ITPRIPL1 binds CD3ε to impede T cell activation and enable tumor immune evasion.

Cancer immunotherapy has transformed treatment possibilities, but its effectiveness differs significantly among patients, indicating the presence of alternative pathways for immune evasion. Here, we show that ITPRIPL1 functions as an inhibitory ligand of CD3ε, and its expression inhibits T cells in the tumor microenvironment. The binding of ITPRIPL1 extracellular domain to CD3ε on T cells significantly decreased calcium influx and ZAP70 phosphorylation, impeding initial T cell activation. Treatment with a neutralizing antibody against ITPRIPL1 restrained tumor growth and promoted T cell infiltration in mouse models across various solid tumor types. The antibody targeting canine ITPRIPL1 exhibited notable therapeutic efficacy against naturally occurring tumors in pet clinics. These findings highlight the role of ITPRIPL1 (or CD3L1, CD3ε ligand 1) in impeding T cell activation during the critical "signal one" phase. This discovery positions ITPRIPL1 as a promising therapeutic target against multiple tumor types.

Effects of 3-Hydroxy-3-methylglutaryl-CoA Reductase Inhibitors on Cholesterol Metabolism in Laying Hens.

This study aimed to investigate the effect of HMGCR inhibitors on egg yolk cholesterol content and its biological mechanisms. Four groups of 180-day-old laying hens (n = 8 cages/group, 6 laying hens/cage) were fed a corn/soybean-based diet (control) and the control diet supplemented with an HMGCR inhibitor at 60, 150, and 300 mg/kg for 4 weeks. The experimental results showed that adding HMGCR inhibitors of 150 mg/kg or more can significantly reduce the cholesterol content in the liver, yolk, serum, and pectoral muscles of laying hens. The RNA-seq results showed that compared with the control group, the addition of HMGCR inhibitors of 150 mg/kg or more to the diet significantly upregulated genes related to cholesterol synthesis in the liver, and the genes involved in steroid synthesis and metabolism, sterol synthesis and metabolism, and cholesterol synthesis and metabolism were all affected by the HMGCR inhibitors. In summary, adding HMGCR inhibitors of 150 mg/kg or more to the diet of hens can significantly reduce the cholesterol content in egg yolk. After the HMGCR inhibitors inhibited the activity of the liver HMGCR, they also altered the expression of genes related to cholesterol synthesis, bile acid synthesis, and cholesterol transport in the liver, and ultimately reduced cholesterol synthesis and cholesterol transport to the egg yolk.

Effects of Methyl Sulfonyl Methane and Selenium Yeast on Fatty Liver Syndrome in Laying Hens and Their Biological Mechanisms.

The purpose of this study was to explore the effects of MSM and Se-Y on FLS in laying hens during the late peak laying period and the underlying biological mechanisms. Therefore 240 55-week-old Jing-fen No. 6 laying hens were randomly divided into five groups, with eight replicates in each group and six laying hens in each replicate. The hens were fed a basal diet (Control) and diets supplemented with 350 and 700 mg/kg MSM and 25 and 50 mg/kg Se-Y, respectively, for four weeks. The results showed that MSM and Se-Y had no significant effects on the performance of laying hens. With the increasing dosage of MSM and Se-Y, the symptoms of liver steatosis in laying hens were reduced, and MSM and Se-Y could significantly reduce the content of malondialdehyde (MDA) in serum and liver (p < 0.05) and increase the contents of total superoxide dismutase (T-SOD) and glutathione peroxidase (GPX) in serum and liver (p < 0.05). The RNA-seq results showed that 700 mg/kg MSM significantly downregulated the expression levels of the ATP5I, ATP5G1, CYCS, and UQCRQ genes in the liver, and 50 mg/kg Se-Y significantly downregulated the expression levels of MAPK10, SRC, BMP2, and FGF9 genes in the liver. In conclusion, dietary supplementation with MSM and Se-Y can effectively reduce the FLS of laying hens in the late peak laying period and increase their antioxidant capacity. The underlying biological mechanism may be related to the downregulation of genes involved in liver oxidative phosphorylation and inflammation-related pathways.

Spatial scale effects of integrated landscape indicators on river water quality in Chaohu Lake basin, China.

Climate change and rapid urbanization have changed the characteristics of basin landscapes. Non-point-source (NPS) pollution affects river water quality. Exploring the impact of landscapes on river water quality is crucial for the control of water pollution in a basin. Current researchers focus on the impact of landscape pattern change on NPS pollution in the basin, but few consider climate, terrain, soil, and other geographical factors. In this study, we selected a subtropical agricultural basin in China named Chaohu Lake basin as the study area, added precipitation, soil erosion resistance, and slope to the original landscape pattern indicators. We quantified the spatial scale effect and seasonal dependence of integrated landscape indicators on water quality and comprehensively analyzed the optimal spatial scale and key landscape indicators. According to the nonlinear relationship between the key landscape indicators and river nutrients, we also determined the Type-1 threshold values of key landscape indicators for water quality protection in the basin. The results showed that the rivers in Chaohu Lake basin were mainly polluted by nitrogen and phosphorus. The strength of interpretation of the integrated landscape indicators of river water quality increased with riparian zone width. We determined the subbasin scale to be the optimal spatial scale. The key landscape indicators affecting water quality in the wet season at the optimal scale were precipitation and aggregation index of construction land (AIbul), whereas those in the dry season were AIbul and COHESION. The interpretation of the key landscape indicators in the wet season was slightly higher than that in the dry season. The above conclusions provide a scientific reference for NPS pollution control and water quality protection in subtropical agricultural basins.

Effect of zearalenone on aflatoxin B1-induced intestinal and ovarian toxicity in pregnant and lactating rats.

Aflatoxin B1 (AFB1) and zearalenone (ZEN) cause serious damage to mammals, but few studies have investigated the impacts of these toxins on pregnant and lactating mammals. This study investigated the effects of ZEN on AFB1-induced intestinal and ovarian toxicity in pregnant and lactating rats. Based on the results, AFB1 reduces the digestion, absorption, and antioxidant capacity in the intestine, increases intestinal mucosal permeability, destroys intestinal mechanical barriers, and increases pathogenic bacteria' relative abundances. Simultaneously, ZEN can exacerbate the intestinal injury caused by AFB1. The intestines of the offspring were also damaged, but the damage was less severe than that observed for the dams. While AFB1 activates various signalling pathways in the ovary and affects genes related to endoplasmic reticulum stress, apoptosis, and inflammation, ZEN may exacerbate or antagonize the AFB1 toxicity on gene expression in the ovary through key node genes and abnormally expressed genes. Our study found that mycotoxins can not only directly damage the ovaries and affect gene expression in the ovaries but can also impact ovarian health by disrupting intestinal microbes. Mycotoxins are an important environmental pathogenic factor for intestinal and ovarian disease in pregnancy and lactation mammals.

A peptidic inhibitor for PD-1 palmitoylation targets its expression and functions.

Programmed cell death protein 1 (PD-1) is a crucial anticancer target, but the relatively low response rate and acquired resistance to existing antibody drugs highlight an urgent need to develop alternative targeting strategies. Here, we report the palmitoylation of PD-1, discover the main DHHC enzyme for this modification, reveal the mechanism of its effect on PD-1 protein stability, and rationally develop a peptide for targeting PD-1 expression. Palmitoylation promoted the trafficking of PD-1 to the recycling endosome, thus preventing its lysosome-dependent degradation. Palmitoylation of PD-1, but not of PD-L1, promoted mTOR signaling and tumor cell proliferation, and targeting palmitoylation displayed significant anti-tumor effects in a three-dimensional culture system. A peptide was designed to competitively inhibit PD-1 palmitoylation and expression, opening a new route for developing PD-1 inhibitors and combinatorial cancer immunotherapy.

THADA drives Golgi residency and upregulation of PD-L1 in cancer cells and provides promising target for immunotherapy.

BACKGROUND: The abnormal upregulation of programmed death-ligand 1 (PD-L1) in cancer cells inhibits T cell-mediated cytotoxicity, but the molecular mechanisms that drive and maintain PD-L1 expression are still incompletely understood. METHODS: Combined analyses of genomes and proteomics were applied to find potential regulators of PD-L1. In vitro experiments were performed to investigate the regulatory mechanism of PD-L1 by thyroid adenoma associated gene (THADA) using human colorectal cancer (CRC) cells. The prevalence of THADA was analyzed using CRC tissue microarrays by immunohistochemistry. T cell killing assay, programmed cell death 1 binding assay and MC38 transplanted tumor models in C57BL/6 mice were developed to investigate the antitumor effect of THADA. RESULTS: THADA is critically required for the Golgi residency of PD-L1, and this non-redundant, coat protein complex II (COPII)-associated mechanism maintains PD-L1 expression in tumor cells. THADA mediated the interaction between PD-L1 as a cargo protein with SEC24A, a module on the COPII trafficking vesicle. Silencing THADA caused absence and endoplasmic reticulum (ER) retention of PD-L1 but not major histocompatibility complex-I, inducing PD-L1 clearance through ER-associated degradation. Targeting THADA substantially enhanced T cell-mediated cytotoxicity, and increased CD8+ T cells infiltration in mouse tumor tissues. Analysis on clinical tissue samples supported a potential role of THADA in upregulating PD-L1 expression in cancer. CONCLUSIONS: Our data reveal a crucial cellular process for PD-L1 maturation and maintenance in tumor cells, and highlight THADA as a promising target for overcoming PD-L1-dependent immune evasion.

Repurposing screen identifies Amlodipine as an inducer of PD-L1 degradation and antitumor immunity.

Cancer cell expression of PD-L1 leads to T cells exhaustion by transducing co-inhibitory signal, and further understanding the regulation of PD-L1 in cancer cells may provide additional therapeutic strategies. Here by drug repurposing screen, we identified amlodipine as a potent inhibitor of PD-L1 expression in cancer cells. Further survey of calcium-associated pathways revealed calpain-dependent stabilization of the PD-L1 protein. Intracellular calcium delivered an operational signal to calpain-dependent Beclin-1 cleavage, blocking autophagic degradation of PD-L1 accumulated on recycling endosome (RE). Blocking calcium flux by amlodipine depleted PD-L1 expression and increased CD8+ T-cell infiltration in tumor tissues but not in myocardium, causing dose-dependent tumor suppression in vivo. Rescuing PD-L1 expression eliminated the effects of amlodipine, suggesting the PD-L1-dependent effect of amlodipine. These results reveal a calcium-dependent mechanism controlling PD-L1 degradation, and highlight calcium flux blockade as a potential strategy for combinatorial immunotherapy.

SPATS2, negatively regulated by miR-145-5p, promotes hepatocellular carcinoma progression through regulating cell cycle.

Spermatogenesis associated serine rich 2 (SPATS2) has been reported to contribute to the tumorigenesis of multiple malignancies. The molecular function of SPATS2 in hepatocellular carcinoma (HCC) is still not fully understood. In this study, we aimed to investigate the expression pattern and function roles of SPATS2 in HCC. The regulation of SPATS2 expression was also explored. We found that SPATS2 was highly expressed in HCC tissues in comparison with that in adjacent normal tissues. High expression of SPATS2 was associated with vascular invasion, advanced TNM stages, tumor multiplicity, and poor survival. Functionally, SPATS2 was found to promote the proliferation and metastasis of HCC cells both in vitro and in vivo, while knockdown of SPATS2 enhanced apoptosis and G1 arrest of HCC cells in vitro. Mechanistically, bioinformatics analysis revealed that MiR-145-5p directly targeted SPATS2 and functional rescue experiments verified that MiR-145-5p overexpression could abolish the effect of SPATS2 on the regulation of HCC malignant phenotype. Taken together, our findings suggest that SPATS2 functions as an oncogene in HCC. The MiR-145-5p/SPATS2 axis provides a novel mechanism underlying HCC progression and may serve as a potential therapeutic target for HCC.

Lysosome as the Black Hole for Checkpoint Molecules.

Lysosomes, as digestive organelles full of hydrolases, have complex functions and play an important role in cellular physiological and pathological processes. In normal physiological conditions, lysosomes can sense the nutritional state and be responsible for recycling raw materials to provide nutrients, affecting cell signaling pathways and regulating cell proliferation. Lysosomes are related to many diseases and associated with metastasis and drug resistance of tumors. In recent years, much attention has been paid to the tumor immunotherapy especially immune checkpoint blockade therapy. Accumulating data suggest that lysosomes may serve as a major destruction for immune checkpoint molecules, and secretory lysosomes can temporarily store immune checkpoint proteins. Once activated, the compounds contained in secretory lysosomes are released to the surface of cell membrane rapidly. Inhibitions of lysosomes can overcome the chemoresistance of some tumors and enhance the efficacy of immunotherapy.

Author Correction: Inhibiting PD-L1 palmitoylation enhances T-cell immune responses against tumours.

In the version of this Article originally published, 'palmitoyltransferase ZDHHC3 (DHHC3)' was incorrectly referred to as an 'acetyltransferase' rather than an as an 'acyltransferase'; this has now been corrected in five instances. In Fig. 3a, the label for the bottom row of the blots was mistakenly written as 'GAPHD'; it should have read 'GAPDH'. In the two right-most panels of Fig. 4j, the antibody labels 'α-PD-L1' for the reciprocal co-immunoprecipitation of DHHC3 were incorrect; they should have been 'α-DHHC3'. These errors have been corrected in all versions of the Article.

Inhibiting PD-L1 palmitoylation enhances T-cell immune responses against tumours.

Checkpoint blockade therapy targeting the programmed-death ligand 1 (PD-L1) and its receptor programmed cell death 1 promotes T-cell-mediated immunosurveillance against tumours, and has been associated with marked clinical benefit in cancer patients. Antibodies against PD-L1 function by blocking PD-L1 on the cell surface, but intracellular storage of PD-L1 and its active redistribution to the cell membrane can minimize the therapeutic benefits, which highlights the importance of targeting PD-L1 throughout the whole cell. Here, we show that PD-L1 is palmitoylated in its cytoplasmic domain, and that this lipid modification stabilizes PD-L1 by blocking its ubiquitination, consequently suppressing PD-L1 degradation by lysosomes. We identified palmitoyltransferase ZDHHC3 (DHHC3) as the main acetyltransferase required for the palmitoylation of PD-L1, and show that the inhibition of PD-L1 palmitoylation via 2-bromopalmitate, or the silencing of DHHC3, activates antitumour immunity in vitro and in mice bearing MC38 tumour cells. We also designed a competitive inhibitor of PD-L1 palmitoylation that decreases PD-L1 expression in tumour cells to enhance T-cell immunity against the tumours. These findings suggest new strategies for overcoming PD-L1-mediated immune evasion in cancer.

HIP1R targets PD-L1 to lysosomal degradation to alter T cell-mediated cytotoxicity.

Expression of programmed cell death 1 (PD-1) ligand 1 (PD-L1) protects tumor cells from T cell-mediated immune surveillance, and immune checkpoint blockade (ICB) therapies targeting PD-1 and PD-L1 have exhibited significant clinical benefits. However, the relatively low response rate and observed ICB resistance highlight the need to understand the molecular regulation of PD-L1. Here we show that HIP1R targets PD-L1 to lysosomal degradation to alter T cell-mediated cytotoxicity. HIP1R physically interacts with PD-L1 and delivers PD-L1 to the lysosome through a lysosomal targeting signal. Depletion of HIP1R in tumor cells caused PD-L1 accumulation and suppressed T cell-mediated cytotoxicity. A rationally designed peptide (PD-LYSO) incorporating the lysosome-sorting signal and the PD-L1-binding sequence of HIP1R successfully depleted PD-L1 expression in tumor cells. Our results identify the molecular machineries governing the lysosomal degradation of PD-L1 and exemplify the development of a chimeric peptide for targeted degradation of PD-L1 as a crucial anticancer target.

Cancer Cell-Intrinsic PD-1 and Implications in Combinatorial Immunotherapy.

Programmed death 1 (PD-1) and its two natural ligands PD-L1 and PD-L2 are responsible for delivering inhibitory signals that regulate the balance between T cell activation, tolerance, and immunopathology. In previous studies, PD-1 was found only expressed on the surface of immune cells, such as T cells and B cells while PD-1's ligands PD-L1 and PD-L2 were found expressed in some tumor cells. However, recent studies revealed intrinsic expression of PD-1 in melanoma and some other cancers. In melanoma cells, PD-1 can be activated by its ligand PD-L1 expressed by tumor cells, modulating downstream mammalian target of rapamycin signaling and promoting tumor growth independent of adaptive immunity. In addition to melanoma, PD-1 was also detected in liver cancer cells as well as in non-small lung cancer cells. Unlike its oncogenic functions in melanoma and hepatic carcinoma cells, PD-1 seemed to play a distinct role in lung cancer, as blockade of PD-1 instead promoted tumor cells proliferation. Tumor-intrinsic PD-1 expression seems to be widespread in many tumor types, according to our reanalysis on cancer transcriptomic and proteomic data. The multifaceted roles of PD-1 in tumor cells beyond immune checkpoint signaling may explain the differential therapeutic effects of anti-PD-1 and anti-PD-L1 drugs and provide crucial information when developing combinatorial approaches to enhance antitumor immunity.

Regulation of PD-L1: Emerging Routes for Targeting Tumor Immune Evasion.

Immune checkpoint blockade therapies (ICBTs) targeting programmed cell death 1 (PD-1) and its ligand programmed death ligand-1 (PD-L1/B7-H1/CD274) have exhibited momentous clinical benefits and durable responses in multiple tumor types. However, primary resistance is found in considerable number of cancer patients, and most responders eventually develop acquired resistance to ICBT. To tackle these challenges, it is essential to understand how PD-L1 is controlled by cancer cells to evade immune surveillance. Recent research has shed new light into the mechanisms of PD-L1 regulation at genetic, epigenetic, transcriptional, translational, and posttranslational levels. In this work, we systematically discuss the mechanisms that control the gene amplification, epigenetic alteration, transcription, subcellular transportation and posttranscriptional modification of PD-L1 in cancer cells. We further categorize posttranscriptional PD-L1 regulations by the molecular modification of PD-L1, including glycosylation, phosphorylation, ubiquitination, deubiquitination, and lysosomal degradation. These findings may provide new routes for targeting tumor immune escape and catalyze the development of small molecular inhibitors of PD-L1 in addition to existing antibody drugs.

A Designed Peptide Targets Two Types of Modifications of p53 with Anti-cancer Activity.

Many cancer-related proteins are controlled by composite post-translational modifications (PTMs), but prevalent strategies only target one type of modification. Here we describe a designed peptide that controls two types of modifications of the p53 tumor suppressor, based on the discovery of a protein complex that suppresses p53 (suppresome). We found that Morn3, a cancer-testis antigen, recruits different PTM enzymes, such as sirtuin deacetylase and ubiquitin ligase, to confer composite modifications on p53. The molecular functions of Morn3 were validated through in vivo assays and chemico-biological intervention. A rationally designed Morn3-targeting peptide (Morncide) successfully activated p53 and suppressed tumor growth. These findings shed light on the regulation of protein PTMs and present a strategy for targeting two modifications with one molecule.

ASAP3 regulates microvilli structure in parietal cells and presents intervention target for gastric acidity.

Gastric acidity-associated disorders such as peptic ulcer and reflux diseases are widespread, and the reported resistance and side effects of currently used medicines suggest an urgent requirement for alternative therapeutic approaches. Here we demonstrate a critical role of ASAP3 in regulating the microvilli structure of parietal cells in vivo, and reveal the feasibility of controlling gastric acidity by targeting ASAP3. Conditional knockout of ASAP3 in mice caused elongation and stacking of microvilli in parietal cells, and substantially decreased gastric acid secretion. These were associated with active assembly of F-actin caused by a higher level of GTP-bound Arf6 GTPase. Consistently, a small molecular compound QS11 inhibited ASAP3 function and significantly reduced gastric acidity in vivo. Of note, the expression of ASAP3 was positively correlated with gastric acid secretion in 90 human cases, and high expression of ASAP3 was associated with reflux disease and peptic ulcer. These results reveal for the first time that ASAP3 regulates the microvilli structures in parietal cells. Our data also suggest ASAP3 as a feasible and drugable therapeutic target for gastric acidity-associated diseases.

Scaffold Proteins in Gastrointestinal Tumors as a Shortcut to Oncoprotein Activation.

BACKGROUND: The development of cancer involves uncontrolled cell proliferation, and multiple signaling pathways that regulate cell proliferation have been found to be dysregulated in cancers. Extracellular signal-regulated protein kinase (ERK) is one of three major subtypes in the mitogen-activated protein kinase (MAPK) families. The MAPK/ERK pathway (RAS/RAF1/MEK/ERK) plays an important part in promoting cell proliferation in response to growth factors, thereby serving as a driving signal in gastrointestinal (GI) tumors. In contrast, the p53 tumor suppressor functions as a "guardian of the genome" and stops cell proliferation when oncogenic signaling is activated. SUMMARY: Both pathways constrain each other in healthy GI epithelium, facilitating controlled proliferation that is essential for tissue repair and regeneration. However, in GI tumors, the MAPK/ERK and p53 pathways are commonly dysregulated, in part due to abnormal posttranslational modifications. Hyperphosphorylation of the ERK protein causes sustained activation of cell proliferation, whereas hypoacetylation of the p53 protein impairs its transcriptional function and blocks cell apoptosis. Multiple scaffold proteins have been found to regulate the posttranslational modifications of ERK and p53 proteins in GI tumors. KEY MESSAGE: Abnormal expression of scaffold proteins may contribute to the dysregulation of the MAPK and p53 signaling pathways and thereby contribute to the development of GI tumors. PRACTICAL IMPLICATIONS: Scaffold proteins are potential biomarkers and therapeutic targets in GI tumors.

PD-L2 expression in colorectal cancer: Independent prognostic effect and targetability by deglycosylation.

Colorectal cancer (CRC) is the second leading cause of cancer death worldwide, and immune checkpoint blockade therapy provides an opportunity for improving the outcome of CRC patients. Recent studies suggest that programmed death ligand-1 (PD-L1) is only expressed in 12% of CRCs. Here, we demonstrate that PD-L2 is expressed in approximately 40% CRCs, and its expression independently associates with poor survival of CRC patients. By detection of PD-L2 expression by immunofluorescence in 124 CRC cases with 10-y survival data, we found significant association between PD-L2 overexpression in cancer cells and worse overall survival (46.3 vs 69.1 mo; p = 0.0004). The association remained significant in multivariate COX regression analysis (hazard ratio = 2.778, 95% confidence interval [CI] = 1.668-4.627; p < 0.0001). In the validation CRC data set, significant association between PD-L2 overexpression and poor survival was supported by the univariate analysis (27.1 vs. 88.9 mo; p = 0.0002) and multivariate model (hazard ratio = 7.09, 95%CI 1.78-28.16; p = 0.005). Western Blot revealed strong induction of PD-L2 expression by interferon-γ (IFNγ) in CRC cells, and the mRNA levels of both genes were significantly correlated in CRC tissue samples. Suppression of glycosylation with tunicamycin caused a shift in molecular weight and significant decrease in the expression of PD-L2 protein. In conclusion, PD-L2 overexpression in CRC cells, under the regulation by IFNγ and glycosylation, associates with poor survival of patients with colorectal cancer. These findings highlight PD-L2 as a promising therapeutic target in CRC and suggest potential routes to control PD-L2 expression in CRC cells.

OncoBinder facilitates interpretation of proteomic interaction data by capturing coactivation pairs in cancer.

High-throughput methods such as co-immunoprecipitationmass spectrometry (coIP-MS) and yeast 2 hybridization (Y2H) have suggested a broad range of unannotated protein-protein interactions (PPIs), and interpretation of these PPIs remains a challenging task. The advancements in cancer genomic researches allow for the inference of "coactivation pairs" in cancer, which may facilitate the identification of PPIs involved in cancer. Here we present OncoBinder as a tool for the assessment of proteomic interaction data based on the functional synergy of oncoproteins in cancer. This decision tree-based method combines gene mutation, copy number and mRNA expression information to infer the functional status of protein-coding genes. We applied OncoBinder to evaluate the potential binders of EGFR and ERK2 proteins based on the gastric cancer dataset of The Cancer Genome Atlas (TCGA). As a result, OncoBinder identified high confidence interactions (annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) or validated by low-throughput assays) more efficiently than co-expression based method. Taken together, our results suggest that evaluation of gene functional synergy in cancer may facilitate the interpretation of proteomic interaction data. The OncoBinder toolbox for Matlab is freely accessible online.