title: Nemo-like kinase as a negative regulator of nuclear receptor Nurr1 gene transcription in prostate cancer.
abstract: BACKGROUND
  Nurr1, a member of the orphan receptor family, plays an important role in several types of cancer. Our previous work demonstrated that increased expression of Nurr1 plays a significant role in the initiation and progression of prostate cancer (PCa), though the mechanisms for regulation of Nurr1 expression remain unknown. In this study, we investigated the hypothesis that Nemo-like kinase (NLK) is a key regulator of Nurr1 expression in PCa.
  
  
  METHODS
  Immunohistochemistry and Western blot analysis were used to evaluate levels of NLK and Nurr1 in prostatic tissues and cell lines. The effects of overexpression or knockdown of Nurr1 were evaluated in PCa cells through use of PCR, Western blots and promoter reporter assays. The role of Nurr1 promoter cis element was studied by creation of two mutant Nurr1 promoter luciferase constructs, one with a mutated NF-κB binding site and one with a mutated CREB binding site. In addition, three specific inhibitors were used to investigate the roles of these proteins in transcriptional activation of Nurr1, including BAY 11-7082 (NF-κB inhibitor), KG-501 (CREB inhibitor) and ICG-001 (CREB binding protein, CBP, inhibitor). The function of CBP in NLK-mediated regulation of Nurr1 expression was investigated using immunofluorescence, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation assays (ChIPs).
  
  
  RESULTS
  NLK expression was inversely correlated with Nurr1 expression in prostate cancer tissues and cell lines. Overexpression of NLK suppressed Nurr1 promoter activity, leading to downregulation of Nurr1 expression. In contrast, knockdown of NLK demonstrated opposite results, leading to upregulation of Nurr1. When compared with the wild-type Nurr1 promoter, mutation of NF-κB- and CREB-binding sites of the Nurr1 promoter region significantly reduced the upregulation of Nurr1 induced by knockdown of NLK in LNCaP cells; treatment with inhibitors of CREB, CBP and NF-κB led to similar results. We also found that NLK directly interacts with CBP, that knockdown of NLK significantly increases the recruitment of CBP to both NF-κB- and CREB-binding sites, and that regulation of NLK on Nurr1 expression is abrogated by knockdown of CBP.
  
  
  CONCLUSIONS
  Our results suggest that NLK inhibits transcriptional activation of Nurr1 gene by impeding CBP's role as a co-activator of NF-κB and CREB in prostate cancer.
authors.affiliation: Sino-American Cancer Research Institute, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan Scientific Research Center, Guangdong Medical University, 1 Xincheng Road, Dongguan, 523808, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Obstetrics and Gynecology, Longgang District Central Hospital of Shenzhen, 1228 Longgang Road, Shenzhen, 518116, China.
authors.forename: Zhi-Hong
authors.initials: ZH
authors.lastname: Yang
authors.affiliation: Sino-American Cancer Research Institute, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan Scientific Research Center, Guangdong Medical University, 1 Xincheng Road, Dongguan, 523808, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Chen
authors.affiliation: Sino-American Cancer Research Institute, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan Scientific Research Center, Guangdong Medical University, 1 Xincheng Road, Dongguan, 523808, China.
authors.forename: Hua-Hui
authors.initials: HH
authors.lastname: Li
authors.affiliation: Sino-American Cancer Research Institute, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan Scientific Research Center, Guangdong Medical University, 1 Xincheng Road, Dongguan, 523808, China.
authors.forename: Li-Yong
authors.initials: LY
authors.lastname: Chen
authors.affiliation: Sino-American Cancer Research Institute, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan Scientific Research Center, Guangdong Medical University, 1 Xincheng Road, Dongguan, 523808, China.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Zhu
authors.affiliation: Sino-American Cancer Research Institute, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan Scientific Research Center, Guangdong Medical University, 1 Xincheng Road, Dongguan, 523808, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zou
authors.affiliation: Sino-American Cancer Research Institute, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan Scientific Research Center, Guangdong Medical University, 1 Xincheng Road, Dongguan, 523808, China.
authors.forename: Cong-Cong
authors.initials: CC
authors.lastname: Ding
authors.affiliation: Department of Biochemistry, Liaoning Medical University, 40 Songpo Road, Jinzhou, 121001, China. lnmuyangjing@126.com.
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: Sino-American Cancer Research Institute, Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan Scientific Research Center, Guangdong Medical University, 1 Xincheng Road, Dongguan, 523808, China. zhiweihe66832@126.com.
authors.forename: Zhi-Wei
authors.initials: ZW
authors.lastname: He
mesh_terms: D001665:Binding Sites; D050882:CREB-Binding Protein / Q000037:antagonists & inhibitors; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000037:antagonists & inhibitors; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D016328:NF-kappa B / Q000235:genetics; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; C539882:NR4A2 protein, human; D057126:Nuclear Receptor Subfamily 4, Group A, Member 2; D050882:CREB-Binding Protein; C470551:NLK protein, human; D017346:Protein Serine-Threonine Kinases
keywords: CBP; CREB; NF-κB; NLK; Nurr1; Prostate cancer