptor-1 (CB1) antagonist, and fully reversed by cotreatment with both agents. We further show that MAGL is part of a gene signature correlated with epithelial-to-mesenchymal transition and the stem-like properties of cancer cells, supporting a role for this enzyme in protumorigenic metabolism that, for prostate cancer, involves the dual control of endocannabinoid and fatty acid pathways.

A High-Resolution C. elegans Essential Gene Network Based on Phenotypic Profiling of a Complex Tissue

High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach-profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes-pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.