CRISPR/Cas9 screen reveals that targeting TRIM34 enhances ferroptosis sensitivity and augments immunotherapy efficacy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent malignancy characterized by complex heterogeneity and drug resistance. Resistance to ferroptosis is closely related to the progression of HCC. While HCC tumors vary in their sensitivity to ferroptosis, the precise factors underlying this heterogeneity remain unclear. In this study, we sought to elucidate the mechanisms that contribute to ferroptosis resistance in HCC. Whole-genome CRISPR/Cas9 screen using a subtoxic concentration (IC20) of ferroptosis inducer erastin in the HCC cell line Huh7 revealed TRIM34 as a critical driver of ferroptosis resistance in HCC. Further investigation revealed that TRIM34 suppresses ferroptosis in HCC cells, promoting their proliferation, migration, and invasion both in vitro and in vivo. Furthermore, TRIM34 expression is elevated in HCC tumor tissues, correlating with a poor prognosis. Mechanistically, TRIM34 directly interacts with Up-frameshift 1 (UPF1), a core component of the nonsense-mediated mRNA decay (NMD) pathway, to promote its ubiquitination and degradation. This interaction suppresses GPX4 transcript degradation, thus promoting the protein levels of this critical ferroptosis suppressor in HCC. In light of the close crosstalk between ferroptosis and the adaptive immune response in cancer, HCC cells with targeting knockdown of TRIM34 exhibited an improved response to anti-PD-1 treatment. Taken together, the TRIM34/UPF1/GPX4 axis mediates ferroptosis resistance in HCC, thereby promoting malignant phenotypes. Targeting TRIM34 may thus represent a promising new strategy for HCC treatment.

Crosstalk Between circRNA and Tumor Microenvironment of Hepatocellular Carcinoma: Mechanism, Function and Applications.

Hepatocellular carcinoma (HCC) is one of the most common aggressive tumors in the world. Despite the availability of various treatments, its prognosis remains poor due to the lack of specific diagnostic indicators and the high heterogeneity of HCC cases. CircRNAs are noncoding RNAs with stable and highly specific expression. Extensive research evidence suggests that circRNAs mediate the pathogenesis and progression of HCC through acting as miRNA sponges, protein modulators, and translation templates. Tumor microenvironment (TME) has become a hotspot of immune-related research in recent years due to its effects on metabolism, secretion and immunity of HCC. Accordingly, understanding the role played by circRNAs in TME is important for the study of HCC. This review will discuss the crosstalk between circRNAs and TME in HCC. In addition, we will discuss the current deficiencies and controversies in research on circRNAs and predict future research directions.

GLIS1 intervention enhances anti-PD1 therapy for hepatocellular carcinoma by targeting SGK1-STAT3-PD1 pathway.

BACKGROUND: GLI-similar 1 (GLIS1) is one of of Krüppel-like zinc finger proteins, which are either stimulators or inhibitors of genetic transcription. Nevertheless, its effects on T cell were elusive. METHODS: In this study, we intend to explore the effects of GLIS1 on modulating the anticancer potency of CD8+ T cells in hepatocellular carcinoma (HCC). The expression of GLIS1 in CD8 peripheral blood mononuclear cell and CD8 tumor-infiltrating lymphocytes of HCC tissues was validated by quantificational real-time-PCR and flow cytometry. The anticancer potency of CD8+ T cells with GLIS1 knock down was confirmed in C57BL/6 mouse model and HCC patient-derived xenograft mice model. GLIS1-/- C57BL/6 mice was applied to explore the effects GLIS1 on tumor immune microenvironment. Chromatin immunoprecipitation and RNA transcriptome sequencing analysis were both performed in GLIS1-knock down of CD8+ T cells. RESULTS: GLIS1 was upregulated in exhausted CD8+ T cells in HCC. GLIS1 downregulation in CD8+ T cells repressed cancer development, elevated the infiltrate ability of CD8+ T cells, mitigated CD8+ T cell exhaustion and ameliorated the anti-PD1 reaction of CD8+ T cells in HCC. The causal link beneath this included transcriptional regulation of SGK1-STAT3-PD1 pathway by GLIS1, thereby maintaining the abundant PD1 expression on the surface of CD8+ T cells. CONCLUSION: Our study revealed that GLIS1 promoted CD8+ T cell exhaustion in HCC through transcriptional regulating SGK1-STAT3-PD1 pathway. Downregulating the expression of GLIS1 in CD8+ T cells exerted an effect with anti-PD1 treatment synergistically, revealing a prospective method for HCC immune therapy.

Inhibition of APOC1 promotes the transformation of M2 into M1 macrophages via the ferroptosis pathway and enhances anti-PD1 immunotherapy in hepatocellular carcinoma based on single-cell RNA sequencing.

Single-cell RNA-sequencing (scRNA-seq) presents better insights into cell behavior in the context of a complex tumor microenvironment by profiling single-cell populations. However, the mechanisms underlying treatment failure in hepatocellular carcinoma (HCC) are poorly understood. In this study, we performed deep scRNA-seq on immune cells under the isolation in peripheral blood, cancer tissues, and nearby common tissues of four HCC cases and two non-cancer controls, and 212,494 cells were included in the analysis. We identified distinct immune cell subtypes, enriched pathways for differential genes, and delineated associated developmentally relevant trajectories. APOC1 was found over-expressed in tumor-associated macrophages (TAMs) of HCC tissues than in normal tissues. Inhibition of APOC1 reversed the M2 phenotype to the M1 phenotype via the ferroptosis pathway in TAMs from HCC. Tumors in APOC1 -/- C57BL/6 mice demonstrated consistent attenuation compared to wild-type (WT) mice. Mass spectrometry results revealed that the relative proportion of M2 macrophages, B cells, and CD4+ T cells in the APOC1 -/- group exhibited a downward expression compared with the WT group, whereas CD8+ T cells, M1 macrophages, and NK cells exhibited an upward trend. Finally, APOC1 was found to be negatively correlated with the expression of PD1/PD-L1 in human HCC samples. In conclusion, the present study demonstrated that inhibiting APOC1 can promote the transformation of M2 macrophages into M1 macrophages via the ferroptosis pathway, thereby reshaping the tumor immune microenvironment and improving the anti-PD1 immunotherapy for HCC, providing a new strategy for improving the therapeutic effect of anti-PD1, and bringing new hope to HCC patients.

Inhibition of APOE potentiates immune checkpoint therapy for cancer.

Checkpoint immunotherapy is capable of unleashing T cells for controlling tumor, whereas it is destroyed by immunosuppressive myeloid cell. Apoprotein E (APOE) refers to a ligand in terms of the members of low-density lipoprotein (LDL) receptor family for mediating Apoprotein B-involving atherogenic lipoprotein clearance. Besides, tumor-infiltration macrophage can express APOE. The present study reported Apoe-/- mice to exhibit higher resistance toward the development of three types of carcinomas as compared with mice with wild type and to have greater responses to αPD-1 (anti-PD-1) immunotherapy. Moreover, treatment by exploiting APOE inhibitor (COG 133TFA, αAPOE) was capable of curbing tumor development and fostering regression if in combination of αPD-1. According to single-cell RNA sequencing (scRNA-seq), Apoe deletion was correlated with the decline of C1QC+ and CCR2+ macrophage within tumor infiltration, and mass spectrometry results noticeably showed down-regulated the number of M2 macrophages as well. Furthermore, APOE expression in cancer patients resistant to αPD-1 treatment significantly exceeded that in the sensitive group. For this reason, APOE is likely to be targeted for modifying tumor macrophage infiltrate and augmenting checkpoint immunotherapy.

CircPAK1 promotes the progression of hepatocellular carcinoma via modulation of YAP nucleus localization by interacting with 14-3-3ζ.

BACKGROUND: Circular RNA (circRNA), a new class of non-coding RNA, has obvious correlations with the occurrence and development of many diseases, including tumors. This study aimed to investigate the potential roles of circPAK1 in hepatocellular carcinoma (HCC). METHODS: High-throughput sequencing was performed on 3 pairs of HCC and matched normal tissues to determine the upregulated circRNAs. The expression level of circPAK1 was detected by qRT-PCR in HCC and paired with normal liver tissue samples. The effects of circPAK1 on proliferation, invasion, metastasis and apoptosis of HCC cells were evaluated by in vitro and in vivo experiments. We also constructed Chitosan/si-circPAK1 (CS/si-circPAK1) nanocomplexes using Chitosan material to evaluate its in vivo therapeutic effect on HCC. High-throughput sequencing, RNA-sequencing, RNA probe pull-down, RNA immunoprecipitation and Co-Immunoprecipitation assays were performed to explore the relationship between circPAK1, 14-3-3ζ, p-LATS1 and YAP. Exosomes isolated from lenvatinib-resistant HCC cell lines were used to evaluate the relationship between exosomal circPAK1 and lenvatinib resistance. RESULTS: CircPAK1, a novel circRNA, is highly expressed in HCC tumor tissues and cell lines as well as correlated with poor outcomes in HCC patients. Functionally, circPAK1 knockdown inhibited HCC cell proliferation, migration, invasion and angiogenesis while circPAK1 overexpression promoted HCC progression. The tumor-promoting phenotypes of circPAK1 on HCC were also confirmed by animal experiments. Importantly, the application of CS/si-circPAK1 nanocomplexes showed a better therapeutic effect on tumor growth and metastasis. Mechanistically, circPAK1 enhanced HCC progression by inactivating the Hippo signaling pathway, and this kind of inactivation is based on its competitively binding of 14-3-3 ζ with YAP, which weakens the recruitment and cytoplasmic fixation of 14-3-3 ζ to YAP, thus promoting YAP nucleus localization. Additionally, circPAK1 could be transported by exosomes from lenvatinib-resistant cells to sensitive cells and induce lenvatinib resistance of receipt cells. CONCLUSION: CircPAK1 exerts its oncogenic function by competitively binding 14-3-3 ζ with YAP, thus promoting YAP nucleus localization, leading to the inactivation of a Hippo signaling pathway. Exosomal circPAK1 may drive resistance to lenvatinib, providing a potential therapeutic target for HCC patients.

E2F1-mediated GINS2 transcriptional activation promotes tumor progression through PI3K/AKT/mTOR pathway in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has high morbidity and mortality rates. It is therefore imperative to study the underlying mechanism of HCC to identify potential prognostic biomarkers and therapeutic targets. Recently, GINS2 has been identified to be a cancer-promoting gene in different cancer types. Nevertheless, the exact mechanism of GINS2 in HCC remains to be elucidated. To systematically explore the significance of GINS2, we first assessed the relative expression of GINS2 in pan-cancers based on data obtained from the HCCDB, TIMER, and TCGA databases. Then, we explored the clinical significance of GINS2 in HCC through Kaplan-Meier method as well as univariate and multivariate cox regression analysis. Additionally, functional enrichment analysis of GINS2 was done through GO, KEGG, PPI network, and immune cell infiltration analyses. Functional experiments were also conducted to investigate the biological significance of GINS2 in HCC cell lines. Our research revealed that GINS2 is involved in HCC progression and highlighted its potential value as a crucial diagnostic and therapeutic target for HCC.

MGP promotes CD8+ T cell exhaustion by activating the NF-κB pathway leading to liver metastasis of colorectal cancer.

Matrix Gla protein (MGP) was originally reported as a physiological suppressor of ectopia calcification and has also been reported to be associated with cancer. However, the relation between the biological functions of MGP and the immune response in colorectal cancer (CRC) remains unclear. Here, we investigated the regulatory role of MGP in the immune microenvironment of CRC. MGP expression in CRC samples was assessed by single-cell RNA sequencing and the Gene Expression Omnibus (GEO) database, and confirmed by quantitative real-time Polymerase Chain Reaction (qRT-PCR) and immunohistochemistry analysis of human CRC samples. The effect of MGP on proliferation and invasion of CRC cells was evaluated by in vitro assays involving MGP knockdown and overexpression. Luciferase reporter assay and chromatin immunoprecipitation (ChIP)-qPCR assay were performed to identify transcriptional regulatory sites of the nuclear factor kappa-B (NF-κB) and programmed cell death ligand 1 (PD-L1). In vivo experiments were performed in mouse model of CRC liver metastasis established via spleen injection. The results revealed that MGP was significantly upregulated in cancer cell clusters from the primary CRC or liver metastases, compared with that in the corresponding paracancerous tissues via single-cell RNA sequencing. MGP enriched intracellular free Ca2+ levels and promoted NF-κB phosphorylation, thereby activated PD-L1 expression to promote CD8+ T cell exhaustion in CRC. The luciferase reporter assay and ChIP-qPCR assay indicated that the transcriptional regulation of NF-κB upregulated PD-L1 expression. In vivo, MGP inhibition significantly decreased the rate of CRC liver metastasis, which was further reduced after combined therapy with αPD1 (anti-PD1). In conclusions, this study revealed that MGP can facilitate CD8+ T cell exhaustion by activating the NF-κB pathway, leading to liver metastasis of CRC. The combination of MGP knockdown and αPD1 can synergistically resist liver metastasis of CRC.

Targeting Immune Cells in the Tumor Microenvironment of HCC: New Opportunities and Challenges.

Immune associated cells in the microenvironment have a significant impact on the development and progression of hepatocellular carcinoma (HCC) and have received more and more attention. Different types of immune-associated cells play different roles, including promoting/inhibiting HCC and several different types that are controversial. It is well known that immune escape of HCC has become a difficult problem in tumor therapy. Therefore, in recent years, a large number of studies have focused on the immune microenvironment of HCC, explored many mechanisms worth identifying tumor immunosuppression, and developed a variety of immunotherapy methods as targets, laying the foundation for the final victory in the fight against HCC. This paper reviews recent studies on the immune microenvironment of HCC that are more reliable and important, and provides a more comprehensive view of the investigation of the immune microenvironment of HCC and the development of more immunotherapeutic approaches based on the relevant summaries of different immune cells.

STK39 enhances the progression of Cholangiocarcinoma via PI3K/AKT pathway.

Serine/threonine kinase 39 (STK39) is overexpressed in various tumor tissues and plays an essential role in tumor progression. In this study, we investigated the clinical value, as well as the potential functions and mechanisms of STK39 in cholangiocarcinoma (CCA). The results showed that STK39 was overexpressed in CCA and negatively associated with the prognosis of patients with CCA. Functionally, STK39 knockdown suppressed cell proliferation, migration, and invasion, while STK39 overexpression facilitated tumor aggressiveness. The tumor-promoting effects of STK39 in CCA were also validated by in vivo experiments. Mechanistically, RNA-seq analysis identified that STK39 enhanced the progression of CCA by activating PI3K/AKT signaling pathway. Furthermore, overexpression of STK39 could induce gemcitabine resistance in CCA cells. Moreover, the increased expression of STK39 may be mediated by the dysregulation of miR-26a-5p. In summary, STK39 could be served as a valuable prognostic candidate and a potential therapeutic target of CCA.

m6A modification of circHPS5 and hepatocellular carcinoma progression through HMGA2 expression.

N6-methyladenosine (m6A) is capable of mediating circRNA generation in carcinoma biology. Nevertheless, the posttranscriptional systems of m6A and circRNA in hepatocellular carcinoma (HCC) development are still unclear. The present study identified a circRNA with m6A modification, circHPS5, which was increased in neoplasm HCC tissues and indicated poor patient survival. Silencing of circHPS5 inhibited epithelial-mesenchymal transition (EMT) and cancer stem-like cell (CSC) phenotypes. Notably, METTL3 could direct the formation of circHPS5, and specific m6A controlled the accumulation of circHPS5. YTHDC1 facilitated the cytoplasmic output of circHPS5 under m6A modification. In addition, we demonstrated that circHPS5 can act as a miR-370 sponge to regulate the expression of HMGA2 and further accelerate HCC cell tumorigenesis. Accordingly, the m6A modification of circHPS5 was found to modulate cytoplasmic output and increase HMGA2 expression to facilitate HCC development. The new regulatory model of "circHPS5-HMGA2" provides a new perspective for circHPS5 as an important prognostic marker and therapeutic target in HCC and provides mechanistic insight for exploring the carcinogenic mechanism of circHPS5 in HCC.

Laparoscopic splenectomy via the spleen bed in combination with selective esophagogastric devascularization for patients with cirrhotic portal hypertension: a single-institution experience.

INTRODUCTION: The safety and feasibility of laparoscopic splenectomy plus selective esophagogastric devascularization (LSSD) via the spleen bed for cirrhotic portal hypertension have not been well studied. AIM: To assess the safety and feasibility of LSSD via the spleen bed for patients with cirrhotic portal hypertension. MATERIAL AND METHODS: From June 2012 to December 2017, 423 patients suffering from portal hypertension and hypersplenism with liver cirrhosis underwent surgery in our department. One hundred and sixty-seven of these patients received totally LSSD, and the others received open splenectomy and esophagogastric devascularization (OSD). The characteristics, intraoperative and postoperative details and complications of the two groups were compared. RESULTS: The operations were successfully performed in all patients. Intraoperative blood loss volume and blood transfusion were similar between the two groups (all p-values > 0.05). Postoperative length of hospital stay and time to oral intake were significantly shorter, but operation time was longer in the LSSD group compared with the OSD group (all p < 0.05). However, postoperative portal vein diameter was significantly smaller in the LSSD group (p < 0.001). The postoperative grade of varices was significantly lower in the LSSD group (p = 0.030). No significant differences were detected between the two groups regarding postoperative liver function, but the incidences of pancreatic leakage, pleural effusion, and wound infections were higher in the OSD group (all p < 0.05). CONCLUSIONS: LSSD via the spleen bed is safe and feasible for liver cirrhosis and portal hypertension.