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It has been reported that tumor-associated macrophages (TAMs) play a complicated role in cancer occurrence and development, immune escape, and immune checkpoint blockade (ICB) resistance. However, the role of granulin precursor (GRN) highly expressed macrophages (hereafter refer to GRN + macrophages) in hepatocellular carcinoma (HCC) remains poorly understood. Herein, we systematically integrated multiomics analysis of human tumor tissues to illustrate the functional role of GRN + macrophages in HCC. GRN is selectively expressed by TAMs in different type of cancers including HCC, and was significantly associated with poor prognosis in several type of cancer. GRN was closely correlated with infiltration levels of most immune cells, especially the M2 macrophage cells in various cancers. In particular, both mRNA and protein expression level of GRN was significantly upregulated in HCC. Compared with tumor tissue, GRN was more significantly expressed in the stroma area between HCC tissues and adjacent non-tumor tissues. High expression of GRN was significantly correlated with M2-polarization of macrophages and T-cell exhaustion in HCC. GRN + macrophages communicated with intratumoral immune cells, especially CD8 + T cells. Functionally, GRN + macrophages contacted with CD8 + T cells, which inducing T-cell exhaustion. Our study offers a comprehensive understanding of the clinical relevance and immunological role of GRN + macrophages in HCC, indicating its potential role as a promising target for immunotherapeutic strategies.

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN -mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases. Progranulin-deficieny results in gangliosidosis due to reduced lysosomal lipids (BMP) required for ganglioside degradation. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012) for cholangiocarcinoma (CCA) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam and Cambodia. We exploited this link to explore the role of the secreted growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions by undertaking programmed CRISPR/Cas9 knockout of the Ov-GRN-1 gene from the liver fluke genome. Deep sequencing of amplicon libraries from genomic DNA of gene-edited parasites revealed Cas9-catalyzed mutations within Ov-GRN-1. Gene editing resulted in rapid depletion of Ov-GRN-1 transcripts and the encoded Ov-GRN-1 protein. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, but the infection resulted in reduced pathology as evidenced by attenuated biliary hyperplasia and fibrosis. Not only does this report pioneer programmed gene-editing in parasitic flatworms, but also the striking, clinically-relevant pathophysiological phenotype confirms the role for Ov-GRN-1 in virulence morbidity during opisthorchiasis. In the rural regions alongside the Mekong River in South East Asia, traditional cuisines often use uncooked or under cooked fish, many of which carry a worm known as Opisthorchis viverrini. Once inside the body, this parasite settles in the human liver, causing a tropical disease known as liver fluke infection. Out of the 10 million people affected by O. viverrini, thousands will also develop a type of liver cancer that is triggered by the presence of the worm. In particular, the parasite secretes a protein known as granulin that may encourage certain liver cells to multiply, potentially raising the risk for cancer. A gene editing technique called CRISPR/Cas9 allows scientist to precisely target and then deactivate the genetic information a cell needs to produce a given protein. While the tool has been used in other species before, it was unknown if it could be applied to O. viverrini. Here, Arunsan et al. harnessed CRISPR/Cas9 to deactivate the gene that codes for granulin and create parasites that can only produce very little of the protein. Hamsters infected with the gene-edited worms had fewer symptoms of liver fluke infection compared to those carrying normal O. viverrini. The animals with parasites that cannot produce granulin also had fewer changes to the liver that are associated with cancer. These findings confirm that granulin has a role in promoting liver fluke infection and liver cancer. Alongside this work, Ittiprasert et al. used CRISPR/Cas9 to inactivate a gene in a species of worm that causes a human disease called schistosomiasis. Together, these findings demonstrate for the first time that the gene editing method can be adapted for use in parasitic worms, which are a major public health problem in tropical climates. This tool should help scientists understand how the parasites invade and damage our bodies, and provide new ideas for treatment and disease control.

Cisplatin is one of the most potent chemotherapy drugs widely used for cancer treatment. However, due to resistance and toxicity, the application of cisplatin for the treatment of hepatocellular carcinoma (HCC) is limited. Our previous study has shown that granulin A (GRN A), an anticancer peptide, is able to interact with enolase1 (ENO1) and inhibit the growth of HCC in vitro. In the present study, we studied the synergistic effect of the combination of cisplatin and GRN A for the inhibitory effect on HCC. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and Chou-Talalay approaches revealed that the combination of GRN A and cisplatin displayed potent synergistic effect. The colony formation and cell viability of HCC cells were inhibited significantly in cells treated with the combination of cisplatin and GRN A, compared with cells treated with cisplatin or GRN A alone. Overexpression of ENO1 diminished the synergistic effect of GRN A and cisplatin in HCC cells. The combination of the two drugs exhibited a more obvious inhibitory effect on cancer cell apoptosis, as analyzed by the cytometry flow, mitochondrial membrane potential (MMP) and western blot analysis. An in vivo study confirmed that the combined use of the two drugs displayed more potent antitumor activity compared to mice treated with cisplatin and GRN A alone; the inhibitory rate of tumor growth was 65.46% and 68.94%, respectively, in mice treated with GRN A and cisplatin. However, the inhibitory rate increased to 86.63% in mice treated with the combination of the two drugs. This study provides evidence that the combination of GRN A and cisplatin is able to sensitize the liver cancer to cisplatin, and that targeting ENO1 is a promising approach for enhancing the antitumor activity of cisplatin.

The biological functions of growth factor, such as granulins, have been explored in parasites, and we elucidated that Clonorchis sinensis granulin ( Cs GRN) promoted the metastasis of hepatocellular carcinoma in our previous study. However, it is still unclear for the malignant transformation role of Cs GRN in normal human hepatocytes. In this study, by transfecting pEGFP-C1- Cs GRN eukaryotic expression plasmid, a cell line with stable overexpression of Cs GRN in normal hepatocyte (LO2-GRN cells) was constructed. The effects on cell proliferation were detected by carrying out cell counting kit-8 (CCK8) assay and colony formation assay. Additionally, we conducted flow cytometry analysis to determine whether the proliferation of Cs GRN was due to cell cycle arrest. Subsequently, the migration ability and the invasion ability of LO2-GRN cells were evaluated through wound-healing assay and transwell assay. Meanwhile, the levels of the markers of RAS/MAPK/ERK and PI3K/Akt signaling pathways activation in LO2-GRN cells were assessed by quantitative RT-PCR and Western blot. Our results indicated that Cs GRN promoted the proliferation of LO2 cells by regulating the expression of cell-cycle-related genes. Moreover, the overexpression of Cs GRN regulates malignant metastasis of liver cells by inducing the upregulation of epithelial–mesenchymal transition (EMT) marker proteins. Furthermore, both mRNA and protein expression levels of p-EGFR, RAS, p-ERK, p-AKT, p-PI3K, and p-braf have been enhanced by Cs GRN. These results showed that Cs GRN promoted the malignant transformation of hepatocytes by regulating epidermal growth factor receptor (EGFR)-mediated RAS/MAPK/ERK and PI3K/Akt signaling pathways, which suggested that Cs GRN could serve as a novel oncoprotein during Clonorchis sinensis –associated malignant transformation of hepatocytes.

The progressive failure of protein homeostasis is a hallmark of aging and a common feature in neurodegenerative disease. As the enzymes executing the final stages of autophagy, lysosomal proteases are key contributors to the maintenance of protein homeostasis with age. We previously reported that expression of granulin peptides, the cleavage products of the neurodegenerative disease protein progranulin, enhance the accumulation and toxicity of TAR DNA binding protein 43 (TDP-43) in Caenorhabditis elegans (C. elegans). In this study we show that C. elegans granulins are produced in an age- and stress-dependent manner. Granulins localize to the endolysosomal compartment where they impair lysosomal protease expression and activity. Consequently, protein homeostasis is disrupted, promoting the nuclear translocation of the lysosomal transcription factor HLH-30/TFEB, and prompting cells to activate a compensatory transcriptional program. The three C. elegans granulin peptides exhibited distinct but overlapping functional effects in our assays, which may be due to amino acid composition that results in distinct electrostatic and hydrophobicity profiles. Our results support a model in which granulin production modulates a critical transition between the normal, physiological regulation of protease activity and the impairment of lysosomal function that can occur with age and disease.

Progranulin (PGRN) plays a crucial role in diverse biological processes, including cell proliferation and embryonic development. PGRN can be cleaved by neutrophil elastase to release granulin (GRN). PGRN has been found to inhibit inflammation. Whereas, GRN plays a role as a pro-inflammatory factor. However, the pathophysiological roles of PGRN and GRN, at early stages after cerebral ischemia, have not yet been fully understood. The aim of this study was to obtain further insight into the pathologic roles of PGRN and GRN. We demonstrated that the amount of PGRN was significantly increased in microglial cells after cerebral ischemia in rats and that neutrophil elastase activity was also increased at an early stage after cerebral ischemia, resulting in the production of GRN. The inhibition of neutrophil elastase activity suppressed PGRN cleavage and GRN production, as well as the increase in pro-inflammatory cytokines, after cerebral ischemia. The administration of an elastase inhibitor decreased the number of injured cells and improved the neurological deficits test scores. Our findings suggest that an increase in the activity of elastase to cleave PGRN, and to produce GRN, was involved in an inflammatory response at the early stages after cerebral ischemia, and that inhibition of elastase activity could suppress the progression of cerebral ischemic injury.

Progranulin (PGRN) haploinsufficiency associated with loss-of-function mutations in the granulin gene causes frontotemporal dementia (FTD). This suggests that increasing PGRN levels could have promising therapeutic implications for patients carrying GRN mutations. In this study, we explored the therapeutic potential of sortilin1 (SORT1), a clearance receptor of PGRN, by generating and characterizing monoclonal antibodies against SORT1. Anti-SORT1 monoclonal antibodies were generated by immunizing Sort1 knockout mice with SORT1 protein. The antibodies were classified into 7 epitope bins based on their competitive binding to the SORT1 protein and further defined by epitope bin-dependent characteristics, including SORT1-PGRN blocking, SORT1 down-regulation, and binding to human and mouse SORT1. We identified a positive correlation between PGRN up-regulation and SORT1 down-regulation. Furthermore, we also characterized K1-67 antibody via SORT1 down-regulation and binding to mouse SORT1 in vivo and confirmed that K1-67 significantly up-regulated PGRN levels in plasma and brain interstitial fluid of mice. These data indicate that SORT1 down-regulation is a key mechanism in increasing PGRN levels via anti-SORT1 antibodies and suggest that SORT1 is a potential target to correct PGRN reduction, such as that in patients with FTD caused by GRN mutation.

Background Clonorchis sinensis granulin ( Cs GRN), a component of the excretory/secretory products of this species, is a multifunctional growth factor that can promote the metastasis of cholangiocarcinoma cells. However, the effect of Cs GRN on human intrahepatic biliary epithelial cells (HIBECs) is unclear. Here, we investigated the effect of Cs GRN on the malignant transformation of HIBECs and its possible underlying mechanism. Methods The malignant transformation phenotypes of HIBECs after Cs GRN treatment were estimated by EdU-488 incorporation assay, colony formation assay, wound-healing assay, Transwell assay and western blot. The biliary damage of Cs GRN-treated mice was detected by western blot, immunohistochemical staining and hematoxylin and eosin staining. The phenotypes of the macrophages [human monocytic leukemia cell line (THP-1)] were analyzed by flow cytometry, immunofluorescence and immunohistochemistry, both in vitro and in vivo. A co-culture system was developed to explore the interaction between THP-1 and HIBECs in Cs GRN-containing medium. Enzyme-linked immunosorbent assay and western blot were used to detected the activation of interleukin 6 (IL-6), phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. An inhibitor of the MEK/ERK pathway, PD98059, was used to determine whether this pathway is involved in Cs GRN-mediated cell interactions as well as in STAT3 phosphorylation and malignant transformation of HIBECs. Results Excessive hyperplasia and abnormal proliferation of HIBECs, enhanced secretion of hepatic pro-inflammatory cytokines and chemokines, as well as biliary damage, were observed in vitro and in vivo after treatment with Cs GRN. The expression of the markers of M2 macrophages significantly increased in Cs GRN-treated THP-1 cells and biliary duct tissues compared with the controls. Moreover, following treatment with Cs GRN, the HIBECs underwent malignant transformation in the THP-1-HIBECs co-culture group. In addition, high expression of IL-6 was observed in the Cs GRN-treated co-culture media, which activated the phosphorylation of STAT3, JAK2, MEK and ERK. However, treatment with an inhibitor of the MEK/ERK pathway, PD98059, decreased expression of p-STAT3 in Cs GRN-treated HIBECs and further repressed the malignant transformation of HIBECs. Conclusions Our results demonstrated that, by inducing the M2-type polarization of macrophages and activating the IL-6/JAK2/STAT3 and MEK/ERK pathways in HIBECs, Cs GRN promoted the malignant transformation of the latter. Graphical abstract

Parkinson’s disease is increasingly prevalent. It progresses from the pre-motor stage (characterised by non-motor symptoms like REM sleep behaviour disorder), to the disabling motor stage. We need objective biomarkers for early/pre-motor disease stages to be able to intervene and slow the underlying neurodegenerative process. Here, we validate a targeted multiplexed mass spectrometry assay for blood samples from recently diagnosed motor Parkinson’s patients ( n  = 99), pre-motor individuals with isolated REM sleep behaviour disorder (two cohorts: n  = 18 and n  = 54 longitudinally), and healthy controls ( n  = 36). Our machine-learning model accurately identifies all Parkinson patients and classifies 79% of the pre-motor individuals up to 7 years before motor onset by analysing the expression of eight proteins—Granulin precursor, Mannan-binding-lectin-serine-peptidase-2, Endoplasmatic-reticulum-chaperone-BiP, Prostaglaindin-H2-D-isomaerase, Interceullular-adhesion-molecule-1, Complement C3, Dickkopf-WNT-signalling pathway-inhibitor-3, and Plasma-protease-C1-inhibitor. Many of these biomarkers correlate with symptom severity. This specific blood panel indicates molecular events in early stages and could help identify at-risk participants for clinical trials aimed at slowing/preventing motor Parkinson’s disease. Parkinson’s disease is lacking easily accessible biomarkers. Here the authors show, that targeted blood proteomics is feasible to identify the patients and to predict the phenoconvertion in prodromal subjects up to 7 years before symptom onset.