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Abstract Phagocytosis, a vital defense mechanism, involves the recognition and elimination of foreign substances by cells. Phagocytes, such as neutrophils and macrophages, rapidly respond to invaders; macrophages are especially important in later stages of the immune response. They detect “find me” signals to locate apoptotic cells and migrate toward them. Apoptotic cells then send “eat me” signals that are recognized by phagocytes via specific receptors. “Find me” and “eat me” signals can be strategically harnessed to modulate antitumor immunity in support of cancer therapy. These signals, such as calreticulin and phosphatidylserine, mediate potent pro‐phagocytic effects, thereby promoting the engulfment of dying cells or their remnants by macrophages, neutrophils, and dendritic cells and inducing tumor cell death. This review summarizes the phagocytic “find me” and “eat me” signals, including their concepts, signaling mechanisms, involved ligands, and functions. Furthermore, we delineate the relationships between “find me” and “eat me” signaling molecules and tumors, especially the roles of these molecules in tumor initiation, progression, diagnosis, and patient prognosis. The interplay of these signals with tumor biology is elucidated, and specific approaches to modulate “find me” and “eat me” signals and enhance antitumor immunity are explored. Additionally, novel therapeutic strategies that combine “find me” and “eat me” signals to better bridge innate and adaptive immunity in the treatment of cancer patients are discussed.

BackgroundRegorafenib, a tyrosine kinase inhibitor (TKI), is used in the treatment of unresectable hepatocellular carcinoma (HCC). However, the occurrence of acquired resistance limits its antitumor efficacy. While multiple studies have highlighted the crucial role of bypass activation in acquired TKI resistance, few have focused on bypass activation in regorafenib resistance in HCC.MethodsHigh-throughput proteomics was used to identify differential proteins associated with bypass activation between acquired regorafenib-resistant cells and parental cells. The ability of epidermal growth factor receptor (EGFR) bypass inhibition to reverse resistance was evaluated both in vitro and in vivo using direct microscopic observation, the CCK-8 assay, colony formation assay, Annexin V-FITC/propidium iodide double staining, cell cycle analysis, western blotting, and a xenograft model.ResultsThe expression of EGFR, a member of the receptor tyrosine kinase (RTK) family, was significantly increased in acquired regorafenib-resistant HCC cells compared with parental cells. Pharmacological inhibition of EGFR with gefitinib restored the sensitivity of regorafenib-resistant HCC cells to regorafenib. In a xenograft mouse model, gefitinib sensitized resistant tumors to regorafenib. Additionally, levels of RAS, RAF, and P-ERK1/2, components of the downstream EGFR signaling pathway, were positively associated with EGFR expression.ConclusionEGFR overexpression promotes acquired resistance to regorafenib through RAS/RAF/ERK bypass activation in HCC. Inhibition of EGFR restores sensitivity to regorafenib, and the combination of gefitinib and regorafenib demonstrates significant antitumor efficacy both in vivo and in vitro. These findings suggest that this combination could be a potential strategy for patients with advanced HCC.

Background and Purpose: Regorafenib was approved by the US Food and Drug Administration (FDA) for hepatocellular carcinoma (HCC) patients showing progress on sorafenib treatment. However, there is an inevitably high rate of drug resistance associated with regorafenib, which reduces its effectiveness in clinical treatment. Thus, there is an urgent need to find a potential way to solve the problem of regorafenib resistance. The metabolite of disulfiram complexed with copper, the Diethyldithiocarbamate-copper complex (CuET), has been found to be an effective anticancer drug candidate. In the present study, we aimed to evaluate the effect of CuET on regorafenib resistance in HCC and uncover the associated mechanism. Experimental Approach: Regorafenib-resistant HCC strains were constructed by applying an increasing concentration gradient. This study employed a comprehensive range of methodologies, including the cell counting kit-8 (CCK-8) assay, colony formation assay, cell cycle analysis, wound healing assay, Transwell assay, tumor xenograft model, and immunohistochemical analysis. These methods were utilized to investigate the antitumor activity of CuET, assess the combined effect of regorafenib and CuET, and elucidate the molecular mechanism underlying CuET-mediated regorafenib resistance. Key Results: The inhibitory effect of regorafenib on cell survival, proliferation and migration was decreased in regorafenib-resistant MHCC-97H (MHCC-97H/REGO) cells compared with parental cells. CuET demonstrated significant inhibitory effects on cell survival, proliferation, and migration of various HCC cell lines. CuET restored the sensitivity of MHCC-97H/REGO HCC cells to regorafenib in vitro and in vivo. Mechanistically, CuET reverses regorafenib resistance in HCC by suppressing epithelial-mesenchymal transition (EMT) through inhibition of the ERK signaling pathway. Conclusion and Implications: Taken together, the results of this study demonstrated that CuET inhibited the activation of the ERK signaling pathway, leading to the suppression of the epithelial-mesenchymal transition (EMT) and subsequently reversing regorafenib resistance in HCC both in vivo and in vitro. This study provides a new idea and potential strategy to improve the treatment of regorafenib-resistant HCC.

The important role of cholesterol in tumor metastasis has been widely studied in recent years. Ezetimibe is currently the only selective cholesterol uptake inhibitor on the market. Here, we explored the effect of ezetimibe on breast cancer metastasis by studying its impact on breast cancer cell migration, invasion, and epithelial‐mesenchymal transition (EMT). Differential gene expression analysis and validation were also carried out to compare ezetimibe‐treated and untreated breast cancer cells. Finally, breast cancer cells overexpressing TGFβ2 were constructed, and the effect of TGFβ2 on the migration and invasion of ezetimibe‐treated breast cancer cells was examined. Our results show that ezetimibe treatment of breast cancer cells inhibited cell migration, invasion, and EMT, and it significantly suppressed the expression of TGFβ2. Overexpression of TGFβ2 reversed the inhibitory effect of ezetimibe on the migration and invasion of breast cancer cells. Taken together, our results suggest that ezetimibe might be a potential candidate for the treatment of breast cancer metastasis.

ABSTRACTColorectal cancer (CRC) is one of the most lethal cancers. Single-cell RNA sequencing (scRNA-seq) and protein-protein interactions (PPIs) have enabled the systematic study of CRC. In our research, the activation of the AKT pathway in CRC was analyzed by KEGG using single-cell sequencing data from the GSE144735 dataset. The correlation and PPIs of MDFI and ITGB4/LAMB3 were examined. The results were verified in the TCGA and CCLE and further tested by coimmunoprecipitation experiments. The effect of MDFI on the AKT pathway via ITGB4/LAMB3 was validated by knockdown and lentiviral overexpression experiments. The effect of MDFI on oxaliplatin/fluorouracil sensitivity was probed by colony formation assay and CCK8 assay. We discovered that MDFI was positively associated with ITGB4/LAMB3. In addition, MDFI was negatively associated with oxaliplatin/fluorouracil sensitivity. MDFI upregulated the AKT pathway by directly interacting with LAMB3 and ITGB4 in CRC cells, and enhanced the proliferation of CRC cells via the AKT pathway. Finally, MDFI reduced the sensitivity of CRC cells to oxaliplatin and fluorouracil. In conclusion, MDFI promotes the proliferation and tolerance to chemotherapy of colorectal cancer cells, partially through the activation of the AKT signaling pathway by the binding to ITGB4/LAMB3. Our findings provide a possible molecular target for CRC therapy.

Abstract Immunotherapy is one of the fastest developing areas in the field of oncology. Many immunological treatment strategies for refractory tumors have been approved and marketed. Nevertheless, much clinical and preclinical experimental evidence has shown that the efficacy of immunotherapy in tumor treatment varies markedly among individuals. The commensal microbiome mainly colonizes the intestinal lumen in humans, is affected by a variety of factors and exhibits individual variation. Moreover, the gut is considered the largest immune organ of the body due to its influence on the immune system. In the last few decades, with the development of next-generation sequencing (NGS) techniques and in-depth research, the view that the gut microbiota intervenes in antitumor immunotherapy through the immune system has been gradually confirmed. Here, we review important studies published in recent years focusing on the influences of microbiota on immune system and the progression of malignancy. Furthermore, we discuss the mechanism by which microbiota affect tumor immunotherapy, including immune checkpoint blockade (ICB) and adoptive T-cell therapy (ACT), and strategies for modulating the microbial composition to facilitate the antitumor immune response. Finally, opportunity and some challenges are mentioned to enable a more systematic understanding of tumor treatment in the future and promote basic research and clinical application in related fields.

Abstract A fundamental goal in cancer-associated genome sequencing is to identify the key genes. Protein–protein interactions (PPIs) play a crucially important role in this goal. Here, human reference interactome (HuRI) map was generated and 64,006 PPIs involving 9094 proteins were identified. Here, we developed a physical link and co-expression combinatory network construction (PLACE) method for genes of interest, which provides a rapid way to analyze genome sequencing datasets. Next, Kaplan‒Meier survival analysis, CCK8 assays, scratch wound assays and Transwell assays were applied to confirm the results. In this study, we selected single-cell sequencing data from patients with hepatocellular carcinoma (HCC) in GSE149614. The PLACE method constructs a protein connection network for genes of interest, and a large fraction (80%) of the genes (screened by the PLACE method) were associated with survival. Then, PLACE discovered that transmembrane protein 14B (TMEM14B) was the most significant prognostic key gene, and target genes of TMEM14B were predicted. The TMEM14B-target gene regulatory network was constructed by PLACE. We also detected that TMEM14B-knockdown inhibited proliferation and migration. The results demonstrate that we proposed a new effective method for identifying key genes. The PLACE method can be used widely and make outstanding contributions to the tumor research field.

Cholesterol levels were strongly associated with tumor progression and metastasis. Targeted cholesterol metabolism has broad prospects in tumor treatment. Ezetimibe, the only FDA-approved inhibitor of cholesterol absorption, has been reported to be able to inhibit angiogenesis in liver cancer. However, the efficacy and specific mechanisms of Ezetimibe in the treatment of Triple-Negative Breast Cancer (TNBC)have not been reported. Our research shows Ezetimibe inhibits TNBC cell proliferation and blocks the cell cycle in the G1 phase. Mechanistically, Ezetimibe inhibits the activation of PDGFRβ/AKT pathway, thereby promoting cell cycle arrest and inhibiting cell proliferation. By overexpressing PDGFRβ in TNBC cells, we found that PDGFRβ significantly reduced the inhibitory effect of Ezetimibe on TNBC cell proliferation and the cell cycle. Similarly, SC79, an AKT agonist, can reduce the proliferation inhibitory and cycle-blocking effects of Ezetimibe on TNBC cells. Furthermore, the AKT inhibitor MK2206 enhanced the inhibitory effect of Ezetimibe on the cell cycle and proliferation ability of TNBC cells overexpressing PDGFRβ. In xenograft tumor models, we also found that Ezetimibe inhibited TNBC growth, an effect that can be blocked by overexpression of PDGFR or activation of AKT. In summary, we have demonstrated that EZ inhibits the PDGFR/AKT pathway, thereby halting TNBC cycle progression and tumor growth.

Peptides can be used as effective molecular tool for covalent modification of proteins and play important roles in ligand directed covalent modification. Tyr-selective protein modifications exert a profound impact on protein functionality. Here, we developed a general strategy that involves nucleophilic addition of alkyne for tyrosine modification. The terminal alkyne of propargyl sulfonium is motivated by the sulfonium center to react with phenolic hydroxyl. This approach provides a straightforward method for tyrosine modification due to its high yield in aqueous solution at physiological temperature. In addition, cyclic peptides could be obtained via adjusting pH to 8.0 from peptides consisting of tyrosine and methionine modified by propargyl bromide, and the resulting cyclic peptides are proved to have better stability, excellent 2-mercaptopyridine resistance and improved cellular uptakes. Furthermore, molecules made from the propargylated sulfonium have the potential to be used as warheads against tyrosine containing biomolecules. Collectively, we develop a direct and uncomplicated technique for modifying tyrosine residues, the strategy concerned can be widely utilized to construct stable peptides and biomolecules imaging.

Abstract Renal clear cell carcinoma (RCCC) is the most common type of renal cell carcinoma, which is also difficult to diagnose and easy to metastasize. Currently, there is still a lack of effective clinical diagnostic indicators and treatment targets. This study aims to find effective diagnostic markers and therapeutic targets from the perspective of noncoding RNA. In this study, we found that the expression of Long noncoding RNA LINC00472 was significantly decreased in RCCC and showed a downward trend with the progression of cancer stage. Patients with low LINC00472 expression have poor prognosis. Inhibition of LINC00472 significantly increased cell proliferation and migration, while overexpression of LINC00472 obviously inhibited cell proliferation and enhanced intercellular adhesion. Transcriptome sequencing analysis demonstrated that LINC00472 was highly correlated with extracellular matrix and cell metastasis-related pathways, and the consistent results were obtained by The Cancer Genome Atlas (TCGA) data analysis. Additionally, we discovered that the integrin family protein ITGB8 is a potential target gene of LINC00472. Mechanistically, we found that the change of LINC00472 affected the acetylation level of H3K27 site in cells, and we speculate that this effect is likely to be generated through the interaction with acetyltransferase P300. In conclusion, LINC00472 has an important impact on the proliferation and metastasis of renal clear cells, and probably participate in the regulation of histone modification, and it may be used as a potential diagnostic marker of RCCC.

ObjectiveThis study aims to identify prognostic factors for low-grade glioma (LGG) via different machine learning methods in the whole genome and to predict patient prognoses based on these factors. We verified the results through in vitro experiments to further screen new potential therapeutic targets.MethodA total of 940 glioma patients from The Cancer Genome Atlas (TCGA) and The Chinese Glioma Genome Atlas (CGGA) were included in this study. Two different feature extraction algorithms – LASSO and Random Forest (RF) – were used to jointly screen genes significantly related to the prognosis of patients. The risk signature was constructed based on these screening genes, and the K-M curve and ROC curve evaluated it. Furthermore, we discussed the differences between the high- and low-risk groups distinguished by the signature in detail, including differential gene expression (DEG), single-nucleotide polymorphism (SNP), copy number variation (CNV), immune infiltration, and immune checkpoint. Finally, we identified the function of a novel molecule, METTL7B, which was highly correlated with PD-L1 expression on tumor cell, as verified by in vitro experiments.ResultsWe constructed an accurate prediction model based on seven genes (AUC at 1, 3, 5 years= 0.91, 0.85, 0.74). Further analysis showed that extracellular matrix remodeling and cytokine and chemokine release were activated in the high-risk group. The proportion of multiple immune cell infiltration was upregulated, especially macrophages, accompanied by the high expression of most immune checkpoints. According to the in vitro experiment, we preliminarily speculate that METTL7B affects the stability of PD-L1 mRNA by participating in the modification of m6A.ConclusionThe seven gene signatures we constructed can predict the prognosis of patients and identify the potential benefits of immune checkpoint inhibitors (ICI) therapy for LGG. More importantly, METTL7B, one of the risk genes, is a crucial molecule that regulates PD-L1 and could be used as a new potential therapeutic target.

Lipotoxicity is an important factor in the development and progression of nonalcoholic steatohepatitis. Excessive accumulation of saturated fatty acids can increase the substrates of the mitochondrial electron transport chain in hepatocytes and cause the generation of reactive oxygen species, resulting in oxidative stress, mitochondrial dysfunction, loss of mitochondrial membrane potential, impaired triphosphate (ATP) production, and fracture and fragmentation of mitochondria, which ultimately leads to hepatocellular inflammatory injuries, apoptosis, and necrosis. In this study, we systematically investigated the effects and molecular mechanisms of empagliflozin on lipotoxicity in palmitic acid-treated LO2 cell lines. We found that empagliflozin protected hepatocytes and inhibited palmitic acid-induced lipotoxicity by reducing oxidative stress, improving mitochondrial functions, and attenuating apoptosis and inflammation responses. The mechanistic study indicated that empagliflozin significantly activated adenosine 5’-monophosphate (AMP)-activated protein kinase alpha (AMPKα) through Calcium/Calmodulin dependent protein kinase kinase beta (CAMKK2) instead of liver kinase B1 (LKB1) or TGF-beta activated kinase (TAK1). The activation of empagliflozin on AMPKα not only promoted FoxO3a phosphorylation and thus forkhead box O 3a (FoxO3a) nuclear translocation, but also promoted Nrf2 nuclear translocation. Furthermore, empagliflozin significantly upregulated the expressions of antioxidant enzymes superoxide dismutase (SOD) and HO-1. In addition, empagliflozin did not attenuate lipid accumulation at all. These results indicated that empagliflozin mitigated lipotoxicity in saturated fatty acid-induced hepatocytes, likely by promoting antioxidant defense instead of attenuating lipid accumulation through enhanced FoxO3a and Nrf2 nuclear translocation dependent on the CAMKK2/AMPKα pathway. The CAMKK2/AMPKα pathway might serve as a promising target in treatment of lipotoxicity in nonalcoholic steatohepatitis.

The microenvironment plays a vital role in tumor progression, and hypoxia is a typical microenvironment feature in nearly all solid tumors. In this study, we focused on elucidating the effect of canagliflozin (CANA), a new class of antidiabetic agents, on hepatocarcinoma (HCC) tumorigenesis under hypoxia, and demonstrated that CANA could significantly inhibit hypoxia-induced metastasis, angiogenesis, and metabolic reprogramming in HCC. At the molecular level, this was accompanied by a reduction in VEGF expression level, as well as a reduction in the epithelial-to-mesenchymal transition (EMT)-related proteins and glycolysis-related proteins. Next, we focused our study particularly on the modulation of HIF-1α by CANA, which revealed that CANA decreased HIF-1α protein level by inhibiting its synthesis without affecting its proteasomal degradation. Furthermore, the AKT/mTOR pathway, which plays an important role in HIF-1α transcription and translation, was also inhibited by CANA. Thus, it can be concluded that CANA decreased metastasis, angiogenesis, and metabolic reprogramming in HCC by inhibiting HIF-1α protein accumulation, probably by targeting the AKT/mTOR pathway. Based on our results, we propose that CANA should be evaluated as a new treatment modality for liver cancer.