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. 2008 Dec;88(12):1340-8.
doi: 10.1038/labinvest.2008.97. Epub 2008 Oct 6.

Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of beta-catenin in prostate cancer

Michelangelo Fiorentino  1 , Giorgia Zadra, Emanuele Palescandolo, Giuseppe Fedele, Dyane Bailey, Christopher Fiore, Paul L Nguyen, Toshiro Migita, Raffaella Zamponi, Dolores Di Vizio, Carmen Priolo, Chandan Sharma, Wanling Xie, Martin E Hemler, Lorelei Mucci, Edward Giovannucci, Stephen Finn, Massimo Loda
Affiliations

Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of beta-catenin in prostate cancer

Michelangelo Fiorentino et al. Lab Invest. 2008 Dec.

Abstract

Fatty acid synthase (FASN), a key metabolic enzyme for liponeogenesis highly expressed in several human cancers, displays oncogenic properties such as resistance to apoptosis and induction of proliferation when overexpressed. To date, no mechanism has been identified to explain the oncogenicity of FASN in prostate cancer. We generated immortalized prostate epithelial cells (iPrECs) overexpressing FASN, and found that (14)C-acetate incorporation into palmitate synthesized de novo by FASN was significantly elevated in immunoprecipitated Wnt-1 when compared to isogenic cells not overexpressing FASN. Overexpression of FASN caused membranous and cytoplasmic beta-catenin protein accumulation and activation, whereas FASN knockdown by short-hairpin RNA resulted in a reduction in the extent of beta-catenin activation. Orthotopic transplantation of iPrECs overexpressing FASN in nude mice resulted in invasive tumors that overexpressed beta-catenin. A strong significant association between FASN and cytoplasmic (stabilized) beta-catenin immunostaining was found in 862 cases of human prostate cancer after computerized subtraction of the membranous beta-catenin signal (P<0.001, Spearman's rho=0.33). We propose that cytoplasmic stabilization of beta-catenin through palmitoylation of Wnt-1 and subsequent activation of the pathway is a potential mechanism of FASN oncogenicity in prostate cancer.

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Figures

Figure 1
Figure 1
Substantiation of the hypothesis that Wnt1 is palmitoylated in iPrEC cells stably overexpressing FASN. (a) Overexpression of FASN led to a specific pattern of palmitoylation in lysates of iPrEC-FASN compared to iPrEC-vector cells pulsed in medium containing 3H-palmitate (left panel). A band of approximately 45 kD corresponding to Wnt1 was revealed after immunoprecipitation (right panel). (b) The Wnt 1 palmitoylation assay showed increased incorporation of labeled acetate in immunoprecipitated Wnt-1 in iPrEC-FASN compared to iPrEC-vector cells. Cpm in Wnt 1 immune complex samples after subtraction of the background (cpm in the negative control sample: rabbit purified igG immunoprecipitation) are shown in the graph. Error bars represent mean standard error (P<0.05). (c) Representative western blotting of immunoprecipitatated Wnt-1 in the same samples used for the palmitoylation assay.
Figure 2
Figure 2
In vitro and in vivo stabilization and activation of β-catenin in immortalized prostate cells stably overexpressing FASN. (a) Immunoblot analysis of FASN and β-catenin in iPrEC-FASN and AR-iPrEC-FASN cell lines and their respective controls. An increase of β-catenin total expression and a slight increase of its activated form were observed in correspondence to FASN overexpression. (b) a predominant cytoplasmic staining for total and active β-catenin was detectable by immunofluorescence in iPrEC-FASN cells (bottom pictures) while the signal in iPrEC-vector (top pictures) was mainly at the membrane (DAPI-Texas Red staining, ×63 magnification). (c) Co-localization between mixed membrane/cytoplasmic staining for β-catenin and FASN was found by immunohistochemistry in AR-iPrEC-FASN xenograft tumors. The upper panel shows intense immunostaining for FASN in the tumor cells and no staining in the normal surrounding mouse prostate tissue. The lower panel shows mixed membrane/cytoplasmic staining for β-catenin in the tumor cells and exclusively membrane staining in the surrounding normal mouse prostate glands (DAB, ×200 magnification).
Figure 3
Figure 3
Activation and expression of β-catenin in FASN-knockdown human prostate tumor cell-lines. Treatment of LNCaP, CL1, PC3 and DU145 prostate cancer cell-lines with a shRNA against FASN resulted in marked down-regulation of FASN. A significant decrease of the active form of β-catenin was found in all the prostate cancer cell-lines after 4 days since the infection with the pKLO.1 plasmid encoding the sh-FASN #2. No significant difference in total β-catenin expression levels was observed after FASN inhibition.
Figure 4
Figure 4
Representative images of immunohistochemistry showing overexpression of FASN (a) and mixed membrane/cytoplasmic expression of β-catenin (b) (DAB, 100X magnification). (c) Pseudo-colorized dual quantum dot imaging of benign prostate epithelium (one star) and prostate adenocarcinoma (two stars). FASN (Qdot 605nm, yellow) and β-catenin (Qdot 655nm, red) were seen to co-localize in the cytoplasm of the prostate adenocarcinoma glands with minimal colocalization and expression in the adjacent benign epithelium (QDots, 100X magnification).
Figure 5
Figure 5
Quantitative assessment of cytoplasmic β-catenin immunostaining by computerized digital analysis in a case of prostate cancer Gleason grade 3. (a) The section stained for β-catenin is digitally acquired. (b) The membranous immunostaining is pseudo-colorized in blue. (c) The cytoplasmic immunostaining is pseudo-colorized in red and then it can be quantified by digital subtraction of the membranous from the total staining. Finally a score based on the area of staining is generated. (d) Correlation between the area of cytoplasmic β-catenin, divided into quartiles, and the area of FASN immunostaining in serial sections of the same cores (100X magnification).
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