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Regulation of Fas Expression by STAT3 and c-Jun Is Mediated by Phosphatidylinositol 3-Kinase-AKT Signaling*

  • Vladimir N. Ivanov
    Affiliations
    From the Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029 and
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  • Ze'ev Ronai
    Correspondence
    To whom correspondence should be addressed: Ruttenberg Cancer Center, Mount Sinai School of Medicine, 1 Gustave Levy Pl., Box 1130, New York, NY 10029. Fax: 212-849-2425;
    Affiliations
    From the Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029 and
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  • Mikhail Krasilnikov
    Affiliations
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  • Author Footnotes
    * This work was supported by NCI, National Institutes of Health, Grants CA51995 and TW00960 and a Sharp Foundation grant (to Z. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:December 03, 2001DOI:https://doi.org/10.1074/jbc.M108233200
      Cooperation between STAT3 and c-Jun results in suppression of Fas Receptor (FasR) transcription, which is often seen in advanced human tumors. To identify requirements for STAT3-Jun cooperation, we elucidated the role of protein kinases that affect both transcription factors. The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway was found capable of down-regulating both STAT3- and c-Jun-dependent transcription, resulting in derepression of FasR transcription. Conversely, inhibition of PI3K-AKT signaling via the specific pharmacological inhibitor LY294002 up-regulated AP1/Jun- and STAT-dependent transcriptional activities, resulting in suppression of the FasR promoter activities and decreased FasR surface expression. PI3K-AKT's ability to affect FasR transcription was not observed in c-jun null fibroblasts, suggesting that c-Jun is required for PI3K/AKT-mediated regulation of FasR transcription. Interestingly, the dominant negative form of Rac1 (RacN17) was also efficient in relieving FasR expression, suggesting that the increase in FasR expression following AKT stimuli could be mediated via AKT ability to elicit suppression of Rac1, which in turn decreases JNK activities and c-Jun phosphorylation. Overall, our findings demonstrate that through its negative effects on both c-Jun and STAT3, the PI3K-AKT pathway disrupts cooperation between c-Jun and STAT3, which is required for silencing the FasR promoter, resulting in increased expression of surface FasR and concomitant sensitization to FasL-mediated programmed cell death.
      Deregulation of cell proliferation and suppression of apoptosis meet the essential requirements for neoplastic development and progression. Understanding the multiple regulatory elements that are impaired in human tumors highlights the complexity of controlled cell growth while pointing to targets that may be impaired during tumor development. Both normal and tumor cells use the PI3K-AKT
      The abbreviations used are:
      PI3K
      phosphatidylinositol 3-kinase
      AKTmyr
      myristoylated AKT
      GFP
      green fluorescent protein
      JNK
      Jun N-terminal kinase
      STAT3
      signal transducer and activator of transcription 3
      IAP
      inhibitor of apoptosis
      FasR
      Fas receptor
      WT
      wild type
      TNF
      tumor necrosis factor
      CHX
      cycloheximide
      PE
      phycoerythrin
      PTEN
      phosphatase and tensin homology deleted on chromosome 10
      1The abbreviations used are:PI3K
      phosphatidylinositol 3-kinase
      AKTmyr
      myristoylated AKT
      GFP
      green fluorescent protein
      JNK
      Jun N-terminal kinase
      STAT3
      signal transducer and activator of transcription 3
      IAP
      inhibitor of apoptosis
      FasR
      Fas receptor
      WT
      wild type
      TNF
      tumor necrosis factor
      CHX
      cycloheximide
      PE
      phycoerythrin
      PTEN
      phosphatase and tensin homology deleted on chromosome 10
      (protein kinase B) as survival pathways that utilize several critical cellular effectors as substrates and modulators of the cell's ability to undergo apoptosis (
      • Coffer P.J.
      • Jin J.
      • Woodgett J.R.
      ,
      • Kandel E.S.
      • Hay N.
      ,
      • Datta S.R.
      • Brunet A.
      • Greenberg M.E.
      ). These include phosphorylation-dependent inhibition of proapoptotic signals of proteins such as BAD, caspase 9, and the family of forkhead transcription factors (
      • Pastorino J.G.
      • Tafani M.
      • Farber J.L.
      ,
      • Rena G.
      • Guo S.
      • Cichy S.C.
      • Unterman T.G.
      • Cohen P.
      ,
      • Brunet A.
      • Bonni A.
      • Zigmond M.J.
      • Lin M.Z.
      • Juo P., Hu, L.S.
      • Anderson M.J.
      • Arden K.C.
      • Blenis J.
      • Greenberg M.E.
      ). Additionally, AKT enhances survival signals via activation of NF-κB-dependent expression of anti-apoptotic genes, including FLIP and c-IAPs, and suppresses mitochondrial pathways required for apoptosis (
      • Madrid L.V.
      • Mayo M.W.
      • Reuther J.Y.
      • Baldwin A.S., Jr.
      ,
      • Suhara T.
      • Mano T.
      • Oliveira B.E.
      • Walsh K.
      ,
      • Plas D.R.
      • Talapatra S.
      • Edinger A.L.
      • Rathmell J.C.
      • Thompson C.B.
      ). Certain tumors increase AKT expression, activity, or both as a means of escaping programmed cell death. For example, AKT was found to be overexpressed in breast cancer cell lines and in ovarian and pancreatic cancers and amplified in gastric adenomas (
      • Bellacosa A.
      • Feo D.D.
      • Godwin A.K.
      • Bell D.W.
      • Cheng J.Q.
      • Altomare D.A.
      • Wan M.
      • Dubeau L.
      • Scambia G.
      • Masciullo V.
      • Ferrandina G.
      • Panici P.B.
      • Mancuso S.
      • Neri G.
      • Testa J.R.
      ,
      • Cheng J.Q.
      • Ruggeri B.
      • Klein W.M.
      • Sonoda G.
      • Altomare D.A.
      • Watson D.K.
      • Testa J.R.
      ,
      • Thompson F.H.
      • Nelson M.A.
      • Trent J.M.
      • Guan X.Y.
      • Liu Y.
      • Yang J.M.
      • Emerson J.
      • Adair L.
      • Wymer J.
      • Balfour C.
      • Massey K.
      • Weinstein R.
      • Alberts D.S.
      • Taetle R.
      ,
      • Staal S.P.
      ). Expression of downstream antiapoptotic factors, such as inhibitors of apoptosis (IAPs), is altered in human tumors; ML-IAP is an example for a member of the family of IAPs, which is preferentially expressed in human melanoma (
      • Vunic D.
      • Stennicke H.R.
      • Pisabarro M.T.
      • Salvesen G.S.
      • Dixit V.M.
      ). Expression of survivin and Bcl2 are also modified in the course of tumor development (
      • Grossman D.
      • Kim P.J.
      • Schechner J.S.
      • Altieri D.C.
      ,
      • Jansen B.
      • Wacheck V.
      • Heere-Ress E.
      • Schlagbauer-Wadl H.
      • Hoeller C.
      • Lucas T.
      • Hoermann M.
      • Hollenstein U.
      • Wolff K.
      • Pehamberger H.
      ). Apaf1, a cell death effector that acts with cytochrome c and caspase 9 to mediate stress-dependent apoptosis, is inactivated in 40% of malignant melanomas, thereby providing another example for a modification of an apoptotic cascade that acquires malignant melanoma with chemoresistance (
      • Soengas M.S.
      • Capodieci P.
      • Polsky D.
      • Mora J.
      • Esteller M.
      • Opitz-Araya X.
      • McCombie R.
      • Herman J.G.
      • Gerald W.L.
      • Lazebnik Y.A.
      • Cordon-Cardo C.
      • Lowe S.W.
      ).
      Changes in the proapoptotic signaling were also documented during tumor development and progression. Each of the six major cell death pathways, TNFR1, FasR, TRAIL-R1, TRAIL-R2, DR3, and DR6, were reported to undergo certain changes in the course of tumor progression (
      • Daniel P.T.
      • Wieder T.
      • Sturm I.
      • Schulze-Osthoff K.
      ). Fas Receptor (Fas, CD95/Apo-1) signaling appears to serve as a primary death cascade in human melanomas (
      • Mouawad R.
      • Khayat D.
      • Soubrane C.
      ). Upon interacting with Fas Ligand, FasR forms a complex with the Fas-associated death domain protein, which directly binds and activates caspase 8, resulting in the induction of apoptosis (
      • Nagata S.
      ,
      • Krammer P.
      ). FasR ligation also induces a rapid and transient tyrosine phosphorylation, which coincides with PI3K/AKT activation and is required for Fas mediated apoptosis (
      • Gulbins E.
      • Hermisson M.
      • Brenner B.
      • Grassme H.U.
      • Linderkamp O.
      • Dichgans J.
      • Weller M.
      • Lang F.
      ). Tyrosine phosphorylation was found essential for FasR-mediated apoptosis (
      • Hehner S.P.
      • Hofmann T.G.
      • Ratter F.
      • Droge W.
      • Schmitz M.L.
      ,
      • Simon H.U.
      • Yousefi S.
      • Dibbert B.
      • Hebestreit H.
      • Weber M.
      • Branch D.R.
      • Blaser K.
      • Levi-Schaffer F.
      • Anderson G.P.
      ).
      Several studies point to the relationship between pro- and antiapoptotic cell death signaling (i.e. PI3K-AKT and Fas). PI3K is among signaling pathways implicated in the regulation of FasR responses (
      • Holmstrom T.H.
      • Eriksson J.E.
      ). UV irradiation-mediated activation of PI3K signaling via epidermal growth factor receptors in human skin provides an example for physiological stimuli that utilize the PI3K/AKT cascade to affect the degree of damage-induced cell death (
      • Wan Y.S.
      • Wang Z.Q.
      • Shao Y.
      • Voorhees J.J.
      • Fisher G.J.
      ). UV, via generation of hydrogen peroxide, induces AKT phosphorylation (
      • Huang C., Li, J.
      • Ding M.
      • Leonard S.S.
      • Wang L.
      • Castranova V.
      • Vallyathan V.
      • Shi X.
      ). Overall increase in basal levels of PI3K/AKT activity coincided with tumor cell ability to exhibit resistance to UV and ionizing radiation (
      • Cataldi A.
      • Zauli G., Di
      • Pietro R.
      • Castorina S.
      • Rana R.
      ,
      • Krasilnikov M.
      • Adler V.
      • Fuchs S.Y.
      • Dong Z.
      • Haimovitz-Friedman A.
      • Herlyn M.
      • Ronai Z.
      ).
      Impaired Fas signaling is frequently observed during tumor progression and has been attributed in most cases to down-regulation of Fas expression (
      • Owen-Schaub L.
      ,
      • Decaudin D.
      • Beurdeley-Thomas A.
      • Nemati F.
      • Miccoli L.
      • Pouillart P.
      • Bourgeois Y.
      • Goncalves R.B.
      • Rouillard D.
      • Poupon M.F.
      ). Loss of Fas function has been implicated in increased resistance of tumors to apoptosis induced by chemical and physical stimuli and is thought to represent one of the mechanisms that enable such tumors to escape immune surveillance as well as to acquire the metastatic phenotype (
      • Hug H.
      ,
      • Owen-Schaub L.B.
      • van Golen K.
      • Hill L.
      • Price J.E.
      ,
      • Shin M.
      • Park W.
      • Kim S.
      • Kim H.
      • Kang S.
      • Song K.
      • Park J.
      • Dong S., Pi, J.
      • Oh R.
      ,
      • Koomagi R.
      • Volm M.
      ,
      • Mottolese M.
      • Buglioni S.
      • Bracalenti C.
      • Cardarelli M.A.
      • Ciabocco L.
      • Giannarelli D.
      • Botti C.
      • Natali P.G.
      • Concetti A.
      • Venanzi F.M.
      ,
      • Mullauer L.
      • Gruber P.
      • Sebinger D.
      • Buch J.
      • Wohlfart S.
      • Chott A.
      ). Whereas regulation offas transcription is mediated by both constitutive and inducible regulatory pathways, down-regulation of Fas expression in advanced human melanoma-derived cell lines was found to be mediated by cooperation of STAT3 with c-Jun, a process that can be most clearly identified in advanced tumors. Interference with such cooperation through the use of dominant negative forms of either STAT3 or c-Jun efficiently restored transcription and surface expression of FasR (
      • Ivanov V.N.
      • Bhoumik a.
      • Krasilnikov M.
      • Raz R.
      • Owen-Schaub L.B.
      • Levy D.
      • Horvath C.M.
      • Ronai Z.
      ). In our search for signaling pathways that could control such cooperation, we discovered the role of PI3K/AKT, which we report here.
      Concomitant with elevated PI3K-AKT activities in human tumors is increased survival, motility, and metastatic capacity (
      • Zhou H., Li, X.M.
      • Meinkoth J.
      • Pittman R.N.
      ,
      • Sheng H.
      • Shao J.
      • DuBois R.N.
      ,
      • Madrid L.V.
      • Wang C.Y.
      • Guttridge D.C.
      • Schottelius A.J.
      • Baldwin A.S., Jr.
      • Mayo M.W.
      ,
      • Miwa W.
      • Yasuda J.
      • Murakami Y.
      • Yashima K.
      • Sugano K.
      • Sekine T.
      • Kono A.
      • Egawa S.
      • Yamaguchi K.
      • Hayashizaki Y.
      • Sekiya T.
      ,
      • Ruggeri B.A.
      • Huang L.
      • Wood M.
      • Cheng J.Q.
      • Testa J.R.
      ,
      • Nakatani K.
      • Thompson D.A.
      • Barthel A.
      • Sakaue H.
      • Liu W.
      • Weigel R.J.
      • Roth R.A.
      ). Here we demonstrate that AKT is also capable of altering the activities of transcription factors that play important roles in regulating FasR expression. In demonstrating that AKT signaling plays an important role in controlling FasR expression, we document a novel layer of regulation that links the PI3K-AKT pathway with Fas-mediated death in tumor cells, which represents an important mechanism in the control of tumor development and progression.

      DISCUSSION

      Advanced tumors often exhibit reduced or complete loss of FasR expression, which coincides with greater resistance to apoptosis and is believed to enable tumors to escape immune surveillance (
      • Ungefroren H.
      • Voss M.
      • Bernstorff W.V.
      • Schmid A.
      • Kremer B.
      • Kalthoff H.
      ). We previously identified cooperation between STAT3 and c-Jun as the mechanism that underlies the loss of FasR transcription and concomitant cell surface expression. Since both STAT3 and c-Jun are ubiquitously expressed, the present study was aimed at understanding the mechanism that enables their cooperation, since it appears to take place in advanced tumors. Our studies, which relied on analysis of intermediate stage melanoma tumor-derived cell lines, led us to identify, albeit surprisingly, the role of the PI3K/AKT signaling pathway in the regulation of FasR expression. Indeed, tumor-derived cell lines including LU1205, WM9, and WM793 exhibit relative high levels of FasR and noticeable levels of AKT in its active form. Constitutively active forms of either AKT or p110, the catalytic subunit of PI3K, further increased FasR transcription, monitored via FasR-luciferase activity, with a concomitant increase in FasR expression on the cell surface. Inhibition of the PI3K/AKT pathway via either the dominant negative form of p85, the regulatory subunit of PI3K, or PTEN or via the pharmacological inhibitor LY294002 resulted in efficient suppression of FasR transcription and cell surface expression. These observations clearly demonstrate the role of PI3K and AKT in the positive regulation of FasR transcription. Further support for the role of PI3K and AKT in FasR-mediated apoptosis was shown in several cell systems, although the prevailing mechanism was not clearly defined (
      • Hausler P.
      • Papoff G.
      • Eramo A.
      • Reif K.
      • Cantrell D.A.
      • Ruberti G.
      ).
      Further analysis revealed that AKT up-regulates FasR transcription via down-regulation of STAT3 and c-Jun transcriptional activities. First, the level of serine-phosphorylated forms of Jun and STAT3 decreases upon AKT treatment, which coincided with decreased binding of the phosphorylated Jun and STAT3 to the FasR promoter sequences in vitro. Noticeable changes in tyrosine phosphorylation of STAT3 were only found with the exogenously expressed form of STAT3β, suggesting that AKT may increase the activity of this dominant negative form of STAT3, thereby providing an additional mechanism to explain inhibition of FasR transcription. Next, AP-1 and STAT-dependent transcription of the luciferase reporter gene decreased in this case, further pointing to the decrease in transcriptional activities of the two proteins. Changes in FasR promoter activities were primarily mediated by c-Jun and STAT3, since promoter sequences that were mutated in the AP1 site or from which AP1 and GAS elements had been deleted no longer responded to AKT signaling. The importance of c-Jun for AKT-mediated derepression of FasR expression is best illustrated in c-Jun null fibroblasts, where the constitutively high level of FasR expression was no longer affected by AKT. Further, a fully functional form of c-Jun was required to decrease FasR expression, since the phospho-mutant form failed to mediate these changes.
      Taken together, these observations establish the important role of c-Jun and STAT3 phosphorylation by their upstream kinases for the regulation of FasR expression. JNK, which is the primary candidate for Jun phosphorylation, is down-regulated upon AKT expression. Along those lines, AKT was previously reported to elicit down-regulation of the stress kinase p38, highlighting another pathway that is subject to negative regulation by AKT (
      • Gratton J.P.
      • Morales-Ruiz M.
      • Kureishi Y.
      • Fulton D.
      • Walsh K.
      • Sessa W.C.
      ). The link between AKT and JNK led us to explore the role of Rac1, which is the target for AKT phosphorylation on Ser71, resulting in Rac1 suppression (
      • Kwon T.
      • Kwon D.Y.
      • Chun J.
      • Kim J.H.
      • Kang S.S.
      ). Indeed, inhibition of Rac1 activities was as efficient in increasing FasR expression as constitutively active forms of AKT or p110. These observations led us to propose that AKT may mediate its effects on JNK via suppression of Rac1 activities. The ability of Rac1 to elicit activation of STAT3 and c-Jun via SEK/MKK4 was demonstrated (
      • Hall A.
      ,
      • Faruqi T.R.
      • Gomez D.
      • Bustelo X.R.
      • Bar-Sagi D.
      • Reich N.C.
      ,
      • Schuringa J.J.
      • Decker L.V.
      • Vellenga E.
      • Kruijer W.
      ,
      • Coso O.A.
      • Chiariello M., Yu, J.C.
      • Teramoto H.
      • Crespo P., Xu, N.
      • Miki T.
      • Gutkind J.S.
      ,
      • Minden A.
      • Lin A.
      • Claret F.X.
      • Abo A.
      • Karin M.
      ,
      • Teramoto H.
      • Coso O.A.
      • Miyata H.
      • Igishi T.
      • Miki T.
      • Gutkind J.S.
      ,
      • Kaminuma O.
      • Deckert M.
      • Elly C.
      • Liu Y.C.
      • Altman A.
      ), suggesting that suppression of Rac1 activities is expected to reduce STAT3 and c-Jun transcriptional activities.
      Although Rac1 was shown capable of altering both Tyr and Ser phosphorylation of STAT3 (
      • Faruqi T.R.
      • Gomez D.
      • Bustelo X.R.
      • Bar-Sagi D.
      • Reich N.C.
      ), our experiments did not reveal that in the melanomas examined here there is an effect on basal levels of Tyr phosphorylation of STAT3; rather, there is a notable effect on the Ser phosphorylation of both STAT3 and c-Jun. This suggests that the primary mechanism by which AKT is capable of altering FasR transcription could be attributed to the serine phosphorylation of c-Jun and STAT3, both are required for their transcriptional activities. The latter is also supported by the notion that an inverse relationship between AKT and STAT3 phosphorylation was found in human cancer cells (
      • Page C.
      • Huang M.
      • Jin X.
      • Cho K.
      • Lilja J.
      • Reynolds R.K.
      • Lin J.
      ). Our findings are also in line with former studies, which demonstrated that the effect of PI3K may not be limited to Tyr phosphorylation of STAT3, since Ser phosphorylation (of STAT1) was also reported (
      • Nguyen H.
      • Ramana C.V.
      • Bayes J.
      • Stark G.R.
      ). Furthermore, Rac1-dependent Ser phosphorylation of STAT3 was also reported (
      • Schuringa J.J.
      • Decker L.V.
      • Vellenga E.
      • Kruijer W.
      ,
      • Schuringa J.J.
      • Jonk L.J.
      • Dokter W.H.
      • Vellenga E.
      • Kruijer W.
      ). Finally, AKT does affect tyrosine phosphorylation of exogenously expressed STAT3β, thereby providing some additional mechanistic insight to the relief of FasR expression in these cells. The effect on STAT3β but not endogenous STAT3 Tyr phosphorylation could be attributed to the amount expressed, sensitivity of detection, or other possible causes, which will be the subject for further studies. It is important to emphasize that impaired STAT3 transcriptional activity may not always depend on the status of its Tyr phosphorylation (
      • Masashi Fukuzawa M.
      • Araki T.
      • Adrian I.
      • Williams J.G.
      ).
      The emerging model based on our studies suggests that PI3K/AKT signaling utilizes Rac1 to down-regulate JNK and consequently c-Jun as well as STAT3 transcriptional activities, which otherwise cooperate to mediate suppression of FasR transcription. As a result of AKT signaling and subsequent Rac1 suppression, the cooperation between STAT3 and c-Jun is impaired, thereby resulting in relief of FasR transcription and increased FasR cell surface expression. According to this model, the increase in AKT signaling, which takes place in response to cytokines, UV (
      • Wan Y.S.
      • Wang Z.Q.
      • Shao Y.
      • Voorhees J.J.
      • Fisher G.J.
      ), or altered PI3K signaling, will result in elevated FasR expression; it is also expected that elevated FasR will result in a concomitant sensitization of cells to FasL-mediated death. This was indeed found to be the case in some (K1735 and SW1) but not in other melanomas (WM9, LU1205, and FEMX). The differences among the two sets of melanomas studied here points to the possible existence of a switch in AKT effector(s) or interference by other signaling cascades, which alter the susceptibility to FasR-FasL-mediated apoptosis. Interference of AKT-mediated increase of FasR is expected to take place during tumor development and progression by any of the multiple signaling cascades that are linked to or interfere with AKT signaling. For example, oncogenic Ras was shown capable of inhibiting FasR expression via its effect on AKT/PI3K pathways (
      • Peli J.
      • Schroter M.
      • Rudaz C.
      • Hahne M.
      • Meyer C.
      • Reichmann E.
      • Tschopp J.
      ,
      • Fenton R.G.
      • Hixon J.A.
      • Wright P.W.
      • Brooks A.D.
      • Sayers T.J.
      ). Similarly, growth factors that are known activators of PI3K/AKT signaling, including hepatocyte growth factor and epidermal growth factor, were shown to be capable of down-regulating FasR-mediated death (
      • Suzuki A.
      • Hayashida M.
      • Kawano H.
      • Sugimoto K.
      • Nakano T.
      • Shiraki K.
      ,
      • Xiao G.H.
      • Jeffers M.
      • Bellacosa A.
      • Mitsuuchi Y.
      • Vande Woude G.F.
      • Testa J.R.
      ,
      • Gibson S., Tu, S.
      • Oyer R.
      • Anderson S.M.
      • Johnson G.L.
      ). Further, a key component in the regulation of PI3K-AKT is PTEN, which is either mutated or down-regulated in many tumor types including melanomas (
      • Di Christofano A.
      • Kotsi P.
      • Peng Y.F.
      • Cordon-Cardo C.
      • Elkon K.B.
      • Pamdolfi P.P.
      ,
      • Poetsch M.
      • Dittberner T.
      • Woenckhaus C.
      ,
      • Hyun T.
      • Yam A.
      • Pece S.
      • Xie X.
      • Zhang J.
      • Miki T.
      • Gutkind J.S.
      • Li W.
      ,
      • Zhou X.P.
      • Gimm O.
      • Hampel H.
      • Niemann T.
      • Walker M.J.
      • Eng C.
      ). The relationship between PTEN and Fas-dependent apoptosis is also illustrated by the finding that PTEN+/− mice exhibit impaired Fas responses, which could be restored upon the use of PI3K inhibitors (
      • Di Christofano A.
      • Kotsi P.
      • Peng Y.F.
      • Cordon-Cardo C.
      • Elkon K.B.
      • Pamdolfi P.P.
      ).
      Another plausible target for changes that are expected to override the AKT effect on FasR expression is c-kit; mutations of c-kit have been identified in a variety of malignancies including melanomas and were recently shown to result in constitutive activation of STAT3 (
      • Ning A.Q., Li, J.
      • McGuinness M.
      • Arceci R.J.
      ). Thus, tumors that harbor mutant c-kit are also expected to exhibit down-regulation of FasR via activated STAT3, which could override the effect of AKT signaling. Finally, one must also consider other regulatory events, which take place between Fas transcription and cell surface expression (i.e. FasR trafficking). Among those, Par4, which is altered in human prostate tumors, was recently found to affect FasR trafficking (
      • Chakraborty M.
      • Qiu S.G.
      • Vasudevan K.M.
      • Rangnekar V.M.
      ).
      Although we expect that K1735 and SW1, which exhibit sensitization to FasL-mediated cell death following AKT-dependent increase in FasR expression, represent a set of tumors, the protection by AKT seen in the other melanomas raises a question as to the possible physiological significance of elevated FasR expression. First, it is likely that activation of AKT by cytokines and DNA damage, as reported for epidermal growth factor receptor and UV irradiation, serves to increase FasR as a part of cell ability to undergo apoptosis, provided that other AKT targets that serve antiapoptotic functions are suppressed. Under constitutive expression of FasR, the scenarios that could be envisioned include the possibility that elevated FasR in parallel with protection from FasR-mediated death may serve to lure the immune system. Along these lines, constitutive expression of FasL in multiple myeloma cells was proposed as a potential mechanism of tumor-induced suppression of immune surveillance (
      • Villunger A.
      • Egle A.
      • Marschitz I.
      • Kos M.
      • Bock G.
      • Ludwig H.
      • Geley S.
      • Kofler R.
      • Greil R.
      ). Alternatively, it is possible that these changes serve other functions required for the aggressively progressing tumors, such as improved vascularization and consequent angiogenic potential. Indeed, elevated vascularization has been reported in tumors that overexpress AKT (
      • Jiang B.H.
      • Zheng J.Z.
      • Aoki M.
      • Vogt P.K.
      ), and AKT-expressing SW1 tumors in vivo exhibit a marked increase in vascularization (data not shown). In contrast, inhibition of STAT3 or c-Jun, which abolish cooperation among the two protooncogenes to silence FasR, results in restored FasR expression and decreases tumor growth in vivo, due to an increased degree of apoptosis (not shown).
      Elevated levels of Rac1 or its isoform expression, which is often reported to take place in human tumors, including human melanomas, is expected to also alter the activities of Rac1 effectors, including JNK and STAT3 kinases, resulting in increased cooperation between STAT3 and c-Jun that inhibits the activities of the FasR promoter. Inasmuch, Rac1 may also serve as a focal point for the signaling cascade reported here. Further, Rac1 has been implicated as an important target of diverse signaling cascades including Rho, Ras, and c-kit, each of which has been shown to undergo modifications during the human tumor development and progression, including melanomas (
      • Wen S.
      • Stolarov J.
      • Myers M.P., Su, J.D.
      • Wigler M.H.
      • Tonks N.K.
      • Durden D.L.
      ,
      • Joneson T.
      • Bar-Sagi D.
      ,
      • Yamaguchi Y.
      • Katah H.
      • Mori K.
      • Negishi M.
      ,
      • Timokhina I.
      • Kissel H.
      • Stella G.
      • Besmer P.
      ). Interestingly, the pathway identified in the present study may be part of a feedback regulatory loop, since STAT3 was reported capable of activating the PI3K cascade (
      • Pfeffer L.M.
      • Mullersmann J.E.
      • Pfeffer S.R.
      • Murti A.
      • Shi W.
      • Yang C.H.
      ). Similarly, Rac2 was shown capable of stimulating AKT in primary mast cells (
      • Yang F.C.
      • Kapur A.J.
      • Tao W.
      • Kim C.
      • Borneo J.
      • Breese R.
      • Marshall M.
      • Dinauer M.C.
      • Williams D.A.
      ), and the Rac1 ability to protect epithelial cells against ankiosis was shown to depend on its activation of PI3K/AKT (
      • Coniglio S.J.
      • Jou T.S.
      • Symons M.
      ). The existence of a positive feedback loop suggests that via STAT3 or other Rac effectors AKT activation may result in either inhibition of Rac1 (as one would expect to take place upon its phosphorylation on Ser71) or constitutive activation, under which AKT-mediated suppression of STAT3 and c-Jun may no longer be seen. It is expected that part(s) of this feedback loop mechanism will be impaired in advanced tumors.

      Acknowledgments

      We thank D. Levy, R. Raz, L. Owen-Schwab, M. Herlyn, and O. Fodstad for melanoma- and STAT3-related cell cultures and Drs. J. Darnell Jr., C. Horvath, L. Williams, P. Tsichlis, R. Jove, and A. Chan for PI3K-, AKT-, and STAT3-related constructs used in this study. We also thank members of the Ronai laboratory for discussions.

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