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

Hydroxychloroquine Destabilizes Phospho-S6 in Human Renal Carcinoma Cells

<div><p>mTOR inhibitors are used to treat metastatic renal cell cancer (RCC), but most patients eventually become resistant. One possible mechanism for resistance is upregulation of autophagy, a pathway that helps recycle intracellular proteins and promotes cell survival. Hydroxychloroquine (HCQ), a potent autophagy inhibitor used to treat malaria and autoimmune disorders, is currently being studied in the context of cancer treatment. Here, we have investigated the effects of HCQ on three different renal carcinoma derived cell lines. We found that HCQ treatment inhibits RCC cell growth, promotes apoptosis, inhibits mitochondrial oxygen consumption, and increases rates of glycolysis. To understand the molecular mechanism behind these effects, we examined various nodes in the mTOR pathway and compared the effects of HCQ with the effects of the mTOR inhibitor RAD001. A key downstream readout of the pathway, phospho-S6 protein, was inhibited by both HCQ and RAD001. However, the upstream kinase, P70S6K was only inhibited by RAD001 and not HCQ, suggesting that the block by HCQ was downstream of P70S6K. Treatment with the proteasome inhibitor bortezomib restored phospho-S6 levels, suggesting that the reduction of phospho-S6 is caused by increased degradation of phospho-S6, but not total S6. Surprisingly, treatment with other autophagy inhibitors did not exhibit the same effects. Our findings suggest that HCQ causes the down-regulation of phospho-S6 in RCC cell lines via a novel mechanism that is not shared with other autophagy inhibitors.</p></div

Effects of HCQ on P70S6K and PP1 activity and localization of S6 and P70S6K.

<p><b>(A)</b><i>In vitro</i> kinase reaction of purified recombinant P70S6K was performed as described in Methods. Levels of phospho and total S6 determined by western blot. Number on top shows amount of HCQ added. Lane labeled RAD is reaction in which 10 μM RAD001 with ATP. <b>(B)</b><i>In vitro</i> kinase activity was performed on immunoprecipitated P70S6K. Extracts were made from either control (C), RAD001 (R), or HCQ (H) treated ACHN cells. Lanes on left show reactions without added ATP. Lane labeled r70 contains recombinant epitope tagged P70S6K, which runs slightly slower than endogenous protein. Reaction mixture was tested for phospho-S6, total S6, phospho-P70S6K, and total P70S6K <b>(C)</b> Immunofluorescence of 769-P cells either treated with nothing, RAD001 or HCQ and stained with indicated antibody. Nuclear DNA was visualized by DAPI. <b>(D)</b> Cells of the indicated type were untreated or treated with RAD001 or HCQ. Extracts were then examined for phospho-S6, total S6, phospho-PP1 or total PP1.</p

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

HCQ effects on cell proliferation and apoptosis.

<p><b>(A)</b> Three different human RCC derived cell lines were seeded on 96-well plates at a concentration of 1x10⁴ cells/well in triplicate and were treated with HCQ from 0 to 125 μM. After 48 hours, cell growth was measured using an MTT assay. Error bars show standard deviation, and the letter at top indicates statistically significant differences between columns with different letters (P<0.05, ANOVA with Tukey post-hoc test). <b>(B)</b> Indicated cell lines were incubated with either nothing, RAD001 (10 μM), HCQ (75 μM) or combination of RAD001 and HCQ for two days. Growth was measured as in (A). <b>(C)</b> RCC cells were cultured in the absence (C) or presence of either 10 μM RAD001 (R) or 75 μM HCQ (H) for two days, and Western analyses was used to measure the levels of autophagy (p62, LC3) and apoptosis (PARP) markers. The different forms of LC3 and PARP are shown. The relative intensities of each protein are assigned as Arbitrary Units obtained by calculating the ratio of test sample divided by a control. <b>(D)</b> Indicated cell lines were treated with either nothing or 75 μM HCQ for 48 hours and then assessed by staining and cell sorting for the apoptotic markers Annexin V-PE and 7-AAD. Bars represent the percentage of early apoptotic cells in each sample.</p

Effect of bortezomib on RAD001 and HCQ treated cells.

<p><b>(A)</b> ACHN cells were treated with either nothing (C), 10 μM RAD001 (R), 100 μM HCQ (H) with or without added bortezomib (B) for 16 hours. Extracts were then analyzed by Western blot for phospho-S6, S6, phospho-P70S6K, P70S6K, p62, beclin 1, LC3, PARP, and actin. <b>(B)</b> For pull-down assay<b>,</b> ACHN cell lysates as described in (A) were incubated with rabbit P-S6 antibody (ser240/244, 1 mg/ml) overnight at 4°C. Immune complexes were analyzed by western analysis using ubiqutin antibody and mouse P-S6 antibody (ser240). <b>(C)</b> ACHN cell lysates were analyzed by Western blot for phospho-PP1 and total PP1.</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

HCQ effects on respiration and glycolysis.

<p>RCC cells were incubated in the absence (control) or presence of HCQ (75 μM) overnight, and the cells were then tested for oxygen consumption using a Seahorse XF96 Analyzer. <b>(A)</b> The respiration assay examines oxygen consumption in the presence of four pharmacologic agents that affect mitochondrial function. These agents are oligomycin (1 μM), FCCP (300 nM), 2-DG (100 μM), and antimycin A/rotenone (1 μM each). The left side of the figure shows the actual OCR consumption measured at each time point. All experiments were done in 16 wells and error bars show standard deviation. On the right is a bar chart showing the derived basal OCR, ATP synthesis, and maximal respiration (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131464#sec002" target="_blank">Methods</a>). Asterisks indicates difference from controls at p<0.001. <b>(B)</b> Glycolysis rates were determined by measuring lactate formation. Cells were grown in the presence of 25 μM glucose for 1 hour. Oligomycin was injected to inhibit mitochondrial ATP production and 2-DG was added to determine background levels of glycolysis. Basal ECAR and glycolytic reserve capacity was determined as described in methods.</p

Rheb activity drives increased TH levels in pupal epidermal cells.

<p>Western blot analysis reveals a robust increase in levels of TH protein, and more modest increase of Yellow protein, in Rheb overexpressing thoraces compared to <i>pannier-Gal4</i> (<i>pnr-G4</i>) line alone (A). TH protein is expressed in a subset of anterior epidermal cells prior to the onset of pigmentation in the P10 stage pupal thorax (B). <i>UAS-Rheb, pannier-Gal4</i> pupa showing increased numbers of TH protein expressing cells along the central dorsal region of the thorax (C), which is suppressed by either <i>raptor^RNAi</i> (D), or <i>s6k1^RNAi</i> (E). Overexpression of Rheb by <i>pannier-Gal4</i> expands the expression of the <i>TH^4.1-LacZ</i> reporter, as shown by β-gal labeling (gray, F, G). Genotypes of flies: <i>Y/w, UAS-dicer2; pannier-Gal4/+</i> (A, B, G), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/+</i> (A, C, G), <i>Y/w, UAS-dicer2; UAS-Rheb/+</i>; <i>pannier-Gal4/UAS-raptor^RNAi</i>(D), <i>Y/w, UAS-dicer2; UAS-Rheb/UAS-s6k1^RNAi</i>; <i>pannier-Gal4/+</i>(E), <i>Y/w, UAS-dicer2;+/TH^4.1-LacZ, pannier-Gal4/+</i> and <i>Y/w, UAS-dicer2; UAS-Rheb/TH^4.1-LacZ</i>; <i>pannier-Gal4/+</i>(F).</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