DRG2 is required for surface localization of PD-L1 and the efficacy of anti-PD-1 therapy.

More than half of tumor patients with high PD-L1 expression do not respond to anti-PD-1/PD-L1 therapy, and the underlying mechanisms are yet to be clarified. Here we show that developmentally regulated GTP-binding protein 2 (DRG2) is required for response of PD-L1-expressing tumors to anti-PD-1 therapy. DRG2 depletion enhanced IFN-γ signaling and increased the PD-L1 level in melanoma cells. However, it inhibited recycling of endosomal PD-L1 and reduced surface PD-L1 levels, which led to defects in interaction with PD-1. Anti-PD-1 did not expand effector-like T cells within DRG2-depleted tumors and failed to improve the survival of DRG2-depleted tumor-bearing mice. Cohort analysis revealed that patients bearing melanoma with low DRG2 protein levels were resistant to anti-PD-1 therapy. These findings identify DRG2 as a key regulator of recycling of endosomal PD-L1 and response to anti-PD-1 therapy and provide insights into how to increase the correlation between PD-L1 expression and response to anti-PD-1 therapy.

FOXO1 Is a Key Mediator of Glucocorticoid-Induced Expression of Tristetraprolin in MDA-MB-231 Breast Cancer Cells.

The mRNA destabilizing factor tristetraprolin (TTP) functions as a tumor suppressor by down-regulating cancer-associated genes. TTP expression is significantly reduced in various cancers, which contributes to cancer processes. Enforced expression of TTP impairs tumorigenesis and abolishes maintenance of the malignant state, emphasizing the need to identify a TTP inducer in cancer cells. To search for novel candidate agents for inducing TTP in cancer cells, we screened a library containing 1019 natural compounds using MCF-7 breast cancer cells transfected with a reporter vector containing the TTP promoter upstream of the luciferase gene. We identified one molecule, of which the enantiomers are betamethasone 21-phosphate (BTM-21-P) and dexamethasone 21-phosphate (BTM-21-P), as a potent inducer of TTP in cancer cells. We confirmed that BTM-21-P, DXM-21-P, and dexamethasone (DXM) induced the expression of TTP in MDA-MB-231 cells in a glucocorticoid receptor (GR)-dependent manner. To identify potential pathways linking BTM-21-P and DXM-21-P to TTP induction, we performed an RNA sequencing-based transcriptome analysis of MDA-MB-231 cells at 3 h after treatment with these compounds. A heat map analysis of FPKM expression showed a similar expression pattern between cells treated with the two compounds. The KEGG pathway analysis results revealed that the upregulated DEGs were strongly associated with several pathways, including the Hippo signaling pathway, PI3K-Akt signaling pathway, FOXO signaling pathway, NF-κB signaling pathway, and p53 signaling pathway. Inhibition of the FOXO pathway using a FOXO1 inhibitor blocked the effects of BTM-21-P and DXM-21-P on the induction of TTP in MDA-MB-231 cells. We found that DXM enhanced the binding of FOXO1 to the TTP promoter in a GR-dependent manner. In conclusion, we identified a natural compound of which the enantiomers are DXM-21-P and BTM-21-P as a potent inducer of TTP in breast cancer cells. We also present new insights into the role of FOXO1 in the DXM-21-P- and BTM-21-P-induced expression of TTP in cancer cells.

Tristetraprolin posttranscriptionally downregulates PFKFB3 in cancer cells.

The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases 3 (PFKFB3) catalyzes the first committed rate-limiting step of glycolysis and is upregulated in cancer cells. The mechanism of PFKFB3 expression upregulation in cancer cells has not been fully elucidated. The PFKFB3 3'-UTR is reported to contain AU-rich elements (AREs) that are important for regulating PFKFB3 mRNA stability. However, the mechanisms by which PFKFB3 mRNA stability is determined by its 3'-UTR are not well known. We demonstrated that tristetraprolin (TTP), an ARE-binding protein, has a critical function regulating PFKFB3 mRNA stability. Our results showed that PFKFB3 mRNA contains three AREs in the 3'-UTR. TTP bound to the 3rd ARE and enhanced the decay of PFKFB3 mRNA. Overexpression of TTP decreased PFKFB3 expression and ATP levels but increased GSH level in cancer cells. Overexpression of PFKFB3 cDNA without the 3'-UTR rescued ATP level and GSH level in TTP-overexpressing cells. Our results suggested that TTP post-transcriptionally downregulated PFKFB3 expression and that overexpression of TTP may contribute to suppression of glycolysis and energy production of cancer cells in part by downregulating PFKFB3 expression.

Doxorubicin inhibits PD-L1 expression by enhancing TTP-mediated decay of PD-L1 mRNA in cancer cells.

Recent research revealed that doxorubicin (DOX) decreased expression of programmed death-ligand 1 (PD-L1) in cancer cells. However, the detailed mechanisms underlying this effect are not well established. Here, we demonstrate that doxorubicin down-regulates PD-L1 expression through induction of AU-rich element (ARE) binding protein tristetraprolin (TTP) in cancer cells. PD-L1 mRNA contain three AREs within its 3'UTR. Doxorubicin induced expression of TTP, increased TTP binding to the 3rd ARE of the PD-L1 3'UTR, and increased decay of PD-L1 mRNA. Inhibition of TTP abrogates the inhibitory effect of doxorubicin on PD-L1 expression. Our data suggest that TTP plays a key role in doxorubicin-mediated down-regulation of PD-L1 by enhancing degradation of PD-L1 mRNA in cancer cells.

DRG2 supports the growth of primary tumors and metastases of melanoma by enhancing VEGF-A expression.

Malignant metastatic melanoma (MM) is the most lethal of all skin cancers, but detailed mechanisms for regulation of melanoma metastasis are not fully understood. Here, we demonstrated that developmentally regulated GTP-binding protein 2 (DRG2) is required for the growth of primary tumors and for metastasis. DRG2 expression was significantly increased in MM compared with primary melanoma (PM) and dysplastic nevi. A correlation between DRG2 expression and poor disease-specific survival in melanoma patients was also identified. Furthermore, inhibition of DRG2 suppressed the binding of Hypoxia-inducible factor 1α to the VEGF-A promoter region, expression of vascular endothelial growth factor (VEGF)-A, and formation of endothelial cell tubes. In experimental mice, DRG2 depletion inhibited the growth of PM and lung metastases and increased survival. These results identify DRG2 as a critical regulator of VEGF-A expression and of growth of PMs and lung metastases.

Agrimonia eupatoria L. (Agrimony) Extract Alters Liver Health in Subjects with Elevated Alanine Transaminase Levels: A Controlled, Randomized, and Double-Blind Trial.

Agrimonia eupatoria L. has been shown to protect against liver injury due to its lipid lowering and antioxidant activities. The aim of this research was to evaluate the effect of A. eupatoria L. aqueous extract (AEE) on 80 subjects with elevated alanine transaminase (ALT) levels in a randomized, double-blind, placebo-controlled, 8-week study. This trial was conducted between January 2013 and July 2013 at the Oriental Medical Hospital (Jecheon) of Semyung University. The trial included subjects aged 20 years or older who were diagnosed with mildly to moderately elevated ALT levels (between 45 and 135 IU/L). Subjects received two capsules of placebo or AEE twice a day for 8 weeks. Adverse events were recorded. Eighty subjects were randomized to placebo or AEE groups who had similar baseline characteristics. During the 8 weeks of treatment, 11 subjects were excluded from the analysis for protocol violation or consent withdrawal; efficacy of treatment was, therefore, evaluated in 69 subjects (placebo = 35, AEE = 34). The AEE group showed a significant reduction in ALT and serum triglyceride (TG) at 8 weeks compared with the placebo group (ALT P = .044, TG P = .020). Significant group and time interactions were found in ALT (P = .038), aspartate aminotransferase (P = .040), and TG (P = .010). Alkaline phosphatase, total bilirubin, and gamma-glutamyl transferase levels were not different between the two groups. There were no reported severe adverse events during this study, and total protein, albumin, blood urea nitrogen, creatine, and total cholesterol levels were normal in both groups. AEE consumption was safe and generally well tolerated without severe adverse events.

Pharmacokinetics and pharmacodynamics of ketoprofen plasters.

Ketoprofen plasters of 70 cm(2) size using DuroTak acrylic adhesive polymers were developed either containing 30 mg (Ketotop-L) or 60 mg drug (Ketotop-P). The in vitro skin permeation profile was obtained in hairless mouse skin and showed the permeation rate of Ketotop-P to be twice that of Ketotop-L. The plasma concentration profile of ketoprofen was determined in Sprague-Dawley rats after applying a 3 x 3 cm(2) plaster. AUC(0-24h) and C(max) of Ketotop-P were 260.92 microg.h/ml and 25.09 microg/ml, respectively, which were about twice the values of Ketotop-L. The hind paw edema induced by carrageenan injection was measured for 6 h after applying a 2 x 2 cm(2) plaster, and the area under the time-response curve (AUR) value was significantly lower in Ketotop-P attached rats (180.70%.h) than in those with the Ketotop-L (298.65%.h) and the control (407.04%.h) groups, indicating a stronger anti-inflammatory action of Ketotop-P. However, the analgesic effect of the two formulations did not show a statistically significant difference. In conclusion, Ketotop-P was able to achieve higher plasma concentration of ketoprofen, thereby exhibiting higher and more constant anti-inflammatory effect compared with Ketotop-L.