EGFR is required for Wnt9a-Fzd9b signalling specificity in haematopoietic stem cells.

Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand-receptor (Wnt-Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit β-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a-Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a-Fzd9b-LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt-Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine

A stromal lysolipid-autotaxin signaling axis promotes pancreatic tumor progression

Pancreatic ductal adenocarcinoma (PDAC) develops a pronounced stromal response reflecting an aberrant wound-healing process. This stromal reaction features transdifferentiation of tissue-resident pancreatic stellate cells (PSC) into activated cancer-associated fibroblasts, a process induced by PDAC cells but of unclear significance for PDAC progression. Here, we show that PSCs undergo a dramatic lipid metabolic shift during differentiation in the context of pancreatic tumorigenesis, including remodeling of the intracellular lipidome and secretion of abundant lipids in the activated, fibroblastic state. Specifically, stroma-derived lysophosphatidylcholines support PDAC cell synthesis of phosphatidylcholines, key components of cell membranes, and also facilitate production of the potent wound-healing mediator lysophosphatidic acid (LPA) by the extracellular enzyme autotaxin, which is overexpressed in PDAC. The autotaxin–LPA axis promotes PDAC cell proliferation, migration, and AKT activation, and genetic or pharmacologic autotaxin inhibition suppresses PDAC growth in vivo. Our work demonstrates how PDAC cells exploit the local production of wound-healing mediators to stimulate their own growth and migration. Significance: Our work highlights an unanticipated role for PSCs in producing the oncogenic LPA signaling lipid and demonstrates how PDAC tumor cells co-opt the release of wound-healing mediators by neighboring PSCs to promote their own proliferation and migration

ABL Tyrosine Kinase Stimulates PUMA Protein Expression

ABL is an ubiquitously expressed non-receptor tyrosine kinase involved in multiple cellular functions including programmed cell death. Upon DNA damage, ABL has been shown to upregulate PUMA, p53 upregulated modulator of apoptosis, and causes downstream mitochondrial intrinsic apoptotic events. However, the mechanism by which ABL regulates PUMA expression remains unknown. We have shown that ABL does not change PUMA protein subcellular localization through immunofluorescence. Through protein and RNA quantification, we showed that overproduction of PUMA RNA leads to an increase in PUMA protein expression. ABL requires a functional kinase domain to stimulate PUMA protein expression. Inhibition of ABL kinase activity using Imatinib diminishes the ability of ABL to induce PUMA protein expression

Actomyosin Spatiotemporally Regulates Par Polarity Dynamics To Create Neural Stem Cell Asymmetry

PDF of dissertation and zipped file of 13 videos.Pattern formation, or specifically symmetry breaking, is a fundamental process essential for proper asymmetric cell division. In asymmetrically dividing stem cells, the evolutionarily conserved Par polarity complex localizes to a discrete Par domain to facilitate unequal distribution of fate determinants into the daughter cells–thereby ensuring a binary cell division outcome where daughters will acquire distinct fates. Hence proper asymmetric cell division requires the spatiotemporal distribution of Par proteins to be precisely coordinated. While a number of studies have been conducted to understand how Par activity creates downstream asymmetry, how the Par complex acquires asymmetry remains unclear. Two standing models exist to explain for how Par proteins can become polarized. In the one-cell stage C. elegans embryo, gradients of contractile force created by the cortical actomyosin cytoskeletal network generates cortical flow towards the anterior pole. Concurrently, symmetrical Par proteins that are entrained within the network becomes advected via bulk motion of the cortex, consequently becoming anteriorly segregated. In Drosophila neuroblasts, Par complex exchanges between the unpolarized, cytoplasmic and polarized, apical states; it is thus thought to become polarized to the apical domain via a direct, asymmetric targeting mechanism. In this dissertation, we examined the spatiotemporal distribution profile of cortical Par proteins and actomyosin in mitotic neuroblasts using a full volume, rapid live imaging approach to capture change in cortical protein distribution as they transition from an unpolarized to a polarized state. In the second chapter, we characterized the Par protein dynamics and investigated if the actomyosin network is essential for Par dynamics. This study demonstrated that Par polarization is a dynamic, multistep process, consisting of asymmetric targeting of cytoplasmic Par into discrete, apical foci and F-actin dependent coalescence of Par foci at the apical pole. In the third chapter, we determined the cortical dynamics of actomyosin and identified that coalescence is spatiotemporally linked to myosin II driven flow. Our studies suggest a conserved role for actomyosin in Par polarity in C. elegans embryos and Drosophila neuroblasts. This dissertation contains previously published and unpublished co-authored material. Live imaging movies of Par proteins and actomyosin are attached in the supplemental files associated with this dissertation.2022-08-2

CRISPR Guide RNA Validation In Vitro

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far. When combined with endonucleases with alternative protospacer adjacent motif site specificities and alternative sgRNAs, this method will streamline genome editing at almost any locus

A cancer cell-intrinsic GOT2-PPARd axis suppresses antitumor immunity

Despite significant recent advances in precision medicine, pancreatic ductal adenocarcinoma (PDAC) remains near uniformly lethal. Although immune-modulatory therapies hold promise to meaningfully improve outcomes for patients with PDAC, the development of such therapies requires an improved understanding of the immune evasion mechanisms that characterize the PDAC microenvironment. Here, we show that cancer cell-intrinsic glutamic-oxaloacetic transaminase 2 (GOT2) shapes the immune microenvironment to suppress antitumor immunity. Mechanistically, we find that GOT2 functions beyond its established role in the malate-aspartate shuttle and promotes the transcriptional activity of nuclear receptor peroxisome proliferator-activated receptor delta (PPARδ), facilitated by direct fatty acid binding. Although GOT2 is dispensable for cancer cell proliferation in vivo, the GOT2-PPARδ axis promotes spatial restriction of both CD4+ and CD8+ T cells from the tumor microenvironment. Our results demonstrate a noncanonical function for an established mitochondrial enzyme in transcriptional regulation of immune evasion, which may be exploitable to promote a productive antitumor immune response.SignificancePrior studies demonstrate the important moonlighting functions of metabolic enzymes in cancer. We find that the mitochondrial transaminase GOT2 binds directly to fatty acid ligands that regulate the nuclear receptor PPARδ, and this functional interaction critically regulates the immune microenvironment of pancreatic cancer to promote tumor progression. See related commentary by Nwosu and di Magliano, p. 2237.. This article is highlighted in the In This Issue feature, p. 2221