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Phosphatidylinositol 3-Kinase Function Is Required for Transforming Growth Factor β-mediated Epithelial to Mesenchymal Transition and Cell Migration*

Open AccessPublished:November 24, 2000DOI:https://doi.org/10.1074/jbc.M005912200
      We have studied the role of phosphatidylinositol 3-OH kinase (PI3K)-Akt signaling in transforming growth factor β (TGFβ)-mediated epithelial to mesenchymal transition (EMT). In NMuMG mammary epithelial cells, exogenous TGFβ1 induced phosphorylation of Akt at Ser-473 and Akt in vitro kinase activity against GSK-3β within 30 min. These responses were temporally correlated with delocalization of E-cadherin, ZO-1, and integrin β1 from cell junctions and the acquisition of spindle cell morphology. LY294002, an inhibitor of the p110 catalytic subunit of PI3K, and a dominant-negative mutant of Akt blocked the delocalization of ZO-1 induced by TGFβ1, whereas transfection of constitutively active p110 induced loss of ZO-1 from tight junctions. In addition, LY294002 blocked TGFβ-mediated C-terminal phosphorylation of Smad2. Consistent with these data, TGFβ-induced p3TP-Lux and p(CAGA)12-Lux reporter activities were inhibited by LY294002 and transiently expressed dominant-negative p85 and Akt mutants in NMuMG and 4T1 cells. Dominant-negative RhoA inhibited TGFβ-induced phosphorylation of Akt at Ser-473, whereas constitutively active RhoA increased the basal phosphorylation of Akt, suggesting that RhoA in involved in TGFβ-induced EMT. Finally, LY294002 and neutralizing TGFβ1 antibodies inhibited ligand-independent constitutively active Akt as well as basal and TGFβ-stimulated migration in 4T1 and EMT6 breast tumor cells. Taken together, these data suggest that PI3K-Akt signaling is required for TGFβ-induced transcriptional responses, EMT, and cell migration.
      TGFβ
      transforming growth factor β
      TβRI
      TGFβ type I
      EMT
      epithelial to mesenchymal transition
      PI3K
      phosphatidylinositol 3-OH kinase
      EGF
      epidermal growth factor
      DMEM
      Dulbecco's modified Eagle's medium
      FBS
      fetal bovine serum
      PAGE
      polyacrylamide gel electrophoresis
      PBS
      phosphate-buffered saline
      MT
      microtubule
      ca
      constitutively active
      dn
      dominant-negative
      PKB
      protein kinase B
      SARA
      Smad activator for receptor activation
      FYVE domain
      domain found in Fab1p, YOTB, Vac1p, and EEA1 proteins
      The transforming growth factor β (TGFβ)1 family of secreted factors is involved in the control of different biological processes including cell proliferation, differentiation, and apoptosis (
      • Massague J.
      ). TGFβ signals through the activation of heteromeric complexes of TGFβ type I (TβRI) and type II (TβRII) receptors (
      • Massague J.
      ,
      • Roberts A.B.
      ). Activated TβRI phosphorylates receptor-associated Smads (Smad2 and Smad3), which then bind Smad4 and translocate to the nucleus where they regulate transcription of target genes (
      • Zhang Y.
      • Derynck R.
      ,
      • Heldin C.H.
      • Miyazono K.
      • ten Dijke P.
      ). TGFβ exhibits a tumor suppressor activity, and components of its signaling pathway are frequently mutated or silenced in colon and pancreatic cancers (
      • Massague J.
      ,
      • Gold L.I.
      ). However, accumulating data indicate that TGFβ can positively affect tumorigenesis and contribute to the progression and invasiveness of tumors (
      • Gold L.I.
      ,
      • Cui W.
      • Fowlis D.J.
      • Bryson S.
      • Duffie E.
      • Ireland H.
      • Balmain A.
      • Akhurst R.J.
      ,
      • Hojo M.
      • Morimoto T.
      • Maluccio M.
      • Asano T.
      • Morimoto K.
      • Lagman M.
      • Shimbo T.
      • Suthanthiran M.
      ,
      • Oft M.
      • Peli J.
      • Rudaz C.
      • Schwarz H.
      • Beug H.
      • Reichmann E.
      ). Moreover, it was recently reported that inhibition of autocrine TGFβ signaling in carcinoma cells reduces cell invasiveness and tumor metastases (
      • Oft M.
      • Heider K.H.
      • Beug H.
      ,
      • Bandyopadhyay A.
      • Zhu Y.
      • Cibull M.L.
      • Bao L.
      • Chen C.
      • Sun L.
      ). These effects of TGFβ are associated with its ability to induce an epithelial to mesenchymal transition (EMT) and stimulate cell migration.
      The EMT induced by TGFβ results in the disruption of the polarized morphology of epithelial cells, formation of actin stress fibers, and enhancement of cell migration (
      • Oft M.
      • Peli J.
      • Rudaz C.
      • Schwarz H.
      • Beug H.
      • Reichmann E.
      ,
      • Oft M.
      • Heider K.H.
      • Beug H.
      ). Two species of TβRI, Alk2 and Alk5, have been implicated in the induction of EMT by TGFβ in mammary epithelial cells (
      • Miettinen P.J.
      • Ebner R.
      • Lopez A.R.
      • Derynck R.
      ,
      • Piek E.
      • Moustakas A.
      • Kurisaki A.
      • Heldin C.H.
      • ten Dijke P.
      ). It has also been reported that high levels of ectopic Smad2 and Smad3 can induce some features of EMT in mammary epithelial cells in the context of expression of an activated type I receptor (
      • Piek E.
      • Moustakas A.
      • Kurisaki A.
      • Heldin C.H.
      • ten Dijke P.
      ). However, considering the complexity of TGFβ signaling (
      • Zhang Y.
      • Derynck R.
      ,
      • Zhang Y.
      • Feng X.H.
      • Derynck R.
      ,
      • Wong C.
      • Rougier-Chapman E.M.
      • Frederick J.P.
      • Datto M.B.
      • Liberati N.T.
      • Li J.M.
      • Wang X.F.
      , ,
      • Pardali E.
      • Xie X.Q.
      • Tsapogas P.
      • Itoh S.
      • Arvanitidis K.
      • Heldin C.H.
      • ten Dijke P.
      • Grundstrom T.
      • Sideras P.
      ), it is conceivable that other molecules can also contribute to EMT. For example, members of the AP-1 family of transcription factors have been shown to induce EMT and promote tumor invasiveness (
      • Kustikova O.
      • Kramerov D.
      • Grigorian M.
      • Berezin V.
      • Bock E.
      • Lukanidin E.
      • Tulchinsky E.
      ,
      • Lamb R.F.
      • Hennigan R.F.
      • Turnbull K.
      • Katsanakis K.D.
      • MacKenzie E.D.
      • Birnie G.D.
      • Ozanne B.W.
      ). AP-1 complexes can be activated in response to TGFβ (
      • Sano Y.
      • Harada J.
      • Tashiro S.
      • Gotoh-Mandeville R.
      • Maekawa T.
      • Ishii S.
      ,
      • Tang W.
      • Yang L.
      • Yang Y.C.
      • Leng S.X.
      • Elias J.A.
      ,
      • Ravanti L.
      • Hakkinen L.
      • Larjava H.
      • Saarialho-Kere U.
      • Foschi M.
      • Han J.
      • Kahari V.M.
      ), physically interact with Smads (
      • Zhang Y.
      • Feng X.H.
      • Derynck R.
      ,
      • Wong C.
      • Rougier-Chapman E.M.
      • Frederick J.P.
      • Datto M.B.
      • Liberati N.T.
      • Li J.M.
      • Wang X.F.
      ), and cooperate with Smads in the control of gene expression (
      • Sano Y.
      • Harada J.
      • Tashiro S.
      • Gotoh-Mandeville R.
      • Maekawa T.
      • Ishii S.
      ,
      • Tang W.
      • Yang L.
      • Yang Y.C.
      • Leng S.X.
      • Elias J.A.
      ,
      • Ravanti L.
      • Hakkinen L.
      • Larjava H.
      • Saarialho-Kere U.
      • Foschi M.
      • Han J.
      • Kahari V.M.
      ). In addition, several other downstream signaling pathways can also be activated by TGFβ, including p38Mapk (
      • Ravanti L.
      • Hakkinen L.
      • Larjava H.
      • Saarialho-Kere U.
      • Foschi M.
      • Han J.
      • Kahari V.M.
      ), c-jun N-terminal kinase (
      • Atfi A.
      • Djelloul S.
      • Chastre E.
      • Davis R.
      • Gespach C.
      ,
      • Engel M.E.
      • McDonnell M.A.
      • Law B.K.
      • Moses H.L.
      ), and phosphatidylinositol 3-OH kinase (PI3K) (
      • Krymskaya V.P.
      • Hoffman R.
      • Eszterhas A.
      • Ciocca V.
      • Panettieri R.A.
      ,
      • Higaki M.
      • Shimokado K.
      ). These signaling pathways can potentially contribute to TGFβ1-mediated EMT, but their significance for EMT and cell migration mediated by TGFβ remains unclear.
      In this study, we used the NMuMG mammary epithelial cell line as a model for TGFβ1-induced EMT (
      • Miettinen P.J.
      • Ebner R.
      • Lopez A.R.
      • Derynck R.
      ). Two metastatic breast tumor cell lines, 4T1 and EMT6, that express high levels of TGFβ ligands and TGFβ receptors were used in transcription and migration studies. We report that TGFβ-induced EMT and cell migration depend on the PI3K-Akt pathway. We also show that the phosphorylation of Smad2 and transcriptional responses induced by TGFβ are inhibited by pharmacological and molecular antagonists of the PI3K-Akt pathway. TGFβ1 can induce phosphorylation and activation of Akt/PKB in a PI3K-dependent manner, and this activation requires the Rho GTPase function. Taken together, our data suggest that PI3K-Akt signaling is required for the morphogenic, transcriptional, and migratory activities of TGFβ.

      DISCUSSION

      The tumor-promoting activity of TGFβ1 associated with the induction of EMT has been documented for different tumor types (
      • Gold L.I.
      ,
      • Cui W.
      • Fowlis D.J.
      • Bryson S.
      • Duffie E.
      • Ireland H.
      • Balmain A.
      • Akhurst R.J.
      ,
      • Hojo M.
      • Morimoto T.
      • Maluccio M.
      • Asano T.
      • Morimoto K.
      • Lagman M.
      • Shimbo T.
      • Suthanthiran M.
      ,
      • Oft M.
      • Peli J.
      • Rudaz C.
      • Schwarz H.
      • Beug H.
      • Reichmann E.
      ,
      • Oft M.
      • Heider K.H.
      • Beug H.
      ). Several reports have shown that TGFβ can induce a reversible mesenchymal transition in mammary epithelial NMuMG cells (
      • Miettinen P.J.
      • Ebner R.
      • Lopez A.R.
      • Derynck R.
      ,
      • Piek E.
      • Moustakas A.
      • Kurisaki A.
      • Heldin C.H.
      • ten Dijke P.
      ). In this study, we present data to support the role of the PI3K-Akt pathway in TGFβ-mediated EMT. We found that either the blockade of PI3K activity by a synthetic inhibitor, LY294002, or by expression of dn-Akt significantly inhibited EMT (Fig. 2). These observations led us to hypothesize that the PI3K-Akt pathway is directly involved in this transition. Similar to TGFβ1, forced expression of constitutively active PI3K (ca-p110) was sufficient to promote the disruption of cellular junctions but did not induce per se the changes in cell morphology associated with EMT (Fig. 2 B). The dissolution of tight junctions and the disruption of adherent junctions induced by TGFβ1 are relatively early processes, occurring within 4–8 h after the addition of TGFβ1, whereas changes in the cell shape occur later. This result suggests that PI3K function is required for the early changes during TGFβ-mediated EMT but that other events associated with the reorganization of cytoskeleton leading to changes in cell morphology may not depend on the PI3K-Akt pathway. The observed delocalization of E-cadherin, integrin β1, and ZO-1 from cellular junctions occurred without detectable changes in their cellular content, suggesting that these TGFβ-mediated effects may involve PI3K-dependent endocytosis. These observations are consistent with the studies implicating PI3K in endocytosis and vesicular trafficking (
      • Li G.
      • D'Souza-Schorey C.
      • Barbieri M.A.
      • Roberts R.L.
      • Klippel A.
      • Williams L.T.
      • Stahl P.D.
      ,
      • Kapeller R.
      • Chakrabarti R.
      • Cantley L.
      • Fay F.
      • Corvera S.
      ,
      • Shpetner H.
      • Joly M.
      • Hartley D.
      • Corvera S.
      ). Similar to TGFβ, hepatocyte growth factor can also disrupt epithelial cell-cell junctions and induce the delocalization of E-cadherin from cell junctions (
      • Kamei T.
      • Matozaki T.
      • Sakisaka T.
      • Kodama A.
      • Yokoyama S.
      • Peng Y.F.
      • Nakano K.
      • Takaishi K.
      • Takai Y.
      ). In this process, hepatocyte growth factor induces the delocalization of both E-cadherin and the hepatocyte growth factor receptor, c-Met, via PI3K-mediated co-endocytosis (
      • Kamei T.
      • Matozaki T.
      • Sakisaka T.
      • Kodama A.
      • Yokoyama S.
      • Peng Y.F.
      • Nakano K.
      • Takaishi K.
      • Takai Y.
      ). This co-endocytosis can be blocked by dominant-negative mutants of RhoA and Rab5, a component of early endosomes (
      • Kamei T.
      • Matozaki T.
      • Sakisaka T.
      • Kodama A.
      • Yokoyama S.
      • Peng Y.F.
      • Nakano K.
      • Takaishi K.
      • Takai Y.
      ). In addition, Rab5-mediated endocytosis is also regulated by Akt/PKB (
      • Barbieri M.A.
      • Kohn A.D.
      • Roth R.A.
      • Stahl P.D.
      ). Thus, TGFβ-mediated delocalization of epithelial markers from cell junctions may involve the function of PI3K-Akt and Rho-like GTPases.
      The activation of PI3K in response to TGFβ has been reported in two other cell systems (
      • Krymskaya V.P.
      • Hoffman R.
      • Eszterhas A.
      • Ciocca V.
      • Panettieri R.A.
      ,
      • Higaki M.
      • Shimokado K.
      ). In NMuMG cells, TGFβ1 induced phosphorylation and activation of Akt/PKB with kinetics similar to the C-terminal phosphorylation of Smad2 (Figs. 3 and 5). Activation of Akt depends on PI3K, since it can be blocked by a synthetic inhibitor of PI3K (Figs. 3 and 5) and by expression of dn-p85 (Fig. 2 B, inset). These results suggest that the PI3K-Akt pathway is activated directly by TGFβ1. This conclusion is further supported by recent reports showing co-precipitation of p85, the regulatory subunit of PI3K, with TGFβ receptors and stimulation of PI3K activity by TGFβ1 in other cell types (
      • Krymskaya V.P.
      • Hoffman R.
      • Eszterhas A.
      • Ciocca V.
      • Panettieri R.A.
      ,
      • Higaki M.
      • Shimokado K.
      ). We also confirmed a direct association p85 with both type I and type II TGFβ receptors in NMuMG cells.
      N. Dumont and A. Bakin, unpublished data.
      Because of the reported role of Rho family GTPases in TGFβ1 signaling and their interaction with the PI3K pathway (
      • Ren X.D.
      • Schwartz M.A.
      ), we tested the role of the RhoA GTPase in TGFβ-mediated activation of Akt. Expression of dominant-negative N19RhoA mutant disrupted ligand-induced phosphorylation of Akt at Ser-473. On the other hand, expression of a constitutively active mutant, Q63LRhoA, resulted in an increase of the basal phosphorylation of Akt. These findings suggest that RhoA GTPase is involved in TGFβ1-mediated activation of Akt, which is consistent with recent reports that Rho-like GTPases can synergize with TGFβ signaling (
      • Atfi A.
      • Djelloul S.
      • Chastre E.
      • Davis R.
      • Gespach C.
      ,
      • Engel M.E.
      • McDonnell M.A.
      • Law B.K.
      • Moses H.L.
      ). Therefore, RhoA may function as an upstream effector of Akt activation in response to TGFβ1.
      Using two reporter constructs, p3TP-Lux and p(CAGA)12-Lux, we found that TGFβ1 transcriptional responses in NMuMG and two tumor cell lines are inhibited by both pharmacological and molecular antagonists of the PI3K-Akt pathway, including dominant-negative p85 and Akt mutants (Fig. 4, A–D). The fact that a blockade of the PI3K-Akt pathway affected Smad-dependent transcriptional responses suggested the involvement of PI3K and Akt in TGFβ intracellular signal transduction. Consistent with this idea, we found that LY294002 significantly reduced TGFβ-mediated C-terminal phosphorylation of Smad2 in NMuMG cells (Fig. 5). However, neither PI3K nor Akt is involved in C-terminal phosphorylation of Smad2, since introduction of ca-p110 or dn-Akt did not affect it. These results, coupled with the inhibitory effect of LY294002 on Smad2 phosphorylation (Fig. 5), suggest that PI3K is involved indirectly in TGFβ-mediated C-terminal phosphorylation of Smad2.
      PI3K activity may also be required for the function of intracellular mediators of TGFβ signaling. Recently, two factors regulating C-terminal phosphorylation of Smad2 were described (
      • Tsukazaki T.
      • Chiang T.A.
      • Davison A.F.
      • Attisano L.
      • Wrana J.L.
      ,
      • Dong C.
      • Li Z.
      • Alvarez R.
      • Feng X.H.
      • Goldschmidt-Clermont P.J.
      ). First, the intracellular localization of Smad2 is controlled by SARA, a recently cloned Smad2-binding protein (
      • Tsukazaki T.
      • Chiang T.A.
      • Davison A.F.
      • Attisano L.
      • Wrana J.L.
      ). SARA co-localizes with EEA1, an early endosome marker,
      J. Wrana (University of Toronto), personal communication.
      and this co-localization depends on the FYVE domain of SARA, which binds phosphatidylinositol 3-phosphates (
      • Cantley L.C.
      • Neel B.G.
      ,
      • Fruman D.A.
      • Rameh L.E.
      • Cantley L.C.
      ). It has been shown that deletion of the FYVE domain results in the mislocalization of Smad2 and inhibition of TGFβ transcriptional responses (
      • Tsukazaki T.
      • Chiang T.A.
      • Davison A.F.
      • Attisano L.
      • Wrana J.L.
      ). We found that Smad2 co-localizes with EEA1 in the absence of TGFβ in NMuMG cells.
      A. Bakin, unpublished data.
      Thus, it is conceivable that the blockade of PI3K activity in NMuMG cells with LY294002, similar to wortmannin (
      • Nobes C.D.
      • Hawkins P.
      • Stephens L.
      • Hall A.
      ), will reduce the levels of phosphatidylinositol 3-phosphate, resulting in the mislocalization of Smad2. This is a potential explanation of the inhibitory effect of LY294002 on TGFβ-induced phosphorylation of Smad2 (Fig. 5 A), whereas neither ca-p110 nor dn-Akt can directly modulate Smad2 phosphorylation (Fig. 5 C, D). In addition, a recent report provides evidence that endogenous Smad2, Smad3, and Smad4 are stored in the MT network (
      • Dong C.
      • Li Z.
      • Alvarez R.
      • Feng X.H.
      • Goldschmidt-Clermont P.J.
      ). It has been suggested that upon TGFβ treatment, Smad2 and Smad3 dissociate from MT, become phosphorylated by TβRI, and translocate to the nucleus where they regulate the transcription of TGFβ target genes. Moreover, destabilization of MTs with nocodazole can facilitate Smad-mediated TGFβ transcriptional responses per se in the absence of exogenous TGFβ1 (
      • Dong C.
      • Li Z.
      • Alvarez R.
      • Feng X.H.
      • Goldschmidt-Clermont P.J.
      ). On the other hand, TGFβ has been reported to stabilize MTs (
      • Gundersen G.G.
      • Kim I.
      • Chapin C.J.
      ), potentially limiting Smad signaling. PI3K has also been shown to control the dynamics of the MT network, which is important for intracellular trafficking, cell motility, and other cell functions (
      • Waterman-Storer C.M.
      • Salmon E.
      ). Therefore, PI3K antagonists may affect the MT network and interfere with TGFβ signaling. To formally demonstrate that PI3K blockade inhibits TGFβ signaling via its effects on MTs will require further investigation.
      Both TGFβ and PI3K have been implicated in chemotaxis and cell migration (
      • Hojo M.
      • Morimoto T.
      • Maluccio M.
      • Asano T.
      • Morimoto K.
      • Lagman M.
      • Shimbo T.
      • Suthanthiran M.
      ,
      • Postlethwaite A.E.
      • Keski-Oja J.
      • Moses H.L.
      • Kang A.H.
      ,
      • Ashcroft G.S.
      • Yang X.
      • Glick A.B.
      • Weinstein M.
      • Letterio J.L.
      • Mizel D.E.
      • Anzano M.
      • Greenwell-Wild T.
      • Wahl S.M.
      • Deng C.
      • Roberts A.B.
      ,
      • Thelen M.
      • Uguccioni M.
      • Bosiger J.
      ,
      • Vanhaesebroeck B.
      • Jones G.E.
      • Allen W.E.
      • Zicha D.
      • Hooshmand-Rad R.
      • Sawyer C.
      • Wells C.
      • Waterfield M.D.
      • Ridley A.J.
      ,
      • Hirsch E.
      • Katanaev V.L.
      • Garlanda C.
      • Azzolino O.
      • Pirola L.
      • Silengo L.
      • Sozzani S.
      • Mantovani A.
      • Altruda F.
      • Wymann M.P.
      ). Here, we show that pm concentrations of TGFβ1 enhanced the basal migration of tumor cells, whereas blockade of PI3K with LY294002 reduced both basal and TGFβ-stimulated cell migration (Fig. 6, A and B). These data are in agreement with a critical role of PI3K in cell motility and migration via the modulation of cytoskeletal organization (
      • Cantley L.C.
      • Neel B.G.
      ,
      • Waterman-Storer C.M.
      • Salmon E.
      ). These results were generated with tumor cells that exhibit high levels of TGFβ expression and TGFβ receptors as well as constitutive activation of Akt in the absence of added TGFβ ligand. Similar to LY294002, TGFβ1-neutralizing monoclonal antibodies reduced basal cell migration and Ser-473 phosphorylation of Akt, suggesting an association between autocrine TGFβ signaling with both constitutively activated Akt/PKB and cell invasiveness. Neither exogenous TGFβ, anti-TGFβ antibodies, nor LY294002 had any effect on 4T1 or EMT6 cell proliferation. These data coupled with the transcription data using TGFβ reporters in 4T1 and EMT6 cells imply that EMT can be dissociated from the anti-mitogenic effects of TGFβ. In summary, the results presented provide evidence that the PI3K-Akt pathway is causally involved in the morphogenic, transcriptional, and migratory activities of TGFβ.

      Acknowledgements

      We thank Teresa Dugger and Sorena Nadaf for excellent technical assistance, Michael Engel for critical reading of the manuscript, W. Ogawa for the adenovirus vectors, C. Kumar for the Rac1 inhibitor SCH51344, C. L. Van Den Berg for the GST-GSK expression construct, P. N. Tsichlis for the mutant AktK179M plasmid, and J. Massague and J.-M. Gauthier for the TGFβ reporter constructs.

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