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4-Hydroxyphenyl Retinamide Is a Highly Selective Activator of Retinoid Receptors*

Open AccessPublished:September 13, 1996DOI:https://doi.org/10.1074/jbc.271.37.22441
      Retinoids have shown promise as anti-cancer and cancer preventative agents. All-trans-N-(4-hydroxyphenyl)retinamide (4HPR) belongs to a new group of retinoids that not only inhibit the proliferation of cancer cells but also can induce apoptosis in certain cancer cells. Because of its increased efficacy against cancer cells and its low toxicity it has been entered into a number of clinical trials. However, its mechanism of action is not known, and it had been assumed that it is not a true retinoid. Here we analyze its ability to function as an activator of nuclear retinoid receptors (RARs and RXRs). We observe that, in transactivation assays, 4HPR is a potent transactivator with RARγ and a moderate activator with RARβ but is not an activator with RARα and RXRα. Furthermore, RARγ-selective transactivation by 4HPR is enhanced on some response elements and reduced on others when compared to natural retinoids. In contrast to transactivation, 4HPR in transrepression assays functions mostly with RARα, RARβ, and RXRα. Optimal receptor activation is seen at 4HPR concentrations at which it is a potent growth inhibitor and inducer of apoptosis. We conclude that 4HPR is a highly selective activator of retinoid receptors. We propose that this selective activation of the nuclear receptors is likely to be the basis for its specific biological activities and its favorable pharmaceutical properties.

      INTRODUCTION

      Retinoic acid (RA)
      The abbreviations used are: RA
      retinoic acid
      RAR
      retinoic acid receptor
      RXR
      retinoic acid X receptor
      RARE
      retinoic acid response element
      tRA
      all-trans-RA
      4HPR
      all-trans-N-(4-hydroxyphenyl)retinamide
      4MPR
      inert metabolite of 4HPR
      TPA
      12-O-tetradecanoyl-phorbol-13-acetate
      FCS
      fetal calf serum
      BrdU
      bromodeoxyuridine
      ELISA
      enzyme-linked immunosorbent assay
      DR
      direct repeat.
      and its synthetic analogs, the retinoids, exert diverse biological effects and control normal growth, fetal development, differentiation, morphogenesis, metabolism, and homeostasis (
      • Lotan R.
      ,
      ,
      • Sporn M.B.
      • Roberts A.B.
      • Goodman O.S.
      ). Clinically, RA and some synthetic retinoids are known to be effective against a number of skin diseases (
      • Schaefer H.
      • Reichert U.
      ) and have shown promise for the treatment and prevention of several cancers (
      • Smith M.A.
      • Parkinson D.R.
      • Cheson B.D.
      • Friedman M.A.
      ). Besides their beneficial effects retinoids also induce a number of undesirable side effects, limiting their clinical applications. In addition, the natural retinoids show only limited activity against most types of cancer cells. A major goal has been to develop retinoids with a higher therapeutic index and more targeted biological activities. Although the unraveling of the molecular mechanism (
      • Pfahl M.
      • Apfel R.
      • Bendik I.
      • Fanjul A.
      • Graupner G.
      • Lee M-O.
      • La-Vista N.
      • Lu X-P.
      • Piedrafita J.
      • Ortiz A.
      • Salbert G.
      • Zhang X-K.
      ,
      • Chambon P.
      ,
      • Mangelsdorf D.J.
      • Umesono K.
      • Evans R.M.
      ) has now made it possible to define retinoids that activate selectively certain receptors and/or pathways (
      • Lehmann J.M.
      • Dawson M.I.
      • Hobbs P.D.
      • Husmann M.
      • Pfahl M.
      ,
      • Graupner G.
      • Malle G.
      • Maignan J.
      • Lange G.
      • Prunieras M.
      • Pfahl M.
      ,
      • Lehmann J.M.
      • Jong L.
      • Fanjul A.N.
      • Cameron J.F.
      • Lu X-P.
      • Haefner P.
      • Dawson M.I.
      • Pfahl M.
      ,
      • Fanjul A.N.
      • Hobbs P.D.
      • Jong L.
      • Cameron J.F.
      • Harlev E.
      • Graupner G.
      • Lux X.-P.
      • Dawson M.I.
      • Pfahl M.
      ,
      • Chen J-Y.
      • Penco S.
      • Ostrowski J.
      • Balaguer P.
      • Pons M.
      • Starrett J.E.
      • Reczed P.
      • Chambon P.
      • Gronemeyer H.
      ,
      • Nagpal S.
      • Athanikar J.
      • Chandraratna R.A.S.
      ), such retinoids have not yet been clinically evaluated. On the other hand, other synthetic retinoids that were found a number of years ago have entered extensive clinical evaluations, although their mechanisms of action are not understood. This is in particular the case for all-trans-N-(4-hydroxyphenyl)retinamide (4HPR or fenretinide) which has been entered into a number of clinical trials and which shows promise for the treatment and prevention of breast cancer (
      • Kelloff G.J.
      • Boone C.W.
      ,
      • De Palo G.
      • Veronesi U.
      • Camerini T.
      • Formelli F.
      ,
      • Dowlatshahi K.
      • Mehta R.G.
      • Thomas C.F.
      • Dinger N.M.
      • Moon R.C.
      ,
      • Moon R.C.
      • Kellof G.J.
      • Detrisac C.J.
      • Steele V.E.
      • Thomas C.F.
      • Sigman C.C.
      ) as well as several other cancers (
      • Pollard M.
      • Luckert P.H.
      ,
      • Pienta J.K.
      • Nguyen N.M.
      • Lehr J.E.
      ). 4HPR was selected for clinical trials because of its low overall toxicity, and has been successfully used as a chemopreventive agent alone and in combination with tamoxifen in high risk breast cancer patients (
      • Modiano M.R.
      • Dalton W.S.
      • Lippman S.M.
      • Joffe L.
      • Booth A.R.
      ,
      • Rotmensz N.
      • DePalo G.
      • Formelli F.
      • Costa A.
      • Marubini E.
      ), against superficial bladder cancer (
      • Decensi A.
      • Bruno S.
      • Giaretti W.
      • Torrisi R.
      • Curotto A.
      ), and oral lichen planus and leukoplakia (
      • Chiesa F.
      • Tradini N.
      • Marazza M.
      • Rossi N.
      • Boracchi P.
      • Clerici M.
      ,
      • Tradini N.
      • Chiesa F.
      • Rossi N.
      • Grigolato R.
      • Formelli F.
      • Costa A.
      • DePalo G.
      ). 4HPR is also a highly effective growth inhibitor of neuroblastoma cell lines (
      • Ponzoni M.
      • Bocca P.
      • Chiesa V.
      • Decensi A.
      • Pistoia V.
      • Raffaghello L.
      • Rozzo C.
      • Montaldo P.G.
      ), malignant hemopoietic cell lines (
      • Delia D.
      • Aiello A.
      • Lombardi L.
      • Pellici P.G.
      • Grignani F.
      • Grignani F.
      • Formelli F.
      • Menard S.
      • Costa A.
      • Veronesi U.
      • Pierotti M.A.
      ), small cell lung cancer cell lines (

      Kalemkerian, G., Slusher, R., Ramalingam, S., Mabry, M., (1995) Proceedings of the American Association for Cancer Research (A.A.C.R.), Toronto, Canada, March 18-22, 1995 abstract).

      ), and a variety of breast cancer cell lines (
      • Bhatnagar D.L.
      • Abou-Issa H.
      • Curley Jr., R.W.
      • Koolemans-Beynen A.
      • Moeschberger M.L.
      • Webb T.E.
      ).
      The mechanism of action of 4HPR is unknown (
      • Sani B.P.
      ). Although 4HPR does not have a terminal carboxyl group, believed to be an essential feature for active retinoids, it has been shown to have certain biological activities that are typical for retinoids. 4HPR inhibits ODC activity induction (
      • Wille J.J.
      • Chopra D.
      • Shealy Y.F.
      ) and prostaglandin synthesis and enhances the immune system (
      • Dillehay D.L.
      • Jiang X.L.
      • Lamon E.W.
      ,
      • Villa M.L.
      • Ferrario E.
      • Trabattoni D.
      • Formelli F.
      • DePalo G.
      • Magni A.
      • Veronesi U.
      • Clerici E.
      ). On the other hand, 4HPR does not induce cell differentiation as RA does, but induces programmed cell death, i.e. apoptosis (
      • Ponzoni M.
      • Bocca P.
      • Chiesa V.
      • Decensi A.
      • Pistoia V.
      • Raffaghello L.
      • Rozzo C.
      • Montaldo P.G.
      ,
      • Delia D.
      • Aiello A.
      • Lombardi L.
      • Pellici P.G.
      • Grignani F.
      • Grignani F.
      • Formelli F.
      • Menard S.
      • Costa A.
      • Veronesi U.
      • Pierotti M.A.
      ,
      • Delia D.
      • Aiello A.
      • Formelli F.
      • Fontanella E.
      • Costa A.
      • Miyashita T.
      • Reed J.C.
      • Pierotti M.A.
      ,
      • Pellegrini R.
      • Mariotti A.
      • Tagliabue E.
      • Bressan R.
      • Bunone G.
      • Coradini D.
      • Della Valle G.
      • Formelli F.
      • Cleris L.
      • Radice P.
      • Pierotti M.A.
      • Colnaghi M.I.
      • Menard S.
      ), while RA usually does not effectively induce apoptosis. Thus, the mechanism of action of fenretinide on cancer cells appears to differ from that of RA. To gain insight into the molecular mechanisms that allow 4HPR to function as an apoptosis-inducing anti-cancer agent with reduced toxicity, we have analyzed its effect on the nuclear retinoid receptors.
      Retinoid signal transduction is mediated by a complex network of nuclear receptor interactions which has been extensively reviewed elsewhere (
      • Pfahl M.
      • Apfel R.
      • Bendik I.
      • Fanjul A.
      • Graupner G.
      • Lee M-O.
      • La-Vista N.
      • Lu X-P.
      • Piedrafita J.
      • Ortiz A.
      • Salbert G.
      • Zhang X-K.
      ,
      • Chambon P.
      ,
      • Mangelsdorf D.J.
      • Umesono K.
      • Evans R.M.
      ). Briefly, six retinoid receptors have been identified that fall into two classes, the RARs (α, β, and γ) and the RXRs (α, β, and γ). These receptors function as transcriptional activators that bind as RAR/RXR heterodimers or 9-cis-RA-induced RXR/RXR homodimers to specific RA response elements (RARE), usually found in the 5′-flanking region of responsive genes. The structure of the RARE can further modulate the heterodimer response (
      • La Vista-Picard N.
      • Hobbs P.D.
      • Pfahl M.
      • Dawson M.I.
      • Pfahl M.
      ). RARs and RXRs in addition can interact with the transcription factor AP-1 and inhibit its activity. This mechanism of transrepression is important since it allows retinoids to directly interfere with cell proliferation signals mediated by the transcription factor AP-1 (reviewed in Ref.
      • Pfahl M.
      ). Transactivation and transrepression by retinoids can be separated, such that certain synthetic retinoids function only as transrepressors, but not as transactivators (
      • Fanjul A.N.
      • Hobbs P.D.
      • Jong L.
      • Cameron J.F.
      • Harlev E.
      • Graupner G.
      • Lux X.-P.
      • Dawson M.I.
      • Pfahl M.
      ).
      We have investigated here the transactivation and transrepression properties of 4HPR with the different retinoid receptors. We observe that 4HPR shows anti-AP-1 activity mostly with RARα, RARβ, and RXRα but activates transcription only via RARγ and RARβ. In addition, transactivation activity in the presence of RARγ/RXR heterodimers varies on different RAREs. Optimal receptor activation is observed at concentrations at which 4HPR shows strong antiproliferative activity and induces apoptosis in a breast cancer cell line.
      We propose that the high therapeutic index of 4HPR may depend, at least in part, on the ability of 4HPR to activate only a selected portion of the broad retinoid receptor response.

      DISCUSSION

      4HPR belongs to a special class of retinoids that can induce apoptosis in cancer cells as shown here and as reported previously (
      • Ponzoni M.
      • Bocca P.
      • Chiesa V.
      • Decensi A.
      • Pistoia V.
      • Raffaghello L.
      • Rozzo C.
      • Montaldo P.G.
      ,
      • Delia D.
      • Aiello A.
      • Lombardi L.
      • Pellici P.G.
      • Grignani F.
      • Grignani F.
      • Formelli F.
      • Menard S.
      • Costa A.
      • Veronesi U.
      • Pierotti M.A.
      ,
      • Delia D.
      • Aiello A.
      • Formelli F.
      • Fontanella E.
      • Costa A.
      • Miyashita T.
      • Reed J.C.
      • Pierotti M.A.
      ). Advances made in recent years in the deciphering of the molecular retinoid signaling pathways has revealed a complex network of receptor dimerizations and interactions with RAREs as well as other proteins, in particular the transcription factor AP-1. The natural retinoids tRA, and even more so 9-cis-RA, can elicit very broad and complex changes in gene expression. This is consistent with their broad biological activities. Synthetic retinoids can have limited activities and act only on selected receptors and response pathways therefore can be expected to induce different biological responses that may also be limited to certain cell types.
      We show here that 4HPR can function as a selective retinoid that induces transcriptional activation preferentially by RARγ and to some extent by RARβ but not by RARα. Transactivation by RXR/RARγ heterodimers is further modulated by response elements. For instance, RARγ activation can be stronger than that seen in the presence of 9-cis-RA or tRA on certain response elements, while it is weaker on others. It has previously been shown that certain retinoids differ in their transactivation and transrepression activities (
      • Fanjul A.N.
      • Hobbs P.D.
      • Jong L.
      • Cameron J.F.
      • Harlev E.
      • Graupner G.
      • Lux X.-P.
      • Dawson M.I.
      • Pfahl M.
      ,
      • Chen J-Y.
      • Penco S.
      • Ostrowski J.
      • Balaguer P.
      • Pons M.
      • Starrett J.E.
      • Reczed P.
      • Chambon P.
      • Gronemeyer H.
      ,
      • Nagpal S.
      • Athanikar J.
      • Chandraratna R.A.S.
      ). Here we demonstrate that 4HPR is such a retinoid, since it shows anti-AP-1 activity (transrepression) mostly with RARα, RARβ, and RXRα. Thus, although 4HPR does not carry a terminal carboxyl group it activates certain nuclear retinoid receptors. Interaction of 4HPR with the receptors may be optimal under in vivo conditions when the receptor is already complexed with other receptors or proteins and/or DNA, consistent with our recent observation (
      • La Vista-Picard N.
      • Hobbs P.D.
      • Pfahl M.
      • Dawson M.I.
      • Pfahl M.
      ) that the ligand response of the retinoid receptors is modulated by the DNA elements they bind to. It is also conceivable that the 4HPR response of a retinoid receptor-DNA complex can be further influenced by the specific promoter contexts. Thus, our analysis leads us to propose that 4HPR is capable of inducing a very specific pattern of modulation of gene expression that can result in cellular responses very distinct from those observed in the presence of tRA and 9-cis-RA and which can include apoptosis.
      The strong antiproliferative/apoptosis inducing activity we observed here, occurs at 4HPR concentrations where this retinoid shows optimal receptor activation capacity. It is therefore most likely that the biological response seen on cell cultures, in animals, and in patients are, or at least include, specific retinoid receptor mediated responses, resulting from retinoid receptor mediated changes in gene expression. The antiproliferative/apoptosis inducing activity we observed here at 10-5 M and above is thus likely to be a specific receptor mediated response. At this point it cannot be excluded that the biological effects observed, including receptor activation, are caused by 4HPR metabolites other than 4MPR. Such unknown metabolites could be generated to various degrees in different cell types. Interestingly another synthetic retinoid that can induce apoptosis, CD437 (
      • Shao Z-M.
      • Dawson M.I.
      • Li X.S.
      • Rishi A.
      • Sheikh M.S.
      • Han Q-X.
      • Ordonez V.
      • Shroot B.
      • Fontana J.
      ),
      A. N. Fanjul and M. Pfahl, unpublished results.
      is also a selective activator of RARγ and, to a lesser extent, of RARβ (
      • Reichert U.
      • Bernardon J.M.
      • Charpentier B.
      • Nedoncelle P.
      • Martin B.
      • Bernard B.A.
      ). Those findings would suggest that selective activation of RARγ could lead to activation of the apoptosis pathway. Increased RARγ expression induced by 4HPR could further enhance this mechanism. However, overexpression of certain RARs alone has also been reported to induce apoptosis (
      • Liu Y.
      • Lee M.-O.
      • Wang H.-G.
      • Li Y.
      • Hashimoto Y.
      • Klaus M.
      • Reed J.C.
      • Zhang X.-K.
      ). In conclusion, 4HPR shows a selective retinoid receptor activation profile that could be the basis for its special biological activity.

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