Oroxylin a promoted apoptotic extracellular vesicles transfer of glycolytic kinases to remodel immune microenvironment in hepatocellular carcinoma model.

Although oroxylin A, a natural flavonoid compound, suppressed progression of hepatocellular carcinoma, whether the tumor microenvironment especially the communication between cancer cells and immune cells was under its modulation remained obscure. Here we investigated the effect of extracellular vesicles from cancer cells elicited by oroxylin A on macrophages in vitro. The data shows oroxylin A elicits apoptosis-related extracellular vesicles through caspase-3-mediated activation of ROCK1in HCC cells, which regulates M1-like polarization of macrophage. Moreover, oroxylin A downregulates the population of M2-like macrophage and promotes T cells infiltration in tumor microenvironment, accompanied by suppression of HCC development and enhancement of immune checkpoint inhibitor treatment in mice model. Mechanistically, glycolytic proteins enriched in oroxylin A-elicited extracellular vesicles from HCC cells are transferred to macrophages where ROS-dependent NLRP3 inflammasome is activated, therefore contributing to anti-tumor phenotype of macrophage. Taken together, this study highlights that oroxylin A promotes metabolic shifts between tumor cells and immune cells, facilitates to inhibit tumor development, and improves immunotherapy response in HCC model.

ENPP2 inhibitor improves proliferation in AOM/DSS-induced colorectal cancer mice via remodeling the gut barrier function and gut microbiota composition.

In our previous multicenter study, we delineated the inherent metabolic features of colorectal cancer (CRC). Therein, we identified a member of the ectonucleotide pyrophosphatase/ phosphodiesterase family (ENPP2) as a significant differential metabolite of CRC. In this study, the role of ENPP2 in CRC has been demonstrated using established in vitro and in vivo models including ENPP2 gene knockdown, and use of the ENPP2 inhibitor, GLPG1690. We found that CRC proliferation was decreased after either ENPP2 gene knockdown or use of ENPP2 inhibitors. We further evaluated the role of GLPG1690 in AOM/DSS-induced CRC mice via intestinal barrier function, macrophage polarization, inflammatory response and microbial homeostasis. Results of immunofluorescence staining and Western blotting showed that GLPG1690 can restore gut-barrier function by increasing the expression of tight junction proteins, claudin-1, occludin and ZO-1. M2 tumor-associated macrophage polarization and colonic inflammation were attenuated after treatment with GLPG1690 using the Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) model. Moreover, 16 S rDNA pyrosequencing and metagenomic analysis showed that GLPG1690 could alleviate gut dysbiosis in mice. Furthermore, administration of GLPG1690 with antibiotics as well as fecal microbiota transplantation assays demonstrated a close link between the efficacy of GLPG1690 and the gut microbiota composition. Finally, results of metabolomic analysis implicated mainly the gut microbiota-derived metabolites of aromatic amino acids in CRC progression. These findings may provide novel insights into the development of small-molecule ENPP2 inhibitors for the treatment of CRC.

Elevated serum ß-hydroxybutyrate, a circulating ketone metabolite, accelerates colorectal cancer proliferation and metastasis via ACAT1.

Colorectal cancer (CRC) ranks third in incidence and second in mortality worldwide. Metabolic disorders are known to be closely associated with CRC. Functional metabolomics aims to translate metabolomics-derived biomarkers to disease mechanisms. Previous work based on untargeted liquid chromatography identified 30 differential metabolites of CRC. Among them, only ß-hydroxybutyrate (BHB) was elevated in CRC. Here, we first confirm the increased level of ß-hydroxybutyrate by targeted metabolomic analysis using an independent cohort of 400 serum samples by UPLC-QQQ-MS/MS analysis. Using appropriate cell and animal models, we find that treatment with pathological levels of ß-hydroxybutyrate expedites CRC proliferation and metastasis. Out of four major rate-limiting enzymes of ketolysis, only acetyl-coenzyme A acetyltransferase1 (ACAT1) expression is increased in paired human CRC tissues. These findings suggest probable clinical relevance for the functional implications of ß-hydroxybutyrate in CRC. We demonstrate that ß-hydroxybutyrate may exert its tumorigenic effects via regulation of ACAT1, due to induction of downstream isocitrate dehydrogenase1 (IDH1) acetylation. Genetic silencing of ACAT1 significantly suppresses the progression of CRC and abrogates the effects of ß-hydroxybutyrate both in vitro and in vivo. Overall, this study suggests that targeting ß-hydroxybutyrate and its major rate-limiting enzyme ACAT1 may provide a new avenue for therapeutic intervention in CRC.

2 Hydroxybutyric Acid-Producing Bacteria in Gut Microbiome and Fusobacterium nucleatum Regulates 2 Hydroxybutyric Acid Level In Vivo.

2-hydroxybutyric acid (2HB) serves as an important regulatory factor in a variety of diseases. The circulating level of 2HB in serum is significantly higher in multiple diseases, such as cancer and type 2 diabetes (T2D). However, there is currently no systematic study on 2HB-producing bacteria that demonstrates whether gut bacteria contribute to the circulating 2HB pool. To address this question, we used BLASTP to reveal the taxonomic profiling of 2HB-producing bacteria in the human microbiome, which are mainly distributed in the phylum Proteobacteria and Firmicutes. In vitro experiments showed that most gut bacteria (21/32) have at least one path to produce 2HB, which includes Aspartic acid, methionine, threonine, and 2-aminobutyric acid. Particularly, Fusobacterium nucleatum has the strongest ability to synthesize 2HB, which is sufficient to alter colon 2HB concentration in mice. Nevertheless, neither antibiotic (ABX) nor Fusobacterium nucleatum gavage significantly affected mouse serum 2HB levels during the time course of this study. Taken together, our study presents the profiles of 2HB-producing bacteria and demonstrates that gut microbiota was a major contributor to 2HB concentration in the intestinal lumen but a relatively minor contributor to serum 2HB concentration.

The involvement of lipid raft pathway in suppression of TGFß-mediated metastasis by tolfenamic acid in hepatocellular carcinoma cells.

TGFß signaling plays an important role in orchestrating a favorable microenvironment for tumor cell growth and promoting epithelial-mesenchymal transition. As a conventional nonsteroidal anti-inflammation drugs, tolfenamic acid (TA) has been previously reported to exhibit anti-cancer activity. Herein, we investigated the effect of TA on TGFß-mediated pro-metastatic activity and the underlying mechanisms in hepatocellular carcinoma (HCC). As a result, TA suppresses TGFß-induced migration and glycolysis in HCC cells, which is accompanied with reduced Smad phosphorylation and subsequent nuclear transcription activity. Mechanistically, TA promotes lipid raft-caveolar internalization pathway of TGFß receptor, therefore leading to its rapid turnover. Consistently, TA inhibits constitutively active TGFß type I receptor induced Smad phosphorylation and EMT markers, whereas ectopic expression of TGFß type II receptor could partially rescue TGFß-mediated Smad2 phosphorylation and downstream genes expression in the presence of TA. Furthermore, TA inhibited HCC cells invasion in nude mice, associated with the alteration of characteristics related with EMT and glycolysis of cancer cells. Our study suggests TA could activate lipid raft pathway and modulate TGFß mediated metastasis, implicating the potential application of TA as a modulator of tumor microenvironment in HCC.

Design, synthesis and evaluation of derivatives based on pyrimidine scaffold as potent Pan-Raf inhibitors to overcome resistance.

Simutaneous targeting all Raf isoforms offers the prospect of enhanced efficacy as well as reduced potential for resistance. Described herein is the discovery and characterization of a series of pyrimidine scaffold with DFG-out conformation as potent Pan-Raf inhibitors. Among them, I-41 with excellent Pan-Raf potency demonstrates inhibitory activity against BRafWT phenotypic melanoma and BRafV600E phenotypic colon cells. The western blot results for the Erk inhibition in human melanoma SK-Mel-2 cell lines showed I-41 inhibited the proliferation of SK-Mel-2 cell lines without paradoxical activation of Erk, which supported I-41 may become a good candidate compound to overcome the resistance of melanoma against the current BRafV600E inhibitor therapy. I-41 also have a favorable pharmacokinetic profile in rat. Synthesis, SAR, lead selection, and evaluation of the key compounds studies are described.