FoxO restricts growth and differentiation of cells with elevated TORC1 activity under nutrient restriction

<div><p>TORC1, a central regulator of cell survival, growth, and metabolism, is activated in a variety of cancers. Loss of the tumor suppressors PTEN and Tsc1/2 results in hyperactivation of TORC1. Tumors caused by the loss of PTEN, but not Tsc1/2, are often malignant and have been shown to be insensitive to nutrient restriction (NR). In <i>Drosophila</i>, loss of PTEN or Tsc1 results in hypertrophic overgrowth of epithelial tissues under normal nutritional conditions, and an enhanced TORC1-dependent hyperplastic overgrowth of <i>PTEN</i> mutant tissue under NR. Here we demonstrate that epithelial cells lacking Tsc1 or Tsc2 also acquire a growth advantage under NR. The overgrowth correlates with high TORC1 activity, and activating TORC1 downstream of Tsc1 by overexpression of <i>Rheb</i> is sufficient to enhance tissue growth. In contrast to cells lacking PTEN, <i>Tsc1</i> mutant cells show decreased PKB activity, and the extent of <i>Tsc1</i> mutant overgrowth is dependent on the loss of PKB-mediated inhibition of the transcription factor FoxO. Removal of FoxO function from <i>Tsc1</i> mutant tissue induces massive hyperplasia, precocious differentiation, and morphological defects specifically under NR, demonstrating that FoxO activation is responsible for restricting overgrowth of <i>Tsc1</i> mutant tissue. The activation status of FoxO may thus explain why tumors caused by the loss of Tsc1–in contrast to PTEN–rarely become malignant.</p></div

FoxO restricts growth and differentiation of cells with elevated TORC1 activity under nutrient restriction

<div><p>TORC1, a central regulator of cell survival, growth, and metabolism, is activated in a variety of cancers. Loss of the tumor suppressors PTEN and Tsc1/2 results in hyperactivation of TORC1. Tumors caused by the loss of PTEN, but not Tsc1/2, are often malignant and have been shown to be insensitive to nutrient restriction (NR). In <i>Drosophila</i>, loss of PTEN or Tsc1 results in hypertrophic overgrowth of epithelial tissues under normal nutritional conditions, and an enhanced TORC1-dependent hyperplastic overgrowth of <i>PTEN</i> mutant tissue under NR. Here we demonstrate that epithelial cells lacking Tsc1 or Tsc2 also acquire a growth advantage under NR. The overgrowth correlates with high TORC1 activity, and activating TORC1 downstream of Tsc1 by overexpression of <i>Rheb</i> is sufficient to enhance tissue growth. In contrast to cells lacking PTEN, <i>Tsc1</i> mutant cells show decreased PKB activity, and the extent of <i>Tsc1</i> mutant overgrowth is dependent on the loss of PKB-mediated inhibition of the transcription factor FoxO. Removal of FoxO function from <i>Tsc1</i> mutant tissue induces massive hyperplasia, precocious differentiation, and morphological defects specifically under NR, demonstrating that FoxO activation is responsible for restricting overgrowth of <i>Tsc1</i> mutant tissue. The activation status of FoxO may thus explain why tumors caused by the loss of Tsc1–in contrast to PTEN–rarely become malignant.</p></div

<i>Tsc1 FoxO</i> double mutant discs exhibit misfoldings due to massive overproliferation under NR.

<p><b>(A)</b> aPKC (in green), phalloidin (in red) and DAPI (in blue) stainings in orthogonal sections of eye discs with control, <i>FoxO</i>, <i>Tsc1</i> and <i>Tsc1 FoxO</i> mutant tissue dissected from larvae reared on normal food and NR <b>(A’)</b>. Scale bars are 25 μm except for <i>Tsc1</i>^<i>Q87X</i> <i>FoxO</i>^<i>25</i> on NR, where they are 75 μm. A lower magnification was used to give a better overview of the misfolded tissue. <b>(B)</b> aPKC (in green) and Dlg (in red) stainings in orthogonal sections of eye discs with hsFlp control, <i>FoxO</i>, <i>Tsc1</i>, and <i>Tsc1 FoxO</i> mutant clones (marked by the absence of GFP) dissected from larvae reared on normal food and NR.</p

Loss of FoxO function leads to enhancement of <i>Tsc1</i> overgrowth under NR.

<p><b>(A)</b> Eyes bearing eyFlp control, <i>Tsc1</i> or <i>PTEN</i> mutant clones (marked by the absence of pigmentation), with or without <i>FoxO</i> mutation of animals reared on normal food or NR. Removal of FoxO caused lethality of <i>Tsc1</i> mutant animals under NR. <b>(B)</b> Eye discs with control, <i>FoxO</i>, <i>Tsc1</i> or <i>Tsc1 FoxO</i> mutant tissue dissected from larvae reared on normal food or NR; <b>(B’)</b> quantification of eye disc size. Scale bars are 250 μm. <b>(C)</b> Nuclear (DAPI, blue) and cell membrane (phalloidin, red) staining in eye discs with hsFlp control, <i>FoxO</i>, <i>Tsc1</i> or <i>Tsc1 FoxO</i> clones (marked by the absence of GFP) dissected from larvae reared on NR. Scale bars are 75 μm.</p

<i>Tsc1 FoxO</i> double mutant discs exhibit misfoldings due to massive overproliferation under NR.

<p><b>(A)</b> aPKC (in green), phalloidin (in red) and DAPI (in blue) stainings in orthogonal sections of eye discs with control, <i>FoxO</i>, <i>Tsc1</i> and <i>Tsc1 FoxO</i> mutant tissue dissected from larvae reared on normal food and NR <b>(A’)</b>. Scale bars are 25 μm except for <i>Tsc1</i>^<i>Q87X</i> <i>FoxO</i>^<i>25</i> on NR, where they are 75 μm. A lower magnification was used to give a better overview of the misfolded tissue. <b>(B)</b> aPKC (in green) and Dlg (in red) stainings in orthogonal sections of eye discs with hsFlp control, <i>FoxO</i>, <i>Tsc1</i>, and <i>Tsc1 FoxO</i> mutant clones (marked by the absence of GFP) dissected from larvae reared on normal food and NR.</p

<i>Tsc1</i> mutant proliferating cells undergo massive apoptosis.

<p><b>(A)</b> Cleaved Caspase-3 antibody staining (in red) of control or <i>Tsc1</i> mutant eye discs dissected at the indicated developmental points from larvae reared on normal food and NR; quantification of eye discs size <b>(A’)</b>, and percentage of apoptotic tissue area with respect to the total area of discs <b>(A”)</b>. Scale bars are 250 μm and 100 μm, respectively.</p

PKB-mediated inhibition of FoxO is lost in <i>Tsc1</i> mutant cells under NR.

<p><b>(A)</b> Phospho-PKB staining (in red) of eyes discs with hsFlp <i>Tsc1</i> and <i>PTEN</i> <b>(A’)</b> mutant clones (marked by the absence of GFP) dissected from larvae reared on normal food and NR. Scale bars are 75 μm. <b>(B)</b> Western blot analysis of proteins extracted from the control or <i>Tsc1</i> mutant eye imaginal discs. The extremely small size of the control eye discs from animals reared on NR precluded the Western analysis. Total levels of PKB were unaltered. β<b>-</b>tubulin served as loading control. <b>(C)</b> FoxO staining (in magenta) of eyes discs with hsFlp control, <i>Tsc1</i> and <i>PTEN</i> clones (marked by the absence of GFP) dissected from larvae reared on normal food and NR. Scale bars are 75 μm. <b>(D)</b> Eyes bearing control, <i>Tsc1</i> knockdown or <i>PTEN</i> knockdown tissue, with or without the overexpression of FoxO, of animals reared on normal food or NR. Scale bars are 100 μm.</p

<i>Tsc1</i> mutant cells have a growth advantage under NR.

<p><b>(A)</b> Third instar eye discs with hsFlp control or <i>Tsc1</i> mutant clones (marked by the absence of GFP) dissected from larvae reared under varying yeast concentrations. <b>(B)</b> Eye discs with hsFlp control or <i>Tsc1</i> mutant clones (marked by the absence of GFP) dissected 72 hours after clone induction from larvae reared on NR. <b>(C)</b> Eyes with eyFlp control or <i>Tsc1</i> mutant clones (marked by the absence of pigmentation) of animals reared on normal food and NR; <b>(C’)</b> quantification of eye sizes. <b>(D)</b> Scanning electron micrographs of eyes composed of control or <i>Tsc1</i> mutant tissue of animals reared on normal food and 20 g/l food, and the quantification of eye size <b>(D’)</b>, ommatidia size (n>260) <b>(D”)</b>, and ommatidia number <b>(D‴).</b></p