Expression of ganglioside GD2, reprogram the lipid metabolism and EMT phenotype in bladder cancer.
ABSTRACT: High-grade Bladder Cancer (BLCA) represents the most aggressive and treatment-resistant cancer that renders the patients with poor survival. However, only a few biomarkers have been identified for the detection and treatment of BLCA. Recent studies show that ganglioside GD2 can be used as cancer biomarker and/or therapeutic target for various cancers. Despite its potential relevance in cancer diagnosis and therapeutics, the role of GD2 is unknown in BLCA. Here, we report for the first time that high-grade BLCA tissues and cell lines have higher expression of GD2 compared to low-grade by high-resolution Mass Spectrometry. The muscle invasive UMUC3 cell line showed high GD2, mesenchymal phenotype, and cell proliferation. Besides, we have shown the cancer stem cells (CSC) property (CD44hiCD24lo) of GD2+ UMUC3 and J82 cells. Also, the evaluation of lipid metabolism in GD2+ BLCA cell lines revealed higher levels of Phosphatidylinositol (PI), Phosphatidic acid (PA), Cardiolipin (CL) and lower levels of Phosphatidylserine (PS), plasmenyl-phosphatidylethanolamines (pPE), plasmenyl-phosphocholines (pPC), sphingomyelins (SM), triglycerides (TGs) and N-Acetylneuraminic acid. These findings are significantly correlated with the tissues of BLCA patients. Based on this evidence, we propose that GD2 may be used as an effective diagnostic and therapeutic target for aggressive BLCA.
Project description:There are still unmet medical needs in the treatment of glioblastoma, the most common and the most aggressive glioma of all brain tumors. Here, we found that O-acetyl GD2 is expressed in surgically resected human glioblastoma tissue. In addition, we demonstrated that 8B6 monoclonal antibody specific for O-acetylat GD2 could effectively inhibit glioblastoma cell proliferation in vitro and in vivo. Taken together, these results indicate that O-acetylated GD2 represents a novel antigen for immunotherapeutic-based treatment of high-grade gliomas.
Project description:To develop biocompatible, tumor-specific multifunctional iron-oxide nanoconstructs targeting neuroblastoma, an aggressive pediatric malignancy.Clinical-grade humanized monoclonal antibody (hu14.18K322A), designed to target GD2 antigen on neuroblastoma with reduced nonspecific immune interactions, was conjugated to hydroxyethyl starch-coated iron-oxide nanoparticles. Targeting capability in vitro and in vivo was assessed by immunofluorescence, electron microscopy, analytical spectrophotometry, histochemistry and magnetic resonance R2* relaxometry.The biocompatible nanoconstructs demonstrated high tumor specificity in vitro and in vivo, and low background uptake in a mouse flank xenograft model. Specific accumulation in tumors enabled particle visualization and quantification by magnetic resonance R2* mapping.Our findings support the further development toward clinical application of this anti-GD2 iron-oxide nanoconstruct as diagnostic and therapeutic scaffold for neuroblastoma and potentially other GD2-positive malignancies.
Project description:BACKGROUND:The prognosis of bladder urothelial carcinoma (BLCA) varies greatly among patients, and conventional pathological predictors are generally inadequate and often inaccurate to predict the heterogeneous behavior of BLCA. This study aims to investigate the prognostic value and function of TOP2A in BLCA. METHODS:TOP2A expression level was examined by RNA-sequencing, quantitative real time polymerase chain reaction and immunohistochemistry from 10, 40 and 209 BLCA samples, respectively. Public databases were analyzed for validation. Cell proliferation, migration, invasion assays were performed to explore potential functions of TOP2A in BLCA. Flow cytometry was performed for cell cycle and apoptosis analysis. Univariable and multivariable Cox regression models were performed to identify independent risk factors for the prognosis of BLCA. RESULTS:We found TOP2A was significantly upregulated in BLCA samples, especially for high-grade and advanced stage tumors, compared with matched normal epithelial tissue. Univariable COX regression analysis revealed high TOP2A expression was significantly associated with poorer cancer-specific, progression-free and recurrence-free survival, but not independently of clinical characteristics in the multivariable models. Knockdown of TOP2A remarkably inhibited the proliferation of BLCA cells and non-cancerous urothelial cells. Furthermore, migration and invasion capacity of BLCA cells were strongly suppressed after TOP2A knockdown. Moreover, flow cytometry suggested TOP2A had anti-apoptotic function, and knockdown of TOP2A could induce resistance to doxorubicin in J82 cells. CONCLUSIONS:In our study, TOP2A was overexpressed in BLCA and could serve as a prognostic biomarker for BLCA. Moreover, TOP2A is functionally important for the proliferation, invasion and survival of BLCA cells.
Project description:Background: Bladder cancer is one of the most common malignancies in urologic system. The glucocorticoid-inducible kinase 2 (SGK2) expression and function were largely unknown in cancers. Current study was aimed to investigate the role of SGK2 in bladder cancer and its potential mechanisms. Methods: SGK2 expression was quantified by western blot (WB) in multiple bladder cancer cell lines (T24, 5637, J82 and UMUC3) compared with normal urothelial cell line (SVHUC). SGK2 knocking down and overexpression model were established by lentivirus transfection. MTT, colony formation, wound healing and transwell assay were used to assess the tumor cell proliferation, migration and invasion abilities, respectively. In addition, molecular function analysis was performed using FunRich software V3. Immunoprecipitation (IP) assay was applied to investigate the interaction between SGK2 and ?-catenin at protein level. TCGA database was retrieved to verify the association between these genes and clinical tumor stage as well as prognosis among bladder cancer patients. Results: SGK2 expression was significantly upregulated in multiple bladder cancer cell lines compared with SVHUC at protein level. Cell proliferation, migration and invasion abilities were significantly decreased after knocking down SGK2 in J82 and UMUC3 cell lines. Inversely, cell aggressive phenotypes were significantly increased after overexpressing SGK2 in T24 cell line. Furthermore, functional analyses of SGK2 based on TCGA database showed that SGK2 related genes were involved in receptor activity, ATP binding, DNA repair protein, trans-membrane receptor activity and lipid binding. In addition, protein interaction analysis identified c-Myc was significantly enriched in SGK2 positively associated genes. The prediction was validated by WB and IP assay that SGK2 could directly bind with ?-catenin at protein level to regulate their downstream gene c-Myc expression in bladder cancer to influence tumor progression. And clinical data generated from TCGA database also identified these downstream genes were significantly associated with tumor stage and survival status of bladder cancer patients. Conclusion: Taken together, our findings suggest SGK2 promotes bladder cancer progression via mediating ?-catenin/c-Myc signaling pathway, which may serve as a potential therapeutic target for bladder cancer patients.
Project description:Target selection is a key feature in cancer immunotherapy, a promising field in cancer research. In this respect, gangliosides, a broad family of structurally related glycolipids, were suggested as potential targets for cancer immunotherapy based on their higher abundance in tumors when compared with the matched normal tissues. GD2 is the first ganglioside proven to be an effective target antigen for cancer immunotherapy with the regulatory approval of dinutuximab, a chimeric anti-GD2 therapeutic antibody. Although the therapeutic efficacy of anti-GD2 monoclonal antibodies is well documented, neuropathic pain may limit its application. O-Acetyl-GD2, the O-acetylated-derivative of GD2, has recently received attention as novel antigen to target GD2-positive cancers. The present paper examines the role of O-acetyl-GD2 in tumor biology as well as the available preclinical data of anti-O-acetyl-GD2 monoclonal antibodies. A discussion on the relevance of O-acetyl-GD2 in chimeric antigen receptor T cell therapy development is also included.
Project description:Sirtuins are emerging players in cancer biology and other age-related disorders, and their putative role in bladder cancer (BlCa) remains elusive. Further understanding of disease biology may allow for generation of more effective pathway-based biomarkers and targeted therapies. Herein, we aimed to illuminate the role of sirtuins' family in BlCa and evaluate their potential as disease biomarkers and therapeutic targets. SIRT1-7 transcripts and protein levels were evaluated in a series of primary BlCa and normal bladder mucosa tissues. SIRT7 knockdown was performed through lentiviral transduction in MGHU3, 5637 and J82 cells and its functional role was assessed. SIRT1, 2, 4 and 5 expression levels were significantly lower in BlCa, whereas SIRT6 and 7 were overexpressed, and these results were corroborated by TCGA cohort analysis. SIRT7 transcript levels were significantly decreased in muscle-invasive vs. papillary BlCa. In vitro studies showed that SIRT7 downregulation promoted cells migration and invasion. Accordingly, increased EMT markers expression and decreased E-Cadherin (CDH1) was observed in those BlCa cells. Moreover, increased EZH2 expression and H3K27me3 deposition in E-Cadherin promoter was found in sh-SIRT7 cells. We demonstrated that sirtuins are globally deregulated in BlCa, and specifically SIRT7 downregulation is implicated in EMT, fostering BlCa invasiveness through EZH2-CDH1 axis.
Project description:There is a crucial need for development of prognostic and predictive biomarkers in human bladder carcinogenesis in order to personalize preventive and therapeutic strategies and improve outcomes. Epigenetic alterations, such as histone modifications, are implicated in the genetic dysregulation that is fundamental to carcinogenesis. Here we focus on profiling the histone modifications during the progression of bladder cancer. Histones were extracted from normal human bladder epithelial cells, an immortalized human bladder epithelial cell line (hTERT), and four human bladder cancer cell lines (RT4, J82, T24, and UMUC3) ranging from superficial low-grade to invasive high-grade cancers. Liquid chromatography-mass spectrometry (LC-MS) profiling revealed a statistically significant increase in phosphorylation of H1 linker histones from normal human bladder epithelial cells to low-grade superficial to high-grade invasive bladder cancer cells. This finding was further validated by immunohistochemical staining of the normal epithelium and transitional cell cancer from human bladders. Cell cycle analysis of histone H1 phosphorylation by Western blotting showed an increase of phosphorylation from G0/G1 phase to M phase, again supporting this as a proliferative marker. Changes in histone H1 phosphorylation status may further clarify epigenetic changes during bladder carcinogenesis and provide diagnostic and prognostic biomarkers or targets for future therapeutic interventions.
Project description:Ganglioside GD2 is a tumor-associated surface antigen found in a broad spectrum of human cancers and stem cells. They include pediatric embryonal tumors (neuroblastoma, retinoblastoma, brain tumors, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma), as well as adult cancers (small cell lung cancer, melanoma, soft tissue sarcomas). Because of its restricted normal tissue distribution, GD2 has been proven safe for antibody targeting. Anti-GD2 antibody is now incorporated into the standard of care for the treatment of high-risk metastatic neuroblastoma. Building on this experience, novel combinations of antibodies, cytokines, cells, and genetically engineered products all directed at GD2 are rapidly moving into the clinic. In this review, past and present immunotherapy trials directed at GD2 will be summarized, highlighting the lessons learned and the future directions.
Project description:We FACS sorted Ras-transformed human mammary epithelial cells (HMLER cells) into GD2+ and GD2- as well as CD44high/CD24low and CD44low/Cd24highcells and comapred the four different population by array. We FACS sorted Ras-transformed human mammary epithelial cells (HMLER cells) into GD2+ and GD2- as well as CD44high/CD24low and CD44low/Cd24highcells and comapred the four different population by array.
Project description:Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M)1-5 are aggressive and universally fatal pediatric brain cancers 6 . Chimeric antigen receptor (CAR)-expressing T cells have mediated impressive clinical activity in B cell malignancies7-10, and recent results suggest benefit in central nervous system malignancies11-13. Here, we report that patient-derived H3-K27M-mutant glioma cell cultures exhibit uniform, high expression of the disialoganglioside GD2. Anti-GD2 CAR T cells incorporating a 4-1BBz costimulatory domain 14 demonstrated robust antigen-dependent cytokine generation and killing of DMG cells in vitro. In five independent patient-derived H3-K27M+ DMG orthotopic xenograft models, systemic administration of GD2-targeted CAR T cells cleared engrafted tumors except for a small number of residual GD2lo glioma cells. To date, GD2-targeted CAR T cells have been well tolerated in clinical trials15-17. Although GD2-targeted CAR T cell administration was tolerated in the majority of mice bearing orthotopic xenografts, peritumoral neuroinflammation during the acute phase of antitumor activity resulted in hydrocephalus that was lethal in a fraction of animals. Given the precarious neuroanatomical location of midline gliomas, careful monitoring and aggressive neurointensive care management will be required for human translation. With a cautious multidisciplinary clinical approach, GD2-targeted CAR T cell therapy for H3-K27M+ diffuse gliomas of pons, thalamus and spinal cord could prove transformative for these lethal childhood cancers.