title: The transcription factor GCF2 is an upstream repressor of the small GTPAse RhoA, regulating membrane protein trafficking, sensitivity to doxorubicin, and resistance to cisplatin.
abstract: Our aim was to explore the involvement of the transcriptional suppressor GCF2 in silencing RhoA, disorganization of the cytoskeleton, mislocalization of MRP1, and sensitivity to anticancer agents as an upstream gene target in cancer therapy. Increased expression of GCF2 was found in human cisplatin-resistant cells, and overexpression in GCF2-transfected cells results in loss of RhoA expression and disruption of the actin/filamin network. In consequence, the membrane transporter MRP1 was internalized from the cell surface into the cytoplasm, rendering cells sensitive to doxorubicin by more than 10-fold due to increased accumulation of doxorubicin in the cells. The GCF2 transfectants also showed reduced accumulation of cisplatin and increased resistance. siRNA targeted to GCF2 suppressed the expression of GCF2 in cisplatin-resistant cells, reactivated RhoA expression, and restored the fine structure of actin microfilaments. MRP1 was also relocated to the cell surface. siRNA targeted to RhoA increased resistance 3-fold in KB-3-1 and KB-CP.5 cells. These data for the first time demonstrate a novel complex regulatory pathway downstream from GCF2 involving the small GTPase RhoA, actin/filamin dynamics, and membrane protein trafficking. This pathway mediates diverse responses to cytotoxic compounds, and also provides a molecular basis for further investigation into the pleiotropic resistance mechanism at play in cisplatin-resistant cells.
authors.affiliation: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
authors.forename: Ding-Wu
authors.initials: DW
authors.lastname: Shen
authors.forename: Lynn M
authors.initials: LM
authors.lastname: Pouliot
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Gillet
authors.forename: Wenting
authors.initials: W
authors.lastname: Ma
authors.forename: Alfred C
authors.initials: AC
authors.lastname: Johnson
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Hall
authors.forename: Michael M
authors.initials: MM
authors.lastname: Gottesman
mesh_terms: D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C114370:LRRFIP1 protein, human; D008565:Membrane Proteins; D016601:RNA-Binding Proteins; D004317:Doxorubicin; D020742:rhoA GTP-Binding Protein; D002945:Cisplatin