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The Tumor Suppressor PTEN Positively Regulates Macroautophagy by Inhibiting the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway*

Open AccessPublished:September 21, 2001DOI:https://doi.org/10.1074/jbc.C100319200
      The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic pathway in the human colon cancer HT-29 cells. Here we demonstrate that the wild-type PTEN is expressed in this cell line. Its overexpression directed by an inducible promoter counteracts the interleukin-13 down-regulation of macroautophagy. This effect was dependent upon the phosphoinositide phosphatase activity of PTEN as determined by using the mutant G129E, which has only protein phosphatase activity. The role of Akt/PKB in the signaling control of interleukin-13-dependent macroautophagy was investigated by expressing a constitutively active form of the kinase (MyrPKB). Under these conditions a dramatic inhibition of macroautophagy was observed. By contrast a high rate of autophagy was observed in cells expressing a dominant negative form of PKB. These data demonstrate that the signaling control of macroautophagy overlaps with the well known PI 3-kinase/PKB survival pathway and that the loss of PTEN function in cancer cells inhibits a major catabolic pathway.
      3-MA
      3-methyladenine
      Akt/PKB
      protein kinase B
      GSK-3β
      glycogen synthase kinase-3β
      HA
      hemagglutinin
      His
      polyhistidine
      IL
      interleukin
      IPTG
      isopropyl-β-d-thiogalactopyranoside
      kd
      kinase dead
      LDH
      lactate dehydrogenase
      Myr
      myristoylated
      PCR
      polymerase chain reaction
      PI 3-K
      phosphatidylinositol 3-kinase
      wt
      wild-type
      Macroautophagy, a multistep process responsible for the degradation of long-lived proteins and organelle renewal, starts with the formation of an autophagosome, which ultimately fuses with the endosomal/lysosomal compartment (
      • Dunn Jr., W.A.
      ,
      • Bohley P.
      • Seglen P.O.
      ). This pathway is known to be important in the maintenance of cell functions during period of nutrient deprivation (
      • Mortimore G.E.
      • Kadowaki M.
      ). However, recent data have shed light on the importance of autophagy in human pathologies, including some forms of cardiomyopathy (Danon's disease) (
      • Tanaka Y.
      • Guhde G.
      • Suter A.
      • Eskelinen E.L.
      • Hartmann D.
      • Lullmann-Rauch R.
      • Janssen P.M.L.
      • Blanz J.
      • von Figura K.
      • Saftig P.
      ) and breast cancer (
      • Liang X.H.
      • Jackson S.
      • Seaman M.
      • Brown K.
      • Kempkes B.
      • Hibshoosh H.
      • Levine B.
      ).
      The recent discovery of apg and aut genes in yeast and the identification of orthologous genes in human cells have increased our knowledge of the molecular machinery responsible for the formation of autophagic vacuoles (
      • Klionsky D.T.
      • Ohsumi Y.
      ,
      • Ohsumi Y.
      ). A better understanding of the control of macroautophagy is also dependent upon the identification of signal transduction pathways that control the formation of autophagosomes (
      • Blommaart E.F.C.
      • Luiken J.J.F.P.
      • Meijer A.J.
      ,
      • Codogno P.
      • Ogier-Denis E.
      • Houri J.J.
      ,
      • Klionsky D.J.
      • Emr S.D.
      ).
      The drug 3-methyladenine (3-MA),1 which inhibits the formation of autophagic vacuoles (
      • Seglen P.O.
      • Gordon P.B.
      ), has been shown to target enzymes of the phosphatidylinositol 3′-kinase family (
      • Blommaart E.F.C.
      • Krause U.
      • Schellens J.P.M.
      • Vreeling-Sindelárová H.
      • Meijer A.J.
      ,
      • Petiot A.
      • Ogier-Denis E.
      • Blommaart E.F.C.
      • Meijer A.J.
      • Codogno P.
      ). Class I and III PI 3-Ks act antagonistically at different steps of autophagy (
      • Petiot A.
      • Ogier-Denis E.
      • Blommaart E.F.C.
      • Meijer A.J.
      • Codogno P.
      ). Class III PI 3-K is probably engaged in the control of the formation of autophagic vacuoles by association with other proteins recruited to cytoplasmic membrane as suggested recently for its yeast homolog VPS34 (
      • Kihara A.
      • Noda T.
      • Ishihara N.
      • Ohsumi Y.
      ). By contrast, the plasma membrane-associated class I PI 3-K would be required to transduce a negative signal for the biogenesis of the autophagic vacuole (
      • Petiot A.
      • Ogier-Denis E.
      • Blommaart E.F.C.
      • Meijer A.J.
      • Codogno P.
      ).
      The tumor suppressor PTEN is a dual protein/lipid phosphatase mutated in a variety of cancers (
      • Di Cristofano A.
      • Pandolfi P.P.
      ,
      • Li J.
      • Yen C.
      • Liaw D.
      • Podsypania K.
      • Bose S.
      • Wag S.I.
      • Puc J.
      • Miliaresis C.
      • Rodgers L.
      • McCombie R.
      • Bigner S.H.
      • Giovanella B.C.
      • Ittmann M.
      • Tycko B.
      • Hibshoosh H.
      • Wigler M.H.
      • Parsons R.
      ,
      • Myers M.P.
      • Stolarov J.P.
      • Eng C.
      • Li J.
      • Wang S.I.
      • Wigler M.H.
      • Parsons R.
      • Tonks N.K.
      ), which has been shown to dephosphorylate the 3′ position of the class I PI 3-K product phosphatidylinositide (3,4,5)P3 (
      • Maehama T.
      • Dixon J.E.
      ) and consequently down-regulates PI 3-K/PKB pathway (
      • Wu X.Y.
      • Senechal K.
      • Neshat M.S.
      • Whang Y.E.
      • Sawyers C.L.
      ). In the present work we demonstrate that PTEN is expressed in human colon cancer HT-29 cells and negatively regulates IL-13-dependent PI 3-K/PKB signaling. Moreover PTEN, via its lipid phosphatase activity, is involved in the signaling control of autophagy, together with the downstream acting Akt/PKB. These results add a new function to the PI 3-K/PTEN/PKB pathway and also provide a new link between the control of autophagy and tumor progression.

      DISCUSSION

      The results presented here demonstrate that the signaling control of autophagy depends upon the activity of the tumor suppressor PTEN. Somatic mutations or deletion of PTEN are frequently observed in a large variety of cancers either at early or late stages of development (
      • Di Cristofano A.
      • Pandolfi P.P.
      ,
      • Cantley L.C.
      • Neel B.G.
      ). Similarly to other colon cancer cell lines, HT-29 cells express wt-PTEN (
      • Li J.
      • Simpson L.
      • Takahashi M.
      • Miliaresis C.
      • Myers M.P.
      • Tonks N.
      • Parsons R.
      ). This result is in line with the observation that PTEN′s loss of function is not a common event in colorectal cancers (
      • Wang Z.J.
      • Taylor F.
      • Churchman M.
      • Norbury G.
      • Tomlinson I.
      ,
      • Guanti G.
      • Resta N.
      • Simone C.
      • Cariola F.
      • Demma I.
      • Fiorente P.
      • Gentile M.
      ). Our data may explain why in contrast to most cancer cells, autophagy is not down-regulated in HT-29 colon cancer cells. However, alterations in PTEN expression and function is not the only cause for the low rate of autophagy in cancer cells. Recently, the protein Beclin 1 has been reported to stimulate autophagy and to suppress tumorigenesis in breast cancer cells (
      • Liang X.H.
      • Jackson S.
      • Seaman M.
      • Brown K.
      • Kempkes B.
      • Hibshoosh H.
      • Levine B.
      ). Interestingly, Beclin 1 interacts with the class III PI 3-K in mammalian cells (
      • Kihara A.
      • Kabeya Y.
      • Ohsumi Y.
      • Yoshimori T.
      ), suggesting that it is probably part of the machinery engaged in the formation of autophagic vacuoles. Together these data show that PTEN and Beclin 1, two proteins with tumor-suppressive properties, control autophagy at different levels,i.e. signaling and autophagosome formation, respectively.
      The role of PTEN in controlling autophagy is dependent upon its lipid phosphatase activity, which antagonizes the inhibitory effect of the PI 3-K/PKB pathway on the autophagic sequestration. These results point to a molecular connection existing between autophagy and cell death, because it is now well established that cell survival signaling is operative through the activation of Akt/PKB (
      • Vanhaesebroeck B.
      • Alessi D.R.
      ). Conversely, expression of wt-PTEN or its forced expression counteracts the Akt/PKB-dependent cell survival (
      • Li J.
      • Simpson L.
      • Takahashi M.
      • Miliaresis C.
      • Myers M.P.
      • Tonks N.
      • Parsons R.
      ,
      • Wang X.
      • Gjörloff-Wingren A.
      • Saxena M.
      • Pathan N.
      • Reed J.C.
      • Mustelin T.
      ). Although the role of autophagy in the execution of a programmed cell death remains to be elucidated, several studies have pointed to its importance in the type II cell death (autophagic cell death) (
      • Zakeri Z.
      • Bursch W.
      • Tenniswood M.
      • Lockshin R.A.
      ) as well as in the modulation of type I cell death (apoptosis) (
      • Lemasters J.J.
      • Nieminen A.L.
      • Qian T.
      • Trost L.C.
      • Elmore S.P.
      • Nishimura Y.
      • Crowe R.A.
      • Cascio W.E.
      • Bradham C.A.
      • Brenner D.A.
      • Herman B.
      ). The recent demonstration that PTEN is essential for embryonic development (
      • Di Cristofano A.
      • Pesce B.
      • Cordon-Cardo C.
      • Pandolfi P.P.
      ) and that a high expression of PTEN was detected in different tissues during human development (
      • Gimm O.
      • Attie-Bitach T.
      • Lees J.A.
      • Vekemans M.
      • Eng C.
      ) give credit to the idea that autophagy is instrumental during development (
      • Clarke P.G.H.
      ) and could utilize some regulatory mechanisms common with those of apoptosis (
      • Lee C.-Y.
      • Baehrecke E.H.
      ).
      The role of Akt/PKB in the negative control of autophagy is compatible with the IL-13 signal transduction pathway and the effect of PTEN. Among the known targets of Akt/PKB several lines of evidence indicate that the kinase target of rapamycin (TOR) occupies a central position in the signaling cascade of autophagy in eucaryotic cells (
      • Blommaart E.F.C.
      • Luiken J.J.F.P.
      • Blommaart P.J.E.
      • Vanwoerkom G.M.
      • Meijer A.J.
      ,
      • Noda T.
      • Ohsumi Y.
      ,
      • Rohde J.
      • Heitman J.
      • Cardenas M.E.
      ). However amino acids, which are physiological inhibitors of autophagy (reviewed in Ref.
      • van Sluijters D.A.
      • Dubbelhuis P.F.
      • Blommaart E.F.C.
      • Meijer A.J.
      ), activate mTOR by an Akt/PKB-independent mechanism in different models including HT-29 cells (
      • Hara K.
      • Yonezawa K.
      • Weng Q.-P.
      • Kozlowski M.T.
      • Belham C.
      • Avruch J.
      ,
      • Kimball S.R.
      • Shantz L.M.
      • Horetsky R.L.
      • Jefferson L.S.
      ).
      S. Arico, A. Petiot, C. Bauvy, P. F. Dubbelhuis, A. J. Meijer, P. Codogno, and E. Ogier-Denis, unpublished data.
      Further studies are needed to elucidate the mechanism involved in the control of autophagy by the PI 3-K/PTEN/PKB pathway. Nevertheless the data reported here point to the molecular connection between the control of a major catabolic route and that of a signaling pathway frequently altered in human cancers.

      Acknowledgments

      We thank A. Minty (Sanofi Elf Biorecherche, Labege, France), T. F. Franke (Columbia University, New York, NY), and P. N. Tsichlis (Fox Chase Cancer Center, Philadelphia, PA) for the gifts of IL-13,MyrPKB and kdPKB, respectively. We also thank Dr. S. E. H. Moore for critical reading of the manuscript.

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