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. 2011 Mar 15;104(6):1027-37.
doi: 10.1038/bjc.2011.37. Epub 2011 Mar 1.

Farnesoid X receptor, overexpressed in pancreatic cancer with lymph node metastasis promotes cell migration and invasion

J Y Lee  1 , K T Lee, J K Lee, K H Lee, K-T Jang, J S Heo, S H Choi, Yil Kim, J C Rhee
Affiliations

Farnesoid X receptor, overexpressed in pancreatic cancer with lymph node metastasis promotes cell migration and invasion

J Y Lee et al. Br J Cancer. .

Erratum in

  • Br J Cancer. 2014 Dec 9;111(12):2381

Abstract

Background: Lymph node metastasis is one of the most important adverse prognostic factors for pancreatic cancer. The aim of this study was to identify novel lymphatic metastasis-associated markers and therapeutic targets for pancreatic cancer.

Methods: DNA microarray study was carried out to identify genes differentially expressed between 17 pancreatic cancer tissues with lymph node metastasis and 17 pancreatic cancer tissues without lymph node metastasis. The microarray results were validated by real-time PCR. Immunohistochemistry and western blotting were used to examine the expression of farnesoid X receptor (FXR). The function of FXR was studied by small interfering RNA and treatment with FXR antagonist guggulsterone and FXR agonist GW4064.

Results: Farnesoid X receptor overexpression in pancreatic cancer tissues with lymph node metastasis is associated with poor patient survival. Small interfering RNA-mediated downregulation of FXR and guggulsterone-mediated FXR inhibition resulted in a marked reduction in cell migration and invasion. In addition, downregulation of FXR reduced NF-κB activation and conditioned medium from FXR siRNA-transfected cells showed reduced VEGF levels. Moreover, GW4064-mediated FXR activation increased cell migration and invasion.

Conclusions: These findings indicated that FXR overexpression plays an important role in lymphatic metastasis of pancreatic cancer and that downregulation of FXR is an effective approach for inhibition of pancreatic tumour progression.

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Figures

Figure 1
Figure 1
Gene expression analysis of selected upregulated genes and immunohistochemical expression of FXR in pancreatic cancer tissues with and without LN metastasis. (A) Upregulated gene analysis between pancreatic cancer tissues with and without LN metastasis by real-time PCR. (B) Real-time PCR validation of microarray data. For each sample, the amount of target and endogenous reference was determined from the appropriate standard curve. The target amount was then divided by an endogenous reference (GAPDH) amount to obtain a normalised target value. The black columns represent the mean of fold (Group II/Group I) values obtained in DNA microarray; the grey columns represent the mean of fold (Group II/Group I) values obtained in real-time PCR; *P<0.05. (C) Overexpression of FXR in pancreatic cancer tissues with LN metastasis. The FXR intensity on tissues was evaluated by immunohistochemical staining. (a) A representative negative staining in pancreatic cancer tissues without LN metastasis. Magnification, × 200. (b) A representative positive staining in pancreatic cancer tissues with LN metastasis. Magnification, × 200. (D) Kaplan–Meier analysis of overall survival for patients with ductal adenocarcinoma (n=34). Farnesoid X receptor expression-positive cases (n=15) had significantly poorer prognosis than expression -negative cases (n=19; P=0.0227, log-rank test).
Figure 2
Figure 2
Farnesoid X receptor expression in pancreatic cancer cell lines. BF, basal FXR; CS, control siRNA; FS, FXR siRNA. (A) Western blot analysis of FXR was carried out on 50 μg of total proteins extracted from five pancreatic cancer cell lines; HepG2 and MCF-7 cells seen as a band at ∼56 kDa; HepG2 and MCF-7 were used as positive controls. β-Actin was used as a loading control. (B, C) MIA-PaCa2 and PANC-1 cells were transfected with control siRNA or FXR siRNA for 72 h. (B) Total RNA was extracted and analysed by TaqMan real-time quantitative RT–PCR. Farnesoid X receptor mRNA values were normalised to GAPDH mRNA. (C) Total proteins were extracted and western blotting analysis was performed. β-Actin was used as a loading control. Band intensities were evaluated in terms of relative density and expressed as percentages of the control, which was assumed to be 100%. Columns, mean of three independent experiments; bars, s.e.m.
Figure 3
Figure 3
Effects of FXR siRNA on pancreatic cancer cell proliferation, migration, and invasion. BF, basal FXR; CS, control siRNA; FS, FXR siRNA; MMC, mitomycin C. (A) MIA-PaCa2 and PANC-1 cells were transfected with control siRNA or FXR siRNA for the indicated time periods before cell number were estimated using CCK-8 assay. (B, C) MIA-PaCa2 and PANC-1 cells transfected with control siRNA or FXR siRNA were cultured in the presence of MMC (μg ml−1) for 48 h. Cell were placed in serum-free culture media and added into the upper compartment of a migration or invasion chamber. After 24 h, cells in the upper chamber were removed and cells that had migrated (B) or invaded (C) onto the lower surface of the membrane were fixed and stained with Wright–Giemsa. The relative-fold migration and invasion values of FXR siRNA-transfected cells was normalised against control siRNA-transfected cells and expressed as percentages of the control, which was assumed to be 100%. Columns, mean of three independent experiments; bars, s.e.m. **P<0.01.
Figure 4
Figure 4
Effects of FXR siRNA on NF-κB p65 and VEGF activities. CS, control siRNA; FS, FXR siRNA. (A) Nuclear extracts were prepared from control siRNA or FXR siRNA-transfected MIA PaCa2 and PANC-1 cells and subjected to analysis for NF-κB p65 activity as measured by Active Motif enzyme-linked immunosorbent assay (ELISA). (B) The culture medium of control siRNA or FXR siRNA-transfected MIA-PaCa2 and PANC-1 cells was used for the detection of VEGF using ELISA, as described under Materials and Methods. The relative-fold change of NF-κB p65 or VEGF activity in FXR siRNA-transfected cells was normalised against control FXR siRNA-transfected cells. Values in control siRNA-transfected cells were arbitrarily set to 1. Columns, mean of three independent experiments; bars, s.e.m. *P<0.05; **P<0.01.
Figure 5
Figure 5
Effects of FXR antagonist guggulsterone on pancreatic cancer cell proliferation, migration, and invasion. DMSO, control; GS, guggulsterone. (A) MIA-PaCa2 and PANC-1 cells were treated with 5, 10, and 20 μM GS or DMSO for the indicated time periods before cell numbers were estimated using CCK-8 assay. (B, C) MIA-PaCa2 and PANC-1 cells were treated with 10 μM GS for 48 h. Cells were placed in serum-free culture media and added into the upper compartment of a migration or invasion chamber. After 24 h, cells in the upper chamber were removed and cells that had migrated (B) or invaded (C) onto the lower surface of the membrane were fixed and stained with Wright–Giemsa. The relative-fold migration and invasion values of GS-treated cells were normalised against DMSO-treated cells and expressed as percentages of the control, which was assumed to be 100%. Columns, mean of three independent experiments; bars, s.e.m. *P<0.05; **P<0.01.
Figure 6
Figure 6
Effect of FXR agonists GW4064 on pancreatic cancer cell migration and invasion. DMSO, control. (A, B) MIA-PaCa2 and PANC-1 cells were treated with 1 μM GW4064 for 48 h. Cells were placed in serum-free culture media and added into the upper compartment of a migration or invasion chamber. After 24 h, cells in the upper chamber were removed and cells that had migrated (A) or invaded (B) onto the lower surface of the membrane were fixed and stained with Wright–Giemsa. The relative-fold migration and invasion values of GW4064-treated cells were normalised against DMSO-treated cells and expressed as percentages of the control, which was assumed to be 100%. Columns, mean of three independent experiments; bars, s.e.m. *P<0.05.
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