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.

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.

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.

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.

The emerging roles of orphan nuclear receptors in prostate cancer.

Orphan nuclear receptors are members of the nuclear receptor (NR) superfamily and are so named because their endogenous physiological ligands are either unknown or may not exist. Because of their important regulatory roles in many key physiological processes, dysregulation of signalings controlled by these receptors is associated with many diseases including cancer. Over years, studies of orphan NRs have become an area of great interest because their specific physiological and pathological roles have not been well-defined, and some of them are promising drug targets for diseases. The recently identified synthetic small molecule ligands, acting as agonists or antagonists, to these orphan NRs not only help to understand better their functional roles but also highlight that the signalings mediated by these ligand-independent NRs in diseases could be therapeutically intervened. This review is a summary of the recent advances in elucidating the emerging functional roles of orphan NRs in cancers, especially prostate cancer. In particular, some orphan NRs, RORγ, TR2, TR4, COUP-IFII, ERRα, DAX1 and SHP, exhibit crosstalk or interference with androgen receptor (AR) signaling in either normal or malignant prostatic cells, highlighting their involvement in prostate cancer progression as androgen and AR signaling pathway play critical roles in this process. We also propose that a better understanding of the mechanism of actions of these orphan NRs in prostate gland or prostate cancer could help to evaluate their potential value as therapeutic targets for prostate cancer.

Orphan nuclear receptor TLX functions as a potent suppressor of oncogene-induced senescence in prostate cancer via its transcriptional co-regulation of the CDKN1A (p21(WAF1) (/) (CIP1) ) and SIRT1 genes.

Oncogene-induced senescence is an important tumour-suppressing mechanism to prevent both premalignant transformation and cancer progression. Overcoming this process is a critical step in early cancer development. The druggable orphan nuclear receptor TLX (NR2E1) is characterized as an important regulator of neural stem cells and is also implicated in the development of some brain tumours. However, its exact functional roles in cancer growth regulation still remain unclear. Here we report that TLX can act as a promoter of tumourigenesis in prostate cancer by suppressing oncogene-induced senescence. We determined that TLX exhibited an increased expression in high-grade prostate cancer tissues and many prostate cancer cell lines. Functional studies revealed that TLX could perform an oncogenic function in prostate cancer cells, as its knockdown triggered cellular senescence and cell growth arrest in vitro and in vivo, whereas its over-expression promoted the malignant growth of prostate cancer cells. Furthermore, enhancement of TLX activity, by either ectopic expression or ligand stimulation, could potently prevent doxorubicin-induced senescence in prostate cancer cells and also allow prostatic epithelial cells to escape oncogene-induced senescence induced either by activated oncogene H-Ras(G12V) or knockdown of tumour suppressor PTEN, via a mechanism of direct but differential transcriptional regulation of two senescence-associated genes, repression of CDKN1A and transactivation of SIRT1. Together, our present study shows, for the first time, that TLX may play an important role in prostate carcinogenesis through its suppression of oncogene-induced senescence, and also suggests that targeting the senescence-regulatory TLX is of potential therapeutic significance in prostate cancer.

Ion channel TRPM8 promotes hypoxic growth of prostate cancer cells via an O2 -independent and RACK1-mediated mechanism of HIF-1α stabilization.

The growth adaptation of cancer cells to a hypoxic tumour microenvironment is mostly regulated by hypoxia-induced transcription factor HIF-1. HIF-1 transcriptional activity is strictly controlled by protein levels of the HIF-1α subunit, which is tightly regulated by a well-characterized O2 -dependent ubiquitin ligase-proteasomal degradation pathway. The cold-sensitive Ca(2+) channel protein TRPM8 exhibits increased expression in advanced prostate cancer. However, its exact functional roles in prostate cancer growth regulation are unclear and controversial. In this work, we show that TRPM8 promotes in vitro hypoxic growth capacities, drug resistance, and in vivo tumourigenicity, accompanied with enhanced HIF-1α protein levels. These effects are further potentiated by TRPM8 agonists but suppressed by TRPM8 gene knockdown and blocking with antagonists or TRPM8 antibody. TRPM8-induced suppression of HIF-1α ubiquitination and enhanced HIF-1 transactivation were attenuated by forced RACK1 expression and TRPM8 overexpression reduced phospho-RACK1 levels, thus affecting its dimerization status, and promoted RACK1 binding to HIF-1α and calcineurin. These data indicate that TRPM8-induced increase of HIF-1α protein in hypoxia- or normoxia-exposed prostate cancer cells was mediated through a newly characterized Ca(2+) -dependent but O2 -independent mechanism involving binding of RACK1 to HIF-1α and RACK1-mediated ubiquitination of HIF-1α. Collectively, our study not only provides a mechanistic insight into how TRPM8 promotes the hypoxic growth adaptation of cancer cells via its promotion of RACK1-mediated stabilization of HIF-1α but also suggests a potential therapeutic strategy for prostate cancer by targeting TRPM8.

ERRα augments HIF-1 signalling by directly interacting with HIF-1α in normoxic and hypoxic prostate cancer cells.

Adaptation of cancer cells to a hypoxic microenvironment is important for their facilitated malignant growth and advanced development. One major mechanism mediating the hypoxic response involves up-regulation of hypoxia-inducible factor 1 (HIF-1) expression, which controls reprogramming of energy metabolism and angiogenesis. Oestrogen-related receptor-α (ERRα) is a pivotal regulator of cellular energy metabolism and many biosynthetic pathways, and has also been proposed to be an important factor promoting the Warburg effect in advanced cancer. We and others have previously shown that ERRα expression is increased in prostate cancer and is also a prognostic marker. Here we show that ERRα is oncogenic in prostate cancer and also a key hypoxic growth regulator. ERRα-over-expressing prostate cancer cells were more resistant to hypoxia and showed enhanced HIF-1α protein expression and HIF-1 signalling. These effects could also be observed in ERRα-over-expressing cells grown under normoxia, suggesting that ERRα could function to pre-adapt cancer cells to meet hypoxia stress. Immunoprecipitation and FRET assays indicated that ERRα could physically interact with HIF-1α via its AF-2 domain. A ubiquitination assay showed that this ERRα-HIF-1α interaction could inhibit ubiquitination of HIF-1α and thus reduce its degradation. Such ERRα-HIF-1α interaction could be attenuated by XCT790, an ERRα-specific inverse agonist, resulting in reduced HIF-1α levels. In summary, we show that ERRα can promote the hypoxic growth adaptation of prostate cancer cells via a protective interaction with HIF-1α, suggesting ERRα as a potential therapeutic target for cancer treatment.

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.].

Off-the-shelf CAR-NK cells targeting immunogenic cell death marker ERp57 execute robust antitumor activity and have a synergistic effect with ICD inducer oxaliplatin.

BACKGROUND: Chimeric antigen receptor natural killer (CAR-NK) therapy holds great promise for treating hematologic tumors, but its efficacy in solid tumors is limited owing to the lack of suitable targets and poor infiltration of engineered NK cells. Here, we explore whether immunogenic cell death (ICD) marker ERp57 translocated from endoplasmic reticulum to cell surface after drug treatment could be used as a target for CAR-NK therapy. METHODS: To target ERp57, a VHH phage display library was used for screening ERp57-targeted nanobodies (Nbs). A candidate Nb with high binding affinity to both human and mouse ERp57 was used for constructing CAR-NK cells. Various in vitro and in vivo studies were performed to assess the antitumor efficacy of the constructed CAR-NK cells. RESULTS: We demonstrate that the translocation of ERp57 can not only be induced by low-dose oxaliplatin (OXP) treatment but also is spontaneously expressed on the surface of various types of tumor cell lines. Our results show that G6-CAR-NK92 cells can effectively kill various tumor cell lines in vitro on which ERp57 is induced or intrinsically expressed, and also exhibit potent antitumor effects in cancer cell-derived xenograft and patient-derived xenograft mouse models. Additionally, the antitumor activity of G6-CAR-NK92 cells is synergistically enhanced by the low-dose ICD-inducible drug OXP. CONCLUSION: Collectively, our findings suggest that ERp57 can be leveraged as a new tumor antigen for CAR-NK targeting, and the resultant CAR-NK cells have the potential to be applied as a broad-spectrum immune cell therapy for various cancers by combining with ICD inducer drugs.

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.

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.

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.

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.

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.

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.

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.

[Cytotoxicity of dichloroacetic acid in lymphocyte and expression of chemokine receptor CXCR2 and chemokine receptor CXCR3 mRNA].

OBJECTIVE: To explore the effects of trichloroethylene (TCE) and its by-products (trichloroacetic acid, TCA; dichloroacetic acid, DCA) on the normal human peripheral blood lymphocyte and the role of DCA in dermatitis medicamentosa- like induced by trichloroethylene (DMLT). METHODS: Lymphocyte was isolated from peripheral venous blood, and cytotoxicity of human lymphocytes treated with different concentrations (0.02 approximately 30.00 mmol/L) of DCA was determined at indicated times (2 h and 4 h) based on the MTS assay. Action of DCA on cell viability, membrane integrity was assessed by neutral red uptake (NRU) assay and lactate dehydrogenase (LDH) release test and measurement of superoxide dismutase (SOD) activity. Fluorescence quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) was employed for detection and quantization of the chemokine receptor CXCR2 and chemokine receptor CXCR3 mRNA in peripheral blood lymphocyte treated with different concentrations of DCA. RESULTS: DCA had a more vital effect on peripheral blood lymphocyte than TCE and TCA. A concentration-dependent release of LDH was observed at 4 h after cells were exposed to different doses of DCA (0.88, 1.75, 3.50 and 7.00 mmol/L) (P < 0.05), and DCA also caused an inhibition of SOD activity in a concentration-dependent manner (P < 0.05). The results of FQ- RT- PCR indicated that CXCR2 and CXCR3 mRNA were all over- expression. At 48 h after the DCA of 0.5 mmol/L and 10.00 mmol/L was used, CXCR2 and CXCR3 mRNA were 10.34, 5.66-fold and 19.43, 8.75-fold of those in the control group (P < 0.01). CONCLUSION: DCA is of a great cytotoxicity and may be one of crucial evocators on DMLT.

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.