title: TDAG51 is an ERK signaling target that opposes ERK-mediated HME16C mammary epithelial cell transformation.
abstract: BACKGROUND
  Signaling downstream of Ras is mediated by three major pathways, Raf/ERK, phosphatidylinositol 3 kinase (PI3K), and Ral guanine nucleotide exchange factor (RalGEF). Ras signal transduction pathways play an important role in breast cancer progression, as evidenced by the frequent over-expression of the Ras-activating epidermal growth factor receptors EGFR and ErbB2. Here we investigated which signal transduction pathways downstream of Ras contribute to EGFR-dependent transformation of telomerase-immortalized mammary epithelial cells HME16C. Furthermore, we examined whether a highly transcriptionally regulated ERK pathway target, PHLDA1 (TDAG51), suggested to be a tumor suppressor in breast cancer and melanoma, might modulate the transformation process.
  
  
  METHODS
  Cellular transformation of human mammary epithelial cells by downstream Ras signal transduction pathways was examined using anchorage-independent growth assays in the presence and absence of EGFR inhibition. TDAG51 protein expression was down-regulated by interfering small hairpin RNA (shRNA), and the effects on cell proliferation and death were examined in Ras pathway-transformed breast epithelial cells.
  
  
  RESULTS
  Activation of both the ERK and PI3K signaling pathways was sufficient to induce cellular transformation, which was accompanied by up-regulation of EGFR ligands, suggesting autocrine EGFR stimulation during the transformation process. Only activation of the ERK pathway was sufficient to transform cells in the presence of EGFR inhibition and was sufficient for tumorigenesis in xenografts. Up-regulation of the PHLDA1 gene product, TDAG51, was found to correlate with persistent ERK activation and anchorage-independent growth in the absence or presence of EGFR inhibition. Knockdown of this putative breast cancer tumor-suppressor gene resulted in increased ERK pathway activation and enhanced matrix-detached cellular proliferation of Ras/Raf transformed cells.
  
  
  CONCLUSIONS
  Our results suggest that multiple Ras signal transduction pathways contribute to mammary epithelial cell transformation, but that the ERK signaling pathway may be a crucial component downstream of EGFR activation during tumorigenesis. Furthermore, persistent activation of ERK signaling up-regulates TDAG51. This event serves as a negative regulator of both Erk activation as well as matrix-detached cellular proliferation and suggests that TDAG51 opposes ERK-mediated transformation in breast epithelial cells.
authors.affiliation: Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Room 1066, Bethesda, MD 20892, USA. michaeloberst@gmail.com
authors.forename: Michael D
authors.initials: MD
authors.lastname: Oberst
authors.forename: Stacey J
authors.initials: SJ
authors.lastname: Beberman
authors.forename: Liu
authors.initials: L
authors.lastname: Zhao
authors.forename: Juan Juan
authors.initials: JJ
authors.lastname: Yin
authors.forename: Yvona
authors.initials: Y
authors.lastname: Ward
authors.forename: Kathleen
authors.initials: K
authors.lastname: Kelly
mesh_terms: D023102:Anoikis; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D004318:Doxycycline / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors; D005260:Female; D006801:Humans; D042361:Mammary Glands, Human / Q000378:metabolism / Q000473:pathology*; D046228:Microarray Analysis; D009154:Mutation; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: C491923:PHLDA1 protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors; D048049:Extracellular Signal-Regulated MAP Kinases; D015689:Oncogene Protein p21(ras); D004318:Doxycycline