Fine particulate matter exposure disturbs autophagy, redox balance and mitochondrial homeostasis via JNK activation to inhibit proliferation and promote EMT in human alveolar epithelial A549 cells.

Epidemiologic studies have demonstrated a direct correlation between fine particulate matter (FPM) exposure and the high risk of respiratory diseases. FPM can penetrate deep into the lung and deposit in the alveoli with breath, where it directly interacts with alveolar epithelial cell (APC). However, we know little about the effects nor mechanisms of FPM on APC. Here, using human APC A549 cells, we found that FPM resulted in blockade of autophagic flux, redox imbalance and oxidative stress, mitochondrial fragmentation, increased mitophagy and impaired mitochondrial respiration. Further we showed that activation of JNK signaling (c-Jun N-terminal kinase) and excessive ROS (reactive oxygen species) release contribute to these adverse effects, with the former being upstream of the latter. More importantly, we found that scavenging ROS or inhibiting JNK activation could restore those effects as well as ameliorate FPM-induced inhibition of cell proliferation, and epithelial-mesenchymal transformation (EMT) in A549 cells. Taken together, our findings indicate that FPM leads to toxicity in alveolar type II cells via JNK activation, and JNK-targeting or antioxidant strategies might be beneficial for prevention or treatment of FPM-related pulmonary diseases.

CD147-specific chimeric antigen receptor T cells effectively inhibit T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is invasive and heterogeneous, and existing therapies are sometimes unsuccessful. Chimeric antigen receptor (CAR) T cell therapy is a breakthrough tumor treatment method, particularly for B cell acute lymphoblastic leukemia. We found that CD147 was highly expressed in tumor T cells of T-ALL patients and T cell lymphoma. Therefore, CD147-CAR T cells that contain a humanized single-chain variable fragment targeting human CD147 and a second-generation CAR frame were constructed for treating T-ALL. CD147-CAR T cells were able to maintain a healthy proliferation rate, preserving a subset of CD62L+/CCR7+ memory T cells. CD147-CAR T cells showed a potent anti-tumor activity against human T-ALL cell line and T-ALL blasts, releasing high level of cytokines in the process. However, CD147-CAR T cells exhibited potential safety toward human normal cells and CD147-deficent cells. NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mice were used to establish a T-ALL xenograft model and CD147-CAR T cells conferred robust protection against T-ALL progression and significantly improved survival in mice. Overall, we found that CD147 is a potential antigen target of CAR T cell therapy for T-ALL.

Pyroptosis-Related LncRNA Signature Predicts Prognosis and Is Associated With Immune Infiltration in Hepatocellular Carcinoma.

Pyroptosis is an inflammatory form of programmed cell death that is involved in various cancers, including hepatocellular carcinoma (HCC). Long non-coding RNAs (lncRNAs) were recently verified as crucial mediators in the regulation of pyroptosis. However, the role of pyroptosis-related lncRNAs in HCC and their associations with prognosis have not been reported. In this study, we constructed a prognostic signature based on pyroptosis-related differentially expressed lncRNAs in HCC. A co-expression network of pyroptosis-related mRNAs-lncRNAs was constructed based on HCC data from The Cancer Genome Atlas. Cox regression analyses were performed to construct a pyroptosis-related lncRNA signature (PRlncSig) in a training cohort, which was subsequently validated in a testing cohort and a combination of the two cohorts. Kaplan-Meier analyses revealed that patients in the high-risk group had poorer survival times. Receiver operating characteristic curve and principal component analyses further verified the accuracy of the PRlncSig model. Besides, the external cohort validation confirmed the robustness of PRlncSig. Furthermore, a nomogram based on the PRlncSig score and clinical characteristics was established and shown to have robust prediction ability. In addition, gene set enrichment analysis revealed that the RNA degradation, the cell cycle, the WNT signaling pathway, and numerous immune processes were significantly enriched in the high-risk group compared to the low-risk group. Moreover, the immune cell subpopulations, the expression of immune checkpoint genes, and response to chemotherapy and immunotherapy differed significantly between the high- and low-risk groups. Finally, the expression levels of the five lncRNAs in the signature were validated by quantitative real-time PCR. In summary, our PRlncSig model shows significant predictive value with respect to prognosis of HCC patients and could provide clinical guidance for individualized immunotherapy.

EYA2 suppresses the progression of hepatocellular carcinoma via SOCS3-mediated blockade of JAK/STAT signaling.

BACKGROUND: Somatic mutations are involved in hepatocellular carcinoma (HCC) progression, but the genetic mechanism associated to hepatocarcinogenesis remains poorly understood. We report that Eyes absent homolog 2 (EYA2) suppresses the HCC progression, while EYA2(A510E) mutation identified by exome sequencing attenuates the tumor-inhibiting effect of EYA2. METHODS: Whole-exome sequencing was performed on six pairs of human HCC primary tumors and matched adjacent tissues. Focusing on EYA2, expression level of EYA2 in human HCC samples was evaluated by quantitative real-time PCR, western blot and immunohistochemistry. Loss- and gain-of-function studies, hepatocyte-specific deletion of EYA2 (Eya2-/-) in mice and RNA sequencing analysis were used to explore the functional effect and mechanism of EYA2 on HCC cell growth and metastasis. EYA2 methylation status was evaluated using Sequenom MassARRAY and publicly available data analysis. RESULTS: A new somatic mutation p.Ala510Glu of EYA2 was identified in HCC tissues. The expression of EYA2 was down-regulated in HCC and associated with tumor size (P = 0.001), Barcelona Clinic Liver Cancer stage (P = 0.016) and tumor differentiation (P = 0.048). High level of EYA2 was correlated with a favorable prognosis in HCC patients (P = 0.003). Results from loss-of-function and gain-of-function experiments suggested that knockdown of EYA2 enhanced, while overexpression of EYA2 attenuated, the proliferation, clone formation, invasion, and migration of HCC cells in vitro. Delivery of EYA2 gene had a therapeutic effect on inhibition of orthotopic liver tumor in nude mice. However, EYA2(A510E) mutation led to protein degradation by unfolded protein response, thus weakening the inhibitory function of EYA2. Hepatocyte-specific deletion of EYA2 in mice dramatically promoted diethylnitrosamine-induced HCC development. EYA2 was also down-regulated in HCC by aberrant CpG methylation. Mechanically, EYA2 combined with DACH1 to transcriptionally regulate SOCS3 expression, thus suppressing the progression of HCC via SOCS3-mediated blockade of the JAK/STAT signaling pathway. CONCLUSIONS: In our study, we identified and validated EYA2 as a tumor suppressor gene in HCC, providing a new insight into HCC pathogenesis.

UBE2S interacting with TRIM28 in the nucleus accelerates cell cycle by ubiquitination of p27 to promote hepatocellular carcinoma development.

Genomic sequencing analysis of tumors provides potential molecular therapeutic targets for precision medicine. However, identifying a key driver gene or mutation that can be used for hepatocellular carcinoma (HCC) treatment remains difficult. Here, we performed whole-exome sequencing on genomic DNA obtained from six pairs of HCC and adjacent tissues and identified two novel somatic mutations of UBE2S (p. Gly57Ala and p. Lys63Asn). Predictions of the functional effects of the mutations showed that two amino-acid substitutions were potentially deleterious. Further, we observed that wild-type UBE2S, especially in the nucleus, was significantly higher in HCC tissues than that in adjacent tissues and closely related to the clinicopathological features of patients with HCC. Functional assays revealed that overexpression of UBE2S promoted the proliferation, invasion, metastasis, and G1/S phase transition of HCC cells in vitro, and promoted the tumor growth significantly in vivo. Mechanistically, UBE2S interacted with TRIM28 in the nucleus, both together enhanced the ubiquitination of p27 to facilitate its degradation and cell cycle progression. Most importantly, the small-molecule cephalomannine was found by a luciferase-based sensitive high-throughput screen (HTS) to inhibit UBE2S expression and significantly attenuate HCC progression in vitro and in vivo, which may represent a promising strategy for HCC therapy.

Fine particulate matter inhibits phagocytosis of macrophages by disturbing autophagy.

Mounting evidence from epidemiological and clinical studies has revealed marked correlations between the air pollutant fine particulate matter (FPM) and respiratory diseases. FPM reaches distal airways and deposits in alveolar regions where it can act directly on alveolar macrophages. However, the detailed effect of FPM on the physiological function of alveolar macrophages and the underlying mechanisms remain unclear. In this study, we showed that exposing THP-1-derived macrophages to FPM led to autophagy dysfunction. FPM activated the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway, which promoted the expression of autophagy-related 2A (ATG2A) and reactive oxygen species generation. The overexpression of ATG2A enhanced the synthesis of autophagic membranes, and the excessive production of reactive oxygen species caused autophagy flux inhibition through disrupting the lysosomal activity. More importantly, FPM impaired the phagocytic ability of macrophages on Escherichia coli and apoptotic neutrophils. Finally, we showed that restoring autophagy rescued the impairment of phagocytic ability induced by FPM. In summary, these results reveal the molecular mechanism of autophagy dysfunction caused by FPM and provide a novel approach to resolve the impaired function of macrophages in respiratory diseases induced by FPM.

Rab11a regulates MMP2 expression by activating the PI3K/AKT pathway in human hepatocellular carcinoma cells.

As a member of the Rab GTPase family, Rab11a plays an important role in vesicle transport and tumor progression. However, it is not clear whether it can also be used as an oncoprotein in hepatocellular carcinoma (HCC). In this study, database and immunohistochemical analyses showed that Rab11a was highly expressed in HCC tissues, and associated with poor clinical prognosis. Rab11a overexpression promoted the proliferation, migration, invasion, and anti-apoptosis of human HCC cell lines, MHCC-97H and HCC-LM3, whereas the downregulation of Rab11a inhibited these biological tumor activities. Nude mice xenograft demonstrated that Rab11a had a positive effect on the growth of hepatocellular carcinoma cells in vivo. Further studies found that the PI3K/AKT pathway and matrix metalloproteinase 2 (MMP2) upregulation can be activated by over-expression of Rab11a. However, MMP2 upregulation induced by Rab11a can be inhibited by the PI3K/AKT pathway inhibitor, LY294002. Altogether, our study established for the first time that Rab11a can play a pro-cancer role in HCC, as a novel oncoprotein, by activating the PI3K/AKT pathway to regulate MMP2 expression.

Blockade of Macrophage CD147 Protects Against Foam Cell Formation in Atherosclerosis.

The persistence of macrophage-derived foam cells in the artery wall fuels atherosclerosis development. However, the mechanism of foam cell formation regulation remains elusive. We are committed to determining the role that CD147 might play in macrophage foam cell formation during atherosclerosis. In this study, we found that CD147 expression was primarily increased in mouse and human atherosclerotic lesions that were rich in macrophages and could be upregulated by ox-LDL. High-throughput compound screening indicated that ox-LDL-induced CD147 upregulation in macrophages was achieved through PI3K/Akt/mTOR signaling. Genetic deletion of macrophage CD147 protected against foam cell formation by impeding cholesterol uptake, probably through the scavenger receptor CD36. The opposite effect was observed in primary macrophages isolated from macrophage-specific CD147-overexpressing mice. Moreover, bioinformatics results indicated that CD147 suppression might exert an atheroprotective effect via various processes, such as cholesterol biosynthetic and metabolic processes, LDL and plasma lipoprotein clearance, and decreased platelet aggregation and collagen degradation. Our findings identify CD147 as a potential target for prevention and treatment of atherosclerosis in the future.

Doxycycline Inducible Chimeric Antigen Receptor T Cells Targeting CD147 for Hepatocellular Carcinoma Therapy.

Chimeric antigen receptor T cell (CAR-T) therapy to hematological malignancies has demonstrated tremendous clinical outcomes. However, the therapeutic efficacy of CAR-T cells in solid tumors remains limited due to the scarcity of tumor-specific antigen targets and the poor infiltration of CAR-T cells into tumor tissue. In this study, we developed a novel inducible CAR-T cell system which targets CD147, a tumor-associated antigen for hepatocellular carcinoma (HCC). To minimize potential toxicities of CAR-T cell therapy, the Tet-On 3G system was introduced to induce CD147CAR expression in the right place at the right time. Specifically, Tet-CD147CAR lentiviral vector (LV-Tet-CD147CAR) was constructed, which comprised CD147CAR controlled by the Tet-On system. Tet-CD147CART cells were successfully generated from activated T cells by infection with LV-Tet-CD147CAR. Proliferation, cytotoxicity, and cytokine secretion of Tet-CD147CART cells were significantly increased against CD147-positive cancer cells in the presence of doxycycline (Dox) compared to Tet-CD147CART cells in the absence of Dox and PBMCs. Consistently, in vivo studies indicated that the tumor growth in nude mice was significantly inhibited by (Dox+) Tet-CD147CART cells through multiple intratumoral administration. Taken together, our results indicated that the expression and activity of CD147CAR were controlled by Dox both in vitro and in vivo, which facilitated decreased toxicity and adverse effects to CAR-T cell therapy. Moreover, this study provides viable evidence in support of the potential benefits and translation of this strategy of CAR-T cells targeting CD147 for the treatment of patients with HCC.

Identification of crucial genes based on expression profiles of hepatocellular carcinomas by bioinformatics analysis.

Hepatocellular carcinoma (HCC) is one of the most heterogeneous malignant cancers with no effective targets and treatments. However, the molecular pathogenesis of HCC remains largely uncertain. The aims of our study were to find crucial genes involved in HCC through multidimensional methods and revealed potential molecular mechanisms. Here, we reported the gene expression profile GSE121248 findings from 70 HCC and 37 adjacent normal tissues, all of which had chronic hepatitis B virus (HBV) infection, we were seeking to identify the dysregulated pathways, crucial genes and therapeutic targets implicated in HBV-associated HCC. We found 164 differentially expressed genes (DEGs) (92 downregulated genes and 72 upregulated genes). Gene ontology (GO) analysis of DEGs revealed significant functional enrichment of mitotic nuclear division, cell division, and the epoxygenase P450 pathway. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEGs were mainly enriched in metabolism, cell cycle regulation and the p53 signaling pathway. The Mcode plugin was calculated to construct a module complex of DEGs, and the module was mainly enriched in cell cycle checkpoints, RHO GTPase effectors and cytochrome P450. Considering a weak contribution of each gene, gene set enrichment analysis (GSEA) was performed, revealing results consistent with those described above. Six crucial proteins were selected based on the degree of centrality, including NDC80, ESR1, ZWINT, NCAPG, ENO3 and CENPF. Real-time quantitative PCR analysis validated the six crucial genes had the same expression trend as predicted. Furthermore, the methylation data of The Cancer Genome Atlas (TCGA) with HCC showed that mRNA expression of crucial genes was negatively correlated with methylation levels of their promoter region. The overall survival reflected that high expression of NDC80, CENPF, ZWINT, and NCAPG significantly predicted poor prognosis, whereas ESR1 high expression exhibited a favorable prognosis. The identification of the crucial genes and pathways would contribute to the development of novel molecular targets and biomarker-driven treatments for HCC.

Gamma-secretase complex-dependent intramembrane proteolysis of CD147 regulates the Notch1 signaling pathway in hepatocellular carcinoma.

The γ-secretase complex is a presenilin-dependent aspartyl protease involved in the intramembranous cleavage of various type I transmembrane proteins. As a type I transmembrane protein, CD147 is highly expressed in hepatoma cells and promotes cell proliferation, migration, and invasion. However, the direct underlying mechanism of how CD147 promotes cancer cell proliferation is unknown. Here, we demonstrated that CD147 undergoes an intramembranous cleavage by the γ-secretase at lysine 231 to release its intracellular domains (ICDs). The nuclear translocation of the CD147ICD regulated Notch1 expression by directly binding to the NOTCH1 promoter and promoted the activation of the Notch signaling pathway. Simultaneously, overexpression of CD147ICD promoted cancer cell proliferation via Notch1 signaling. In 102 cases of human hepatocellular carcinoma (HCC) tissues, patients with a high positive rate of nuclear CD147ICD expression had a significantly poor overall survival compared with patients with a low positive rate of nuclear CD147ICD expression. We confirmed that nuclear CD147ICD predicted a poor prognosis in human HCC. The combined therapy of the γ-secretase complex inhibitor and CD147-directed antibody showed better efficacy than monotherapy in orthotopic transplantation HCC mouse models. In conclusion, CD147 is cleaved by the γ-secretase and releases CD147ICD to the cell nucleus, promoting Notch1 expression via direct binding to the NOTCH1 promoter. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CD147 overexpression promotes tumorigenicity in Chinese hamster ovary cells.

CD147 overexpresses in many epithelium-originated tumors and plays an important role in tumor migration and invasion. Most studies aim at the role of CD147 in tumor progression using tumor cell models. However, the influence of abnormal overexpression of CD147 on neoplastic transformation of normal cells is unknown. Here, the role of CD147 in malignant phenotype transformation in CHO cells was investigated. Three CHO cell lines that stably overexpressed CD147 (CHO-CD147), EGFP-CD147 (CHO-EGFP-CD147), and EGFP (CHO-EGFP) were generated by transfection of plasmids containing human CD147, EGFP-human CD147, and EGFP genes into CHO cells. Cell migration and invasion were detected by wound healing and transwell matrix penetration assay. Trypan blue exclusion, MTT, cell cycle analysis, and BrdU cell proliferation assay were used to detect cell viability and cell proliferation. Annexin V-FITC analysis was performed to detect apoptosis. We found that CD147 overexpression promoted the migration and invasion of CHO cells. CD147 accelerated the G1 to S phase transition and enhanced the CHO cell proliferation. Overexpression of CD147 inhibited both early- and late-stages of apoptosis of CHO-CD147 cells, which is caused by serum deprivation. CHO-EGFP-CD147 cells showed an increased anchorage-independent growth compared with CHO-EGFP cells as detected by soft-agar colony formation assay. The tumors formed by CHO-CD147 cells in nude mice were larger and coupled with higher expression of proliferating cell nuclear antigen and Ki-67 than that of CHO cells. In conclusion, human CD147 overexpression induces malignant phenotype in CHO cells.