Macrophage migration inhibitory factor - a therapeutic target in gallbladder cancer

Abstract

Background: Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer.

Methods: Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA.

Results: The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells.

Conclusions: Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.

Fig. 1

Experimental design and proteomic resulta Invasive property of GBC cell lines - TGBC24TKB - non-invasive.; OCUG-1 - moderately invasive; NOZ – moderately invasive; GB-d1- highly invasive. b Workflow for quantitative proteomic analysis of GBC cell line using iTRAQ labeling. c Venn diagrams depicting the overlap of the differentially expressed proteins in the three invasive cell lines, OCUG-1, NOZ and GB-d1

Fig. 2

Representative MS/MS spectra of overexpressed proteins and validation of MIF by immunohistochemistry. Representative MS/MS spectra of overexpressed proteins in invasive GBC cell lines, OCUG-1, NOZ and GB-d1 as compared with the non-invasive GBC cell line, TGBC24TKB. a Macrophage migration inhibitory factor (MIF). b CD74 molecule, major histocompatibility complex, class II invariant chain (CD74). c CD44 antigen (CD44). d Validation of MIF by IHC. Representative sections from cholecystitis tissues (weak staining) – (i) stained with hematoxylin and eosin; (ii) probed with anti-MIF antibody. Representative sections from gallbladder adenocarcinoma tissue (strong staining) - (iii) stained with hematoxylin and eosin; (iv) probed with anti-MIF antibody

Fig. 3

MIF affects the colony forming ability of the GBC cells – a-i Expression of MIF across a panel of GBC cell lines. Western blot analysis was performed using anti-MIF antibody. β-Actin was used as loading control. Top panel: GBC whole cell lysates, Middle panel: β-Actin, Bottom panel: GBC cell secretome. a-ii A graphical representation of MIF expression in GBC cell lines compared to β-Actin. b-i Colony forming ability of GBC cell lines was decreased post-transfection with MIF siRNA. b-ii A graphical representation of the same *P < 0.05. c-i Inhibition of MIF in GBC cell lines with ISO-1 (50 μM) and 4-IPP (5 μM) led to a decrease in the colony forming ability of the cells. c-ii A graphical representation of the same *P < 0.05

Fig. 4

Inhibition of MIF decreases the invasive property of the GBC cells. a-i siRNA mediated silencing of MIF decreases the invasive property of GBC cells. a-ii A graphical representation of the same *P < 0.05. b-i Inhibition of MIF using ISO-1 (50 μM) and 4-IPP (5 μM) lead to a decrease in the invasive ability of the GBC cells. b-ii A graphical representation of the same *P < 0.05