Convergent and complementary selection shaped gains and losses of eusociality in sweat bees.

Sweat bees have repeatedly gained and lost eusociality, a transition from individual to group reproduction. Here we generate chromosome-length genome assemblies for 17 species and identify genomic signatures of evolutionary trade-offs associated with transitions between social and solitary living. Both young genes and regulatory regions show enrichment for these molecular patterns. We also identify loci that show evidence of complementary signals of positive and relaxed selection linked specifically to the convergent gains and losses of eusociality in sweat bees. This includes two pleiotropic proteins that bind and transport juvenile hormone (JH)-a key regulator of insect development and reproduction. We find that one of these proteins is primarily expressed in subperineurial glial cells that form the insect blood-brain barrier and that brain levels of JH vary by sociality. Our findings are consistent with a role of JH in modulating social behaviour and suggest that eusocial evolution was facilitated by alteration of the proteins that bind and transport JH, revealing how an ancestral developmental hormone may have been co-opted during one of life's major transitions. More broadly, our results highlight how evolutionary trade-offs have structured the molecular basis of eusociality in these bees and demonstrate how both directional selection and release from constraint can shape trait evolution.

Autophagy in PDGFRα+ mesenchymal cells is essential for intestinal stem cell survival.

Autophagy defects are a risk factor for inflammatory bowel diseases (IBDs) through unknown mechanisms. Whole-body conditional deletion of autophagy-related gene (Atg) Atg7 in adult mice (Atg7Δ/Δ) causes tissue damage and death within 3 mo due to neurodegeneration without substantial effect on intestine. In contrast, we report here that whole-body conditional deletion of other essential Atg genes Atg5 or Fip200/Atg17 in adult mice (Atg5Δ/Δ or Fip200Δ/Δ) caused death within 5 d due to rapid autophagy inhibition, elimination of ileum stem cells, and loss of barrier function. Atg5Δ/Δ mice lost PDGFRα+ mesenchymal cells (PMCs) and Wnt signaling essential for stem cell renewal, which were partially rescued by exogenous Wnt. Matrix-assisted laser desorption ionization coupled to mass spectrometry imaging (MALDI-MSI) of Atg5Δ/Δ ileum revealed depletion of aspartate and nucleotides, consistent with metabolic insufficiency underlying PMC loss. The difference in the autophagy gene knockout phenotypes is likely due to distinct kinetics of autophagy loss, as deletion of Atg5 more gradually extended lifespan phenocopying deletion of Atg7 or Atg12. Thus, autophagy is required for PMC metabolism and ileum stem cell and mammalian survival. Failure to maintain PMCs through autophagy may therefore contribute to IBD.

Spatially resolved isotope tracing reveals tissue metabolic activity.

Isotope tracing has helped to determine the metabolic activities of organs. Methods to probe metabolic heterogeneity within organs are less developed. We couple stable-isotope-labeled nutrient infusion to matrix-assisted laser desorption ionization imaging mass spectrometry (iso-imaging) to quantitate metabolic activity in mammalian tissues in a spatially resolved manner. In the kidney, we visualize gluconeogenic flux and glycolytic flux in the cortex and medulla, respectively. Tricarboxylic acid cycle substrate usage differs across kidney regions; glutamine and citrate are used preferentially in the cortex and fatty acids are used in the medulla. In the brain, we observe spatial gradations in carbon inputs to the tricarboxylic acid cycle and glutamate under a ketogenic diet. In a carbohydrate-rich diet, glucose predominates throughout but in a ketogenic diet, 3-hydroxybutyrate contributes most strongly in the hippocampus and least in the midbrain. Brain nitrogen sources also vary spatially; branched-chain amino acids contribute most in the midbrain, whereas ammonia contributes in the thalamus. Thus, iso-imaging can reveal the spatial organization of metabolic activity.

Activation of Oxidative Stress Response in Cancer Generates a Druggable Dependency on Exogenous Non-essential Amino Acids.

Rewiring of metabolic pathways is a hallmark of tumorigenesis as cancer cells acquire novel nutrient dependencies to support oncogenic growth. A major genetic subtype of lung adenocarcinoma with KEAP1/NRF2 mutations, which activates the endogenous oxidative stress response, undergoes significant metabolic rewiring to support enhanced antioxidant production. We demonstrate that cancers with high antioxidant capacity exhibit a general dependency on exogenous non-essential amino acids (NEAAs) that is driven by the Nrf2-dependent secretion of glutamate through system xc- (XCT), which limits intracellular glutamate pools that are required for NEAA synthesis. This dependency can be therapeutically targeted by dietary restriction or enzymatic depletion of individual NEAAs. Importantly, limiting endogenous glutamate levels by glutaminase inhibition can sensitize tumors without alterations in the Keap1/Nrf2 pathway to dietary restriction of NEAAs. Our findings identify a metabolic strategy to therapeutically target cancers with genetic or pharmacologic activation of the Nrf2 antioxidant response pathway by restricting exogenous sources of NEAAs.

The spliceosomal proteins PPIH and PRPF4 exhibit bi-partite binding.

Pre-mRNA splicing is a dynamic, multistep process that is catalyzed by the RNA (ribonucleic acid)-protein complex called the spliceosome. The spliceosome contains a core set of RNAs and proteins that are conserved in all organisms that perform splicing. In higher organisms, peptidyl-prolyl isomerase H (PPIH) directly interacts with the core protein pre-mRNA processing factor 4 (PRPF4) and both integrate into the pre-catalytic spliceosome as part of the tri-snRNP (small nuclear RNA-protein complex) subcomplex. As a first step to understand the protein interactions that dictate PPIH and PRPF4 function, we expressed and purified soluble forms of each protein and formed a complex between them. We found two sites of interaction between PPIH and the N-terminus of PRPF4, an unexpected result. The N-terminus of PRPF4 is an intrinsically disordered region and does not adopt secondary structure in the presence of PPIH. In the absence of an atomic resolution structure, we used mutational analysis to identify point mutations that uncouple these two binding sites and find that mutations in both sites are necessary to break up the complex. A discussion of how this bipartite interaction between PPIH and PRPF4 may modulate spliceosomal function is included.

Nuclear cyclophilins affect spliceosome assembly and function in vitro.

Cyclophilins are ubiquitously expressed proteins that bind to prolines and can catalyse cis/trans isomerization of proline residues. There are 17 annotated members of the cyclophilin family in humans, ubiquitously expressed and localized variously to the cytoplasm, nucleus or mitochondria. Surprisingly, all eight of the nuclear localized cyclophilins are found associated with spliceosomal complexes. However, their particular functions within this context are unknown. We have therefore adapted three established assays for in vitro pre-mRNA splicing to probe the functional roles of nuclear cyclophilins in the context of the human spliceosome. We find that four of the eight spliceosom-associated cyclophilins exert strong effects on splicing in vitro. These effects are dose-dependent and, remarkably, uniquely characteristic of each cyclophilin. Using both qualitative and quantitative means, we show that at least half of the nuclear cyclophilins can act as regulatory factors of spliceosome function in vitro. The present work provides the first quantifiable evidence that nuclear cyclophilins are splicing factors and provides a novel approach for future work into small molecule-based modulation of pre-mRNA splicing.

Critical role of O-Linked ß-N-acetylglucosamine transferase in prostate cancer invasion, angiogenesis, and metastasis.

Cancer cells universally increase glucose and glutamine consumption, leading to the altered metabolic state known as the Warburg effect; one metabolic pathway, highly dependent on glucose and glutamine, is the hexosamine biosynthetic pathway. Increased flux through the hexosamine biosynthetic pathway leads to increases in the post-translational addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) to various nuclear and cytosolic proteins. A number of these target proteins are implicated in cancer, and recently, O-GlcNAcylation was shown to play a role in breast cancer; however, O-GlcNAcylation in other cancers remains poorly defined. Here, we show that O-GlcNAc transferase (OGT) is overexpressed in prostate cancer compared with normal prostate epithelium and that OGT protein and O-GlcNAc levels are elevated in prostate carcinoma cell lines. Reducing O-GlcNAcylation in PC3-ML cells was associated with reduced expression of matrix metalloproteinase (MMP)-2, MMP-9, and VEGF, resulting in inhibition of invasion and angiogenesis. OGT-mediated regulation of invasion and angiogenesis was dependent upon regulation of the oncogenic transcription factor FoxM1, a key regulator of invasion and angiogenesis, as reducing OGT expression led to increased FoxM1 protein degradation. Conversely, overexpression of a degradation-resistant FoxM1 mutant abrogated OGT RNAi-mediated effects on invasion, MMP levels, angiogenesis, and VEGF expression. Using a mouse model of metastasis, we found that reduction of OGT expression blocked bone metastasis. Altogether, these data suggest that as prostate cancer cells alter glucose and glutamine levels, O-GlcNAc modifications and OGT levels become elevated and are required for regulation of malignant properties, implicating OGT as a novel therapeutic target in the treatment of cancer.

Hypoxia suppression of Bim and Bmf blocks anoikis and luminal clearing during mammary morphogenesis.

Proper adhesion to extracellular matrix is critical for epithelial cell survival. Detachment from matrix signals results in apoptosis, referred to as anoikis. Selective apoptosis of cells that become detached from matrix is associated with the formation of a lumen in three-dimensional mammary epithelial acinar structures in vitro. Because early breast cancer lesions such as carcinoma in situ, characterized by ducts exhibiting lumens filled with cells, are often associated with hypoxic markers, we sought to examine the role of hypoxia in anoikis and lumen formation in mammary epithelial cells. Here, we show that hypoxic conditions inhibit anoikis and block expression of proapoptotic BH3-only family members Bim and Bmf in epithelial cells. Hypoxia-mediated anoikis protection is associated with increased activation of the epidermal growth factor receptor-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase (Erk) kinase pathway and requires the hypoxia-activated transcription factor. Consistent with these data, hypoxic conditions inhibit luminal clearing during morphogenesis in human mammary epithelial acini when grown in three-dimensional cultures and are associated with decreased expression of Bim and Bmf as well as Erk activation. We show that hypoxia regulates specific cell survival pathways that disrupt tissue architecture related to clearing of luminal space during mammary morphogenesis and suggest that hypoxia-mediated anoikis resistance may contribute to cancer progression.

ErbB2 requires integrin alpha5 for anoikis resistance via Src regulation of receptor activity in human mammary epithelial cells.

ErbB2, a receptor tyrosine kinase highly expressed in many tumors, is known to inhibit apoptotic signals. Overexpression of ErbB2 causes anoikis resistance that contributes to luminal filling in three-dimensional mammary epithelial acinar structures in vitro. Given that integrins and growth factor receptors are highly interdependent for function, we examined the role of integrin subunits in ErbB2-mediated survival signaling. Here, we show that MCF-10A cells overexpressing ErbB2 upregulate integrin alpha5 via the MAP-kinase pathway in three-dimensional acini and found elevated integrin alpha5 levels associated with ErbB2 status in human breast cancer. Integrin alpha5 is required for ErbB2-mediated anoikis resistance and for optimal ErbB2 signaling to the Mek-Erk-Bim axis as depletion of integrin alpha5 reverses anoikis resistance and Bim inhibition. Integrin alpha5 is required for full activation of ErbB2 tyrosine phosphorylation on Y877 and ErbB2 phosphorylation is associated with increased activity of Src in the absence of adhesion. Indeed, we show that blocking elevated Src activity during cell detachment reverses ErbB2-mediated survival and Bim repression. Thus, integrin alpha5 serves as a key mediator of Src and ErbB2-survival signaling in low adhesion states, which are necessary to block the pro-anoikis mediator Bim, and we suggest that this pathway represents a potential novel therapeutic target in ErbB2-positive tumors.