title: Co-targeting of FAK and MDM2 triggers additive anti-proliferative effects in mesothelioma via a coordinated reactivation of p53.
abstract: BACKGROUND
  Improved mesothelioma patient survival will require development of novel and more effective pharmacological interventions. TP53 genomic mutations are uncommon in mesothelioma, and recent data indicate that p53 remains functional, and therefore is a potential therapeutic target in these cancers. In addition, the tumour suppressor NF2 is inactivated by genomic mechanisms in more than 80% of mesothelioma, causing upregulation of FAK activity. Because FAK is a negative regulator of p53, NF2 regulation of FAK-p53-MDM2 signalling loops were evaluated.
  
  
  METHODS
  Interactions of FAK-p53 or NF2-FAK were evaluated by phosphotyrosine-p53 immunoaffinity purification and tandem mass spectrometry, and p53, FAK, and NF2 immunoprecipitations. Activation and/or expression of FAK, p53, and NF2 were also evaluated in mesotheliomas. Effects of combination MDM2 and FAK inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, expression of cell cycle checkpoints, and cell cycle alterations.
  
  
  RESULTS
  We observed constitutive activation of FAK, a known negative regulator of p53, in each of 10 mesothelioma cell lines and each of nine mesothelioma surgical specimens, and FAK was associated with p53 in five of five mesothelioma cell lines. In four mesotheliomas with wild-type p53, FAK silencing by RNAi induced expression and phosphorylation of p53. However, FAK regulation of mesothelioma proliferation was not restricted to p53-dependent pathways, as demonstrated by immunoblots after FAK knockdown in JMN1B mesothelioma cells, which have mutant/inactivated p53, compared with four mesothelioma cell lines with nonmutant p53. Additive effects were obtained through a coordinated reactivation of p53, by FAK knockdown/inhibition and MDM2 inhibition, as demonstrated by immunoblots, cell viability, and cell-cycle analyses, showing increased p53 expression, apoptosis, anti-proliferative effects, and cell-cycle arrest, as compared with either intervention alone. Our results also indicate that NF2 regulates the interaction of FAK-p53 and MDM2-p53.
  
  
  CONCLUSIONS
  These findings highlight novel therapeutic opportunities in mesothelioma.
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.|Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.|Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing, Zhejiang, China.
authors.forename: Wen-Bin
authors.initials: WB
authors.lastname: Ou
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Minmin
authors.initials: M
authors.lastname: Lu
authors.affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: Grant
authors.initials: G
authors.lastname: Eilers
authors.affiliation: Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing, Zhejiang, China.
authors.forename: Hailong
authors.initials: H
authors.lastname: Li
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Jiongyan
authors.initials: J
authors.lastname: Ding
authors.affiliation: Department of Breast Cancer Surgery, Zhejiang Cancer Hospital, Hangzhou, China.
authors.forename: Xuli
authors.initials: X
authors.lastname: Meng
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Yuehong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: He
authors.affiliation: Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Sheng
authors.affiliation: Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing, Zhejiang, China.
authors.forename: Hai-Meng
authors.initials: HM
authors.lastname: Zhou
authors.affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Fletcher
mesh_terms: D059447:Cell Cycle Checkpoints / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics; D051417:Focal Adhesion Kinase 1 / Q000235:genetics*; D016147:Genes, Tumor Suppressor / Q000502:physiology; D006801:Humans; D008654:Mesothelioma / Q000235:genetics* / Q000473:pathology; D009154:Mutation / Q000235:genetics; D025581:Neurofibromin 2 / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics*; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D025581:Neurofibromin 2; D034741:RNA, Small Interfering; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human