PPP1R15A-expressing monocytic MDSCs promote immunosuppressive liver microenvironment in fibrosis-associated hepatocellular carcinoma.

Background & Aims: Recent studies demonstrated the importance of fibrosis in promoting an immunosuppressive liver microenvironment and thereby aggressive hepatocellular carcinoma (HCC) growth and resistance to immune checkpoint blockade (ICB), particularly via monocyte-to-monocytic myeloid-derived suppressor cell (M-MDSC) differentiation triggered by hepatic stellate cells (HSCs). We thus aimed to identify druggable targets in these immunosuppressive myeloid cells for HCC therapy. Methods: M-MDSC signature genes were identified by integrated transcriptomic analysis of a human HSC-monocyte culture system and tumor-surrounding fibrotic livers of patients with HCC. Mechanistic and functional studies were conducted using in vitro-generated and patient-derived M-MDSCs. The therapeutic efficacy of a M-MDSC targeting approach was determined in fibrosis-associated HCC mouse models. Results: We uncovered over-expression of protein phosphatase 1 regulatory subunit 15A (PPP1R15A), a myeloid cell-enriched endoplasmic reticulum stress modulator, in human M-MDSCs that correlated with poor prognosis and ICB non-responsiveness in patients with HCC. Blocking TGF-ß signaling reduced PPP1R15A expression in HSC-induced M-MDSCs, whereas treatment of monocytes by TGF-ß upregulated PPP1R15A, which in turn promoted ARG1 and S100A8/9 expression in M-MDSCs and reduced T-cell proliferation. Consistently, lentiviral-mediated knockdown of Ppp1r15a in vivo significantly reduced ARG1+S100A8/9+ M-MDSCs in fibrotic liver, leading to elevated intratumoral IFN-γ+GZMB+CD8+ T cells and enhanced anti-tumor efficacy of ICB. Notably, pharmacological inhibition of PPP1R15A by Sephin1 reduced the immunosuppressive potential but increased the maturation status of fibrotic HCC patient-derived M-MDSCs. Conclusions: PPP1R15A+ M-MDSC cells are involved in immunosuppression in HCC development and represent a novel potential target for therapies. Impact and implications: Our cross-species analysis has identified PPP1R15A as a therapeutic target governing the anti-T-cell activities of fibrosis-associated M-MDSCs (monocytic myeloid-derived suppressor cells). The results from the preclinical models show that specific inhibition of PPP1R15A can break the immunosuppressive barrier to restrict hepatocellular carcinoma growth and enhance the efficacy of immune checkpoint blockade. PPP1R15A may also function as a prognostic and/or predictive biomarker in patients with hepatocellular carcinoma.

Targeting PPAR-gamma counteracts tumour adaptation to immune-checkpoint blockade in hepatocellular carcinoma.

OBJECTIVE: Therapy-induced tumour microenvironment (TME) remodelling poses a major hurdle for cancer cure. As the majority of patients with hepatocellular carcinoma (HCC) exhibits primary or acquired resistance to antiprogrammed cell death (ligand)-1 (anti-PD-[L]1) therapies, we aimed to investigate the mechanisms underlying tumour adaptation to immune-checkpoint targeting. DESIGN: Two immunotherapy-resistant HCC models were generated by serial orthotopic implantation of HCC cells through anti-PD-L1-treated syngeneic, immunocompetent mice and interrogated by single-cell RNA sequencing (scRNA-seq), genomic and immune profiling. Key signalling pathway was investigated by lentiviral-mediated knockdown and pharmacological inhibition, and further verified by scRNA-seq analysis of HCC tumour biopsies from a phase II trial of pembrolizumab (NCT03419481). RESULTS: Anti-PD-L1-resistant tumours grew >10-fold larger than parental tumours in immunocompetent but not immunocompromised mice without overt genetic changes, which were accompanied by intratumoral accumulation of myeloid-derived suppressor cells (MDSC), cytotoxic to exhausted CD8+ T cell conversion and exclusion. Mechanistically, tumour cell-intrinsic upregulation of peroxisome proliferator-activated receptor-gamma (PPARγ) transcriptionally activated vascular endothelial growth factor-A (VEGF-A) production to drive MDSC expansion and CD8+ T cell dysfunction. A selective PPARγ antagonist triggered an immune suppressive-to-stimulatory TME conversion and resensitised tumours to anti-PD-L1 therapy in orthotopic and spontaneous HCC models. Importantly, 40% (6/15) of patients with HCC resistant to pembrolizumab exhibited tumorous PPARγ induction. Moreover, higher baseline PPARγ expression was associated with poorer survival of anti-PD-(L)1-treated patients in multiple cancer types. CONCLUSION: We uncover an adaptive transcriptional programme by which tumour cells evade immune-checkpoint targeting via PPARγ/VEGF-A-mediated TME immunosuppression, thus providing a strategy for counteracting immunotherapeutic resistance in HCC.

Aberrant cholesterol metabolic signaling impairs antitumor immunosurveillance through natural killer T cell dysfunction in obese liver.

Obesity is a major risk factor for cancers including hepatocellular carcinoma (HCC) that develops from a background of non-alcoholic fatty liver disease (NAFLD). Hypercholesterolemia is a common comorbidity of obesity. Although cholesterol biosynthesis mainly occurs in the liver, its role in HCC development of obese people remains obscure. Using high-fat high-carbohydrate diet-associated orthotopic and spontaneous NAFLD-HCC mouse models, we found that hepatic cholesterol accumulation in obesity selectively suppressed natural killer T (NKT) cell-mediated antitumor immunosurveillance. Transcriptome analysis of human liver revealed aberrant cholesterol metabolism and NKT cell dysfunction in NAFLD patients. Notably, cholesterol-lowering rosuvastatin restored NKT expansion and cytotoxicity to prevent obesogenic diet-promoted HCC development. Moreover, suppression of hepatic cholesterol biosynthesis by a mammalian target of rapamycin (mTOR) inhibitor vistusertib preceded tumor regression, which was abolished by NKT inactivation but not CD8+ T cell depletion. Mechanistically, sterol regulatory element-binding protein 2 (SREBP2)-driven excessive cholesterol production from hepatocytes induced lipid peroxide accumulation and deficient cytotoxicity in NKT cells, which were supported by findings in people with obesity, NAFLD and NAFLD-HCC. This study highlights mTORC1/SREBP2/cholesterol-mediated NKT dysfunction in the tumor-promoting NAFLD liver microenvironment, providing intervention strategies that invigorating NKT cells to control HCC in the obesity epidemic.

Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis.

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.

Triptolide impairs thioredoxin system by suppressing Notch1-mediated PTEN/Akt/Txnip signaling in hepatocytes.

Triptolide (TP) is the main ingredient of Chinese herb Tripterygium wilfordii Hook f. (TWHF). Despite of its multifunction in pharmaceutics, accumulating evidences showed that TP caused obvious hepatotoxicity in clinic. The current study investigated the role of Notch1 signaling in TP-induced hepatotoxicity. Our data indicated that TP inhibited the protein expression of Notch1 and its active form Notch intracellular domain (NICD) leading to increased PTEN (phosphatase and tensin homolog deleted on chromosome ten) expression. Moreover, PTEN triggered Txnip (thioredoxin-interacting protein) activation by inhibiting Akt phosphorylation, which resulted in reduction of Trx (thioredoxin). In conclusion, TP caused liver injury through initiating oxidative stress in hepatocyte. This study indicated the potency of Notch1 to protect against TP-induced hepatotoxicity.

Multifunctionalization of cotton fabrics with polyvinylsilsesquioxane/ZnO composite coatings.

Ultraviolet (UV) shielding, superhydrophobic and antimicrobial cotton fabrics were fabricated using functional coatings combined with advantages of polyvinylsilsesquioxane and ZnO nanoparticles by solution immersion. The influence of composite coatings on surface morphology, water-repellence, UV shielding property, mechanical property, thermal degradation behavior and antibacterial property of the cotton fabrics was investigated respectively. It is evidently found that the cotton fabrics functionalized by composite coatings exhibited excellent UV shielding, durable superhydrophobic and antimicrobial properties as compared to the reference materials. Most notably, the mechanical properties of cotton fabrics was significantly improved by surface treatment of the composite coatings without compromising their thermal stability as compared to the pristine cotton fabric. This strategy for fabricating UV shielding and superhydrophobic cotton fabrics will guide for developing advanced functional textile in the future work, which will likely be found in many applications such as advanced protective textiles, oil/water separation, water-proof, antibacterial and self-cleaning fields.

Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition.

Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3ß (GSK-3ß) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3ß in TP-induced cardiotoxicity. Inhibition of GSK-3ß activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3ß overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3ß overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3ß interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3ß inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3ß could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP.

Activation of SIRT3 attenuates triptolide-induced toxicity through closing mitochondrial permeability transition pore in cardiomyocytes.

Triptolide (TP), an active component of the traditional Chinese herb Tripterygium wilfordii Hook f. (TWHF), has multiple pharmacological effects. However, the severe toxicity of TP greatly restricts its clinical applications. Although TP exposure causes serious heart injury, the mechanism underlying TP-induced cardiotoxicity has rarely been investigated. In previous studies, we found that TP-induced oxidative stress was involved in the mitochondria-dependent apoptosis of cardiomyocytes. Opening of the mitochondrial permeability transition pore (mPTP) is the key to the mitochondrial dysfunction in cardiac toxicity. The aim of this study was to investigate the potential cardioprotective effects of sirtuin 3 (SIRT3) on the mPTP. In the present study, the cytotoxicity of TP was accompanied by the up-regulation of the SIRT3 protein level and its rapid aggregation in nuclei and mitochondria. The SIRT3-FOXO3 signaling pathway was activated simultaneously, resulting in increased transcription of manganese superoxide dismutase (MnSOD) and catalase (CAT) for the elimination of reactive oxygen species (ROS). In addition, augmentation of the SIRT3 level via the overexpression plasmid SIRT3-Flag provided resistance to TP-induced cellular damage, whereas knocking down the SIRT3 level via siRNA accelerated the damage. Because it is an activator of SIRT3, the protective effect of resveratrol was also evaluated in H9c2 cells. In conclusion, the current results suggest that activation of SIRT3 substantially ameliorates the detrimental effects of TP by closing the mPTP.

Resveratrol protects against triptolide-induced cardiotoxicity through SIRT3 signaling pathway in vivo and in vitro.

Clinical application of triptolide (TP), a main active ingredient of the traditional Chinese herb Tripterygium wilfordii Hook f. (TWHF), is limited by a series of severe toxicities, including cardiotoxicity. In previous studies, we found the activation of sirtuin 3 (SIRT3) attenuated TP-induced toxicity in cardiomyocytes. Resveratrol (RSV), a polyphenol from the skins of grapes and red wine, is an activator of SIRT3. The current study aimed to investigate the protective effect of RSV against TP-induced cardiotoxicity and the underlying mechanisms. Mice were treated with a single dose of TP (2.5 mg/kg) via the intragastric (i.g.) route. After 24 h, TP induced abnormal changes of serum biochemistry, activity decrease of antioxidant enzymes and damage of heart tissue such as myocardial fiber rupture, cell swelling and interstitial congestion. In contrast, administration with RSV (50 mg/kg i.g. 12 h before and 2 h after the administration of TP) attenuated the detrimental effects induced by TP in BALB/c mice. Moreover, the cardiomyocyte protective effects of RSV on TP-induced heart injury were associated with the activation of SIRT3 and its downstream targets. In vitro study also indicated that RSV counteracted TP-induced cardiotoxicity through SIRT3-FOXO3 signaling pathway in H9c2 cells. Collectively, these findings suggest the potential of RSV as a promising agent in protecting heart from TP-induced damage.