title: The antiproliferative response of indole-3-carbinol in human melanoma cells is triggered by an interaction with NEDD4-1 and disruption of wild-type PTEN degradation.
abstract: Human melanoma cells displaying distinct PTEN genotypes were used to assess the cellular role of this important tumor-suppressor protein in the antiproliferative response induced by the chemopreventative agent indole-3-carbinol (I3C), a natural indolecarbinol compound derived from the breakdown of glucobrassicin produced in cruciferous vegetables such as broccoli and Brussels sprouts. I3C induced a G1-phase cell-cycle arrest and apoptosis by stabilization of PTEN in human melanoma cells that express wild-type PTEN, but not in cells with mutant or null PTEN genotypes. Importantly, normal human epidermal melanocytes were unaffected by I3C treatment. In wild-type PTEN-expressing melanoma xenografts, formed in athymic mice, I3C inhibited the in vivo tumor growth rate and increased PTEN protein levels in the residual tumors. Mechanistically, I3C disrupted the ubiquitination of PTEN by NEDD4-1 (NEDD4), which prevented the proteasome-mediated degradation of PTEN without altering its transcript levels. RNAi-mediated knockdown of PTEN prevented the I3C-induced apoptotic response, whereas knockdown of NEDD4-1 mimicked the I3C apoptotic response, stabilized PTEN protein levels, and downregulated phosphorylated AKT-1 levels. Co-knockdown of PTEN and NEDD4-1 revealed that I3C-regulated apoptotic signaling through NEDD4-1 requires the presence of the wild-type PTEN protein. Finally, in silico structural modeling, in combination with isothermal titration calorimetry analysis, demonstrated that I3C directly interacts with purified NEDD4-1 protein.
  
  
  CONCLUSIONS
  This study identifies NEDD4-1 as a new I3C target protein, and that the I3C disruption of NEDD4-1 ubiquitination activity triggers the stabilization of the wild-type PTEN tumor suppressor to induce an antiproliferative response in melanoma. Mol Cancer Res; 12(11); 1621-34. ©2014 AACR.
authors.affiliation: Department of Molecular and Cell Biology and The Cancer Research Laboratory, Univ. of California at Berkeley, Berkeley, CA 94720-3200.
authors.forename: Ida
authors.initials: I
authors.lastname: Aronchik
authors.affiliation: Department of Molecular and Cell Biology and The Cancer Research Laboratory, Univ. of California at Berkeley, Berkeley, CA 94720-3200.
authors.forename: Aishwarya
authors.initials: A
authors.lastname: Kundu
authors.affiliation: Department of Molecular and Cell Biology and The Cancer Research Laboratory, Univ. of California at Berkeley, Berkeley, CA 94720-3200.
authors.forename: Jeanne G
authors.initials: JG
authors.lastname: Quirit
authors.affiliation: Department of Molecular and Cell Biology and The Cancer Research Laboratory, Univ. of California at Berkeley, Berkeley, CA 94720-3200.
authors.forename: Gary L
authors.initials: GL
authors.lastname: Firestone
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002151:Calorimetry; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003198:Computer Simulation; D015536:Down-Regulation / Q000187:drug effects; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007211:Indoles / Q000494:pharmacology*; D008545:Melanoma / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D008819:Mice, Nude; D008958:Models, Molecular; D000075702:Nedd4 Ubiquitin Protein Ligases; D051059:PTEN Phosphohydrolase / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D017433:Protein Structure, Secondary; D059748:Proteolysis / Q000187:drug effects*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D007211:Indoles; C016517:indole-3-carbinol; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D046988:Proteasome Endopeptidase Complex