FKBP51 increases the tumour-promoter potential of TGF-beta

Abstract

Background: FKBP51 (FKBP5 Official Symbol) is a large molecular weight component of the family of FK506 binding proteins (FKBP). In recent years, research studies from our laboratory highlighted functions for FKBP51 in the control of apoptosis and melanoma progression. FKBP51 expression correlated with the invasiveness and aggressiveness of melanoma. Since a role for TGF-β in the enhanced tumorigenic potential of melanoma cells is widely described, we hypothesized a cooperative effect between FKBP51 and TGF-β in melanoma progression.

Methods: SAN and A375 melanoma cell lines were utilized for this study. Balb/c IL2γ NOD SCID served to assess the ability to colonize organs and metastasize of different cell lines, which was evaluated by in vivo imaging. Realtime PCR and western blot served for measurement of mRNA and protein expression, respectively.

Results: By comparing the metastatic potential of two melanoma cell lines, namely A375 and SAN, we confirmed that an increased capability to colonize murine organs was associated with increased levels of FKBP51. A375 melanoma cell line expressed FKBP51 mRNA levels 30-fold higher in comparison to the SAN mRNA level and appeared more aggressive than SAN melanoma cell line in an experimental metastasis model. In addition, A375 expressed, more abundantly than SAN, the TGF-β and the pro angiogenic TGF-β receptor type III (TβRIII) factors. FKBP51 silencing produced a reduction of TGF-β and TβRIII gene expression in A375 cell line, in accordance with previous studies. We found that the inducing effect of TGF-β on Sparc and Vimentin expression was impaired in condition of FKBP51 depletion, suggesting that FKBP51 is an important cofactor in the TGF-β signal. Such a hypothesis was supported by co immunoprecipitation assays, showing that FKBP51 interacted with either Smad2,3 and p300. In normal melanocytes, FKBP51 potentiated the effect of TGF-β on N-cadherin expression and conferred a mesenchymal-like morphology to such round-shaped cells.

Conclusions: Overall, our findings show that FKBP51 enhances some pro oncogenic functions of TGF-β, suggesting that FKBP51-overexpression may help melanoma to take advantage of the tumor promoting activities of the cytokine.

Figure 1

High FKBP51 expression is accompanied by increased metastatic potential. A, Real-time PCR measurement of FKBP51, TβRIII, and TGF-β mRNAs in A375 melanoma cells, which were silenced (FKBP51 siRNA) or not (NS RNA) for FKBP51. The relative change of expression in A375 samples was estimated relative to SAN samples (expression = 1). Reduced mRNA levels in FKBP51-silenced A375 confirmed that the immunophilin regulated the expression of TβRIII, and TGF-β in melanoma. B, FDG PET coronal views of mouse models of melanoma metastasis, 21 days after iv injection of two different cell lines: A375 (left) and SAN (right). FDG PET imaging show a diffuse and more prominent FDG uptake (mostly at lung and liver level, yellow squares) in the left mouse, suggesting that A375 have enhanced tumorigenic potential compared with SAN.

Figure 2

FKBP51 interacts with the general transcriptional co-activator p300 and the TGF-β transcription factor Smad2/3. Left; FKBP51 co immunoprecipitates with p300. Right; p300 co immunoprecipitates with FKBP51. Total cell lysates were prepared by SAN melanoma cells transfected with FKBP51/Flag. Cell lysates were immunoprecipitated with anti-Kat3B/p300 (IP p300) or anti-Flag (IP FKBP51). Immunoprecipitated and total lysates were then subjected to Western blot with anti-FKBP51, anti-p300 or anti-Smad 2/3. Smad 2/3 co immunoprecipitate with either p300 (left) and FKBP51 (right).

Figure 3

FKBP51 enhances expression of pro oncogenic factors in SAN melanoma cells stimulated with TGF-β. A, upper Western blot assay of Sparc levels in cell lysates obtained from the melanoma cell line SAN transfected with a specific FKBP51 shRNA (SH-FKBP51), or a non-silencing (SH-NS) shRNA as control, in the absence or the presence of 10 ng/ml TGF-β. After a 18 h culture, cell was harvested for lysates preparation. A, lower Flow cytometric histograms of Vimentin expression in SH-FKBP51 or SH-NS, in the absence or the presence of 10 ng/ml TGF-β. Cell was harvested after a 18 h culture. Vimentin was measured by indirect immunofluorescence, in fixed and permeabilized cells. B Effect of FKBP51 siRNA on VIM and SLUG expression levels. Normalized expression rates (mean±s. d.) of VIM (upper), SLUG (intermediate) and FKBP51 (lower) mRNA levels. NS RNA-treated sample expression=1 (N=2). Melanoma cell line SAN was transfected with a specific FKBP51 siRNA, or a non-silencing (NS) RNA as control. After 24 h from transfection 10 ng/ml TGF-β was added to the cultures. Cell was harvested after further 18 h and total RNA was extracted.

Figure 4

FKBP51 enhances expression of pro oncogenic factors in A375 melanoma cells stimulated with TGF-β. A Normalized expression rates (mean±s. d.) of SLUG (upper), and FKBP51 (lower) mRNA levels. SH-NS sample expression=1 (N=2). RNA was extracted from the melanoma cell line A375 stably transfected with a specific FKBP51 shRNA (SH-FKBP51), or a non-silencing (SH-NS) shRNA as control, in the absence or the presence of 10 ng/ml TGF-β. After a 18 h culture, cell was harvested. B Western blot assay of vimentin expression in whole cell lysates prepared from SH-NS and SH-FKBP51 cells cultured in the absence or the presence of 10 ng/ml TGF-β for 18 h. TGF-β stimulated an increase in vimentin level in SH-NS but not SH-FKBP51 cells. Reduced basal levels of vimentin were observed in SH-FKBP51 cells.

Figure 5

Mechanism proposed for FKBP51 enhancement of TGF-β pro-oncogenic signal. Left, FKBP51 facilitates Smad recruitment to coactivators. Right, FKBP51 takes part to the transcriptional complex formed by P300 and Smad 2,3 Increase in FKBP51, as it occurs in melanoma, generates an auto regulatory loop of TGF-β signaling, which in turn promotes tumour progression.

Figure 6

FKBP51 modulates the TGF-β signal in normal melanocytes. A, left Real-time PCR measurement of FKBP51, mRNAs in melanocytes, which were transfected with FKBP51 plasmid or the empty vector (EV) as control. The enhancement of FKBP51 transcript validated the efficacy of transfection. A, right Real-time PCR measurement of Cyclin B and CDH2 mRNAs in melanocytes, FKBP51- or EV-transfected, cultured in the absence or the presence of 10 ng/ml TGF-β for 3 days. The relative change of expression in different samples was estimated relative to EV sample (expression = 1). B, Phase contrast microscopy of melanocytes FKBP51- or EV-transfected, cultured in the absence or the presence of 10 ng/ml TGF-β for 3 days. Upper; EV- or FKBP51-melanocytes presented rounded/polygonal morphology. Lower left; EV- melanocytes cultured with TGF-β presented slender spindle shape with multiple dendritic extensions. Lower right; FKBP51-melanocytes cultured with TGF-β displayed elongated shape and bipolar spindle morphology.