Interleukin-6-driven progranulin expression increases cholangiocarcinoma growth by an Akt-dependent mechanism - PubMed (original) (raw)

doi: 10.1136/gutjnl-2011-300643. Epub 2011 Nov 7.

Pietro Invernizzi, Francesca Bernuzzi, Hae Yong Pae, Matthew Quinn, Darijana Horvat, Cheryl Galindo, Li Huang, Matthew McMillin, Brandon Cooper, Lorenza Rimassa, Sharon DeMorrow

Affiliations

Interleukin-6-driven progranulin expression increases cholangiocarcinoma growth by an Akt-dependent mechanism

Gabriel Frampton et al. Gut. 2012 Feb.

Abstract

Background and objectives: Cholangiocarcinoma is a devastating cancer of biliary origin with limited treatment options. The growth factor, progranulin, is overexpressed in a number of tumours. The study aims were to assess the expression of progranulin in cholangiocarcinoma and to determine its effects on tumour growth.

Methods: The expression and secretion of progranulin were evaluated in multiple cholangiocarcinoma cell lines and in clinical samples from patients with cholangiocarcinoma. The role of interleukin 6 (IL-6)-mediated signalling in the expression of progranulin was assessed using a combination of specific inhibitors and shRNA knockdown techniques. The effect of progranulin on proliferation and Akt activation and subsequent effects of FOXO1 phosphorylation were assessed in vitro. Progranulin knockdown cell lines were established, and the effects on cholangiocarcinoma growth were determined.

Results: Progranulin expression and secretion were upregulated in cholangiocarcinoma cell lines and tissue, which were in part via IL-6-mediated activation of the ERK1/2/RSK1/C/EBPβ pathway. Blocking any of these signalling molecules, by either pharmacological inhibitors or shRNA, prevented the IL-6-dependent activation of progranulin expression. Treatment of cholangiocarcinoma cells with recombinant progranulin increased cell proliferation in vitro by a mechanism involving Akt phosphorylation leading to phosphorylation and nuclear extrusion of FOXO1. Knockdown of progranulin expression in cholangiocarcinoma cells decreased the expression of proliferating cellular nuclear antigen, a marker of proliferative capacity, and slowed tumour growth in vivo.

Conclusions: Evidence is presented for a role for progranulin as a novel growth factor regulating cholangiocarcinoma growth. Specific targeting of progranulin may represent an alternative for the development of therapeutic strategies.

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Figures

Figure 1

Figure 1

Progranulin (PGRN) expression and secretion is increased in cholangiocarcinoma. PGRN levels were assessed in four cholangiocarcinoma cell lines as well as non-malignant cholangiocyte cell lines, H69 and HIBEC, by real time PCR and immunoblotting (A). For real time PCR, data are expressed as mean±SEM (n=4) (*p<0.05 compared with PGRN in H69 cells). Representative PGRN immunoblots are shown (lower panel). β-Actin is shown as a loading control. PGRN levels were also assessed in biopsy samples from 48 patients with cholangiocarcinoma and non-malignant controls by immunohistochemistry. Representative photomicrographs of the PGRN immunoreactivity are shown (B; magnification ×40). Staining intensity was assessed as described in the methods and expressed as mean±SEM for all patients with cholangiocarcinoma compared with control samples (B; *p<0.05 compared with PGRN immunoreactivity in control biopsy samples). PGRN levels in the supernatant of cell suspensions of cholangiocarcinoma cell lines and the non-malignant cholangiocyte cell lines, H69 and HIBEC, were determined by enzyme immunoassay (EIA) after 6 h (C). Data are expressed as mean±SEM PGRN concentration (ng/ml; n=3; *p<0.05 compared with PGRN levels secreted from H69 cells). PGRN levels in bile samples from cholangiocarcinoma and intrahepatic cholelithiasis patients was assayed by EIA (D). Data are expressed as mean±SEM PGRN concentration (ng/ml).

Figure 2

Figure 2

Interleukin (IL)-6 regulates progranulin (PGRN) expression. Mz-ChA-1 cells were treated with recombinant IL-6 (10 ng/ml) for various time points up to 4 h (A). In parallel, stably transfected cell lines were established by transfecting Mz-ChA-1 cells with IL-6 shRNA vector. The resulting cell line (Mz-IL-6 shRNA) had only 4% of the IL-6 expression compared with the mock-transfected cell line (Mz-neo neg) (B). PGRN expression was assessed by real time PCR and immunoblotting in these treatment groups. Real time PCR data are expressed as mean±SEM (n=4) (*p<0.05 compared with PGRN in control samples). Representative PGRN immunoblots are shown. β-Actin was used as the loading control.

Figure 3

Figure 3

Interleukin (IL)-6 drives progranulin (PGRN) expression via a mechanism involving extracellular signal-regulated kinase (ERK)1/2 and ribosomal protein S6 kinase-1 (RSK1). Mz-ChA-1 cells were treated with rIL-6 (10 ng/ml) in the presence or absence of the ERK1/2 inhibitor, PD98059 (10 µM), or the RSK1 inhibitor, SL-0101-1 (0.2 µM). PGRN expression was assessed by real time PCR (A). ERK1/2 activation was assessed in Mz-ChA- 1 cells after treatment with recombinant (r)IL-6 (10 ng/ml) for 1 h by immunoblotting using a phosphospecific ERK1/2 antibody (B). Data are expressed as relative phospho-ERK (pERK) levels after normalisation of the data to total ERK (tERK) levels in each sample. RSK1 activity was assessed in Mz-ChA-1 cells treated with rIL-6 (10 ng/ml) in the absence or presence of PD98059 (10 µM) for 1 h, by enzyme immunoassay activity kit (C). In parallel, basal ERK1/2 (D) and RSK1 (E) activity was assessed in Mz-IL-6 shRNA cells (lane 2) compared with the mock-transfected Mz-neo neg cell line (lane 1). All data are expressed as mean±SEM (n=3) (*p<0.05 compared with control samples).

Figure 4

Figure 4

CCAAT-enhancer binding protein β (C/EBPβ) is a downstream modulator of interleukin (IL)-6-driven progranulin (PGRN) expression. Mz-ChA-1 cells were treated with rIL-6 (10 ng/ml) in the presence or absence of the extracellular signal-regulated kinase (ERK)1/2 inhibitor PD98059 (10 µM) or the ribosomal protein S6 kinase-1 (RSK1) inhibitor SL-0101-1 (0.2 µM). C/EBPβ activation was assessed by immunoblotting using a phosphospecific C/EBPβ antibody (A). Data are expressed as relative phospho- C/EBPβ (pC/EBPβ) levels after normalisation of the data to total C/EBPβ (tC/EBPβ) levels in each sample. In parallel, basal C/EBPβ activation was assessed in Mz-IL6 shRNA compared with Mz-neo neg cells (B). The relative amount of C/EBPβ bound to the PGRN promoter was assessed in Mz-IL-6 shRNA and Mz-neo neg cells by chromatin immunoprecipitation (C) using a specific C/EBPβ-specific antibody to precipitate the complex followed by real time PCR using specific primers for the PGRN promoter region. The effect of IL-6 on PRGN expression in cells with C/EBPβ expression suppressed was determined. C/EBPβ expression was knocked down in Mz-ChA-1 cells, and the resulting cell line (Mz-C/EBPβ shRNA) was treated with recombinant IL-6 (10 ng/ml) for 4 h. PGRN expression was assessed by real time PCR (D). All data are expressed as mean±SEM (n=3; *p<0.05 compared with control samples).

Figure 5

Figure 5

Progranulin (PGRN) exerts proliferative effects on cholangiocarcinoma (A). Four cholangiocarcinoma cell lines were treated with various concentrations of recombinant (r)PGRN for 48 h. (B) Mz-ChA-1 cells were treated with rPGRN (1 µg/ml) in the absence or presence of the Akt inhibitor, FPA124 (1 µM). Cell proliferation was assessed using an MTS cell proliferation assay. Data are expressed as fold change in proliferation (mean±SEM; n=7; *p<0.05 compared with basal treatment within each cell line).

Figure 6

Figure 6

Progranulin (PGRN) increases FOXO1 phosphorylation and nuclear extrusion. Mz-ChA-1 cells were treated with recombinant (r)PGRN (1 µg/ml) for various times up to 24 h. The levels of phospho FOXO1 and total FOXO1 were assessed by enzyme immunoassay-based activity kits (A). Data are expressed as fold increase of phospho FOXO1 (mean±SEM; n=4; *p<0.05 compared with basal treatment). The subcellular location of FOXO1 was determined by immunofluorescence microscopy in Mz-ChA-1 cells treated with rPGRN (1 µg/ ml) in the presence or absence of the Akt inhibitor, FPA124 (1 µM; B). FOXO1 immunoreactivity is seen in red, and nuclei were counterstained with DAPI (blue). Scale bar=20 µm.

Figure 7

Figure 7

Suppression of progranulin (PGRN) expression slows the rate of cholangiocarcinoma growth. PGRN expression was knocked down in Mz-ChA-1 cells, and the expression of proliferating cellular nuclear antigen (PCNA), as a marker of proliferative capacity, was assessed in the resulting cell line (Mz-PGRN shRNA), the parental cell line (Mz-ChA-1), and the mock-transfected control cell line (Mz-neo neg) immunoblots (A). Data are expressed as mean±SEM (n=4) after normalisation to loading with β-actin (*p<0.05 compared with PCNA in Mz-ChA-1 cells). Representative PCNA immunoblots are shown. β-Actin is shown as a loading control. (B) Mz-ChA-1, Mz-Neo neg or Mz-PGRN shRNA cells were seeded into a 96-well plate and allowed to adhere overnight. Once adhered, the number of cells was counted in three non-overlapping fields per well and then again 24 h later. Data are expressed as fold increase in cell number per field (mean±SEM; *p<0.05). (C) In vivo, Mz-ChA-1 and Mz-PGRN shRNA cells were injected into the flank of athymic mice. After tumours were established (10 days), tumour volume was assessed, which was then considered to be 100%. Tumour volume was then assessed for a further 26 days, and the percentage increase in tumour volume determined (n=6).

Comment in

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