Glutathione peroxidase 8 expression on cancer cells and cancer-associated fibroblasts facilitates lung cancer metastasis.

Lung cancer is the leading cause of cancer death worldwide, of which lung adenocarcinoma (LUAD) is the most common subtype. Metastasis is the major cause of poor prognosis and mortality for lung cancer patients, which urgently needs great efforts to be further explored. Herein, glutathione peroxidase 8 (GPX8) was identified as a novel potential pro-metastatic gene in LUAD metastatic mice models from GEO database. GPX8 was highly expressed in tumor tissues, predicting poor prognosis in LUAD patients. Knockdown of GPX8 inhibited LUAD metastasis in vitro and in vivo, while it did not obviously affect tumor growth. Knockdown of GPX8 decreased the levels of p-FAK and p-Paxillin and disturbed the distribution of focal adhesion. Furthermore, GPX8 was overexpressed in cancer-associated fibroblast (CAF) and associated with CAF infiltration in tumor microenvironment of lung cancer. GPX8 silence on fibroblasts suppressed lung cancer cell migration in the coculture system. BRD2 and RRD4 were the potential transcriptionally regulators for GPX8. Bromodomain extra-terminal inhibitor JQ1 downregulated GPX8 expression and suppressed lung cancer cell migration. Our findings indicate that highly expressed GPX8 in lung cancer cells and fibroblasts functions as a pro-metastatic factor in lung cancer. JQ1 is identified as a potential inhibitor against GPX8-mediated lung cancer metastasis.

c-MYC-mediated TRIB3/P62+ aggresomes accumulation triggers paraptosis upon the combination of everolimus and ginsenoside Rh2.

The mammalian target of rapamycin (mTOR) pathway is abnormally activated in lung cancer. However, the anti-lung cancer effect of mTOR inhibitors as monotherapy is modest. Here, we identified that ginsenoside Rh2, an active component of Panax ginseng C. A. Mey., enhanced the anti-cancer effect of the mTOR inhibitor everolimus both in vitro and in vivo. Moreover, ginsenoside Rh2 alleviated the hepatic fat accumulation caused by everolimus in xenograft nude mice models. The combination of everolimus and ginsenoside Rh2 (labeled Eve-Rh2) induced caspase-independent cell death and cytoplasmic vacuolation in lung cancer cells, indicating that Eve-Rh2 prevented tumor progression by triggering paraptosis. Eve-Rh2 up-regulated the expression of c-MYC in cancer cells as well as tumor tissues. The increased c-MYC mediated the accumulation of tribbles homolog 3 (TRIB3)/P62+ aggresomes and consequently triggered paraptosis, bypassing the classical c-MYC/MAX pathway. Our study offers a potential effective and safe strategy for the treatment of lung cancer. Moreover, we have identified a new mechanism of TRIB3/P62+ aggresomes-triggered paraptosis and revealed a unique function of c-MYC.

Myricetin inhibits interferon-γ-induced PD-L1 and IDO1 expression in lung cancer cells.

Programmed death ligand-1 (PD-L1) and indoleamine 2, 3-dioxygenase 1 (IDO1) are immune checkpoints induced by interferon-γ (IFN-γ) in the tumor microenvironment, leading to immune escape of tumors. Myricetin (MY) is a flavonoid distributed in many edible and medicinal plants. In this study, MY was identified to inhibit IFN-γ-induced PD-L1 expression in human lung cancer cells. It also reduced the expression of IDO1 and the production of kynurenine which is the product catalyzed by IDO1, while didn't show obvious effect on the expression of major histocompatibility complex-I (MHC-I), a crucial molecule for antigen presentation. In addition, the function of T cells was evaluated using a co-culture system consist of lung cancer cells and the Jurkat-PD-1 T cell line overexpressing PD-1. MY restored the survival, proliferation, CD69 expression and interleukin-2 (IL-2) secretion of Jurkat-PD-1 T cells suppressed by IFN-γ-treated lung cancer cells. Mechanistically, IFN-γ up-regulated PD-L1 and IDO1 at the transcriptional level through the JAK-STAT-IRF1 axis, which was targeted and inhibited by MY. Together, our research revealed a new mechanism of MY mediated anti-tumor activity and highlighted the potential implications of MY in tumor immunotherapy.

Regulation of CD47 expression by interferon-gamma in cancer cells.

The anti-phagocytosis signal, CD47, prevents phagocytosis when it interacts with signal-regulatory protein alpha (SIRPα) on macrophages. Given the vital role of CD47 in immune response, further investigation on the regulation of CD47 in tumor microenvironment is needed. Herein, we identified that interferon-gamma (IFN-γ), one of the most important cytokines in the immune and inflammatory response, up-regulated CD47 expression in cancer cells and this effect could be inhibited by the JAK1/2 inhibitor ruxolitinib, as well as siRNA-mediated silencing of JAK1, STAT1, and IRF1. The IFN-γ-induced surface expression of CD47 contributed to a stronger binding affinity to SIRPα and a decrease in phagocytosis of cancer cells by macrophages. Knockdown of JAK1, STAT1, or IRF1 by siRNA reversed the decreased phagocytosis caused by IFN-γ. Besides, analysis from TCGA revealed that IFNG had a positive correlation with CD47 in various types of cancer, which was supported by the increased surface CD47 expression after IFN-γ treatment in different types of cancer cells. The discovery of IFN-γ-induced up-regulation of CD47 in cancer cells unveils another feedback inhibitory mechanism of IFN-γ, thus providing insights into cancer immunotherapy targeting CD47.

Cytotoxic effects of flavonoids from root of Sophora flavescens in cancer cells.

One new flavanonol 4 H-​1-​benzopyran-​4-​one,2-​(4-​hydroxyphenyl)​-​3,​7-​dihydroxy-​5-​methoxy-​8-​[5-​methyl-​2-​(1-​methylethenyl)​-​4-​hexenyl]​(21), and twenty-six known compounds 1-20, 22-27 were isolated from the dried root of Sophora flavescens (S. flavescens) in this chemical study. Their structures were elucidated according to the spectroscopic and spectrometric methods. All the isolated compounds were evaluated for their cytotoxicity against lung cancer A549 cells and colon cancer HCT116 cells. Among them, compound 21 showed relatively predominant cell proliferation inhibition effect on the two tumor cell lines. Moreover, it induced cells apoptosis as evidenced by the Annexin V/PI double staining assay as well as the increased cleaved-PARP expression. The aforementioned data indicated that the flavonoids of S. flavescens have potential anti-cancer effect.

TGFß2-mediated epithelial-mesenchymal transition and NF-κB pathway activation contribute to osimertinib resistance.

Osimertinib (AZD9291) has been widely used for the treatment of EGFR mutant non-small cell lung cancer. However, resistance to osimertinib is inevitable. In this study we elucidated the molecular mechanisms of resistance in osimertinib-resistant NCI-H1975/OSIR cells. We showed that NCI-H1975/OSIR cells underwent epithelial-mesenchymal transition (EMT), which conferred sensitivity to the GPX4 inhibitor 1S, 3R-RSL3 to induce ferroptotic cell death. The EMT occurrence resulted from osimertinib-induced upregulation of TGFß2 that activated SMAD2. On the other hand, we revealed that NCI-H1975/OSIR cells were highly dependent on NF-κB pathway for survival, since treatment with the NF-κB pathway inhibitor BAY 11-7082 or genetic silence of p65 caused much greater cell death as compared with the parental NCI-H1975 cells. In NCI-H1975 cells, osimertinib activated NF-κB pathway, evidenced by the increased p65 nuclear translocation, which was abolished by knockdown of TGFß2. In the cancer genome atlas lung adenocarcinoma data, TGFB2 transcript abundance significantly correlated with EMT-associated genes and NF-κB pathway. In addition, coexistence of EMT and activation of NF-κB pathway was observed in several NCI-H1975/OSIR clones. These findings shed new light on distinct roles of TGFß2 in osimertinib-resistant cells and provide new strategies for treatment of this resistant status.

Licochalcone A inhibits interferon-gamma-induced programmed death-ligand 1 in lung cancer cells.

BACKGROUND: Programmed death-ligand 1 (PD-L1), which can be induced by interferon-gamma (IFN-γ) in the tumor microenvironment, is a critical immune checkpoint in cancer immunotherapy. Natural products which reduce IFN-γ-induced PD-L1 might be exert immunotherapy effect. Licochalcone A (LCA), a natural compound derived from the root of Glycyrrhiza inflata Batalin. (Fabaceae), was found to interfere IFN-γ-induced PD-L1. PURPOSE: The aim of this study is to further clarify the effect and the mechanism of LCA on inhibiting IFN-γ-induced PD-L1 in lung cancer cells. METHODS: The expression levels of PD-L1 were evaluated by flow cytometry, western blot and qRT-PCR. Click-iT protein synthesis assay and luciferase assay were used to identify the effect of LCA on protein synthesis. Jurkat T cell proliferation and apoptosis in the co-culture system were detected by flow cytometry. Flow cytometry was also applied to evaluate reactive oxygen species (ROS) generation. RESULTS: LCA downregulated IFN-γ-induced PD-L1 protein expression and membrane localization in human lung cancer cells, regardless of inhibiting PD-L1 mRNA level or promoting its protein degradation. LCA decreased apoptosis and proliferative inhibition of Jurkat T cells caused by IFN-γ-induced PD-L1-expressing in A549 cells in the co-culture system. Strikingly, LCA was verified as a protein synthesis inhibitor, which reduced both cap-dependent and -independent translation. LCA inhibited PD-L1 translation, likely due to inhibition of 4EBP1 phosphorylation (Ser 65) and activation of PERK-eIF2α pathway. Furthermore, LCA induced ROS generation in a time-dependent manner in lung cancer cells. N-acetyl-L-cysteine (NAC) not only revered ROS generation triggered by LCA but also restored IFN-γ-induced expression of PD-L1. Both the inhibition of 4EBP1 phosphorylation (Ser 65) and activation of PERK-eIF2α axis triggered by LCA was restored by co-treatment with NAC. CONCLUSION: LCA abrogated IFN-γ-induced PD-L1 expression via ROS generation to abolish the protein translation, indicating that LCA has the potential to be applied in cancer immunotherapy.

Nagilactone E increases PD-L1 expression through activation of c-Jun in lung cancer cells.

Nagilactone E (NLE), a natural product with anticancer activities, is isolated from Podocarpus nagi. In this study, we reported that NLE increased programmed death ligand 1 (PD-L1) expressions at both protein and mRNA levels in human lung cancer cells, and enhanced its localization on the cell membrane. Mechanistically, NLE increased the phosphorylation and expression of c-Jun, and promoted the localization of c-Jun in the nucleus, while silencing of c-Jun by small interfering RNA (siRNA) reduced NLE-induced PD-L1. Further study showed that NLE activated the c-Jun N-terminal kinases (JNK), the upstream of c-Jun, and its inhibitor SP600125 reversed the NLE-increased PD-L1. Moreover, NLE-induced PD-L1 increased the binding intensity of PD-1 on the cell surface. In summary, NLE upregulates the expression of PD-L1 in lung cancer cells through the activation of JNK-c-Jun axis, which has the potential to combine with the PD-1/PD-L1 antibody therapies in lung cancer.

Platycodin D triggers the extracellular release of programed death Ligand-1 in lung cancer cells.

Programmed death ligand-1 (PD-L1) is an important immune checkpoint for cancer immunotherapy in clinic. In this study, we reported that platycodin D, a natural product isolated from an edible and medicinal plant Platycodon grandiflorus (Jacq.) A. DC., down-regulated the protein level of PD-L1 in lung cancer cells. Flow cytometry and immunofluorescence assay showed a weaker surface PD-L1 signal in NCI-H1975 cells after the incubation with platycodin D (10 µM) for 15 min compared to the control group. Jurkat T cells showed enhancive interleukin-2 secretion when co-cultured with platycodin D-treated NCI-H1975 cells, suggesting that platycodin D-induced PD-L1 reduction increases the activation of Jurkat T cells. An augmentation of PD-L1 protein was detected in the cell culture medium from platycodin D treatment group. Chlorpromazine (60 µM) almost abolished the platycodin D-mediated PD-L1 extracellular release and restored the membrane PD-L1. Finally, hemolysis assay exhibited that platycodin D-triggered PD-L1 extracellular release was independent of the hemolytic mechanism. Taken together, our study demonstrates that platycodin D reduces the protein level of PD-L1 in lung cancer cells via triggering its release into the cell culture medium, which sheds new light for the application of natural products in cancer immunotherapy.