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, 10 (8), 847-56

Altered Fibroblast Growth Factor Receptor 4 Stability Promotes Prostate Cancer Progression

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Altered Fibroblast Growth Factor Receptor 4 Stability Promotes Prostate Cancer Progression

Jianghua Wang et al. Neoplasia.

Abstract

Fibroblast growth factor receptor 4 (FGFR-4) is expressed at significant levels in almost all human prostate cancers, and expression of its ligands is ubiquitous. A common polymorphism of FGFR-4 in which arginine (Arg(388)) replaces glycine (Gly(388)) at amino acid 388 is associated with progression in human prostate cancer. We show that the FGFR-4 Arg(388) polymorphism, which is present in most prostate cancer patients, results in increased receptor stability and sustained receptor activation. In patients bearing the FGFR-4 Gly(388) variant, expression of Huntingtin-interacting protein 1 (HIP1), which occurs in more than half of human prostate cancers, also results in FGFR-4 stabilization. This is associated with enhanced proliferation and anchorage-independent growth in vitro. Our findings indicate that increased receptor stability and sustained FGFR-4 signaling occur in most human prostate cancers due to either the presence of a common genetic polymorphism or the expression of a protein that stabilizes FGFR-4. Both of these alterations are associated with clinical progression in patients with prostate cancer. Thus, FGFR-4 signaling and receptor turnover are important potential therapeutic targets in prostate cancer.

Figures

Figure 1
Figure 1
Increased stability of the FGFR-4 Arg388 variant after ligand stimulation. 293T cells were transfected with V5-tagged FGFR-4 Arg388 or Gly388 and cell surface receptors labeled with biotin. Cells were then stimulated with FGF2 and lysed at the indicated time. Labeled receptors were then immunoprecipitated with streptavidin-agarose and FGFR-4 detected by Western blot of the immunoprecipitates with anti-V5 antibody. Western blot of an aliquot of the lysate used for immunoprecipitation with anti-β-actin antibody is shown.
Figure 2
Figure 2
Sustained phosphorylation of the FGFR-4 Arg388 variant after ligand stimulation. (A) PNT1A cells expressing V5-tagged FGFR-4 Arg388 or Gly388 were seeded at 2.5 x 106 in 60-mm diameter culture dishes in complete medium. Cells were gently scraped with a plastic tip. The medium was removed, and cells were washed twice with PBS. Complete medium was added, and cells were allowed to scatter/migrate into the area of clearing for a total of 40 hours, and photomicrographs were taken at 0-, 24-, and 40-hour time points. Scratch assays were performed four times, and representative results are shown. (B) PNT1A cells expressing either V5-tagged FGFR-4 Arg388 (AA) or Gly388 (GG) were plated. After overnight incubation in medium with insulin as the only growth factor, cells were stimulated with FGF2 and lysates were prepared at the indicated times. Tagged FGFR-4 was immunoprecipitated with an anti-V5 antibody, and the phosphorylated receptor was detected by Western blot of immunoprecipitates with anti-phospho-FGF-R antibody (Tyr641/642). Western blot with anti-V5 antibody is shown to confirm equivalent immunoprecipitation.
Figure 3
Figure 3
Quantitation of HIP1 transcripts by quantitative RT-PCR in prostate cancer. Quantitative RT-PCR was performed on RNA extracted from benign peripheral zone tissue from radical prostatectomies (PZ) or prostate cancers from patients with no PSA recurrence within 5 years of surgery (Non-recur) or with PSA recurrence (Recur) within 5 years of surgery. β-Actin transcript levels were used for normalization. Mean ± SD is shown.
Figure 4
Figure 4
Stabilization of FGFR-4 by HIP1 in prostate epithelial and prostate cancer cells. Immortalized normal prostate epithelial cells (PNT1A) or prostate cancer cells (LNCaP) were transfected with HIP1 in the TOPO-V5 expression vector and stable cells lines selected. (A) Expression of HIP1 and FGFR-4 protein was evaluated by Western blot with β-actin as a loading control. (B) Expression of FGFR-4 mRNA was determined by quantitative RT-PCR with normalization to β-actin transcript levels.
Figure 5
Figure 5
Stabilization of FGFR-4 Gly388 by HIP1. 293T cells were transfected with V5-tagged FGFR-4 Arg388 or Gly388 with or without HIP1, and cell surface receptors were labeled with biotin. Cells were then stimulated with FGF2 and lysed at the indicated time. Labeled receptors were then immunoprecipitated with streptavidin- agarose, and FGFR-4 was detected by Western blot of the immunoprecipitates with anti-V5 antibody.
Figure 6
Figure 6
Direct interaction of FGFR-4 and HIP1. (A) 293T cells were transfected with Flag-tagged HIP1 or N-terminally truncated HIP1 (-183) with or without either FGFR-4 Arg388 or Gly388 (both V5-tagged). Vector control (Tag2B) is also shown. Cell lysates were then analyzed by Western blot using anti-V5 or anti-Flag antibody or were used for reciprocal immunoprecipitation and Western blot analysis with the two antibodies. (B) Map of HIP1 showing major domains and deletion fragments used to prepare bacterial fusion proteins. (C) Purified HIP1 and HIP1 deletion bacterial fusion proteins on Coomassie blue-stained polyacrylamide gel after electrophoreisis. (D) 293T cells were transfected with V5-tagged FGFR-4 and lysates prepared and incubated with purified HIP1 and HIP1 deletion constructs. Complexes were then immunoprecipitated with streptavidin beads, and Western blot was performed with anti-V5 antibody. Control is streptavidin beads but no purified protein.
Figure 7
Figure 7
Biologic affects of HIP1 in prostate and prostate cancer cell lines. (A) Prostate (PNT1A) or prostate cancer (DU145) cell lines expressing full-length HIP1, an amino terminal-truncated HIP1 (NT) or vector only were plated, and growth was determined in a defined medium containing FGF2 and insulin as the only growth factors. Cell number was determined at 2-day intervals. Mean ± SD of triplicates is shown. (B) PNT1A cells overexpressing HIP1 were infected with lentivirus expressing ShRNA targeting HIP1 or FGFR-4, and stable expressors were selected and pooled. Quantitative RT-PCR showed 70% and 60% knockdown of HIP1 and FGFR-4 mRNA, respectively (data not shown). Cells were then plated, and grown was determined in defined medium with FGF2 and insulin as the only growth factors or serum-containing medium. Cell number was determined at 2-day intervals by cell counting. Mean ± SD of triplicates is shown. (C) PNT1A cells as described in (B) were plated in soft agar. Colony formation was evaluated by counting. Mean ± SD of triplicates is shown.

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