Detection of HBV DNA integration in plasma cell-free DNA of different HBV diseases utilizing DNA capture strategy.

The landscape of hepatitis B virus (HBV) integration in the plasma cell-free DNA (cfDNA) of HBV-infected patients with different stages of liver diseases [chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC)] remains unclear. In this study, we developed an improved strategy for detecting HBV DNA integration in plasma cfDNA, based on DNA probe capture and next-generation sequencing. Using this optimized strategy, we successfully detected HBV integration events in chimeric artificial DNA samples and HBV-infected HepG2-NTCP cells at day one post infection, with high sensitivity and accuracy. The characteristics of HBV integration events in the HBV-infected HepG2-NTCP cells and plasma cfDNA from HBV-infected individuals (CHB, LC, and HCC) were further investigated. A total of 112 and 333 integration breakpoints were detected in the HepG2-NTCP cells and 22 out of 25 (88%) clinical HBV-infected samples, respectively. In vivo analysis showed that the normalized number of support unique sequences (nnsus) in HCC was significantly higher than in CHB or LC patients (P values â€‹< â€‹0.05). All integration breakpoints are randomly distributed on human chromosomes and are enriched in the HBV genome around nt 1800. The majority of integration breakpoints (61.86%) are located in the gene-coding region. Both non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) interactions occurred during HBV integration across the three different stages of liver diseases. Our study provides evidence that HBV DNA integration can be detected in the plasma cfDNA of HBV-infected patients, including those with CHB, LC, or HCC, using this optimized strategy.

SPOP promotes CREB5 ubiquitination to inhibit MET signaling in liver cancer.

Liver cancer is ranked as the sixth most prevalent from of malignancy globally and stands as the third primary contributor to cancer-related mortality. Metastasis is the main reason for liver cancer treatment failure and patient deaths. Speckle-type POZ protein (SPOP) serves as a crucial substrate junction protein within the cullin-RING E3 ligase complex, acting as a significant tumor suppressor in liver cancer. Nevertheless, the precise molecular mechanism underlying the role of SPOP in liver cancer metastasis remain elusive. In the current study, we identified cAMP response element binding 5 (CREB5) as a novel SPOP substrate in liver cancer. SPOP facilitates non-degradative K63-polyubiquitination of CREB5 on K432 site, consequently hindering its capacity to activate receptor tyrosine kinase MET. Moreover, liver cancer-associated SPOP mutant S119N disrupts the SPOP-CREB5 interactions and impairs the ubiquitination of CREB5.This disruption ultimately leads to the activation of the MET signaling pathway and enhances metastatic properties of hepatoma cells both in vitro and in vivo. In conclusion, our findings highlight the functional significance of the SPOP-CREB5-MET axis in liver cancer metastasis.

Protective Effects of NaHS/miR-133a-3p on Lipopolysaccharide-Induced Cardiomyocytes Injury.

Objective: The aim of this study was to investigate the effects of sodium hydrosulfide (NaHS) on Lipopolysaccharide (LPS)-induced cardiomyocyte injury in H9c2 cells. Methods: H9c2 cardiomyocytes cultivated with medium containing 10 µg/mL LPS were used to recapitulate the phenotypes of those in sepsis. Two sequential experiments were performed. The first contained a control group, a LPS group, and a LPS + NaHS group, with the aim to assure the protective effects of NaHS on LPS-treated cardiomyocytes. The second experiment added a fourth group, the LPS + NaHS + miR-133a-3p inhibition group, with the aim to preliminarily explore whether miR-133-3p exerts a protective function downstream of NaHS. The adenosine triphosphate (ATP) kit was used to detect ATP content; real-time quantitative polynucleotide chain reaction (qPCR) was used to measure the levels of mammalian targets of rapamycin (mTOR), AMP-dependent protein kinase (AMPK), and miR-133a-3p, and Western blot (WB) was used to detect protein levels of mTOR, AMPK, myosin-like Bcl2 interacting protein (Beclin-1), microtubule-associated protein 1 light chain 3 (LC3I/II), and P62 (sequestosome-1, sqstm-1/P62). Results: Compared with the control group, the expressions of miR-133a-3p (P < 0.001), P62 (P < 0.001), and the content of ATP (P < 0.001) decreased, while the expressions of Beclin-1 (P = 0.023) and LC3I/II (P = 0.048) increased in the LPS group. Compared with the LPS group, the expressions of miR-133a-3p (P < 0.001), P62 (P < 0.001), and the content of ATP (P < 0.001) in the NaHS + LPS group increased, while the expressions of Beclin-1 (P = 0.023) and LC3I/II (P = 0.022) decreased. Compared with the NaHS + LPS group, the expression levels of miR-133a-3p (P < 0.001), P62 (P = 0.001), and the content of ATP (P < 0.001) in the LPS + NaHS + miR-133a-3p inhibition group were downregulated, and the expression levels of Beclin-1 (P = 0.012) and LC3I/II (P = 0.010) were upregulated. The difference was statistically significant. There was no significant difference in the expression of AMPK and mTOR between groups. Conclusion: Our research demonstrated that NaHS relieved LPS-induced myocardial injury in H9c2 by promoting the expression of miR-133a-3p, inhibiting autophagy in cardiomyocytes, and restoring cellular ATP levels.

Engineering irradiated tumor-derived microparticles as personalized vaccines to enhance anti-tumor immunity.

The inadequate activation of antigen-presenting cells, the entanglement of T cells, and the highly immunosuppressive conditions in the tumor microenvironment (TME) are important factors that limit the effectiveness of cancer vaccines. Studies show that a personalized and broad antigen repertoire fully activates anti-tumor immunity and that inhibiting the function of transforming growth factor (TGF)-ß facilitates T cell migration. In our study, we introduce a vaccine strategy by engineering irradiated tumor cell-derived microparticles (RT-MPs), which have both personalized and broad antigen repertoire, to induce comprehensive anti-tumor effects. Encouraged by the proinflammatory effects of the spike protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the high affinity between TGF-ß receptor 2 (TGFBR2) and TGF-ß, we develop RT-MPs with the SARS-CoV-2 spike protein and TGFBR2. This spike protein and high TGFBR2 expression induce the innate immune response and ameliorate the immunosuppressive TME, thereby promoting T cell activation and infiltration and ultimately inhibiting tumor growth. Our study provides a strategy for producing an effective personalized anti-tumor vaccine.

MCC950 Ameliorates Acute Exogenous Lipoid Pneumonia Induced by Sewing Machine Oil in Rats via the NF-κB/NLRP3 Inflammasome Pathway.

BACKGROUND/AIM: Acute exogenous lipoid pneumonia (AELP) is a rare disorder caused by intake of lipid formulations and is often underdiagnosed. Meanwhile, the mechanism of AELP is still underlying. MCC950, was previously found to significantly suppress the release of inflammatory cytokines IL-18 and IL-1ß. However, the effect of MCC950 on AELP induced by sewing machine oil has not been reported. MATERIALS AND METHODS: The NLRP3, NF-[Formula: see text]B p65, caspase-1 and IL-1ß expression in lung tissues were compared between a rat model of AELP and control rats using western blotting and real-time quantitative assay. Moreover, haematoxylin and eosin (H&E) staining was performed to elucidate the mechanisms by which MCC950 ameliorates sewing machine oil-induced AELP in vivo. RESULTS: MCC950 reduced the expression of NF-[Formula: see text]B p65 in the lung samples of the treatment group and further down-regulated the NLRP3 and caspase-1 levels while inhibited the production of IL-1ß. Besides, decreases in inflammatory cell infiltration in the lung were shown using H&E staining. CONCLUSION: MCC950 ameliorates sewing machine oil-induced acute exogenous lipoid pneumonia in rats through inhibition of the NF-[Formula: see text]B/NLRP3 inflammasome pathway.

High dietary fructose promotes hepatocellular carcinoma progression by enhancing O-GlcNAcylation via microbiota-derived acetate.

Emerging studies have addressed the tumor-promoting role of fructose in different cancers. The effects and pathological mechanisms of high dietary fructose on hepatocellular carcinoma (HCC) remain unclear. Here, we examined the effects of fructose supplementation on HCC progression in wild-type C57BL/6 mice using a spontaneous and chemically induced HCC mouse model. We show that elevated uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) and O-GlcNAcylation levels induced by high dietary fructose contribute to HCC progression. Non-targeted metabolomics and stable isotope tracing revealed that under fructose treatment, microbiota-derived acetate upregulates glutamine and UDP-GlcNAc levels and enhances protein O-GlcNAcylation in HCC. Global profiling of O-GlcNAcylation revealed that hyper-O-GlcNAcylation of eukaryotic elongation factor 1A1 promotes cell proliferation and tumor growth. Targeting glutamate-ammonia ligase or O-linked N-acetylglucosamine transferase (OGT) remarkably impeded HCC progression in mice with high fructose intake. We propose that high dietary fructose promotes HCC progression through microbial acetate-induced hyper-O-GlcNAcylation.

The association of adverse reactions and depression in cervical cancer patients treated with radiotherapy and/or chemotherapy: moderated mediation models.

Objective: Several studies reported that adverse reactions to treatment, neuroticism, marital relations, and quality of life may impact the development of depression in cervical cancer patients treated with radiotherapy and/or chemotherapy, but the associations between them remained unclear. This study investigated the associations between these factors using moderated mediation models. Methods: Data were extracted from a survey involving cervical cancer patients treated with radiotherapy and/or chemotherapy at five tertiary hospitals in Hubei Province, China, from June to December 2022. The SPSS-PROCESS program was used to develop a moderated mediation model to study the roles of neuroticism, quality of life, and marital relations in the association between adverse reactions and depression in the study population. Results: A total of 802 cervical cancer patients treated with radiotherapy and/or chemotherapy (54.84 ± 9.68 years) were recruited. The prevalence of depression among these patients was 72.72%, with four symptom clusters of dizziness-ringing in the ears, digestive system-related symptoms, skin dryness and itching, and urinary frequency-urgency-leakage. Adverse reactions directly and positively affected the occurrence of depression, neuroticism mediated the association between adverse reactions and depression, while this association varied according to the quality of life and marital relations. Conclusion: Our findings suggest that depression is common among cervical cancer patients receiving radiotherapy and/or chemotherapy. Intervention targets for depression in cervical cancer patients should be precisely selected and targeted according to the quality of life and marital relations differences in patients, taking into account the cost of the intervention and the benefit to the patient.

ROS impairs tumor vasculature normalization through an endocytosis effect of caveolae on extracellular SPARC.

BACKGROUND: The accumulation of reactive oxygen species (ROS) in tumor microenvironment (TME) is an important player for tumorigenesis and progression. We aimed to explore the outcomes of ROS on tumor vessels and the potential regulated mechanisms. METHODS: Exogenous H2O2 was adopted to simulate the ROS setting. Immunofluorescence staining and ultrasonography were used to assess the vascular endothelial coverage and perfusions in the tumors inoculated with Lewis lung cancer (LLC) and melanoma (B16F10) cells of C57BL/6 mice, respectively. ELISA and western-blot were used to detect the expression of secreted acidic and cysteine-rich protein (SPARC) and Caveale-1 in human umbilical vein endothelial cells (HUVEC) extra- and intracellularly. Intracellular translocation of SPARC was observed using electron microscopy and immunofluorescence approaches. RESULT: Under the context of oxidative stress, the pericyte recruitment of neovascularization in mouse lung cancer and melanoma tissues would be aberrated, which subsequently led to the disruption of the tumor vascular architecture and perfusion dysfunction. In vitro, HUVEC extracellularly SPARC was down-regulated, whereas intracellularly it was up-regulated. By electron microscopy and immunofluorescence staining, we observed that SPARC might undergo transmembrane transport via caveale-1-mediated endocytosis. Finally, the binding of SPARC to phosphorylated-caveale-1 was also detected in B16F10 tissues. CONCLUSION: In the oxidative stress environment, neovascularization within the tumor occurs structural deterioration and decreased perfusion capacity. One of the main regulatory mechanisms is the migration of extracellular SPARC from the endothelium to intracellular compartments via Caveolin-1 carriers.

Small cytosolic double-stranded DNA represses cyclic GMP-AMP synthase activation and induces autophagy.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a major mediator of inflammation following stimulation with >45 bp double-stranded DNA (dsDNA). Herein, we identify a class of ∼20-40 bp small cytosolic dsDNA (scDNA) molecules that compete with long dsDNA (200-1,500 bp herring testis [HT]-DNA) for binding to cGAS, thus repressing HT-DNA-induced cGAS activation. The scDNA promotes cGAS and Beclin-1 interaction, releasing Rubicon, a negative regulator of phosphatidylinositol 3-kinase class III (PI3KC3), from the Beclin-1-PI3KC3 complex. This leads to PI3KC3 activation and induces autophagy, causing degradation of STING and long cytosolic dsDNA. Moreover, DNA damage decreases, and autophagy inducers increase scDNA levels. scDNA transfection and treatment with autophagy inducers attenuate DNA damage-induced cGAS activation. Thus, scDNA molecules serve as effective brakes for cGAS activation, preventing excessive inflammatory cytokine production following DNA damage. Our findings may have therapeutic implications for cytosolic DNA-associated inflammatory diseases.

Risk Factors for Postoperative Puncture Site Bleeding after Interventional Treatment of Cerebrovascular Disease via Common Femoral Artery Puncture: A Retrospective Analysis of 710 Cases.

This study aimed to identify the risk factors associated with puncture site bleeding following percutaneous puncture of the common femoral artery during interventional treatment of cerebrovascular disease (CVD). A retrospective analysis was conducted on 710 patients who underwent interventional treatment for CVD via femoral artery puncture. Among them, 26 individuals (3.66%) experienced bleeding at the femoral artery puncture site. Binary logistic regression analysis was performed to identify risk factors for puncture site bleeding. The impact of salt bag compression on postoperative bleeding was evaluated in patients with intermediate to high bleeding risk scores. The bleeding group showed higher blood pressure, lower platelet counts, longer prothrombin time and activated partial thromboplastin time, as well as a higher prevalence of larger vascular sheath sizes and variations in the timing of anti-coagulant and anti-platelet therapy administration. The bleeding risk score was higher in the bleeding group, indicating its predictive value for bleeding risk. Higher bleeding risk score, unstable blood pressure, repeated puncture, and serious vascular conditions were significant risk factors for puncture site bleeding. Application of salt bag compression for a duration of 2 hours reduced postoperative puncture site bleeding in patients with intermediate to high bleeding risk scores. Our study identified several significant risk factors for puncture site bleeding after cerebral vascular intervention via femoral artery puncture, including the bleeding risk score, blood pressure, repeated puncture, and vascular conditions. Implementing salt bag compression as a preventive measure can help mitigate bleeding complications in these high-risk patients.

Immune cell identity behind the Ktrans mapping of mouse glioblastoma.

Dynamic contrast-enhanced MR imaging (DCE-MRI) can assess the integrity of the blood brain barrier (BBB) and has been used in GBM patients to determine glioma grade, predict prognosis, evaluate treatment response, and differentiate treatment-induced effect from recurrence. The volume transfer constant Ktrans is the most frequently used metric in tumor assessment. Based on previous studies that a higher WHO grade of brain tumor was associated with greater impairments of immunity and that Ktrans value was associated with the pathological grading, the relationship between differential composition of immune cells in GBM tissue and dynamic changes in Ktrans mapping was anticipated in this study. The present study utilized an orthotopic allograft model of GBM in which mouse GL26 cells are implanted into Ccr2RFP/wtCx3cr1GFP/wt mice on a C57 background. The brain tumors exhibited heterogenous Ktrans values with the coefficients of variation (CV) above 75%, or relatively homogeneous Ktrans maps with CV values below 50%. The Ktrans values of homogeneous tumors ranged between 0.02/min-0.32/min with a median value of 0.10/min. The immune cell composition defined by quantitative immunohistochemistry and cell sorting was compared between the tumors with Ktrans values above 0.10/min (higher Ktrans) or below 0.10/min (lower Ktrans). Histological analysis showed that tumors with higher Ktrans values exhibited greater numbers of CCR2pos cells (257.60 ± 16.42/mm2 vs 203.23 ± 12.20/mm2, p = 0.04) and an increased ratio of CCR2pos cells to CX3CR1pos cells (1.20 ± 0.02 vs 0.38 ± 0.04, p = 0.001), the numbers of CX3CR1pos cells did not differ significantly based on Ktrans values (219.70 ± 16.20/mm2 vs 250.38 ± 21.20/mm2, p = 0.19). Flowcytometry analysis showed that tumors with higher Ktrans values (above 0.1/min) were associated with greater numbers of both overall monocytes (54.93 ± 6.81% vs 29.75 ± 3.54%, p = 0.01) and inflammatory monocytes (72.38 ± 1.49% vs 59.52 ± 2.44%, p = 0.001). In contrast, tumors with lower Ktrans values (below 0.1/min) exhibited greater numbers of patrolling monocytes (75.65 ± 4.14% vs 63 ± 6.94%, p = 0.05). In the tumors with lower Ktrans values, all three types of tumor associated cells, including patrolling monocytes, inflammatory monocytes, and microglia cells possessed a higher proportion of cells at pro-inflammatory status (41.77 ± 6.13% vs 25.06 ± 6.72%, p = 0.05; 27.50 ± 2.11% vs 20.62 ± 1.87%, p = 0.03; and 55.80 ± 9.88% vs 31.12 ± 7.31%, p = 0.05), inflammatory monocytes showed fewer anti-inflammatory cells (1.25 ± 0.62% vs 3.16 ± 3.56%, p = 0.04). Taken together, differences in Ktrans values were associated with differential immune cell phenotypes and polarizations. Ktrans mapping may therefore represent a novel approach for defining the immune status of GBM.

Case report: Anterior mediastinal mass in a patient with pleural effusion and dyspnea.

Background: Compound lymphoma is an uncommon type of lymphoid malignancy, and those consisting of concurrent B- and T-cell tumors are relatively rare. Case Summary: A 41-year-old man was presented with a 1-month history of progressively worsening cough, chest tightness, and dyspnea after exercise, which could be relieved following rest. Contrast-enhanced computed tomography scan demonstrated a 7.4 × 4.9 cm2 heterogeneous mass in the anterior mediastinum, where a large area of cystic liquid existed, and multiple enlarged lymph nodes in the mediastinum. Since the biopsy failed to yield an exact diagnosis and there was no sign of metastasis, the tumor was surgically resectioned. Surgical findings included obscure boundaries and consistent tumor stiffness with pericardial and pleural invasion. Further pathological examination combined with immunophenotype and gene rearrangement test found the mass composite of angioimmunoblastic T-cell lymphoma (AITL) and B-cell lymphoma. The patient recovered well after R0 resection and received chemotherapy with four cycles of CHOP combined with chidamide 2 weeks after surgery. The patient has had a complete response for over 60 months. Conclusion: In conclusion, we reported a composite lymphoma of AITL combined with B-cell lymphomas. Our experience provides the first successful attempt to treat this rare disease with combined surgery and chemotherapy.

Neoadjuvant chemoradiotherapy plus tislelizumab followed by surgery for esophageal carcinoma (CRISEC study): the protocol of a prospective, single-arm, phase II trial.

BACKGROUND: The failure rate after neoadjuvant chemoradiotherapy followed by surgery is approximately 34.6%-48% for resectable esophageal carcinoma. Pathologic complete response after neoadjuvant chemoradiotherapy is an important factor in predicting lower recurrence and better survival. Whether the sequential addition of immunotherapy to neoadjuvant chemoradiotherapy will be beneficial to improving the pathologic complete response rate is unknown. METHODS: Patients with pathologically confirmed thoracic esophageal squamous cell carcinoma and at clinical T1-2N1-3M0 or T3-4aN0-3M0 (stage II-IVA) according to the eighth edition of American Joint Committee on Cancer staging will be allocated to receive neoadjuvant radiotherapy (41.4 Gy with 23 fractions to planning target volume) with concurrent chemotherapy (albumin-bound paclitaxel, 100 mg/m2, once weekly for five weeks; carboplatin, area under the curve of 2 mg/mL/min, once weekly for five weeks) plus tislelizumab monotherapy sequentially (200 mg every three weeks for three cycles, beginning from the first to the 14th day after the end of radiotherapy). Then, subtotal esophagectomy with two-field lymphadenectomy, including the whole mediastinum and abdomen, will be performed. The primary endpoint for this study is the pathologic complete response rate after neoadjuvant chemoradiotherapy plus tislelizumab. DISCUSSION: The optimal timing of the combination of immunotherapy and neoadjuvant chemoradiotherapy in esophageal carcinoma is not determined. The results of this phase II trial will be helpful to clarify the safety and efficacy of the sequential addition of tislelizumab after neoadjuvant chemoradiotherapy for locally advanced resectable esophageal carcinoma. TRIAL REGISTRATION: This study was approved on January 26, 2021 and retrospectively registered with ClinicalTrials.gov ( NCT04776590 ) on March 1, 2021.

VE-822 upregulates the deubiquitinase OTUD1 to stabilize FHL1 to inhibit the progression of lung adenocarcinoma.

BACKGROUND: The deubiquitinase ovarian tumor domain-containing 1 (OTUD1) has been considered as a tumor suppressor in many tumors, but there is minimal research on the role of OTUD1 in lung adenocarcinoma (LUAD) pathogenesis. METHODS: Bioinformatics analyses and western blot were applied for investigating OTUD1 expression in lung cancer and the drug that upregulated OTUD1. Kaplan-Meier analysis with log-rank test was used for survival analyses. IP-MS and co-IP were performed for identifying potential protein interactions with OTUD1. In vitro and in vivo assays were used for exploring the function of OTUD1 during the progression of LUAD. RESULTS: OTUD1 was dramatically downregulated in tumors and cell lines of human lung cancer. OTUD1 inhibited proliferation and migration of lung cancer cells in vitro. Moreover, OTUD1 inhibited growth of xenografts in nude mice and formation of primary lung tumors in urethane-induced lung cancer model. Mechanistically, we showed that OTUD1 deubiquitinated and stabilized FHL1. Furthermore, we listed and identified VE-822 as a candidate agonist for OTUD1. VE-822 inhibited proliferation of lung adenocarcinoma both in vitro and in vivo. CONCLUSION: These results indicated that the deubiquitinase OTUD1, which was upregulated by VE-822, inhibited the progression of LUAD in vitro and in vivo by deubiquitinating and stabilizing FHL1.

Microglial voltage-dependent anion channel 1 signaling modulates sleep deprivation-induced transition to chronic postsurgical pain.

STUDY OBJECTIVES: This study verified that sleep deprivation before and after skin/muscle incision and retraction (SMIR) surgery increased the risk of chronic pain and investigated the underlying roles of microglial voltage-dependent anion channel 1 (VDAC1) signaling. METHODS: Adult mice received 6 hours of total sleep deprivation from 1 day prior to SMIR until the third day after surgery. Mechanical and heat-evoked pain was assessed before and within 21 days after surgery. Microglial activation and changes in VDAC1 expression and oligomerization were measured. Minocycline was injected to observe the effects of inhibiting microglial activation on pain maintenance. The VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and oligomerization inhibitor VBIT-4 were used to determine the roles of VDAC1 signaling on microglial adenosine 5' triphosphate (ATP) release, inflammation (IL-1ß and CCL2), and chronicity of pain. RESULTS: Sleep deprivation significantly increased the pain duration after SMIR surgery, activated microglia, and enhanced VDAC1 signaling in the spinal cord. Minocycline inhibited microglial activation and alleviated sleep deprivation-induced pain maintenance. Lipopolysaccharide (LPS)-induced microglial activation was accompanied by increased VDAC1 expression and oligomerization, and more VDAC1 was observed on the cell membrane surface compared with control. DIDS and VBIT-4 rescued LPS-induced microglial ATP release and IL-1ß and CCL2 expression. DIDS and VBIT-4 reversed sleep loss-induced microglial activation and pain chronicity in mice, similar to the effects of minocycline. No synergistic effects were found for minocycline plus VBIT-4 or DIDS. CONCLUSIONS: Perioperative sleep deprivation activated spinal microglia and increases the risk of chronic postsurgical pain in mice. VDAC1 signaling regulates microglial activation-related ATP release, inflammation, and chronicity of pain.

Upregulation of TUBG1 expression promotes hepatocellular carcinoma development.

Tubulin γ-1 (TUBG1) is a highly conserved component of the centrosome and its deregulation is involved in the development of several types of cancer. However, the role of TUBG1 in hepatocellular carcinoma (HCC) remains unclear. In this study, we found that TUBG1 was upregulated in human HCC cells and tissues and that TUBG1 upregulation was associated with promoter hypomethylation in HCC tissues. TUBG1 knockdown suppressed the proliferation, invasion, and migration of HCC cells. While TUBG1 expression was positively correlated with CD4 + memory T lymphocyte infiltration, it was negatively correlated with CD4 + regulatory T-cell infiltration in human HCC tissues. Furthermore, TUBG1 expression was positively correlated with the expression of genes involved in cell division. Noticeably, high expression of TUBG1 was associated with poor prognosis in patients with HCC. Overall, our findings revealed that TUBG1 promotes hepatocarcinogenesis by increasing proliferation, invasion, and migration of HCC cells and may regulate T lymphocyte infiltration. The current findings provide important insights into TUBG1 regulation in HCC, which could provide new therapeutic targets for hepatocarcinoma which has a very high incidence and mortality rate worldwide.

Downregulation of phosphoserine phosphatase potentiates tumor immune environments to enhance immune checkpoint blockade therapy.

BACKGROUND: Effects of immune checkpoint blockade (ICB) treatment in hepatocellular carcinoma (HCC) are limited. The current study explored the possibility of exploiting tumor metabolic switches to enhance HCC sensitivity to immune therapies. METHODS: Levels of one-carbon (1C) metabolism and the expression of phosphoserine phosphatase (PSPH), an upstream enzyme of 1C pathway, were evaluated in paired non-tumor and tumor tissues from HCC. Underlying mechanisms mediating the role of PSPH in regulating the infiltration of monocytes/macrophages and CD8+ T lymphocytes were studied through both in vitro and in vivo experiments. RESULTS: PSPH was significantly upregulated in tumor tissues of HCC and its levels were positively correlated with disease progression. PSPH knockdown inhibited tumor growth in immunocompetent mice, but not in those with macrophage or T lymphocyte deficiencies, indicating the pro-tumor effects of PSPH were dependent on both immune components. Mechanistically, PSPH facilitated monocytes/macrophages infiltration by inducing the production of C-C motif chemokine 2 (CCL2), while at the same time reduced CD8+ T lymphocytes recruitment through inhibiting the production of C-X-C Motif Chemokine 10 (CXCL10) in tumor necrosis factor alpha (TNF-α)-conditioned cancer cells. Glutathione and S-adenosyl-methionine were partially involved in regulating the production of CCL2 and CXCL10, respectively. shPSPH (short hairpin RNA) transfection of cancer cells enhanced tumor sensitivity to anti-programmed cell death protein 1 (PD-1) therapy in vivo, and interestingly, metformin could inhibit PSPH expression in cancer cells and mimic the effects of shPSPH in sensitizing tumors to anti-PD-1 treatment. CONCLUSIONS: By tilting the immune balance towards a tumor-friendly composition, PSPH might be useful both as a marker in stratifying patients for ICB therapy, and as an attractive therapeutic target in the treatment of human HCC.

O-GlcNAcylation of SPOP promotes carcinogenesis in hepatocellular carcinoma.

Aberrantly elevated O-GlcNAcylation level is commonly observed in human cancer patients, and has been proposed as a potential therapeutic target. Speckle-type POZ protein (SPOP), an important substrate adaptor of cullin3-RING ubiquitin ligase, plays a key role in the initiation and development of various cancers. However, the regulatory mechanisms governing SPOP and its function during hepatocellular carcinoma (HCC) progression remain unclear. Here, we show that, in HCC, SPOP is highly O-GlcNAcylated by O-GlcNAc transferase (OGT) at Ser96. In normal liver cells, the SPOP protein mainly localizes in the cytoplasm and mediates the ubiquitination of the oncoprotein neurite outgrowth inhibitor-B (Nogo-B) (also known as reticulon 4 B) by recognizing its N-terminal SPOP-binding consensus (SBC) motifs. However, O-GlcNAcylation of SPOP at Ser96 increases the nuclear positioning of SPOP in hepatoma cells, alleviating the ubiquitination of the Nogo-B protein, thereby promoting HCC progression in vitro and in vivo. In addition, ablation of O-GlcNAcylation by an S96A mutation increased the cytoplasmic localization of SPOP, thereby inhibiting the Nogo-B/c-FLIP cascade and HCC progression. Our findings reveal a novel post-translational modification of SPOP and identify a novel SPOP substrate, Nogo-B, in HCC. Intervention with the hyper O-GlcNAcylation of SPOP may provide a novel strategy for HCC treatment.

Molecular Identity Changes of Tumor-Associated Macrophages and Microglia After Magnetic Resonance Imaging-Guided Focused Ultrasound-Induced Blood-Brain Barrier Opening in a Mouse Glioblastoma Model.

An orthotopically allografted mouse GL26 glioma model (Ccr2RFP/wt-Cx3cr1GFP/wt) was used to evaluate the effect of transient, focal opening of the blood-brain barrier (BBB) on the composition of tumor-associated macrophages and microglia (TAMs). BBB opening was induced by magnetic resonance imaging (MRI)-guided focused ultrasound (MRgFUS) combined with microbubbles. CX3CR1-GFP cells and CCR2-RFP cells in brain tumors were quantified in microscopic images. Tumors in animals treated with a single session of MRgFUS did not exhibit significant changes in cell numbers when compared with tumors in animals not receiving FUS. However, tumors that received two or three sessions of MRgFUS had significantly increased amounts of both CX3CR1-GFP and CCR2-RFP cells. The effect of MRgFUS on immune cell composition was also characterized and quantified using flow cytometry. Glioma implantation resulted in increased amounts of lymphocytes, monocytes and neutrophils in the brain parenchyma. Tumors administered MRgFUS exhibited increased numbers of monocytes and monocyte-derived TAMs. In addition, MRgFUS-treated tumors exhibited more CD80+ cells in monocytes and microglia. In summary, transient, focal opening of the BBB using MRgFUS combined with microbubbles can activate the homing and differentiation of monocytes and induce a shift toward a more pro-inflammatory status of the immune environment in glioblastoma.