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Background: Von Hippel–Lindau (VHL) disease is an autosomal dominant hereditary tumor syndrome with an incidence of approximately 1/36,000. VHL disease-associated clear cell renal cell carcinoma (ccRCC) is the most common congenital RCC. Although recent advances in treating RCC have improved the long-term prognosis of patients with VHL disease, kidney cancer is still the leading cause of death in these patients. Therefore, finding new targets for diagnosing and treating VHL disease-associated ccRCC is still essential. Methods: In this study, we collected matched tumor tissues and normal samples from 25 patients with VHL disease-associated ccRCC, diagnosed and surgically treated in the Department of Urology, Peking University First Hospital. After screening, we performed whole genome bisulfite sequencing (WGBS) on 23 pairs of tissues and RNA-seq on 6 pairs of tissues. And we also compared the VHL disease-associated ccRCC transcriptome data with the sporadic ccRCC transcriptome data from the The Cancer Genome Atlas (TCGA) public database Results: We found that the methylation level of VHL disease-associated ccRCC tumor tissues was significantly lower than that of normal tissues. The tumor tissues showed a difference in the copy number of 3p loss and 5q and 7q gain compared with normal tissues. We integrated RNA-seq and WGBS data to reveal methylation candidate genes associated with VHL disease-associated ccRCC; our results showed 124 hypermethylated and downregulated genes, and 245 hypomethylated and upregulated genes. By comparing the VHL disease-associated ccRCC transcriptome data with the sporadic ccRCC transcriptome data from the TCGA public database, we found that the major pathways of differential gene enrichment differed between them. Conclusions: Our study mapped the multiomics of copy number variation, methylation and mRNA level changes in tumor and normal tissues of clear cell renal cell carcinoma with VHL syndrome, which provides a solid foundation for the mechanistic study, biomarker screening, and therapeutic target discovery of clear cell renal cell carcinoma. [ABSTRACT FROM AUTHOR]

A lipidome comprises thousands of lipid species, many of which are isomers and isobars. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), although widely used for lipidomic profiling, faces challenges in differentiating lipid isomers. Herein, we address this issue by leveraging the orthogonal separation capabilities of hydrophilic interaction liquid chromatography (HILIC) and trapped ion mobility spectrometry (TIMS). We further integrate isomer-resolved MS/MS methods onto HILIC-TIMS, which enable pinpointing double bond locations in phospholipids and sn-positions in phosphatidylcholine. This system profiles phospholipids at multiple structural levels with short analysis time (<10 min per LC run), high sensitivity (nM detection limit), and wide coverage, while data analysis is streamlined using a home-developed software, LipidNovelist. Notably, compared to our previous report, the system doubles the coverage of phospholipids in bovine liver and reveals uncanonical desaturation pathways in RAW 264.7 macrophages. Relative quantitation of the double bond location isomers of phospholipids and the sn-position isomers of phosphatidylcholine enables the phenotyping of human bladder cancer tissue relative to normal control, which would be otherwise indistinguishable by traditional profiling methods. Our research offers a comprehensive solution for lipidomic profiling and highlights the critical role of isomer analysis in studying lipid metabolism in both healthy and diseased states. The existence of large number of isomers poses challenges for lipidomic analysis. The authors integrate hydrophilic interaction liquid chromatography, trapped ion mobility, and isomer-resolved MS/MS into a single system, enabling deep profiling of phospholipidomes at fast speed and wide coverage. [ABSTRACT FROM AUTHOR]

Ubiquitin-conjugating enzyme E2T (UBE2T), a member of the E2 family, has been reported to be overexpressed in certain tumor types and to have an important role in the Fanconi anemia pathway. However, the role of UBE2T in clear cell renal cell carcinoma (ccRCC) has not been clarified. MicroRNAs (miRNAs) participate in tumorigenesis by binding to genes and proteins that regulate cell proliferation or cell apoptosis. The aim of this study was to determine the role of UBE2T and the relationship between miR-182-5p and UBE2T in ccRCC. In the present study, UBE2T expression levels in ccRCC tissues and cells were assessed using real-time quantitative PCR (RT-qPCR) and western blotting. UBE2T protein expression was assessed in a total of 93 ccRCC patients from Peking University First Hospital (PKU) via immunohistochemistry (IHC). The effects of UBE2T knockdown on ccRCC cells were assessed with MTS assays, wound healing assays, Transwell invasion assays and flow cytometry. The effects of in vivo treatment were evaluated through xenograft experiments. The relationship between miR-182-5p and UBE2T was verified with a dual-luciferase reporter gene assay. We found that UBE2T was highly expressed in ccRCC cells and tissues. High UBE2T expression was positively correlated with advanced pathological stage, histological grade, maximum tumor diameter and distant metastasis. Multivariate analysis revealed that UBE2T expression was an independent risk factor for overall survival (OS) and recurrence-free survival (RFS) in patients with ccRCC. Knockdown of UBE2T significantly suppressed RCC cell proliferation, migration and invasion. Flow cytometry analysis showed that UBE2T knockdown promoted RCC cell cycle arrest at G2/M phase and increased cell apoptosis. The xenograft model confirmed that suppression of UBE2T significantly delayed tumor formation and growth in vivo. In addition, miR-182-5p inhibited UBE2T protein expression by targeting UBE2T mRNA and then inhibited the proliferation, migration and invasion of ccRCC cell. Our research reveals that UBE2T likely plays a critical role in ccRCC progression and may be a potential therapeutic target for ccRCC. [ABSTRACT FROM AUTHOR]

Purpose: Nowadays, it is necessary to explore effective biomarkers associated with tumor immune microenvironment (TIME) noninvasively. Here, we investigated whether the metabolic parameter from preoperative 2-[18F]FDG PET/CT could provide information related to TIME in patients with clear cell renal cell carcinoma (ccRCC). Methods: Ninety patients with newly diagnosed ccRCC who underwent 2-[18F]FDG PET/CT prior to surgery were retrospectively reviewed. The immunological features included tumor-infiltrating lymphocytes (TILs) density, programmed death-ligand 1 (PD-L1) expression, and tumor immune microenvironment types (TIMTs). TIMTs were classified as TIMT I (positive PD-L1 and high TILs), TIMT II (negative PD-L1 and low TILs), TIMT III (positive PD-L1 and low TILs), and TIMT IV (negative PD-L1 and high TILs). The relationship between maximum standardized uptake value (SUVmax) in the primary lesion from 2-[18F]FDG PET/CT and immunological features was analyzed. Cox proportional hazards analyses were performed to identify the prognostic factors for disease-free survival (DFS) after nephrectomy. Results: Tumors with high TILs infiltration showed remarkable correlation with elevated SUVmax and aggressive clinicopathological characteristics, such as high World Health Organization/International Society of Urological Pathology (WHO/ISUP) grade. PD-L1 expression on tumor cells was positively associated with WHO/ISUP grade and negatively correlated with body mass index (BMI). However, no correlation was observed between SUVmax and PD-L1 expression, regardless of its spatial tissue distribution. SUVmax of TIMT I and IV was higher than that of TIMT II, but there was remarkable difference merely between TIMT II and IV. In multivariate analysis, SUVmax (P = 0.022, HR 3.120, 95% CI 1.175–8.284) and WHO/ISUP grade (P = 0.046, HR 2.613, 95% CI 1.017–6.710) were the significant prognostic factors for DFS. Six cases (16.2%) with normal SUVmax showed disease progression, while 25 cases (71.4%) with elevated SUVmax experienced disease progression. Conversely, the immunological features held no prognostic value. Conclusions: Our findings demonstrated that 2-[18F]FDG PET/CT could provide metabolic information of TIME for ccRCC patients and develop image-guided therapeutic strategies accordingly. Patients with elevated preoperative SUVmax should be seriously considered, and perioperative immunotherapy might be beneficial for them. [ABSTRACT FROM AUTHOR]

Immunologically-cold tumors including glioblastoma (GBM) are refractory to checkpoint blockade therapy, largely due to extensive infiltration of immunosuppressive macrophages (Mϕs). Consistent with a pro-tumor role of IL-6 in alternative Mϕs polarization, we here show that targeting IL-6 by genetic ablation or pharmacological inhibition moderately improves T-cell infiltration into GBM and enhances mouse survival; however, IL-6 inhibition does not synergize PD-1 and CTLA-4 checkpoint blockade. Interestingly, anti-IL-6 therapy reduces CD40 expression in GBM-associated Mϕs. We identify a Stat3/HIF-1α-mediated axis, through which IL-6 executes an anti-tumor role to induce CD40 expression in Mϕs. Combination of IL-6 inhibition with CD40 stimulation reverses Mϕ-mediated tumor immunosuppression, sensitizes tumors to checkpoint blockade, and extends animal survival in two syngeneic GBM models, particularly inducing complete regression of GL261 tumors after checkpoint blockade. Thus, antibody cocktail-based immunotherapy that combines checkpoint blockade with dual-targeting of IL-6 and CD40 may offer exciting opportunities for GBM and other solid tumors. Glioblastomas are generally resistant to treatment with immune checkpoint inhibitors. Here the authors show that IL6 blockade, in combination with a CD40 agonist, overcomes macrophage-mediated immunosuppression and sensitizes glioblastoma to immune checkpoint blockade in preclinical models. [ABSTRACT FROM AUTHOR]

The microRNA let-7d has been reported to be a tumor suppressor in renal cell carcinoma (RCC). Tumor-associated macrophages (TAM) are M2-polarized macrophages that can enhance tumor growth and angiogenesis in many human cancers. However, the role of let-7d in TAM-associated RCC progression remains elusive. First, we observed a strongly inverse correlation between let-7d expression and microvessel density in RCC tissues. Furthermore, the proliferation, migration, and tube formation of HUVECs were significantly inhibited by conditioned medium from a coculture system of the phorbol myristate acetate pretreated human THP-1 macrophages and let-7d-overexpressing RCC cells. Moreover, the proportion of M2 macrophages was significantly lower in the group that was cocultured with let-7d-overexpressing RCC cells. Subcutaneous xenografts formed by the injection of let-7d-overexpressing RCC cells together with THP-1 cells resulted in a significant decrease in the M2 macrophage ratio and microvessel density compared with those formed by the injection of control RCC cells with THP-1 cells. In silico and experimental analysis revealed interleukin-10 (IL-10) and IL-13 as let-7d target genes. Importantly, the addition of IL-10 and IL-13 counteracted the inhibitory effects of the conditioned medium from the coculture system with let-7d-overexpressing RCC cells in vitro. Additionally, overexpression of IL-10 and IL-13 reversed the effects of let-7d on macrophage M2 polarization and tumor angiogenesis in vivo. Finally, the expression of IL-10 and IL-13 were inversely correlated with the expression of let-7d in RCC clinical specimens. These results suggest that let-7d may inhibit intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-10 and IL-13. [ABSTRACT FROM AUTHOR]

Original magnification: × 20. e, f Tube formation assay showed that the number of tubes formed by HUVEC cells in conditioned medium from let-7d-overex-pressing 786O and 769P cells was not significantly different from those in conditioned medium from respective control cells. Correction to: Let-7d inhibits intratumoral macrophage M2 polarization and subsequent tumor angiogenesis by targeting IL-13 and IL-10 1 The conditioned medium from let-7d-overexpressing RCC cells had no effects on the proliferation, migration and tube formation of HUVECs. a, b The cell proliferation assay showed that conditioned medium from let-7d transfected 786O and 769P cells had no significant influence on proliferation of HUVEC cells compared with that from vehicle control transfected cells. [Extracted from the article]

Purpose: Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries, with incidence in Chinese populations also increasing. CLL involves an accumulation of abnormal B cells which result in dysregulation of cell proliferation and apoptosis rates. The calcyclin-binding protein/Siah-1-interacting protein (CacyBP/SIP) plays a pivotal role in tumorigenicity and cell apoptosis. Here, we investigated the function of CacyBP/SIP in CLL cell proliferation and apoptosis. Methods: CacyBP/SIP expression levels were measured in peripheral blood mononuclear cells from 23 Chinese CLL patients and three healthy donors by western blotting. Correlation analysis was performed to assess associations between CacyBP/SIP expression and clinical stage, chromosome abnormalities and zeta-chain-associated protein kinase 70 (ZAP-70) expression. We silenced CacyBP/SIP expression in MEC-1 cells using a lentivirus system and analyzed cell vitality, cell cycle and tumorigenicity. Apoptosis was also analyzed following the upregulation of CacyBP/SIP expression in MEC-1 cells. Results: Downregulation of CacyBP/SIP expression in CLL patients was negatively correlated with CLL clinical stage, but not with patient sex, age, del(13q14) or del(17q-) presence, or ZAP-70 expression. CacyBP/SIP silencing significantly enhanced cell proliferation and tumorigenicity. CacyBP/SIP silencing promoted accumulation of cells in S phase by upregulation of β-catenin, cyclin D1 and cyclin E, and downregulation of p21. Moreover, CacyBP/SIP overexpression facilitated CLL apoptosis through the activation of pro-caspase-3. Conclusion: CacyBP/SIP is a useful indicator of CLL disease processes and plays an important role in sustaining the balance of cell proliferation and apoptosis. [ABSTRACT FROM AUTHOR]

The tumour vascular microenvironment supports tumorigenesis not only by supplying oxygen and diffusible nutrients but also by secreting soluble factors that promote tumorigenesis. Here we identify a feedforward mechanism in which endothelial cells (ECs), in response to tumour-derived mediators, release angiocrines driving aberrant vascularization and glioblastoma multiforme (GBM) progression through a hypoxia-independent induction of hypoxia-inducible factor (HIF)-1α. Phosphorylation of profilin-1 (Pfn-1) at Tyr 129 in ECs induces binding to the tumour suppressor protein von Hippel-Lindau (VHL), and prevents VHL-mediated degradation of prolyl-hydroxylated HIF-1α, culminating in HIF-1α accumulation even in normoxia. Elevated HIF-1α induces expression of multiple angiogenic factors, leading to vascular abnormality and tumour progression. In a genetic model of GBM, mice with an EC-specific defect in Pfn-1 phosphorylation exhibit reduced tumour angiogenesis, normalized vasculature and improved survival. Moreover, EC-specific Pfn-1 phosphorylation is associated with tumour aggressiveness in human glioma. These findings suggest that targeting Pfn-1 phosphorylation may offer a selective strategy for therapeutic intervention of malignant solid tumours. [ABSTRACT FROM AUTHOR]

Angiogenesis, the formation of new blood vessels, is fundamental to development and post-injury tissue repair. Vascular endothelial growth factor (VEGF)-A guides and enhances endothelial cell migration to initiate angiogenesis. Profilin-1 (Pfn-1) is an actin-binding protein that enhances actin filament formation and cell migration, but stimulus-dependent regulation of Pfn-1 has not been observed. Here, we show that VEGF-A-inducible phosphorylation of Pfn-1 at Tyr 129 is critical for endothelial cell migration and angiogenesis. Chemotactic activation of VEGF receptor kinase-2 (VEGFR2) and Src induces Pfn-1 phosphorylation in the cell leading edge, promoting Pfn-1 binding to actin and actin polymerization. Conditional endothelial knock-in of phosphorylation-deficient Pfn1Y129F in mice reveals that Pfn-1 phosphorylation is critical for angiogenesis in response to wounding and ischaemic injury, but not for developmental angiogenesis. Thus, VEGFR2/Src-mediated phosphorylation of Pfn-1 bypasses canonical, multistep intracellular signalling events to initiate endothelial cell migration and angiogenesis, and might serve as a selective therapeutic target for anti-angiogenic therapy. [ABSTRACT FROM AUTHOR]

'zebra' mutants have alternating green and chlorotic crossbands on leaf blades and are widely distributed in monocotyledonous crops. Most recently, we cloned the first responsible gene from rice, ZEBRA2, which also leads to the phenotype of rice preharvest sprouting. ZEBRA2, a single-copy gene in the rice genome, encodes a carotenoid isomerase (CRTISO), the key enzyme catalyzing the conversion of cis-lycopene to all- trans lycopene. ZEBRA2 shares high identity with known CRTISOs from other species . Expression analysis via both RT-PCR and ZEBRA2-promoter- β-glucuronidase (GUS) transgenic rice indicates that ZEBRA2 is predominantly expressed in mesophyll cells of mature leaves where active photosynthesis occurs. Consistent with the alteration in agronomic traits, the zebra2 mutant exhibits decreased photosynthetic rate and chlorophyll content. Mutation of the ZEBRA2 gene results in the accumulation of all- trans-lycopene precursor, prolycopene (7Z,9Z,7′Z,9′Z tetra cis-lycopene), in dark-grown zebra2 tissues. Light-grown zebra2 mutant exhibits the characteristic 'zebra' phenotype and decreased level of lutein, the xanthophyll that is essential for efficient chl triplet quenching. More severe phenotype of the zebra2 mutant under high light intensity indicates that 'zebra' phenotype might be caused by photooxidative damages. We conclude that ZEBRA2 is involved in photoprotection in rice. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is a leading cause of death worldwide, largely because efficient interventions to restore cardiac function after MI are currently lacking. Here, we characterize vascular aberrancies induced by MI, and propose to target acquired endothelial cell (EC) changes to normalize vessels and promote cardiac repair after MI. Single-cell transcriptome analyses of MI-associated ECs indicates that ECs acquire mesenchymal gene signature that result in phenotypic and functional changes and lead to vessel abnormalities. We identify a PDGF/NF-κB/HIF-1α axis that induces Snail expression and mesenchymal phenotypes in ECs under hypoxia, altogether causing aberrant vascularization. EC-specific knockout of PDGFR-β, pharmacological PDGFR inhibition or nanoparticle-based targeted PDGFR-β siRNA delivery in mice attenuates vascular abnormalities in the infarcted tissue and improves cardiac repair after MI. These findings illustrate a mechanism controlling aberrant neovascularization after ischemia, and suggest that targeting PDGF/Snail-mediated endothelial plasticity may offer opportunities for normalizing vasculature and treating ischemic heart diseases., Competing Interests: Declaration of Interests The authors declare no competing interests.