Introduction. FGF2 and FGF-Receptors (FGFRs) comprise a signaling system involved in the control of a multitude of fundamental biological processes, ranging from cell proliferation to differentiation, tumorigenesis, and programmed cell death, in developing and adult tissues. In vertebrates, FGFRs are single spanning transmembrane proteins harboring Tyr kinase activity, which are encoded by 5 highly related genes, with exception of FGFR5 which lacks Tyr kinase activity. In addition, alternative splicing generates a diversity of FGFR isoforms, whose expression depends on cell type. We have recently found a high correlation between tumorigenicity and cell death induced by FGF2 in the following mouse lines: Y-1 adrenocortical tumor cells and Balb 3T3 fibroblasts transformed by the H-ras-V12 oncogene.
Objective. Our goal is to elucidate the functional roles of FGFRs by RNA interference in tumor cell proliferation and death induced by exogenously added FGF2 in Y-1 adrenocortical cells and Balb 3T3 transformed fibroblasts.
Methods. a) FGFR isoforms were analyzed by PCR amplification and DNA sequencing. b) FGFR expression was analyzed in clonal sublines derived from, respectively, Y-1 adrenocortical cells and Balb 3T3 fibroblasts transformed by the H-ras-V12 oncogene (3T3-B61 cells). c) To knockdown FGFR and FGF2 by RNAi, we designed specific double strand oligonucleotides and inserted them in a pSUPER vector. d) pSUPER constructions were transfected in Y-1 cells to derive stable clonal sublines. e) FGFRs expression was measured by RT Real-time PCR. f) Clonogenic assays were performed for response towards FGF2.
Results. a) Y-1 tumor cells and Balb 3T3 cells cells display all of the FGFRs. b) Stable Y-1 subline with FGFR2 knockdown presented a general decrease in the expression of all FGFRs. c) Stable Y-1 sublines with FGFR3 knockdown showed increased levels of FGFR1 and decreased levels of FGFR4. d) These Y-1 sublines with FGFR2 and FGFR3 knockdown are more resistant to FGF2 cell death induction than parental Y-1. e) An Y-1 subline resistant to FGF2 (Y-1-FR3) (tumor-negative and FGF2-death-negative) and Y-1 treated with FGF2 show 4 to 5 fold increase in FGFR3 mRNA. f) 3T3-B61 transformed fibroblasts (tumor-positive and FGF2-death-positive) exhibit a decrease in FGFR2, and an increase in FGF2 mRNA.
Conclusion. The phenotype FGF2-death-positive/tumor-positive compared to the phenotype FGF2-death-negative/tumor-negative involves alterations in the repertoire of expressed FGFRs and endogenously expressed FGF2. Besides that, these results suggest an anti-tumorigenic role for FGF2 and FGFRs.
Supported by CNPq and FAPESP.