Genetical inhibition of IRE1α in macrophages suppresses hepatocellular carcinoma growth

Abstract

Introduction The microenvironment of hepatocellular carcinoma (HCC) will be synchronised to tumor-promoting niche by the cytokine and growth factors produced by infiltrated inflammatory cells. Macrophages, in particular, have been regarded as the inflammatory effector in HCC. Inositol-requiring enzyme 1α (IRE1α) is an unfolded protein response sensor, that controls protein regulation, metabolism and inflammation. Inhibition of IRE1α has been reported to inhibit metabolic inflammation through macrophages modulation. Therefore, the aim of this study is to investigate the role of IRE1α in tumour associated macrophages of HCC.

Material and methods Bone marrow isolated mononuclear cells were differentiated by either culturing with M-CSF or tumour supernatant for 7 days. Expressions of mRNAs was detected by quantitative real time PCR; protein expression was detected by immunoblotting assay; gene silencing was performed by RNA interference. HCC proliferation was detected by BrDU incorporation assay. The animal model was established by implanting subcutaneous grown tumour cube to left lobe of mouse liver. The hepatic tumour growth was monitored by in vivo luciferase imaging system.

Results and discussions We observed pharmacological inhibition of IRE1α by genipin, an iridoid aglycone reduced expressions of inflammatory factors such as CCR2, TNFα, CCL5, iNOS and IL6 on tumour supernatant-cultured and M-CSF induced macrophages. LPS-induced inflammatory gene expression in macrophages was similarly suppressed by genipin treatment. The role of IRE1α was confirmed from the observation of suppression of all the inflammatory mediators in IRE1α silencing macrophages. Furthermore, co-incubation of HCC cells with IRE1α silencing macrophages inhibited HCC cells proliferation. Our further in vivo study showed suppression of orthotopic HCC tumour growth after genipin intervention. We observed reduced IRE1α expression in hepatic macrophages after genipin treatment. This was accompanied with consistent reduction of inflammatory markers on the sorted hepatic macrophages.

Conclusion Altogether, our findings unveil the role of IRE1α in modulating inflammation in macrophages and pharmacological or genetical inactivation of IRE1α in macrophages suppressed HCC progression.