Progranulin Promotes Melanoma Progression By Inhibiting Natural Killer Cell Recruitment To The Tumor Microenvironment

Progranulin?PGRN?is a growth factor?also known as acrogranin,granulin/epithelin precursor,proepithelin,PC cell-derived growth factor?and its gene in human encodes a 593 amino acid secreted glycoprotein,progranulin.PGRN is excessively expressed in immune,neuronal and highly proliferative epithelial cells whereas in normal conditions,it doesn't express in fibroblast and endothelial cells.This growth factor has significant biological effects in different types of cancer.PGRN facilitates tumorigenesis by stimulating cell proliferation,migration,invasion,angiogenesis,and transformation.However,its role in melanoma progression has not been explored.In this study,we first analyzed publicly available clinical datasets and showed that high PGRN expression levels were correlated with poor prognosis of human melanoma patients.Expression of PGRN was upregulated in patients with advanced malignant melanoma.Consist with this notion,the survival rate decreased.Further,we demonstrate in the transplanted B16-F10murine melanoma model in which the endogenous Grn gene coding for PGRN has been deleted that tumor-derived,not host-derived PGRN,promotes melanoma growth and metastasis.These results were validated by genetic reconstitution of PGRN expression in the PGRN-deficient tumor,which completely restored the tumor growth to the wild type level.To investigate the function of PGRN in the regulation of tumor metastasis,cell migration and invasion were assessed.PGRN deficiency in the tumor resulted in reduced B16-F10 motility both in vitro and in vivo.Furthermore,immune cell analyses in the tumor and spleen revealed an enhanced infiltration of natural killer?NK?cells,but not T lymphocytes,into PGRN-deficient tumors compared to the wild type control.To test the hypothesis that NK cells are crucial in controlling PGRN-regulated B16-F10 growth,NK cells were depleted in vivo using an antibody,which confirmed the critical role of NK cells in restricting B16-F10 tumor growth.RNA-seq analysis of dissected tumors from mice reveals that 18 pathways were enriched in B16-F10/Grn^-/-vs.WT tumors.The most differently expressed genes were involved in immune response pathways,and a heat map presents 22 genes that were significantly upregulated in the B16-F10/Grn^-/-group in two independent tumors.Notably,several chemokines,including CCL5 were strongly upregulated in B16-F10/Grn^-/-tumor-bearing mice,suggesting that PGRN regulates their expression.CCL5 is one of the most relevant chemokines in recruiting NK cells to the tumor microenvironment.Silencing CCL5expression in PGRN-deficient tumor reduced NK cell recruitment and restored tumor growth to the control level.Moreover,reconstitution of B16-F10/Grn^-/-tumor with mPGRN inhibited CCL5 expression at mRNA and protein levels with or without IFN-?stimulation,pointing to a role of PGRN in the regulation of Ccl5 gene transcription.We corroborated this notion via transient transfection with a luciferase reporter gene driven by the Grn promoter in B16-B10 cells.In summary,this study highlights a novel and critical role of PGRN in melanoma growth and metastasis and suggests that it may represent a tangible therapeutic target for melanoma therapy,in addition to the prospect that PGRN may be a new marker for diagnosis of melanoma at an early stage.