FBXO38 mediates PD-1 ubiquitination and regulates anti-tumour immunity of T cells.

Dysfunctional T cells in the tumour microenvironment have abnormally high expression of PD-1 and antibody inhibitors against PD-1 or its ligand (PD-L1) have become commonly used drugs to treat various types of cancer1-4. The clinical success of these inhibitors highlights the need to study the mechanisms by which PD-1 is regulated. Here we report a mechanism of PD-1 degradation and the importance of this mechanism in anti-tumour immunity in preclinical models. We show that surface PD-1 undergoes internalization, subsequent ubiquitination and proteasome degradation in activated T cells. FBXO38 is an E3 ligase of PD-1 that mediates Lys48-linked poly-ubiquitination and subsequent proteasome degradation. Conditional knockout of Fbxo38 in T cells did not affect T cell receptor and CD28 signalling, but led to faster tumour progression in mice owing to higher levels of PD-1 in tumour-infiltrating T cells. Anti-PD-1 therapy normalized the effect of FBXO38 deficiency on tumour growth in mice, which suggests that PD-1 is the primary target of FBXO38 in T cells. In human tumour tissues and a mouse cancer model, transcriptional levels of FBXO38 and Fbxo38, respectively, were downregulated in tumour-infiltrating T cells. However, IL-2 therapy rescued Fbxo38 transcription and therefore downregulated PD-1 levels in PD-1+ T cells in mice. These data indicate that FBXO38 regulates PD-1 expression and highlight an alternative method to block the PD-1 pathway.

Prediction model for hyperprogressive disease in patients with advanced solid tumors received immune-checkpoint inhibitors: a pan-cancer study.

BACKGROUND: Hyper progressive disease (HPD) describes the phenomenon that patients can't benefit from immunotherapy but cause rapid tumor progression. HPD is a particular phenomenon in immunotherapy but lacks prediction methods. Our study aims to screen the factors that may forecast HPD and provide a predictive model for risky stratifying. METHODS: We retrospectively reviewed advanced-stage tumor patients who received immune checkpoint inhibitors (ICI) in the General PLA Hospital. Subsequently, we calculated the tumor growth kinetics ratio (TGKr) and identified typical HPD patients. Differences analysis of clinical characteristics was performed, and a predictive binary classification model was constructed. RESULTS: 867 patients with complete image information were screened from more than 3000 patients who received ICI between January 2015 and January 2020. Among them, 36 patients were identified as HPD for TGKr > 2. After the propensity score matched, confounding factors were limited. Survival analysis revealed that the clinical outcome of HPD patients was significantly worse than non-HPD patients. Besides, we found that Body Mass Index (BMI), anemia, lymph node metastasis in non-draining areas, pancreatic metastasis, and whether combined with anti-angiogenesis or chemotherapy therapy were closely connected with the HPD incidence. Based on these risk factors, we constructed a visualised predicted nomogram model, and the Area Under Curve (AUC) is 0.850 in the train dataset, whereas 0.812 in the test dataset. CONCLUSION: We carried out a retrospective study for HPD based on real-world patients and constructed a clinically feasible and practical model for predicting HPD incidence, which could help oncologists to stratify risky patients and select treatment strategies.

Potentiating the antitumour response of CD8(+) T cells by modulating cholesterol metabolism.

CD8(+) T cells have a central role in antitumour immunity, but their activity is suppressed in the tumour microenvironment. Reactivating the cytotoxicity of CD8(+) T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8(+) T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1, a key cholesterol esterification enzyme, led to potentiated effector function and enhanced proliferation of CD8(+) but not CD4(+) T cells. This is due to the increase in the plasma membrane cholesterol level of CD8(+) T cells, which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8(+) T cells were better than wild-type CD8(+) T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe, which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile, to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1, an established target for atherosclerosis, is therefore also a potential target for cancer immunotherapy.

SIRT4 is the molecular switch mediating cellular proliferation in colorectal cancer through GLS mediated activation of AKT/GSK3ß/CyclinD1 pathway.

Mitochondria-localized sirtuin 4 (SIRT4) is associated with malignant phenotypes in colorectal cancer (CRC). However, the molecular mechanisms that drive SIRT4-mediated carcinogenesis are unclear. Initially, we confirmed expression of SIRT4 in CRC through public database and in CRC patient tissues using quantitative real-time reverse transcription PCR. We established HCT116 colorectal cells that overexpressed SIRT4 and HT29 cells were transfected with plasmids bearing a small interfering RNA construct to silence SIRT4. Assays to determine the malignant phenotypes (proliferation, invasion and migration) were performed. Xenograft in vivo models were also constructed. A protein interactome network was built using differentially expressed proteins identified using the liquid chromatography/tandem mass spectrophotometry, the findings of which were confirmed using co-immunoprecipitation, western blotting and phenotype rescue experiments. Decreased SIRT4 expression was associated with malignant phenotypes in vitro and in vivo. The ribosomal biogenesis pathway was enriched in the interactome network. SIRT4 suppression activated glutaminase, thereby initiating AKT activation. Our research provided novel insights into the molecular mechanisms underlying CRC, and identified that SIRT4 exerts its antitumor activity in CRC possibly dependent on glutaminase to inhibit proliferation, migration and invasion via the AKT/GSK3ß/CyclinD1 pathway.

Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids.

Ionic protein-lipid interactions are critical for the structure and function of membrane receptors, ion channels, integrins and many other proteins. However, the regulatory mechanism of these interactions is largely unknown. Here we show that Ca(2+) can bind directly to anionic phospholipids and thus modulate membrane protein function. The activation of T-cell antigen receptor-CD3 complex (TCR), a key membrane receptor for adaptive immunity, is regulated by ionic interactions between positively charged CD3ε/ζ cytoplasmic domains (CD3(CD)) and negatively charged phospholipids in the plasma membrane. Crucial tyrosines are buried in the membrane and are largely protected from phosphorylation in resting T cells. It is not clear how CD3(CD) dissociates from the membrane in antigen-stimulated T cells. The antigen engagement of even a single TCR triggers a Ca(2+) influx and TCR-proximal Ca(2+) concentration is higher than the average cytosolic Ca(2+) concentration. Our biochemical, live-cell fluorescence resonance energy transfer and NMR experiments showed that an increase in Ca(2+) concentration induced the dissociation of CD3(CD) from the membrane and the solvent exposure of tyrosine residues. As a consequence, CD3 tyrosine phosphorylation was significantly enhanced by Ca(2+) influx. Moreover, when compared with wild-type cells, Ca(2+) channel-deficient T cells had substantially lower levels of CD3 phosphorylation after stimulation. The effect of Ca(2+) on facilitating CD3 phosphorylation is primarily due to the charge of this ion, as demonstrated by the fact that replacing Ca(2+) with the non-physiological ion Sr(2+) resulted in the same feedback effect. Finally, (31)P NMR spectroscopy showed that Ca(2+) bound to the phosphate group in anionic phospholipids at physiological concentrations, thus neutralizing the negative charge of phospholipids. Rather than initiating CD3 phosphorylation, this regulatory pathway of Ca(2+) has a positive feedback effect on amplifying and sustaining CD3 phosphorylation and should enhance T-cell sensitivity to foreign antigens. Our study thus provides a new regulatory mechanism of Ca(2+) to T-cell activation involving direct lipid manipulation.

The PI3K/AKT cell signaling pathway is involved in regulation of osteoporosis.

BACKGROUND: Osteoporosis is a systemic skeletal disease with the high incidence, serious complications, financial burden, and heavily decrease in living quality. METHODS: Proliferation of osteoblast was tested by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) method, alkaline phosphatase (ALP) activity of osteoblasts was tested by ALP REAGENT, Calcium level was determined by a colorimetric assay, mRNA expression of phosphoinositide-3 kinase (PI3K), 3-phosphoinositide-dependent protein kinase 1 (PDK1), Akt, Caspase-3, Caspase-7, Caspase-9, osteocalcin (OCN), Osterix and Runx2 of osteoblasts was tested by RNA preparation and quantitative reverse transcription polymerase chain reaction (RT-PCR), and protein expression of phospho-PI3K, phospho-PDK1 and phospho-Akt was measured by Western Blot analysis. RESULTS: In osteoporosis model rats, it found that mRNA expression of PI3K, PDK1 and Akt showed no changes while protein expression of phospho-PI3K, phospho-PDK1 and phospho-Akt in bone tissue was decreased dramatically. To further characterize the molecular mechanisms that regulate osteoporosis, we examined the contribution of the PI3K/Akt cell signaling pathway in cultured osteoblasts. It suggested that, the blockade of PI3K activation by LY294002, a specific inhibitor of the PI3K/Akt signaling pathway in osteoblasts, heavily inhibited cell proliferation, ALP activity, calcium accumulation, and mRNA expression of OCN, Osterix and Runx2. However, mRNA expression of Caspase-3 and Caspase-9 was promoted accordingly. CONCLUSION: The in vivo and in vitro studies indicated that the PI3K/Akt cell signaling pathway is involved in the inhibition of osteoporosis through promoting osteoblast proliferation, differentiation and bone formation.

[Effect of Shen warming Pi strengthening method on the expression of serum T cell subsets in IBS-D rats].

OBJECTIVE: To observe the effect of Shen warming Pi strengthening method on expressions of serum T cell subsets (C045+%, C03+%, and C04 +/COB+) in diarrhea-predominant irritable bowel syndrome (IBS-0) rats. Methods An IBS-0 rat model was established referring to AL-Chaer's modeling method combined with tail clamp and intragastric administration of sanna leaf. After modeling 30 SO rats were randomly divided into 6 groups according to random digit table, i.e., the model group, the high, middle, low dose Wenshen Jianpi Recipe (WJR) groups, and the Sishen Pill control group, 6 in each group. A normal control group consisting of 6 SO rats were also set up. Rats in high, middle, low dose WJ R groups were administered by gastrogavage with boil-free WJ R at the daily dose of 3. 100, 1. 550, 0. 775 g/kg, respectively. Rats in the Sis hen Pill control group were administered by gastrogavage with boil-free Sis hen Pill at the daily dose of 0. 736 g/kg. Equal volume of normal saline was given by gastrogavage to rats in the model group and the normal control group. All medication lasted for 2 successive weeks. Rats' general state, expressions of T cell subsets (CD45+%, CD3+%, and CD4+ /CDB+) changes were observed. RESULTS: Compared with the normal control group, expressions of CD45+% and CD3+% increased, but CD4+ /CDB+ decreased with statistical difference (P < 0. 05). Compared with the model group, expressions of CD45+% and CD3+% decreased, but CD4+ ICDB+ increased with statistical difference in high, middle, low dose WJR groups, and the Sis hen Pill control group (P <0. 05). Compared with the Sis hen Pill control group, there was statistical difference in all indices except CD45+ value in the low dose SWPSM group (P <0. 05). Compared with the low dose WJ R group, the expression of CD3+% decreased in high and middle dose WJR groups, and the Sis hen Pill control group; CD4+ /CD8+ increased in the Sishen Pill control group and the high dose SWPSM group (all P < 0. 05). CONCLUSIONS: WJR showed better treatment effect. The mechanism of Shen warming Pi strengthening method might be achieved by regulating expressions of CD45+% and CD3+%, and CD4+ /CD8+ ratios.

[Shen warming Pi strengthening method intervened IBS-D rats: an efficacy assessment].

OBJECTIVE: IBS-D rat model was established to assess the effect of Shen warming Pi strengthening method (SWPSM) for intervening diarrhea-predominant irritable bowel syndrome (IBS-D) by observing rats' general state, stool properties, AWR ranking, and histopathological changes. METHODS: Totally 72 rats were randomly divided into 6 groups, i.e. the normal group, the model group, the high, middle, low dose SWPSM groups, and the control group, 12 in each group. The IBS-D rat model was successfully established referring to AL-Chaer ED's modeling method. After modeling high, middle, and low dose SWPS Recipe boil-free granules were given by gastrogavage to rats in corresponding treatment groups. Sishen Pill boil-free granule was given by gastrogavage to those in the control group. Equal volume of normal saline was given by gastrogavage to rats in the model group. The medication lasted for 2 weeks. Rats' general state, stool properties, abdominal withdrawal reflex (AWR) ranking, and histopathological changes were observed. RESULTS: After treatment, the general state of all rats got im- provement to various degrees. The improvement in the high and middle dose SWPS Recipe groups were superior to that in the low dose SWPS Recipe group and the control group (P < 0.05). There was no statistical difference in the growth rate between after and before treatment in each group (P > 0.05). Compared with the model group and the low dose SWPS Recipe group, the defecation amount within 4 h was less in the high and middle dose SWPS Recipe groups and the control group (P < 0.05). The Bristol ranking score, average ranking of loose stool, ratio of dry stool and wet stool were lower in the high and middle dose SWPS Recipe groups than in the control group and the low dose SWPS Recipe group (P < 0.05). The AWR ranking score was lower in the high and middle dose SWPS Recipe groups than in the control group when the volume of balloon dilation was 1.5 mL. There was no organic change of histological or morphological observation. CONCLUSIONS: High sensitive IBS-D model was proved to be reliable. SWPSM could reduce the quantity of stools, lower Bristol ranking score, average ranking of loose stools as well as ratios of dry stool and wet stool, contributing to reducing the high sensitivity of rats' visceral organs to some extent.

Immunotherapy for advanced non-small cell lung cancer with negative programmed death-ligand 1 expression: a literature review.

Background and Objective: Lung cancer, mainly non-small cell lung cancer (NSCLC), is a serious threat to human life. In particular, the prognosis for advanced patients is poor, with the 5-year survival rate being exceedingly low. In recent years, immune checkpoint inhibition has changed the pattern of the treatment of a variety of cancers, including lung cancer; however, not all patients can benefit from immunotherapy, and thus finding the right biomarkers is particularly important for guiding precise treatment. Programmed death-ligand 1 (PD-L1) expression is one of the most valuable biomarkers for predicting the efficacy of lung cancer immunotherapy. Several studies have confirmed that patients with high PD-L1 expression are more likely to benefit from immunotherapy, but there is a high proportion of people with negative PD-L1 expression constituting a patient population that cannot be ignored. This article reviews the distribution of PD-L1 expression, the methods for evaluating PD-L1, and the effectiveness of immunotherapy for advanced NSCLC with negative PD-L1 expression. Methods: We performed a literature review to identify relevant data published until September 2022. In order to organize related information, we searched for literature in PubMed; abstracts and reports published in the American Society of Clinical Oncology (ASCO), the European Society for Medical Oncology (ESMO), the World Conference on Lung Cancer (WCLC), and other congresses; and clinical trial information registered on ClinicalTrials.gov. Information on the distribution of PD-L1 expression, detection of PD-L1, and immunotherapy efficacy for NSCLC with negative PD-L1 expression was collated and reviewed. Key Content and Findings: The incidence of PD-L1 expression in patients with stage IIIB/IV NSCLC is similar in all regions of the world, but PD-L1 expression level is associated with certain clinicopathological features. The expression of PD-L1 can be evaluated by various detecting methods. Some immunotherapy regimens have better efficacy than traditional chemotherapy in patients with negative PD-L1 expression. Conclusions: Patients with NSCLC and negative PD-L1 expression can receive better survival benefits under some immunotherapy types, and these may represent a better treatment option for this relatively small patient population.

Systematic pan-cancer analysis identified RASSF1 as an immunological and prognostic biomarker and validated in lung cancer.

Background: Ras association domain family member 1 (RASSF1) encodes the RASSF1A protein, serving as a scaffold protein situated at the intersection of a complex signalling network. Aims: To evaluate the immunological and prognostic significance of RASSF1 expression in various types of human cancers, with a specific focus on lung cancer. Methods: Differential expression analysis of RASSF1 was conducted based on data from The Cancer Genome Atlas, Genotype-Tissue Expression, and Cancer Cell Line Encyclopaedia databases. Prognostic analysis was performed using the Cox regression test and Kaplan-Meier test. Spearman's test was utilized for correlation analysis. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) gene sets were employed to enrich the associated signaling pathways. Immunohistochemical staining and quantitative real-time PCR were employed to detect protein and mRNA expression levels, respectively. Results: RASSF1 expression was significantly lower in tumour tissues than in normal tissues in most cancers, and Cox regression analysis demonstrated a significant correlation between RASSF1 expression and the prognosis of over 12 types of cancer. Specifically, high RASSF1 expression was associated with poor OS in nine cancer types, including GBMLGG (HR = 4.98, P = 1.2e-31), LGG (HR = 3.72, P = 2.5e-10), and LAML (HR = 1.48, P = 2.4e-3). Further analysis showed that RASSF1 expression was significantly correlated with immune checkpoint- and immune-related genes. Moreover, RASSF1 expression is involved in tumour microenvironment (TME), RNA modification, genomic heterogeneity, and tumour stemness. GO and KEGG analyses showed that RASSF1 was closely related to tumour immune-related pathways. Finally, RASSF1A was moderately correlated with PD-L1 (R = 0.556), and RASSF1A overexpression significantly affected the expression of several genes involved in the Th17 cell differentiation signalling pathway in lung cancer. Conclusions: RASSF1 was differentially expressed in 29 human cancers and played a critical role in tumour immunity. Thus, RASSF1 has the potential to be used as a prognostic marker and reference for achieving more precise immunotherapy, particularly in lung cancer.