Interferon-induced transmembrane protein-1 competitively blocks Ephrin receptor A2-mediated Epstein-Barr virus entry into epithelial cells.

Epstein-Barr virus (EBV) can infect both B cells and epithelial cells (ECs), causing diseases such as mononucleosis and cancer. It enters ECs via Ephrin receptor A2 (EphA2). The function of interferon-induced transmembrane protein-1 (IFITM1) in EBV infection of ECs remains elusive. Here we report that IFITM1 inhibits EphA2-mediated EBV entry into ECs. RNA-sequencing and clinical sample analysis show reduced IFITM1 in EBV-positive ECs and a negative correlation between IFITM1 level and EBV copy number. IFITM1 depletion increases EBV infection and vice versa. Exogenous soluble IFITM1 effectively prevents EBV infection in vitro and in vivo. Furthermore, three-dimensional structure prediction and site-directed mutagenesis demonstrate that IFITM1 interacts with EphA2 via its two specific residues, competitively blocking EphA2 binding to EBV glycoproteins. Finally, YTHDF3, an m6A reader, suppresses IFITM1 via degradation-related DEAD-box protein 5 (DDX5). Thus, this study underscores IFITM1's crucial role in blocking EphA2-mediated EBV entry into ECs, indicating its potential in preventing EBV infection.

Corrigendum: In vitro and in vivo antitumor activity of cucurbitacin C, a novel natural product from cucumber.

[This corrects the article DOI: 10.3389/fphar.2019.01287.].

PERK-Mediated Cholesterol Excretion from IDH Mutant Glioma Determines Anti-Tumoral Polarization of Microglia.

Isocitrate dehydrogenase (IDH) mutation, a known pathologic classifier, initiates metabolic reprogramming in glioma cells and has been linked to the reaction status of glioma-associated microglia/macrophages (GAMs). However, it remains unclear how IDH genotypes contribute to GAM phenotypes. Here, it is demonstrated that gliomas expressing mutant IDH determine M1-like polarization of GAMs, while archetypal IDH induces M2-like polarization. Intriguingly, IDH-mutant gliomas secrete excess cholesterol, resulting in cholesterol-rich, pro-inflammatory GAMs without altering their cholesterol biosynthesis, and simultaneously exhibiting low levels of tumoral cholesterol due to expression remodeling of cholesterol transport molecules, particularly upregulation of ABCA1 and downregulation of LDLR. Mechanistically, a miR-19a/LDLR axis-mediated novel post-transcriptional regulation of cholesterol uptake is identified, modulated by IDH mutation, and influencing tumor cell proliferation and invasion. IDH mutation-induced PERK activation enhances cholesterol export from glioma cells via the miR-19a/LDLR axis and ABCA1/APOE upregulation. Further, a synthetic PERK activator, CCT020312 is introduced, which markedly stimulates cholesterol efflux from IDH wild-type glioma cells, induces M1-like polarization of GAMs, and consequently suppresses glioma cell invasion. The findings reveal an essential role of the PERK/miR-19a/LDLR signaling pathway in orchestrating gliomal cholesterol transport and the subsequent phenotypes of GAMs, thereby highlighting a novel potential target pathway for glioma therapy.

Safety and antitumor activity of GD2-Specific 4SCAR-T cells in patients with glioblastoma.

BACKGROUND: This study aimed to validate whether infusion of GD2-specific fourth-generation safety-designed chimeric antigen receptor (4SCAR)-T cells is safe and whether CAR-T cells exert anti-glioblastoma (GBM) activity. METHODS: A total of eight patients with GD2-positive GBM were enrolled and infused with autologous GD2-specific 4SCAR-T cells, either through intravenous administration alone or intravenous combined with intracavitary administration. RESULTS: 4SCAR-T cells expanded for 1-3 weeks and persisted at a low frequency in peripheral blood. Of the eight evaluable patients, four showed a partial response for 3 to 24 months, three had progressive disease for 6 to 23 months, and one had stable disease for 4 months after infusion. For the entire cohort, the median overall survival was 10 months from the infusion. GD2 antigen loss and infiltrated T cells were observed in the tumor resected after infusion. CONCLUSION: Both single and combined infusions of GD2-specific 4SCAR-T cells in targeting GBM were safe and well tolerated, with no severe adverse events. In addition, GD2-specific 4SCAR-T cells partially mediate antigen loss and activate immune responses in the tumor microenvironment. Validation of our findings in a larger prospective trial is warranted. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03170141 . Registered 30 May 2017.

Alterations of plasma exosomal proteins and motabolies are associated with the progression of castration-resistant prostate cancer.

BACKGROUND: Current diagnosis tools for prostate cancer (PCa) such as serum PSA detection and prostate biopsy cannot distinguish dormant tumors from invasive malignancies, either be used as prognosis marker for castration resistant prostate cancer (CRPC), the lethal stage of PCa patients. Exosomes have been widely investigated as promising biomarkers for various diseases. We aim to characterize the proteomic and metabolomic profile of exosomes and to evaluate their potential value for the diagnosis of PCa, especially CRPC. We also investigate the functions of some specific exosome biomarkers in the progression of CRPC. METHODS: Integrated proteomics and metabolomics analysis were performed for plasma-derived exosomes collected from tumor-free controls (TFC), PCa and CRPC patients. Expression of specific exosomal proteins were further validated by targeted 4D-parallel reaction monitoring (PRM) mass spectrometry among the three cohorts. Tissue distribution and functional role of exosomal protein LRG1 was studied in clinical PCa tissue samples and cell line models. RESULTS: Three potential exosomal protein markers were identified. The apolipoprotein E level in PCa samples was 1.7-fold higher than that in TFC (receiver operating characteristic value, 0.74). Similarly, the levels of exosome-derived leucine-rich alpha2-glycoprotein 1 (LRG1) and inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) in the CRPC group were 1.7 and 2.04 times, respectively, higher than those in the PCa group (ROC values, 0.84 and 0.85, respectively), indicating that LRG1 and ITIH3 could serve as predictive markers for CRPC. For metabolomic evaluation of exosomes, a series of differentially expressed metabolites were identified, and a combined metabolite panel showed ROC value of 0.94 for distinguishing PCa from TFC and 0.97 for distinguishing CRPC from PCa. Immunohistochemistry of tissue microarray showed that LRG1 protein was significantly upregulated in advanced prostate cancer and functional assay revealed that ectopic expression of LRG1 can significantly enhance the malignant phenotype of prostate cancer cells. More importantly, PCa cell derived LRG1-overexpressed exosomes remarkably promoted angiogenesis. CONCLUSION: Integration of proteomics and metabolomics data generated proteomic and metabolic signatures of plasma exosomes that may facilitate discrimination of CRPC from PCa and TFC patients, suggesting the potential of exosomal proteins and metabolites as CRPC markers. The study also confirmed the important role of exosomal protein LRG1 in PCa malignant progression.

Identification of the Key Genes Involved in the Tumorigenesis and Prognosis of Prostate Cancer.

Background: Prostate cancer (PCa) is a malignant tumor in males, with a majority of the cases advancing to metastatic castration resistance. Metastasis is the leading cause of mortality in PCa. The traditional early detection and prediction approaches cannot differentiate between the different stages of PCa. Therefore, new biomarkers are necessary for early detection and clear differentiation of PCa stages to provide precise therapeutic intervention. Methods: The objective of the study was to find significant differences in genes and combine the three GEO datasets with TCGA-PRAD datasets (DEG). Weighted gene coexpression network analysis (WGCNA) determined the gene set and PCa clinical feature correlation module utilizing the TGGA-PRAD clinical feature data. The correlation module genes were rescreened using the biological information analysis tools, with the three hub genes (TOP2A, NCAPG, and BUB1B) for proper verification. Finally, internal (TCGA) and external (GSE32571, GSE70770) validation datasets were used to validate and predict the value of last hub genes. Results: The hub gene was abnormally upregulated in PCa samples during verification. The expression of each gene was favorably connected with the Gleason score and TN tumor grade in clinical samples but negatively correlated with the overall survival rate. The expression of these genes was linked to CD8 naive cells and macrophages, among other cells. Antitumor immune cells like NK and NKT were favorably and adversely correlated with infiltrating cells, respectively. Simultaneously, the GSCV and GSEA indicated that the hub gene is connected with cell proliferation, death, and androgen receptor, among other signaling pathways. Therefore, these genes could influence the incidence and progression of PCa by participating in or modulating various signaling pathways. Furthermore, using the online tool of CMap, we examined the individual medications for Hughes and determined that tipifarnib could be useful for the clinical therapy of PCa. Conclusion: TOP2A, NCAPG, and BUB1B are important genes intimately linked to the clinical prognosis of PCa and can be employed as reliable biomarkers for early diagnosis and prognosis. Moreover, these genes can provide a theoretical basis for precision differentiation and treatment of PCa.

A novel ferroptosis-related gene prognostic index for prognosis and response to immunotherapy in patients with prostate cancer.

Background: Prostate cancer (PCa) is among the leading causes of cancer death worldwide. Ferroptosis refers to an iron-dependent form of regulated cell death and is involved in prostate tumorigenesis. A few ferroptosis-related gene signatures have been developed to predict the prognosis for PCa patients. However, previous signatures were typically established based on biochemical recurrence-free survival, which has proven not to be a good surrogate for overall survival (OS). This study aimed to construct a novel ferroptosis-related gene prognostic index (FRGPI) to predict disease-free survival (DFS) and response to immunotherapy for PCa patients after radical prostatectomy. Methods: Gene expression and clinicopathological data on PCa patients were obtained from the TCGA database. Ferroptosis-related hub genes associated with DFS of PCa patients were identified by an in-depth bioinformatics analysis using a novel and comprehensive algorithm based on functional enrichment, consensus clustering, weighted gene co-expression network analysis (WGCNA), and protein-protein interaction (PPI) network construction. The FRGPI was established on the basis of the genes selected using multivariate cox regression analysis and further validated in two additional PCa cohorts. Next, the clinicopathological, molecular, and immune profiles were characterized and compared between FRGPI-high and FRGPI-low subgroups. Finally, the predictive role of the FRGPI in response to immunotherapy was estimated using a metastatic urothelial cancer cohort treated with an anti-PD-L1 agent. Results: The FRGPI was constructed based on four genes (E2F1, CDC20, TYMS, and NUP85), and FRGPI-high patients had worse DFS than FRGPI-low patients. Multivariate cox regression analysis revealed that FRGPI could act as an independent prognostic factor for PCa patients after radical prostatectomy. A prognostic nomogram comprising the FRGPI and other clinicopathological parameters was established to predict the DFS for PCa patients quantitatively. In addition, comprehensive results demonstrated that high FRGPI scores showed a significantly positive correlation with worse clinicopathological features, higher mutation counts, increased frequency of copy number variations (CNVs), higher homologous recombination deficiency (HRD) and immune scores, higher mRNAsi, and more importantly, enhanced sensitivity to immunotherapy. Conclusions: FRGPI is not only a promising and robust prognostic biomarker, but also a potential indicator of immunotherapeutic outcomes for PCa patients after radical prostatectomy.

Endothelial nitric oxide synthase (eNOS)-NO signaling axis functions to promote the growth of prostate cancer stem-like cells.

BACKGROUND: Accumulating evidence supports that prostate cancer stem-like cells (PCSCs) play significant roles in therapy resistance and metastasis of prostate cancer. Many studies also show that nitric oxide (NO) synthesized by NO synthases can function to promote tumor progression. However, the exact roles of NOSs and NO signaling in the growth regulation of PCSCs and castration-resistant prostate cancer (CRPC) are still not fully understood. METHODS: The regulatory functions of NOS-NO signaling were evaluated in prostate cancer cells, especially in PCSCs enriched by 3D spheroid culture and CD133/CD44 cell sorting. The molecular mechanisms of NOS-NO signaling in PCSCs growth regulation and tumor metastasis were investigated in PCSCs and mice orthotopic prostate tumor model. RESULTS: Endothelial NOS (eNOS) exhibited a significant upregulation in high-grade prostate cancer and metastatic CRPC. Xenograft models of CRPC exhibited notable increased eNOS expression and higher intracellular NO levels. PCSCs isolated from various models displayed significant enhanced eNOS-NO signaling. Functional analyses demonstrated that increased eNOS expression could promote in vivo tumorigenicity and metastatic potential of prostate cancer cells. Characterization of eNOS-NO involved downstream pathway which confirmed that enhanced eNOS signaling could promote the growth of PCSCs and antiandrogen-resistant prostate cancer cells via an activated downstream NO-sGC-cGMP-PKG effector signaling pathway. Interestingly, eNOS expression could be co-targeted by nuclear receptor ERRα and transcription factor ERG in prostate cancer cells and PCSCs. CONCLUSIONS: Enhanced eNOS-NO signaling could function to promote the growth of PCSCs and also the development of metastatic CRPC. Besides eNOS-NO as potential targets, targeting its upstream regulators (ERRα and ERG) of eNOS-NO signaling could also be the therapeutic strategy for the management of advanced prostate cancer, particularly the aggressive cancer carrying with the TMPRSS2:ERG fusion gene.

Hsa_circ_0003258 promotes prostate cancer metastasis by complexing with IGF2BP3 and sponging miR-653-5p.

BACKGROUND: More and more studies have shown that circular RNAs (circRNAs) play a critical regulatory role in many cancers. However, the potential molecular mechanism of circRNAs in prostate cancer (PCa) remains largely unknown. METHODS: Differentially expressed circRNAs were identified by RNA sequencing. The expression of hsa_circ_0003258 was evaluated using quantitative real-time PCR and RNA in situ hybridization. The impacts of hsa_circ_0003258 on the metastasis of PCa cells were investigated by a series of in vitro and in vivo assays. Lastly, the underlying mechanism of hsa_circ_0003258 was revealed by Western blot, biotin-labeled RNA pulldown, RNA immunoprecipitation, luciferase assays and rescue experiments. RESULTS: Increased expression of hsa_circ_0003258 was found in PCa tissues and was associated with advanced TNM stage and ISUP grade. Overexpression of hsa_circ_0003258 promoted PCa cell migration by inducing epithelial mesenchymal transformation (EMT) in vitro as well as tumor metastasis in vivo, while knockdown of hsa_circ_0003258 exerts the opposite effect. Mechanistically, hsa_circ_0003258 could elevate the expression of Rho GTPase activating protein 5 (ARHGAP5) via sponging miR-653-5p. In addition, hsa_circ_0003258 physically binds to insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) in the cytoplasm and enhanced HDAC4 mRNA stability, in which it activates ERK signalling pathway, then triggers EMT programming and finally accelerates the metastasis of PCa. CONCLUSIONS: Upregulation of hsa_circ_0003258 drives tumor progression through both hsa_circ_0003258/miR-653-5p/ARHGAP5 axis and hsa_circ_0003258/IGF2BP3 /HDAC4 axis. Hsa_circ_0003258 may act as a promising biomarker for metastasis of PCa and an attractive target for PCa intervention.

Orphan nuclear receptor TLX promotes immunosuppression via its transcriptional activation of PD-L1 in glioma.

BACKGROUND: High-grade gliomas are rapidly progressing tumors of the central nervous system, and are associated with poor prognosis and highly immunosuppressive microenvironments. Meanwhile, a better understanding of PD-L1, a major prognostic biomarker for checkpoint immune therapy, regulation may provide insights for developing novel immunotherapeutic strategies for treating gliomas. In the present study, we elucidate the functional significance of the orphan nuclear receptor TLX in human glioma, and its functional role in immune suppression through regulation of PD-L1/PD-1 axis. METHODS: TLX and PD-L1 expression patterns, and their association with clinicopathological parameters and immune phenotypes of glioma were analysed using CIBERSORT algorithm and single-sample gene-set enrichment analysis from The Cancer Genome Atlas (n=695) and Chinese Glioma Genome Atlas (n=1018) databases. Protein expression and cellular localization of TLX, PD-L1, and PD-1, as well as the prevalence of cytotoxic tumor-infiltrating lymphocytes (TILs), and tumor-associated macrophages (TAMs), in the glioma immune microenvironment were analyzed via tissue microarray by immunohistochemistry and multiplex immunofluorescence. Glioma allografts and xenografts with TLX manipulation (knockdown/knockout or reverse agonist) were inoculated subcutaneously, or orthotopically into the brains of immunodeficient and immunocompetent mice to assess tumor growth by imaging, and the immune microenvironment by flow cytometry. PD-L1 transcriptional regulation by TLX was analyzed by chromatin immunoprecipitation and luciferase reporter assays. RESULTS: TLX and PD-L1 expression was positively associated with macrophage-mediated immunosuppressive phenotypes in gliomas. TLX showed significant upregulation and positive correlation with PD-L1. Meanwhile, suppression of TLX significantly inhibited in vivo growth of glioma allografts and xenografts (p<0.05), rescued the antitumoral immune response, significantly decreased the PD-L1+, and glioma-associated macrophage population, and increased cytotoxic lymphocyte infiltration (p<0.05). Mechanistically, TLX binds directly to CD274 (PD-L1) gene promoter and activates CD274 transcription. CONCLUSIONS: TLX contributes to glioma malignancy and immunosuppression through transcriptional activation of PD-L1 ligands that bind to PD-1 expressed on both TILs and TAMs. Thus, targeting the druggable TLX may have potential therapeutic significance in glioma immune therapy.

Orphan nuclear receptors as regulators of intratumoral androgen biosynthesis in castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) almost invariably occurs after androgen-deprivation therapy (ADT) for the advanced metastatic disease. It is generally believed that among multiple mechanisms and signaling pathways, CRPC is significantly driven by the reactivation of androgen receptor (AR) signaling in ADT-treated patients with castrate levels of androgen, partially at least mediated by the androgen biosynthesis within the tumor, also known as intratumoral or intraprostatic androgen biosynthesis. Steroidogenic enzymes, such as CYP11A1, CYP17A1, HSD3B1, AKR1C3 and SRD5A, are essential to catalyze the conversion of the initial substrate cholesterol into potent androgens that confers the CRPC progression. Accumulating evidences indicate that many steroidogenic enzymes are upregulated in the progression setting; however, little is known about the dysregulation of these enzymes in CRPC. Orphan nuclear receptors (ONRs) are members of the nuclear receptor superfamily, of which endogenous physiological ligands are unknown and which are constitutively active independent of any physiological ligands. Studies have validated that besides AR, ONRs could be the potential therapeutic targets for prostate cancer, particularly the lethal CRPC progression. Early studies reveal that ONRs play crucial roles in the transcriptional regulation of steroidogenic enzyme genes. Notably, we and others show that three distinct ONRs, including liver receptor homolog-1 (LRH-1, NR5A2), steroidogenic factor 1 (SF-1, AD4BP, NR5A1) and estrogen-related receptor α (ERRα, NR3B1), can contribute to the CRPC progression by promotion of the intratumoral androgen synthesis via their direct transcriptional regulation on multiple steroidogenic enzymes. This review presents an overview of the current understanding on the intratumoral androgen biosynthesis in CRPC, with a special focus on the emerging roles of ONRs in this process.

Correction: Nuclear receptor HNF4α performs a tumor suppressor function in prostate cancer via its induction of p21-driven cellular senescence.

The original version of this Article contained an error in the spelling of the author Chan FL, which was incorrectly given as leung Chan F. This has now been corrected in both the PDF and HTML versions of the Article.

Nuclear factor IX promotes glioblastoma development through transcriptional activation of Ezrin.

Enhanced migration is pivotal for the malignant development of glioblastoma (GBM), but the underlying molecular mechanism that modulates the migration of the GBM cells remains obscure. Here we show that nuclear factor IX (NFIX) is significantly upregulated in human GBM lesions compared with normal or low-grade gliomas. NFIX deficiency impairs the migration of GBM cells and inhibits the tumor growth in the hippocampus of immunodeficient nude mice. Mechanistically, NFIX silencing suppresses the expression of Ezrin, a protein that crosslinks actin cytoskeleton and plasma membrane, which is also positively correlated with GBM malignancy. NFIX depletion induced migration inhibition of GBM cells can be rescued by the replenishment of Ezrin. Furthermore, we identify a NFIX response element (RE) between -840 and -825 bp in the promoter region of the Ezrin gene. Altogether, our findings show, for the first time that NFIX can transcriptionally upregulate the expression of Ezrin and contribute to the enhanced migration of GBM cells, suggesting that NFIX is a potential target for GBM therapy.

Targeting prostate cancer stem-like cells by an immunotherapeutic platform based on immunogenic peptide-sensitized dendritic cells-cytokine-induced killer cells.

BACKGROUND: Autologous cellular immunotherapy or immune enhancement therapy has demonstrated some promising benefits for prostate cancer. T cell-based immunotherapy or sipuleucel-T therapy has yielded certain beneficial responses and a slight improvement on the overall survival of patients with metastatic castration-resistant prostate cancer (mCRPC) as shown in some clinical trials, suggesting that prostate cancer is immunoresponsive. METHODS: In this study, we developed an adaptive cytokine-induced killer cell (CIK)-based immunotherapeutic application targeting the prostate cancer stem-like cells (PCSCs). In this therapeutic platform, dendritic cells (DC) were isolated from the peripheral blood mononuclear cells (PBMCs) and preloaded or sensitized with immunogenic peptides derived from two PCSC-associated cell membrane molecules, CD44 and EpCAM, followed by co-culture with the expanded peripheral blood lymphocyte (PBL)-derived CIK cells. The in vitro cytotoxic activity of DC-activated CIK cells against PCSCs was determined by CCK8 and TUNEL assays, and the in vivo anti-tumor effect of DC-activated CIK cells on prostate cancer xenograft tumors was evaluated in subcutaneous and orthotopic xenograft models. RESULTS: Our results showed that the peptide-sensitized DC-CIK cell preparation manifested significant in vitro cytotoxic activity against the PCSC-enriched prostatospheroids and also in vivo anti-tumor effect against prostate cancer xenografts derived from the PCSC-enriched prostatospheroids. CONCLUSIONS: Together, our established immunogenic peptide-sensitized DC-CIK-based cell preparation platform manifests its potential immunotherapeutic application in targeting the PCSCs and also prostate cancer.

Nuclear receptor HNF4α performs a tumor suppressor function in prostate cancer via its induction of p21-driven cellular senescence.

Hepatocyte nuclear factor 4α (HNF4α, NR2A1) is a highly conserved member of the nuclear receptor superfamily. Recent advances reveal that it is a key transcriptional regulator of genes, broadly involved in xenobiotic and drug metabolism and also cancers of gastrointestinal tract. However, the exact functional roles of HNF4α in prostate cancer progression are still not fully understood. In this study, we determined the functional significance of HNF4α in prostate cancer. Our results showed that HNF4α exhibited a reduced expression pattern in clinical prostate cancer tissues, prostate cancer cell lines and xenograft model of castration-relapse prostate cancer. Stable HNF4α knockdown not only could promote cell proliferation and suppress doxorubicin (Dox)-induced cellular senescence in prostate cancer cells, but also confer resistance to paclitaxel treatment and enhance colony formation capacity and in vivo tumorigenicity of prostate cancer cells. On the contrary, ectopic overexpression of HNF4α could significantly inhibit the cell proliferation of prostate cancer cells, induce cell-cycle arrest at G2/M phase and trigger the cellular senescence in prostate cancer cells by activation of p21 signal pathway in a p53-independent manner via its direct transactivation of CDKN1A. Together, our results show that HNF4α performs a tumor suppressor function in prostate cancer via a mechanism of p21-driven cellular senescence.

In Vitro and In Vivo Antitumor Activity of Cucurbitacin C, a Novel Natural Product From Cucumber.

Cucurbitacin C (CuC), a novel analogue of triterpenoids cucurbitacins, confers a bitter taste in cucumber. Genes and signaling pathways responsive for biosynthesis of CuC have been identified in the recent years. In the present study, we explored the anti-cancer effects of CuC against human cancers in vitro and in vivo. CuC inhibited proliferation and clonogenic potential of multiple cancer cells in a dose-dependent manner. Low-dose CuC treatment induced cell cycle arrest at G1 or G2/M stage in different cancer lines, whereas high-dose treatment of CuC caused apoptosis in cancer cells. PI3K-Akt signaling pathway was found to be one of the major pathways involved in CuC-induced cell growth arrest and apoptosis by RNA-Seq and Western blotting. Mechanistic dissection further confirmed that CuC effectively inhibited the Akt signaling by inhibition of Akt phosphorylation at Ser473. In vivo CuC treatment (0.1 mg/kg body weight) effectively inhibited growth of cancer cell-derived xenograft tumors in athymic nude mice and caused significant apoptosis. Our findings for the first time demonstrated the potential therapeutic significance of CuC against human cancers.

miR-182-5p affects human bladder cancer cell proliferation, migration and invasion through regulating Cofilin 1.

BACKGROUND: Human bladder cancer is one of the common malignant tumors, and it mainly occurs in men. miR-182-5p, a member of miR-183 family, acts as tumor suppressor or oncogene in various kinds of tumors. In this study, we first investigate that the absence of miR-182-5p in human bladder cancer promotes tumor growth by regulating the expression of Cofilin 1, an actin modulating-protein. METHODS: Human bladder tumor tissue specimens were collected to detect the expression of miR-182-5p and Cofilin 1 by qRT-PCR. Luciferase activity assay was performed to demonstrate the regulation of Cofilin 1 mRNA 3'UTR by miR-182-5p. Then, cell experiments were performed to analysis the effect of miR-182-5p/Cofilin 1 pathway on tumor cell proliferation, migration, invasion and colony forming efficiency. Finally, xenograft tumor models were established to evaluate the role of miR-182-5p in tumorigenesis abilities in vivo. RESULTS: qRT-PCR and Western blotting analysis showed that Cofilin 1 expression was up-regulated in both bladder cancer tissues and cell lines compared with normal. Luciferase activity assay showed that miR-182-5p specifically targets Cofilin 1 mRNA 3'UTR and represses the expression of Cofilin 1. Also, miR-182-5p inhibited bladder tumor cell proliferation, migration, invasion and colony forming efficiency. Furthermore, xenograft tumor model assay showed that miR-182-5p plays a negative role in bladder cancer tumorigenesis abilities in vivo. CONCLUSION: Present results suggest that miR-182-5p could inhibit human bladder tumor growth by repressing Cofilin 1 expression. Our findings may provide a new horizon for exploring therapeutic target of bladder cancer.

Long non-coding RNA HOXA-AS2 promotes the migration, invasion and stemness of bladder cancer via regulating miR-125b/Smad2 axis.

Long non-coding RNA HOXA-AS2 (HOXA cluster antisense RNA 2) has been reported to function as an oncogene in different types of cancers including breast cancer, liver cancer, gastric cancer and colorectal cancer, etc. However, its role in the development and progression of bladder cancer remains unknown. This study aimed to examine the expression of HOXA-AS2 in bladder cancer, to explore its role in the migration, invasion and stemness of bladder cancer cells and to further identify the potential downstream target miRNAs of HOXA-AS2 in this type of cancer. Our results firstly demonstrated the upregulation of HOXA-AS2 in both bladder cancer cells and clinical bladder tumors. Such upregulation was also positively correlated with the advanced stage, invasion and lymph node metastasis of bladder cancer as well as the expression of cancer stem cell marker OCT4 in patients. After knockdown of HOXA-AS2 in bladder cancer 5637 and T24 cells, the migration, invasion and stemness of cancer cells were significantly inhibited, indicating the capability of HOXA-AS2 to promote the migration, invasion and stemness of bladder cancer cells. Knockdown of HOXA-AS2 also suppressed in vivo tumor growth in the nude mice. Furthermore, this study also identified miR-125b as a downstream target of HOXA-AS2 and revealed the downregulation of miR-125b by HOXA-AS2 as well as the involvement of HOXA-AS2/miR-125b/Smad2 interactions in the functional role of HOXA-AS2 in mediating the migration, invasion and stemness of bladder cancer cells. Together, our findings suggest that HOXA-AS2 might be a potential biomarker and target for the diagnosis, monitoring and treatment of bladder cancer.

Development of a novel and economical agar-based non-adherent three-dimensional culture method for enrichment of cancer stem-like cells.

BACKGROUND: Non-adherent or ultra-low attachment three-dimensional (3D) culture, also called sphere formation assay, has been widely used to assess the malignant phenotype and stemness potential of transformed or cancer cells. This method is also popularly used to isolate the cancer stem-like cells (CSCs) or tumor-initiating cells based on their unique anchorage-independent growth or anoikis-resistant capacity. Different non-adhesive coating agents, such as poly-2-hydroxyethyl methacrylate (poly-HEMA) and synthetic hydrogels, have been used in this non-adherent 3D culture. However, preparation of non-adherent culture-ware is labor-intensive and technically demanding, and also costs of commercial non-adherent culture-ware prepared with various coating agents are relatively expensive and the culture-ware cannot be used repeatedly. METHODS: In this study, we developed a non-adherent 3D culture method based on agar coating for growing tumor spheres derived from various cancer cell lines and primary prostate cancer tissues under a non-adherent and serum-free condition. The tumor spheres generated by this 3D culture method were analyzed on their expression profiles of CSC-associated markers by reverse transcription quantitative polymerase chain reaction, presence and relative proportion of CSCs by fluorescence-activated cell sorting (CD133+/CD44+ cell sorting) and also a CSC-visualizing reporter system responsive to OCT4 and SOX2 (SORE6), and in vivo tumorigenicity. The repeated use of agar-coated plates for serial passages of tumor spheres was also evaluated. RESULTS: Our results validated that the multicellular tumor spheres generated by this culture method were enriched of CSCs, as evidenced by their enhanced expression profiles of CSC markers, presence of CD133+/CD44+ or SORE6+ cells, enhanced self-renewal capacity, and in vivo tumorigenicity, indicating its usefulness in isolation and enrichment of CSCs. The agar-coated plates could be used multiple times in serial passages of tumor spheres. CONCLUSIONS: The described agar-based 3D culture method offers several advantages as compared with other methods in isolation of CSCs, including its simplicity and low-cost and repeated use of agar-coated plates for continuous passages of CSC-enriched spheres.

Nuclear receptor ERRα and transcription factor ERG form a reciprocal loop in the regulation of TMPRSS2:ERG fusion gene in prostate cancer.

The TMPRSS2:ERG (T:E) fusion gene is generally believed to be mainly regulated by the activated androgen receptor (AR) signaling in androgen-dependent prostate cancer. However, its persistent expression in castration-resistant and neuroendocrine prostate cancers implies that other transcription factors might also regulate its expression. Here, we showed that up-regulation of nuclear receptor estrogen-related receptor alpha (ERRα) was closely associated with the oncogenic transcription factor ERG expression in prostate cancer, and their increased coexpression patterns were closely associated with high Gleason scores and metastasis in patients. Both ERRα and ERG exhibited a positive expression correlation in a castration-resistant prostate cancer (CRPC) xenograft model VCaP-CRPC. We showed that ERRα could directly transactivate T:E fusion gene in both AR-positive and -negative prostate cancer cells via both ERR-binding element- and AR-binding element-dependent manners. Ectopic T:E expression under ERRα regulation could promote both in vitro invasion and in vivo metastasis capacities of AR-negative prostatic cells. Intriguingly, ERG expressed by the T:E fusion could also transactivate the ERRα (ESRRA) gene. Hereby, ERRα and ERG can synergistically regulate each other and form a reciprocal regulatory loop to promote the advanced growth of prostate cancer. Inhibition of ERRα activity by ERRα inverse agonist could suppress T:E expression in prostate cancer cells, implicating that targeting ERRα could be a potential therapeutic strategy for treating the aggressive T:E-positive prostate cancer.