title: Inhibition of G9a induces DUSP4-dependent autophagic cell death in head and neck squamous cell carcinoma.
abstract: BACKGROUND
  Head and neck squamous cell carcinoma (HNSCC) is a common cancer worldwide. Emerging evidence indicates that alteration of epigenetics might be a key event in HNSCC progression. Abnormal expression of histone methyltransferase G9a, which contributes to transcriptional repression of tumor suppressors, has been implicated in promoting cancerous malignancies. However, its role in HNSCC has not been previously characterized. In this study, we elucidate the function of G9a and its downstream mechanism in HNSCC.
  
  
  METHODS
  We investigated the clinical relevance of G9a in HNSCC using immunohistochemistry (IHC) staining. In vitro cell proliferation and tumorigenesis ability of G9a-manipulated HNSCC cells were examined with MTT assays, clonogenic assays, and soft agar assays. We examined different routes of cell death in HNSCC cells induced by G9a-depletion or enzymatic inhibition by immunoblot, flow cytometry, fluorescent and transmission electron microscopy analysis. Specific targets of G9a were identified by affymetrix microarray and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Lastly, functions of G9a in vivo were confirmed with a xenograft tumor model.
  
  
  RESULTS
  G9a expression is positively correlated to proliferation marker Ki-67 and to poor prognosis in HNSCC patients. Genetic or pharmacological inhibition of G9a reduced cell proliferation without inducing necrosis or apoptosis. Instead, autophagic cell death was the major consequence, and our investigation of mechanisms suggested it is mediated via the dual specificity phosphatase-4 (DUSP4) dependent ERK inactivation pathway. An orthotopic tumor model further confirmed the growth inhibiting effect and induction of autophagy that followed suppression of G9a.
  
  
  CONCLUSIONS
  In this study, we provide evidence that G9a confers the survival advantage of HNSCC. Genetic or pharmacological inhibition of G9a induces autophagic cell death; this finding provides a basis for new therapeutic targets for treating HNSCC.
authors.forename: Kai-Chun
authors.initials: KC
authors.lastname: Li
authors.forename: Kuo-Tai
authors.initials: KT
authors.lastname: Hua
authors.forename: Yi-Shen
authors.initials: YS
authors.lastname: Lin
authors.forename: Chia-Yi
authors.initials: CY
authors.lastname: Su
authors.forename: Jenq-Yuh
authors.initials: JY
authors.lastname: Ko
authors.forename: Michael
authors.initials: M
authors.lastname: Hsiao
authors.forename: Min-Liang
authors.initials: ML
authors.lastname: Kuo
authors.affiliation: Department of Otolaryngology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan. tanct5222@ntu.edu.tw.
authors.forename: Ching-Ting
authors.initials: CT
authors.lastname: Tan
mesh_terms: D000818:Animals; D001343:Autophagy / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D054637:Dual-Specificity Phosphatases / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000473:pathology; D006649:Histocompatibility Antigens / Q000378:metabolism*; D011495:Histone-Lysine N-Methyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000235:genetics* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
chemical_list: D006649:Histocompatibility Antigens; C445405:EHMT2 protein, human; D011495:Histone-Lysine N-Methyltransferase; D054639:Mitogen-Activated Protein Kinase Phosphatases; C490583:DUSP4 protein, human; D054637:Dual-Specificity Phosphatases