Constitutive mTOR activation in TSC mutants sensitizes cells to energy starvation and genomic damage via p53

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102117/1/emboj7601900.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102117/2/emboj7601900-sup-0001.pd

Complementing Cancer Metastasis

Complement is an effector of innate immunity and a bridge connecting innate immunity and subsequent adaptive immune responses. It is essential for protection against infections and for orchestrating inflammatory responses. Recent studies have also demonstrated contribution of the complement system to several homeostatic processes that are traditionally not considered to be involved in immunity. Thus, complement regulates homeostasis and immunity. However, dysregulation of this system contributes to several pathologies including inflammatory and autoimmune diseases. Unexpectedly, studies of the last decade have also revealed that complement promotes cancer progression. Since the initial discovery of tumor promoting role of complement, numerous preclinical and clinical studies demonstrated contribution of several complement components to regulation of tumor growth through their direct interactions with the corresponding receptors on tumor cells or through suppression of antitumor immunity. Most of this work, however, focused on a role of complement in regulating growth of primary tumors. Only recently, a few studies showed that complement promotes cancer metastasis through its contribution to epithelial-to-mesenchymal transition and the premetastatic niche. This latter work has shown that complement activation and generation of complement effectors including C5a occur in organs that are target for metastasis prior to arrival of the very first tumor cells. C5a through its interactions with C5a receptor 1 inhibits antitumor immunity by activating and recruiting immunosuppressive cells from the bone marrow to the premetastatic niche and by regulating function and self-renewal of pulmonary tissue-resident alveolar macrophages. These new advancements provide additional evidence for multifaceted functions of complement in cancer

Surface potential and roughness controlled cell adhesion and collagen formation in electrospun PCL fibers for bone regeneration

Surface potential of biomaterials is a key factor regulating cell responses, driving their adhesion and signaling in tissue regeneration. In this study we compared the surface and zeta potential of smooth and porous electrospun polycaprolactone (PCL) fibers, as well as PCL films, to evaluate their significance in bone regeneration. The ' surface potential of the fibers was controlled by applying positive and negative voltage polarities during the electrospinning. The surface properties of the different PCL fibers and films were measured using X-ray photoelectron spectroscopy (XPS) and Kelvin probe force microscopy (KPFM), and the zeta potential was measured using the electrokinetic technique. The effect of surface potential on the morphology of bone cells was examined using advanced microcopy, including 3D reconstruction based on a scanning electron microscope with a focused ion beam (FIB-SEM). Initial cell adhesion and collagen formation were studied using fluorescence microscopy and Sirius Red assay respectively, while calcium mineralization was confirmed with energy-dispersive x-ray (EDX) and Alzarin Red staining. These studies revealed that cell adhesion is driven by both the surface potential and morphology of PCL fibers. Furthermore, the ability to tune the surface potential of electrospun PCL scaffolds provides an essential electrostatic handle to enhance cell-material interaction and cellular activity, leading to controllable morphological changes

Complement c5a receptor facilitates cancer metastasis by altering t-cell responses in the metastatic niche

The impact of complement on cancer metastasis has not been well studied. In this report, we demonstrate in a preclinical mouse model of breast cancer that the complement anaphylatoxin C5a receptor (C5aR) facilitates metastasis by suppressing effector CD8(+) and CD4(+) T-cell responses in the lungs. Mechanisms of this suppression involve recruitment of immature myeloid cells to the lungs and regulation of TGF beta and IL10 production in these cells. TGF beta and IL10 favored generation of T regulatory cells (T-reg) and Th2-oriented responses that rendered CD8(+) T cells dysfunctional. Importantly, pharmacologic blockade of C5aR or its genetic ablation in C5aR-deficient mice were sufficient to reduce lung metastases. Depletion of CD8(+) T cells abolished this beneficial effect, suggesting that CD8(+) T cells were responsible for the effects of C5aR inhibition. In contrast to previous findings, we observed that C5aR signaling promoted T-reg generation and suppressed T-cell responses in organs where metastases arose. Overall, our findings indicated that the immunomodulatory functions of C5aR are highly context dependent. Furthermore, they offered proof-of-concept for complement-based immunotherapies to prevent or reduce cancer metastasis. (C) 2014 AACR

The TSC1/2 Complex Controls <em>Drosophila</em> Pigmentation through TORC1-Dependent Regulation of Catecholamine Biosynthesis

<div><p>In <em>Drosophila</em>, the pattern of adult pigmentation is initiated during late pupal stages by the production of catecholamines DOPA and dopamine, which are converted to melanin. The pattern and degree of melanin deposition is controlled by the expression of genes such as <em>ebony</em> and <em>yellow</em> as well as by the enzymes involved in catecholamine biosynthesis. In this study, we show that the conserved TSC/TORC1 cell growth pathway controls catecholamine biosynthesis in <em>Drosophila</em> during pigmentation. We find that high levels of Rheb, an activator of the TORC1 complex, promote premature pigmentation in the mechanosensory bristles during pupal stages, and alter pigmentation in the cuticle of the adult fly. Disrupting either melanin synthesis by RNAi knockdown of melanogenic enzymes such as <em>tyrosine hydroxylase</em> (TH), or downregulating TORC1 activity by Raptor knockdown, suppresses the Rheb-dependent pigmentation phenotype in vivo. Increased Rheb activity drives pigmentation by increasing levels of TH in epidermal cells. Our findings indicate that control of pigmentation is linked to the cellular nutrient-sensing pathway by regulating levels of a critical enzyme in melanogenesis, providing further evidence that inappropriate activation of TORC1, a hallmark of the human tuberous sclerosis complex tumor syndrome disorder, can alter metabolic and differentiation pathways in unexpected ways.</p> </div

Rheb drives increased pigmentation of the pupal and adult cuticle.

<p>The evolutionarily conserved TSC pathway regulates protein synthesis and cell growth through activation of TOR complex 1 (TORC1) (A) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048720#pone.0048720-Garami1" target="_blank">[12]</a>. Uniform pigmentation of the adult male thorax in <i>pannier-Gal4/+</i> (we will use the abbreviation “<i>-G4</i>” for Gal4 in this and subsequent figures) (B). Pattern of expression of <i>pannier-Gal4, UAS-Rheb-GFP</i> on the pupal thorax (C). “trident pattern” pigmentation in the posterior thorax <i>UAS-Rheb</i>, <i>pannier-Gal4</i> adult male fly (D). MARCM clones of <i>tsc1^w243x</i> and <i>tsc2¹⁰⁹</i> (E,F), exhibit posterior pigmentation (white arrowheads) in clones (clones marked with GFP, see L­O). <i>UAS-TSC1</i> and <i>UAS-TSC2</i> suppress the increased growth and pigmentation in <i>pannier-Gal4, UAS-Rheb</i> flies (G). <i>UAS-TSC2^RNAi</i> enhances the increased growth and pigmentation in <i>pannier-Gal4, UAS-Rheb</i> flies (H). <i>pannier-Gal4, UAS-Rheb</i> shows premature bristle pigmentation in a dorsal stripe in stage P11 pupa (I). Pupa, stage P10 in wildtype (J) and <i>tsc1^w243x</i> MARCM clones (K-M), GFP-marked (arrowheads) <i>tsc1^w243x</i> bristles pigment prematurely, red in M and O is autofluorescence of the cuticle. Premature pupal bristle pigmentation is suppressed in <i>rheb^2D1, tsc1^R453x</i> clones, marked by arrowheads (N,O) and GFP (green, O). Genotypes of flies: <i>Y/w, UAS-dicer2; pannier-Gal4/+</i>(B), <i>Y/w, UAS-dicer2; UAS-Rheb-GFP/+</i>, <i>pannier-Gal4/+</i>(C), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/+</i>(D,I), <i>w/yw, Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-Tau-GFP/+; FRT82B, tsc1^w243x/FRT82B tub-Gal80</i> (E, K–M), <i>w/yw, Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-tau-GFP/+; tsc2¹⁰⁹ FRT80B/tub-Gal80 FRT80B</i> (F). <i>Y/w; UAS-Rheb/+</i>, <i>pannier-Gal4/UAS-tsc1,UAS-tsc2</i> (G), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>, <i>pannier-Gal4/UAS-tsc2^RNAi</i> (H). <i>w/yw, Ubx-flp</i>; <i>scabrous-Gal4,UAS-actin-GFP/+; FRT82B rheb^2D1, tsc1^R453x</i>/<i>FRT82B tub-Gal80</i> (N,O).</p

TSC1/2 pathway regulates amino acid levels and function upstream of the catecholamine pathway.

<p>The <i>Drosophila</i> melanin biosynthesis pathway (modified from (Wittkopp, True and Carroll, 2002) enzymes in blue, substrates in black; phenol oxidases, aaNAT and NADA sclerotin have been excluded) (A). Pigmentation in MARCM clones of <i>tsc1^R453x</i> (B) is partially suppressed in a <i>yellow</i> background (C, arrowheads indicate clone regions in both B and C). Amino acid and metabolite analysis of heads collected from <i>UAS-Rheb/TM3, Sb</i> and <i>elav-Gal4/UAS-Rheb</i> flies, show statistically significant increases in glutamine, ammonia, lysine, 1-methylhistidine, and asparagine under conditions of neuronal Rheb-overexpression (Student’s T-test-*, D). <i>UAS-TH^RNAi</i> markedly suppressed the <i>UAS-Rheb</i>, <i>pannier-Gal4</i> pigmentation phenotype (E). Genotypes of flies: <i>w/yw,Ubx-flp; scabrous-Gal4,UAS-Pon-GFP,UAS-Tau-GFP/+;FRT82B, tsc1^R453x/FRT82B tub-Gal80</i> (B), y<i>w/yw,Ubx-flp; scabrous-Gal4,UAS-Pon-GFP, UAS-Tau-GFP/+; FRT82B, tsc1^R453/FRT82B tub-Gal80</i> (C), <i>Y/w</i>; UAS-Rheb/TM3, Sb and <i>Y/w</i>; UAS-Rheb/<i>elav-Gal4</i> (D), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/UAS-TH^RNAi</i> (E).</p

TORC1 and S6 kinase-dependent pigmentation of the adult cuticle.

<p>Pigmentation and bristle growth phenotype in <i>UAS-Rheb-GFP</i>, <i>pannier-Gal4</i> is suppressed in <i>tor^Æ</i>^Pclones (A–D). In order to identify clones by expression of fluorescent markers, the epidermis was imaged in P9 pupae prior to the onset of pigmentation (A, B). Clones were identified by lack of Ubi-nls-RFP (red), and expression of Rheb was visualized by GFP (green). After live imaging of fluorescently marked clones (dotted lines) in the pupa, the adult fly was recovered to assess the effect of <i>tor</i> deletion on pigmentation induced by Rheb-GFP (C, D), the location of the clone was identified by it position relative to the large nuclei of macrochaete bristle cells in the pupa (white arrowheads). Expression of either <i>raptor^RNAi</i> (E), or <i>s6k1^RNAi</i> (F). <i>UAS-s6k1^TE</i>, <i>pannier-Gal4</i> flies show mild posterior pigmentation on the thorax (G). The increased pigmentation in the posterior thorax by <i>pannier-Gal4-</i>driven overexpression of both <i>s6k1^TE</i> and eIF4E was fully penetrant, but the darkening of the scutellum in this background was not consistently observed in all flies (H). Genotypes of flies: <i>yw, Ubx-FLP/w; Tor^ΔP FRT40A/Ubi-mRFP.nls FRT40A; pannier-Gal4, UAS-Rheb-GFP/+</i> (A–D). <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/UAS-raptor^RNAi</i> (E), <i>Y/w, UAS-dicer2; UAS-Rheb/UAS-s6k1^RNAi</i>; <i>pannier-Gal4/+</i>(F), <i>Y/w, UAS-dicer2; +/UAS-s6k1^TE</i>; <i>pannier-Gal4/+</i> (G), <i>Y/w, UAS-dicer2; +/UAS-s6k1^TE</i>; <i>pannier-Gal4/UAS-eIF4E</i> raised at 29°C (H).</p