O-GlcNAcylated RALY Contributes to Hepatocellular Carcinoma Cells Proliferation by Regulating USP22 mRNA Nuclear Export.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly tumors; however, its pathogenic mechanism remains largely elusive. In-depth researches are needed to reveal the expression regulatory mechanisms and functions of the RNA-binding protein RALY in HCC. Here, we identify RALY as a highly expressed oncogenic factor that affects HCC cells proliferation both in vitro and in vivo. O-GlcNAcylation of RALY at Ser176 enhances its stability by protecting RALY from TRIM27-mediated ubiquitination, thus maintaining hyper-expression of the RALY protein. Mechanistically, RALY interacts with USP22 messenger RNA, as revealed by RNA immunoprecipitation, to increase their cytoplasmic localization and protein expression, thereby promoting the proliferation of HCC cells. Furthermore, we develop a novel RALY protein degrader based on peptide proteolysis-targeting chimeras, named RALY-PROTAC, which we chemically synthesize by linking a RALY-targeting peptide with the E3 ubiquitin ligase recruitment ligand pomalidomide. In conclusion, our findings demonstrate a novel mechanism by which O-GlcNAcylation/RALY/USP22 mRNA axis aggravates HCC cells proliferation. RALY-PROTACs as degraders of the RALY protein exhibit potential as therapeutic drugs for RALY-overexpressing HCC.

Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression.

More than 90% of hepatocellular carcinoma (HCC) cases develop in the presence of fibrosis or cirrhosis, making the tumor microenvironment (TME) of HCC distinctive due to the intricate interplay between cancer-associated fibroblasts (CAFs) and cancer stem cells (CSCs), which collectively regulate HCC progression. However, the mechanisms through which CSCs orchestrate the dynamics of the tumor stroma during HCC development remain elusive. Our study unveils a significant upregulation of Sema3C in fibrotic liver, HCC tissues, peripheral blood of HCC patients, as well as sorafenib-resistant tissues and cells, with its overexpression correlating with the acquisition of stemness properties in HCC. We further identify NRP1 and ITGB1 as pivotal functional receptors of Sema3C, activating downstream AKT/Gli1/c-Myc signaling pathways to bolster HCC self-renewal and tumor initiation. Additionally, HCC cells-derived Sema3C facilitated extracellular matrix (ECM) contraction and collagen deposition in vivo, while also promoting the proliferation and activation of hepatic stellate cells (HSCs). Mechanistically, Sema3C interacted with NRP1 and ITGB1 in HSCs, activating downstream NF-kB signaling, thereby stimulating the release of IL-6 and upregulating HMGCR expression, consequently enhancing cholesterol synthesis in HSCs. Furthermore, CAF-secreted TGF-ß1 activates AP1 signaling to augment Sema3C expression in HCC cells, establishing a positive feedback loop that accelerates HCC progression. Notably, blockade of Sema3C effectively inhibits tumor growth and sensitizes HCC cells to sorafenib in vivo. In sum, our findings spotlight Sema3C as a novel biomarker facilitating the crosstalk between CSCs and stroma during hepatocarcinogenesis, thereby offering a promising avenue for enhancing treatment efficacy and overcoming drug resistance in HCC.

PHLDA2 reshapes the immune microenvironment and induces drug resistance in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a malignancy known for its unfavorable prognosis. The dysregulation of the tumor microenvironment (TME) can affect the sensitivity to immunotherapy or chemotherapy, leading to treatment failure. The elucidation of PHLDA2's involvement in HCC is imperative, and the clinical value of PHLDA2 is also underestimated. Here, bioinformatics analysis was performed in multiple cohorts to explore the phenotype and mechanism through which PHLDA2 may affect the progression of HCC. Then, the expression and function of PHLDA2 were examined via the qRT-PCR, Western Blot, and MTT assays. Our findings indicate a substantial upregulation of PHLDA2 in HCC, correlated with a poorer prognosis. The methylation levels of PHLDA2 were found to be lower in HCC tissues compared to normal liver tissues. Besides, noteworthy associations were observed between PHLDA2 expression and immune infiltration in HCC. In addition, PHLDA2 upregulation is closely associated with stemness features and immunotherapy or chemotherapy resistance in HCC. In vitro experiments showed that sorafenib or cisplatin significantly up-regulated PHLDA2 mRNA levels, and PHLDA2 knockdown markedly decreased the sensitivity of HCC cells to chemotherapy drugs. Meanwhile, we found that TGF-ß induced the expression of PHLDA2 in vitro. The GSEA and in vitro experiment indicated that PHLDA2 may promote the HCC progression via activating the AKT signaling pathway. Our study revealed the novel role of PHLDA2 as an independent prognostic factor, which plays an essential role in TME remodeling and treatment resistance in HCC.

Ubiquitin specific peptidase 3: an emerging deubiquitinase that regulates physiology and diseases.

Proteins are the keystone for the execution of various life activities, and the maintenance of protein normalization is crucial for organisms. Ubiquitination, as a post-transcriptional modification, is widely present in organisms, and it relies on the sophisticated ubiquitin-proteasome (UPS) system that controls protein quality and modulates protein lifespan. Deubiquitinases (DUBs) counteract ubiquitination and are essential for the maintenance of homeostasis. Ubiquitin specific peptidase 3 (USP3) is a member of the DUBs that has received increasing attention in recent years. USP3 is a novel chromatin modifier that tightly regulates the DNA damage response (DDR) and maintains genome integrity. Meanwhile, USP3 acts as a key regulator of inflammatory vesicles and sustains the normal operation of the innate immune system. In addition, USP3 is aberrantly expressed in a wide range of cancers, such as gastric cancer, glioblastoma and neuroblastoma, implicating that USP3 could be an effective target for targeted therapies. In this review, we retrace all the current researches of USP3, describe the structure of USP3, elucidate its functions in DNA damage, immune and inflammatory responses and the cell cycle, and summarize the important role of USP3 in multiple cancers and diseases.

How CLSPN could demystify its prognostic value and potential molecular mechanism for hepatocellular carcinoma: A crosstalk study.

BACKGROUND & AIMS: CLSPN, a critical component of the S-phase checkpoint in response to DNA replication stress, has been implicated in the pathogenesis of multiple tumor types. The rising incidence of hepatocellular carcinoma (HCC) poses a significant challenge to global public health. Despite this, the specific functions of CLSPN in the development of HCC remain poorly understood. METHODS: We systematically evaluated the expression of CLSPN, prognosis and immune infiltration in patients with HCC and identified a competing endogenous RNA (ceRNA) network by using public database. The RT-qPCR, western blot, CCK8, transwell, flow cytometry, animal experiments, proteasome inhibition experiment, Co-IP assay and mass spectrometry were applied to explore its biological functions, post-transcriptional modifications and potential molecular mechanisms of CLSPN in HCC. RESULTS: We verified the expression of CLSPN, and its high expression is an independent prognostic factor in HCC. The expression of CLSPN is also associated with the immune microenvironment of HCC. CLSPN silencing inhibited the proliferation, migration, invasion and cell cycle progression of HCC cells. We established a PSMA3-AS1/hsa-miR-101-3p/CLSPN regulator axis in HCC. CLSPN was influenced by ubiquitination and was involved in the Wnt/ß-catenin pathway to regulate HCC progression. CONCLUSIONS: It was the first time to comprehensively discover and identify the expression, prognosis, immunotherapy, RNAs regulator, posttranscriptional modification, and molecular mechanisms of CLSPN in HCC. These novel insights have the potential to expedite the development of personalized treatment strategies and translational medicine approaches for HCC patients.

An integrated investigation of sulfotransferases (SULTs) in hepatocellular carcinoma and identification of the role of SULT2A1 on stemness.

The cytosolic sulfotransferases (SULTs) are phase II conjugating enzymes, which are widely expressed in the liver and mainly mediate the sulfation of numerous xenobiotics and endogenous compounds. However, the role of various SULTs genes has not been reported in hepatocellular carcinoma (HCC). This study aims to analyze the expression and potential functional roles of SULTs genes in HCC and to identify the role of SULT2A1 in HCC stemness as well as the possible mechanism. We found that all of the 12 SULTs genes were differentially expressed in HCC. Moreover, clinicopathological features and survival rates were also investigated. Multivariate regression analysis showed that SULT2A1 and SULT1C2 could be used as independent prognostic factors in HCC. SULT1C4, SULT1E1, and SULT2A1 were significantly associated with immune infiltration. SULT2A1 deficiency in HCC promoted chemotherapy resistance and stemness maintenance. Mechanistically, silencing of SULT2A1 activated the AKT signaling pathway, on the one hand, promoted the expression of downstream stemness gene c-Myc, on the other hand, facilitated the NRF2 expression to reduce the accumulation of ROS, and jointly increased HCC stemness. Moreover, knockdown NR1I3 was involved in the transcriptional regulation of SULT2A1 in stemness maintenance. In addition, SULT2A1 knockdown HCC cells promoted the proliferation and activation of hepatic stellate cells (HSCs), thereby exerting a potential stroma remodeling effect. Our study revealed the expression and role of SULTs genes in HCC and identified the contribution of SULT2A1 to the initiation and progression of HCC.

Spotlights on ubiquitin-specific protease 12 (USP12) in diseases: from multifaceted roles to pathophysiological mechanisms.

Ubiquitination is one of the most significant post-translational modifications that regulate almost all physiological processes like cell proliferation, autophagy, apoptosis, and cell cycle progression. Contrary to ubiquitination, deubiquitination removes ubiquitin from targeted protein to maintain its stability and thus regulate cellular homeostasis. Ubiquitin-Specific Protease 12 (USP12) belongs to the biggest family of deubiquitinases named ubiquitin-specific proteases and has been reported to be correlated with various pathophysiological processes. In this review, we initially introduce the structure and biological functions of USP12 briefly and summarize multiple substrates of USP12 as well as the underlying mechanisms. Moreover, we discuss the influence of USP12 on tumorigenesis, tumor immune microenvironment (TME), disease, and related signaling pathways. This study also provides updated information on the roles and functions of USP12 in different types of cancers and other diseases, including prostate cancer, breast cancer, lung cancer, liver cancer, cardiac hypertrophy, multiple myeloma, and Huntington's disease. Generally, this review sums up the research advances of USP12 and discusses its potential clinical application value which deserves more exploration in the future.

Glycosylation-related molecular subtypes and risk score of hepatocellular carcinoma: Novel insights to clinical decision-making.

Background: Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer deaths worldwide, seriously affecting human community health and care. Emerging evidence has shown that aberrant glycosylation is associated with tumor progression and metastasis. However, the role of glycosylation-related genes in HCC has notbeen reported. Methods: Weighted gene coexpression network analysis and non-negative matrix factorization analysis were applied to identify functional modules and molecularm subtypes in HCC. The least absolute shrinkage and selection operator Cox regression was used to construct the glycosylation-related signature. The independent prognostic value of the risk model was confirmed and validated by systematic techniques, including principal component analysis, T-distributed random neighbor embedding analysis, Kaplan-Meier survival analysis, the ROC curve, multivariate Cox regression, the nomogram, and the calibration curve. The single-sample gene set enrichment analysis, gene set variation analysis, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes analyses were evaluated by the immune microenvironment and potential biological processes. The quantitative real-time polymerase chain reaction and immunohistochemistry analysis were used to verify the expression of five genes. Results: We identified the glycosylation-related genes with bioinformatics analysis to construct and validate a five-gene signature for the prognosis of HCC patients. Patients with HCC in the high-risk group had a worse prognosis. The risk score could be an independent factor and was associated with clinical features, such as the grade and stage. The nomogram exhibited an accurate score that included the risk score and clinical parameters. The infiltration levels of antitumor cells were upregulated in the low-risk group, including B_cells, Mast_cells, neutrophils, NK_cells, and T_helper_cells. Moreover, glycosylation was more sensitive to immunotherapy, and may play a critical role in the metabolic processes of HCC, such as bile acid metabolism and fatty acid metabolism. In addition, the five-gene messenger RNA (mRNA) and protein expression were overexpressed in HCC cells and tissues. Conclusions: The glycosylation-related signature is effective for prognostic recognition, immune efficacy evaluation, and substance metabolism in HCC, providing a novel insight for therapeutic target prediction and clinical decision-making.