title: PHLPP is a negative regulator of RAF1, which reduces colorectal cancer cell motility and prevents tumor progression in mice.
abstract: OBJECTIVE
  Hyperactivation of the RAS-RAF signaling pathway in colorectal tumors is associated with metastasis and poor outcomes of patients. Little is known about how RAS-RAF signaling is turned off once activated. We investigated how the pH domain and leucine-rich repeat protein phosphatases (PHLPPs) control RAS-RAF signaling and colorectal cancer (CRC) development.
  
  
  METHODS
  We used co-immunoprecipitation assays to identify substrates of PHLPP1 and PHLPP2. We studied phosphorylation of RAF1 in CRC cells that express exogenous PHLPP1 or PHLPP2, or lentiviral-based small hairpin RNAs against their transcripts; we measured effects on cell motility, migration, and invasion in vitro. Tumor progression and survival were analyzed in Phlpp1(-/-) Apc(Min) and Apc(Min)/Phlpp1(-/-) mice. Microarray datasets of colorectal tumor and nontumor tissues were analyzed for PHLPP gene expression.
  
  
  RESULTS
  PHLPP1 and 2 were found to dephosphorylate RAF1 at S338, inhibiting its kinase activity in vitro and in CRC cells. In cells, knockdown of PHLPP1 or PHLPP2 increased the amplitude and duration of RAF-MEK-ERK signaling downstream of epidermal growth factor receptor and KRAS, whereas overexpression had the opposite effect. In addition, knockdown of PHLPP1 or PHLPP2 caused CRC cells to express markers of the epithelial-mesenchymal transition, and increased cell migration and invasion. Apc(Min)/Phlpp1(-/-) mice had decreased survival and developed larger intestinal and colon tumors compared to Apc(Min) mice. Whereas Apc(Min) mice developed mostly low-grade adenomas, 20% of the tumors that developed in Apc(Min)/Phlpp1(-/-) mice were invasive adenocarcinomas. Normal villi and adenomas of Apc(Min)/Phlpp1(-/-) mice had significantly fewer apoptotic cells than Apc(Min) mice. Human CRC patient microarray data revealed that the expression of PHLPP1 or PHLPP2 is positively correlated with CDH1.
  
  
  CONCLUSIONS
  PHLPP1 and PHLPP2 dephosphorylate RAF1 to reduce its signaling, increase the invasive and migratory activities of CRC cells, and activate the epithelial-mesenchymal transition. In Apc(Min) mice, loss of PHLPP1 promotes tumor progression.
authors: Markey Cancer Center, University of Kentucky, Lexington, Kentucky.
authors: Xin
authors: X
authors: Li
authors: Markey Cancer Center, University of Kentucky, Lexington, Kentucky.
authors: Payton D
authors: PD
authors: Stevens
authors: Markey Cancer Center, University of Kentucky, Lexington, Kentucky.
authors: Jianyu
authors: J
authors: Liu
authors: Department of Oncology, Taizhou Hospital, Taizhou, Zhejian, China.
authors: Haihua
authors: H
authors: Yang
authors: Markey Cancer Center, University of Kentucky, Lexington, Kentucky.
authors: Wei
authors: W
authors: Wang
authors: Markey Cancer Center, University of Kentucky, Lexington, Kentucky.
authors: Chi
authors: C
authors: Wang
authors: Markey Cancer Center, University of Kentucky, Lexington, Kentucky.
authors: Zheng
authors: Z
authors: Zeng
authors: Markey Cancer Center, University of Kentucky, Lexington, Kentucky.
authors: Micheal D
authors: MD
authors: Schmidt
authors: Paul Laurence Dunbar High School, Lexington, Kentucky.
authors: Mike
authors: M
authors: Yang
authors: Department of Pathology, University of Kentucky, Lexington, Kentucky.
authors: Eun Y
authors: EY
authors: Lee
authors: Markey Cancer Center, University of Kentucky, Lexington, Kentucky; Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky. Electronic address: tianyan.gao@uky.edu.
authors: Tianyan
authors: T
authors: Gao
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D000236:Adenoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D000818:Animals; D015703:Antigens, CD; D017209:Apoptosis; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D004195:Disease Models, Animal; D018450:Disease Progression; D004789:Enzyme Activation; D058750:Epithelial-Mesenchymal Transition; D015972:Gene Expression Regulation, Neoplastic; D017491:Genes, APC; D006801:Humans; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D047368:Tumor Burden
chemical_list: D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; D009687:Nuclear Proteins; D051057:Proto-Oncogene Proteins c-akt; D019908:Proto-Oncogene Proteins c-raf; C400601:PHLPP1 protein, human; C519282:PHLPP1 protein, mouse; C541572:PHLPP2 protein, human; D010749:Phosphoprotein Phosphatases
keywords: Colorectal Cancer; Metastasis; Phosphatase; Tumor Suppressor