Phosphoinositide 3-kinase (PI3K) pathway plays a central role in tumor development and progression, and is frequently activated in human breast cancers. However, the mechanism underlying PI3K-mediated regulation of breast cancer growth and metastasis remains poorly understood. Here we show that the activation of PI3K contributes to breast cancer progression by regulating the downstream OGT ( O -linked N-acetylglucosaminyltransferase)-mediated signaling. Mechanistically, PI3K activation enhances O-GlcNAcylation of BRCA1-associated protein 1 (BAP1), a deubiquitinase of OGT, increasing the association of BAP1 with OGT to suppress proteasomal degradation of OGT. Increased enrichment of OGT at the promoter of genes related to cell proliferation and metastasis subsequently elevates gene expressions to promote breast cancer malignancy. Additionally, OGT-mediated O-GlcNAcylation of oncoprotein forkhead (FKH) box protein M1 (FOXM1) on S394 promotes the biocondensate formation by blocking AMPKα2-mediated S376 phosphorylation, thereby activating oncogenic transcription and accelerating breast cancer progression. Dual blockade of BAP1 and FOXM1 O-GlcNAcylation synergistically impairs breast tumor growth and metastasis in mice. Collectively, our study reveals an important signaling event downstream of PI3K activation to drive breast cancer progression, and suggests new therapeutic avenues for treating patients harboring tumorigenic mutations in the PI3K pathway.

The tumor microenvironment is an immunosuppressive niche that contributes to tumor growth by downregulating immune cell functions or restraining immune cell infiltration. The underlying mechanisms are not still poorly understood. Here, we demonstrate that O-linked N-acetylglucosamine (O-GlcNAcylation), a prevalent form of protein glycosylation, contributes to establishing the immunosuppressive niche through regulating the metabolic and non-metabolic functions of uridine diphosphate glucose dehydrogenase (UGDH). Tumor cells carrying O-GlcNAcylation-deficient UGDH showed reduced xenograft tumor growth and improved survival in mice. Cytometry by time-of-flight (CyTOF) analysis suggests UGDH O-GlcNAcylation negatively correlates with cytotoxic CD8+ T cell infiltration. O-GlcNAcylation on serine 350 of UGDH is located within the UDP-binding domain, and the subsequent extensive all-atom molecular dynamics simulations reveal that O-GlcNAcylation reinforces hydrogen-bonding interaction and enzymatic activity of UGDH, leading to enhanced hyaluronic acid (HA) synthesis in the extracellular matrix. Moreover, O-GlcNAcylation of UGDH reduces CD8+ T cell infiltration by decreasing the chemokine CXCL10 expression. Specifically, O-GlcNAcylation enhances UGDH interaction with KPNA2 to compete with STAT1, and suppresses translocation of STAT1 into the nucleus, thereby transcriptionally downregulating CXCL10 expression. Thus, our study identifies UGDH O-GlcNAcylation as a key regulator of tumor immunity and further suggests a potential strategy for enhancing immunotherapy.

Arginine methylation is a common post-translational modification that plays critical roles in many biological processes. However, the existence of arginine demethylases that remove the modification has not been fully established. Here, we report that Myc-induced nuclear antigen 53 (Mina53), a member of the jumonji C (JmjC) protein family, is an arginine demethylase. Mina53 catalyzes the removal of asymmetric dimethylation at arginine 337 of p53. Mina53-mediated demethylation reduces p53 stability and oligomerization and alters chromatin modifications at the gene promoter, thereby suppressing p53-mediated transcriptional activation and cell-cycle arrest. Mina53 represses p53-dependent tumor suppression both in mouse xenografts and spontaneous tumor models. Moreover, downregulation of p53-mediated gene expression is observed in several types of cancer with elevated expression of Mina53. Thus, our study reveals a regulatory mechanism of p53 homeostasis and activity and, more broadly, defines a paradigm for dynamic arginine methylation in controlling important biological functions.

Glycosylation, the covalent attachment of glycans to proteins, lipids, and RNAs, is fundamental in regulating diverse biological processes. Glycosylation patterns are aberrantly altered in the tumor microenvironment and closely associated with tumor immune escape. However, the molecular mechanisms by which glycosylation regulates tumor immune escape are poorly understood. We show that Cluster of Differentiation 47 (CD47), an innate immune checkpoint protein, is highly modified with core fucosylated N-linked glycans. Core fucosylation of CD47 mediated by fucosyltransferase 8 (FUT8) at asparagine 111 (N111) reduces CD47 ubiquitination and degradation. Blockade of N111 glycosylation represses CD47 expression and promotes macrophage phagocytosis of tumor cells. Furthermore, elimination of N111 glycosylation promotes the infiltration of CD103+ dendritic cells (DCs), leading to the increased recruitment of natural killer (NK) cells and inhibition of tumor growth in a murine hepatocellular carcinoma (HCC) model. Combined treatment with core fucosylation inhibitors and an anti-CD47 antibody synergistically promotes therapeutic efficacy in the HCC model. Finally, FUT8 levels in human HCC specimens are positively correlated with CD47 expressions and negatively correlated with the infiltration of CD103+ DC and NK cells. Collectively, this study reveals a mechanism underlying CD47 upregulation in tumor cells and highlights the potential of targeting the FUT8-SMURF1-CD47 axis as a therapeutic strategy to improve anti-tumor immune responses.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with unsatisfactory response to immunotherapy. Pyroptosis, a recently discovered form of regulated cell death (RCD), possesses a huge potential to enhance the immunotherapy efficiency against HCC. To achieve efficient drug delivery and ideal activation of antitumor immunity, an E-selectin modified liposomal nanoplatform co-loading gemcitabine elaidate and BMS-202 (a small molecule programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitor) was designed. Following intravenous injection, the liposomal nanoplatform could efficiently bind to sialylated carbohydrates on the surface of peripheral blood leucocytes via E-selectin, subsequently hitchhiking with leucocytes to realize substantial accumulation in the HCC tissue. After cellular uptake by HCC cells, the released gemcitabine could trigger gasdermin E (GSDME)-dependent pyroptosis with the release of danger-associated molecular patterns (DAMPs) and pro-inflammatory cytokines, thus generating antitumor immunity. The released BMS-202 could further relieve immune suppression by blocking the formation of PD-1/PD-L1 complex. More importantly, gemcitabine-triggered tumor pyroptosis enhanced natural orientation of leucocytes to inflammatory tumor site, further increasing the nanoplatform delivery by facilitating tumor leucocyte infiltration through a positive feedback loop. The in vivo efficacy of the fabricated liposomes demonstrated a favorable antitumor immunity by promoting dendritic cell maturation and T cell activation. In summary, this pyroptosis-enhanced leucocyte-hitchhiking liposomal nanoplatform suggests synergistic antitumor activity and unique ability to modulate drug delivery, showing promise as a highly efficient strategy for potentiated tumor immunotherapy, with a potential for clinical translation.

Aerobic glycolysis and immune evasion are two key hallmarks of cancer. However, how these two features are mechanistically linked to promote tumor growth is not well understood. Here, we show that the glycolytic enzyme enolase-1 (ENO1) is dynamically modified with an O-linked β-N-acetylglucosamine (O-GlcNAcylation), and simultaneously regulates aerobic glycolysis and immune evasion via differential glycosylation. Glycosylation of threonine 19 (T19) on ENO1 promotes its glycolytic activity via the formation of active dimers. On the other hand, glycosylation of serine 249 (S249) on ENO1 inhibits its interaction with PD-L1, decreases association of PD-L1 with the E3 ligase STUB1, resulting in stabilization of PD-L1. Consequently, blockade of T19 glycosylation on ENO1 inhibits glycolysis, and decreases cell proliferation and tumor growth. Blockade of S249 glycosylation on ENO1 reduces PD-L1 expression and enhances T cell-mediated immunity against tumor cells. Notably, elimination of glycosylation at both sites synergizes with PD-L1 monoclonal antibody therapy to promote antitumor immune response. Clinically, ENO1 glycosylation levels are up-regulated and show a positive correlation with PD-L1 levels in human colorectal cancers. Thus, our findings provide a mechanistic understanding of how O-GlcNAcylation bridges aerobic glycolysis and immune evasion to promote tumor growth, suggesting effective therapeutic opportunities.

To the Editor: Hepatocellular carcinoma(HCC)is one of the main causes of cancer related death worldwide,and new cases are expected to continue to increase in future[1].Surgical resection is still the best way to remove the tumor and improve patient's prognosis.However,resection of caudate lobe often presents a technical chal-lenge,even to accomplished hepatic surgeons.Caudate lobe is lo-cated anterior to the inferior vena cava(IVC)and posterior to the bifurcation of the portal vein(PV),generally consists of three re-gions:the paracaval portion,the left Spiegel lobe and the pro-cess portion[2].The anatomy of the paracaval portion includes the liver parenchyma ventral to the hepatic IVC and the area between the Spiegel lobe and the right lobe adjacent to the middle hepatic vein(MHV)ventrally,which was classified by Couinaud as segment Ⅸ[3].Here,we presented a case of HCC with successful down-staging therapy,as well as variation of MHV,which provides a safe path for anterior transection of segment Ⅸ.

To the Editor: Pancreatic cancer is a malignancy characterized by a poor prog-nosis,with a 5-year survival rate of＜10％[1].Furthermore,only a minority of patients(＜20％)qualify for curative-intent resec-tion,and even among those who undergo this procedure,the risk of recurrence within three years remains alarmingly high,reach-ing up to 70％[2].

Deregulated glucose metabolism termed the "Warburg effect" is a fundamental feature of cancers, including the colorectal cancer. This is typically characterized with an increased rate of glycolysis, and a concomitant reduced rate of the tricarboxylic acid (TCA) cycle metabolism as compared to the normal cells. How the TCA cycle is manipulated in cancer cells remains unknown. Here, we show that O-linked N-acetylglucosamine (O-GlcNAc) regulates the TCA cycle in colorectal cancer cells. Depletion of OGT, the sole transferase of O-GlcNAc, significantly increases the TCA cycle metabolism in colorectal cancer cells. Mechanistically, OGT-catalyzed O-GlcNAc modification of c-Myc at serine 415 (S415) increases c-Myc stability, which transcriptionally upregulates the expression of pyruvate dehydrogenase kinase 2 (PDK2). PDK2 phosphorylates pyruvate dehydrogenase (PDH) to inhibit the activity of mitochondrial pyruvate dehydrogenase complex, which reduces mitochondrial pyruvate metabolism, suppresses reactive oxygen species production, and promotes xenograft tumor growth. Furthermore, c-Myc S415 glycosylation levels positively correlate with PDK2 expression levels in clinical colorectal tumor tissues. This study highlights the OGT-c-Myc-PDK2 axis as a key mechanism linking oncoprotein activation with deregulated glucose metabolism in colorectal cancer.

Programmed-death ligand 1 (PD-L1) and its receptor programmed cell death 1 (PD-1) mediate T cell-dependent immunity against tumors. The abundance of cell surface PD-L1 is a key determinant of the efficacy of immune checkpoint blockade therapy targeting PD-L1. However, the regulation of cell surface PD-L1 is still poorly understood. Here, we show that lysosomal degradation of PD-L1 is regu-lated by O-linked N-acetylglucosamine (O-GlcNAc) during the intracellular traffick-ing pathway. O-GlcNAc modifies the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a key component of the endosomal sorting machinery, and subsequently inhibits its interaction with intracellular PD-L1, leading to impaired lysosomal degradation of PD-L1. O-GlcNAc inhibition activates T cell-mediated antitumor immunity in vitro and in immune-competent mice in a manner dependent on HGS glycosylation. Combination of O-GlcNAc inhibition with PD-L1 antibody synergistically promotes antitumor immune response. We also designed a competitive peptide inhibitor of HGS glycosylation that decreases PD-L1 expression and enhances T cell-mediated immunity against tumor cells. Collectively, our study reveals a link between O-GlcNAc and tumor immune evasion, and suggests strategies for improving PD-L1-mediated immune checkpoint blockade therapy.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) plays a key role in hepatocellular carcinoma (HCC) development, and the inhibition of O-GlcNAcylation has therapeutic potential. To decrease the systemic adverse events and increase targeting, we used sialic acid (SA)-decorated liposomes loaded with OSMI-1, an inhibitor of the O-GlcNAcylation, to further improve the anti-HCC effect. Fifty pairs of HCC tissue samples and the cancer genome atlas database were used to analyze the expression of O-GlcNAc transferase (OGT) and its effects on prognosis and immune cell infiltration. OSMI-1 cells were treated with SA and liposomes. Western blotting, immunofluorescence, cell proliferation assay, flow cytometry, enzyme-linked immunosorbent assay, immunohistochemistry, and tumorigenicity assays were used to investigate the antitumor effect of SA-modified OSMI-1 liposomes in vitro and in vivo. OGT was highly expressed in HCC tissues, negatively correlated with the degree of tumor infiltration of CD8+ and CD4+T cells and prognosis, and positively correlated with the degree of Treg cell infiltration. SA-modified OSMI-1 liposome (OSMI-1-SAL) was synthesized with stable hydrodynamic size distribution. Both in vitro and in vivo, OSMI-1-SAL exhibited satisfactory biosafety and rapid uptake by HCC cells. Compared to free OSMI-1, OSMI-1-SAL had a stronger capacity for suppressing the proliferation and promoting the apoptosis of HCC cells. Moreover, OSMI-1-SAL effectively inhibited tumor initiation and development in mice. OSMI-1-SAL also promoted the release of damage-associated molecular patterns, including anticalreticulin, high-mobility-group protein B1, and adenosine triphosphate, from HCC cells and further promoted the activation and proliferation of the CD8+ and CD4+T cells. In conclusion, the OSMI-1-SAL synthesized in this study can target HCC cells, inhibit tumor proliferation, induce tumor immunogenic cell death, enhance tumor immunogenicity, and promote antitumor immune responses, which has the potential for clinical application in the future.

Metabolic reprogramming, such as alterations in glutamine metabolism or glycolysis, is the hallmark of hepatocellular carcinoma (HCC). However, the underlying mechanisms are still incompletely elucidated. Previous studies have identified that methyltransferase SET and MYND domain-containing protein 2(SMYD2) is responsible for the pathogenesis of numerous types of cancer. Here, we innovatively uncover how SMYD2 regulates glutamine metabolism in HCC cells and promotes HCC progression. We identified that SMYD2 expression is upregulated in HCC tissues, which correlates with unfavorable clinical outcomes. Our in vitro and in vivo results showed that the depletion of SMYD2 inhibits HCC cell growth. Mechanistically, c-Myc methylation by SMYD2 increases its protein stability through the ubiquitin–proteasome system. We showed SMYD2 depletion destabilized c-Myc protein by increasing the conjugated K48-linked polyubiquitin chain. SMYD2 increased c-Myc expression and further upregulated glutaminase1 (GLS1), a crucial enzyme that catalyzes the conversion of glutamine to glutamic acid, in HCC cells. GLS1 plays an important role in SMYD2-mediated HCC progression and glutamine metabolism regulation. The knockdown of SMYD2 inhibited glutamine metabolism in HCC cells and overcame their chemoresistance to sorafenib. Collectively, our findings demonstrated a novel mechanism of how SMYD2 promotes HCC progression by regulating glutamine metabolism through the c-Myc/GLS1signaling, implicating the therapeutic potential of targeting SMYD2 in HCC patients.

168 Background: Hepatocellular carcinoma (HCC), as a malignant tumor with high incidence worldwide, faces the challenge of low efficacy of traditional chemotherapy drugs and immunotherapy drugs. Pyroptosis is a type of programmed cell death that can alleviate tumor immunosuppressive microenvironment and promote systemic immune response, which has great potential in improving the efficacy of immunotherapy drugs. The selection of more effective pyroptosis-inducing drugs and immunotherapy drugs, supplemented by more precise drug delivery strategies, is one of the research focuses in the treatment of HCC. Methods: In this study, Western Blot were performed to screen suitable pyroptosis-inducing drugs. A leucocyte-hitchhiking liposome nanodrug delivery system was designed to co-deliver a pyroptosis-inducing drug and a small molecule immunotherapy drug. The ability of the nanocarrier to induce pyroptosis of liver cancer cells and to activate immune cells were investigated in vitro. The targeting ability of the nanocarrier to peripheral blood leucocytes, the function of tumor inhibition and immune activation were evaluated in vivo. Results: In this study, gemcitabine was confirmed to induce pyroptosis in liver cancer cells. E-selectin modified liposomes were designed to co-deliver gemcitabine and the small molecule PD-1/PD-L1 complex inhibitor BMS-202. In vitro experiments confirmed that the nanocarrier could induce pyroptosis in liver cancer cells and activate immune cells. In vitro experiments confirmed the targeting ability of the nanocarrier to leukocytes and its ability to inhibit tumor and activate immunity. Conclusions: Leucocyte-hitchhiking liposomes loading gemcitabine and BMS-202 was successfully synthesized. After intravenous injection, liposomes effectively adhere to the surface of peripheral blood leukocytes mediated by E-selectin, thus hitchhiking with leukocytes to achieve high accumulation at the tumor site. The released gemcitabine can evoke tumor cell pyroptosis and generate anti-tumor immunity, while BMS-202 can alleviate immune suppression by inhibiting the formation of PD-1/PD-L1 complex, further enhance gemcitabine-induced anti-tumor immunity. This leucocyte-hitchhiking dual-delivery liposomal nanoplatform might provide a promising strategy for high-efficiency immunotherapy, exhibiting a great potential for clinical translation of HCC treatment.

Significance Epidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.

Background: Accumulating data have suggested that long non-coding RNAs（lnc RNAs） play important roles in regulating tumor cell growth. This study was designed to investigate the role of SNHG16 in hepatocellular carcinoma（HCC）. Methods: SNHG16 expression was detected with real-time polymerase chain reaction（PCR）. The cutoff value of SNHG16 for tumor-free survival（TFS） was determined with receiver operating characteristic curve analysis. Small interfering RNA was used to inhibit the expression of SNHG16 in HCC cell lines. The biologic behavior of HCC cell was determined with cell viability assay and Transwell assay in vitro. The potential predictive value of SNHG16 on prognosis was analyzed by Kaplan-Meier curves and Cox proportional hazards regression model. Results: SNHG16 expression was upregulated in tumor tissues and HCC cell lines. High expression of SNHG16 was associated with tumor recurrence and poor prognosis after surgery. Multivariate analysis revealed that SNHG16 was an independent prognostic factor for poor recurrence-free survival. Moreover, inhibition of SNHG16 in Hep G2, Hep3 B, and BEL-7402 cells significantly reduced cell invasiveness and proliferation. Mechanistic analyses indicated that the ECM-receptor interaction pathway was remarkably activated by SNHG16. Conclusions: SNHG16 might be a promising biomarker for predicting tumor recurrence in HCC patients after surgery and a potential therapeutic target for HCC.

O-linked N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous post-translational modification of proteins that is essential for cell function. Perturbation of O-GlcNAcylation leads to altered cell-cycle progression and DNA damage response. However, the underlying mechanisms are poorly understood. Here, we develop a highly sensitive one-step enzymatic strategy for capture and profiling O-GlcNAcylated proteins in cells. Using this strategy, we discover that flap endonuclease 1 (FEN1), an essential enzyme in DNA synthesis, is a novel substrate for O-GlcNAcylation. FEN1 O-GlcNAcylation is dynamically regulated during the cell cycle. O-GlcNAcylation at the serine 352 of FEN1 disrupts its interaction with Proliferating Cell Nuclear Antigen (PCNA) at the replication foci, and leads to altered cell cycle, defects in DNA replication, accumulation of DNA damage, and enhanced sensitivity to DNA damage agents. Thus, our study provides a sensitive method for profiling O-GlcNAcylated proteins, and reveals an unknown mechanism of O-GlcNAcylation in regulating cell cycle progression and DNA damage response.

Background: Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity. However, this association could be influenced by the coexisting metabolic abnormalities. This study aimed to investigate the role of obesity and metabolic abnormalities in NAFLD among elderly Chinese.Methods: A cross-sectional study was performed among elderly residents who took their annual health checkups during 2016 in Keqiao District, Shaoxing, China. Results: A total of 3359 elderly adults were retrospectively included in this study. The overall prevalence of NAFLD was 28.7％. The prevalence of NAFLD were 7.14％, 27.92％, 34.80％, and 61.02％ in participants with metabolically healthy normal weight (MHNW), metabolically abnormal normal weight (MANW), metabol- ically healthy obese (MHO), and metabolically abnormal obese (MAO), respectively. NAFLD patients in MHO group had more unfavorable metabolic profiles than those in MHNW group. Logistic regression analysis showed that sex, body mass index (BMI), fasting blood glucose, and serum uric acid were the risk factors of NAFLD. Conclusions: Both obesity and metabolic health were significantly associated with NAFLD in elderly Chi- nese. Screening for obesity and other metabolic abnormalities should be routinely performed for early risk stratification of NAFLD.

Background: Novel immunotherapy is one of the options for advanced biliary tract cancer (BTC) patients who are traditionally intolerant to chemotherapy. However, clinical evidence for single immunotherapy with pembrolizumab or nivolumab is limited. The present study assessed the safety and efficiency of the anti-PD-1 antibody, camrelizumab, as monotherapy in patients with unresectable or recurrent BTC. Methods: A retrospective evaluation was conducted among 4 patients with BTC, including 2 with intrahepatic cholangiocellular carcinoma (ICC), one with extrahepatic bile duct cancer, and one with gallbladder cancer. The patients with unresectable or recurrent BTC were refractory or intolerant to gemcitabine plus cisplatin treatment regimens and received at least one intravenous dose (3 mg/kg) of camrelizumab monotherapy every 3 weeks. Gene sequencing analysis was also performed for biomarker screening. Patient reaction was evaluated according to modified response evaluation criteria in solid tumor (RECIST) version 1.1, progression-free survival (PFS), and toxicity. Results: In this cohort, 1 patient with recurrent ICC had a positive response to treatment, with a substantial tumor size reduction in liver and lung metastases verified using a radiological test after receiving 3 cycles of camrelizumab. The PFS was 4.9 months. The remaining 3 patients showed no response to treatment and experienced disease progression. RNA sequence analysis didn’t found high expression on genes that related to PD-L1, microsatellite instability, tumor mutation burden, and DNA mismatch repair in these patients. Grade 3 treatment-related adverse event was observed in 1 patient. Conclusions: Anti-PD-1 antibody camrelizumab had a manageable safety profile in patients with advanced BTC. This initial assessment of camrelizumab monotherapy provides effective evidence for patients with refractory BTC in biomarker-unselected patients.