An androgen receptor-based signature to predict prognosis and identification of ORC1 as a therapeutical target for prostate adenocarcinoma.

Background: Aberrant activation of androgen receptor (AR) signaling plays a crucial role in the progression of prostate adenocarcinoma (PRAD) and contributes significantly to the development of enzalutamide resistance. In this study, we aimed to identify a novel AR-driven signature that can predict prognosis and endows potentially reveal novel therapeutic targets for PRAD. Methods: The Seurat package was used to preprocess the single-cell RNA sequencing (scRNA-seq). Differentially expressed genes were visualized using limma and pheamap packages. LASSO and multi-variate Cox regression models were established using glmnet package. The package "Consensus Cluster Plus" was utilized to perform the consensus clustering analysis. The biological roles of origin recognition complex subunit 1 (ORC1) in PRAD were determined by gain- and loss-of-function studies in vitro and in vivo. Result: We characterized the scRNA-seq data from GSE99795 and identified 10 AR-associated genes (ARGs). The ARGs model was trained and validated in internal and external cohorts. The ARGs were identified as an independent hazard factor in PRAD and correlated with clinical risk characteristics. In addition, the ARGs were found to be correlated with somatic tumor mutation burden (TMB) levels. Two groups that have distinct prognostic and molecular features were identified through consensus clustering analysis. ORC1 was identified as a critical target among these ARGs, and it ORC1 promoted proliferation and stem-like properties of PRAD cells. Chromatin immunoprecipitation (ChIP)-qPCR assay confirmed that AR could directly bind the promoter of ORC1. Activated AR/ORC1 axis contributed to enzalutamide resistance, and targeting ORC1 rendered PRAD cells more susceptible to enzalutamide. Conclusions: This study defines an AR-driven signature that AR activates ORC1 expressions to promote PRAD progression and enzalutamide resistance, which may provide novel targets for PRAD treatment.

A covalent inhibitor targeting the papain-like protease from SARS-CoV-2 inhibits viral replication.

Covalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-2 have great potential as antivirals, but their non-specific reactivity with thiols has limited their development. In this report, we performed an 8000 molecule electrophile screen against PLpro and identified an α-chloro amide fragment, termed compound 1, which inhibited SARS-CoV-2 replication in cells, and also had low non-specific reactivity with thiols. Compound 1 covalently reacts with the active site cysteine of PLpro, and had an IC50 of 18 µM for PLpro inhibition. Compound 1 also had low non-specific reactivity with thiols and reacted with glutathione 1-2 orders of magnitude slower than other commonly used electrophilic warheads. Finally, compound 1 had low toxicity in cells and mice and has a molecular weight of only 247 daltons and consequently has great potential for further optimization. Collectively, these results demonstrate that compound 1 is a promising lead fragment for future PLpro drug discovery campaigns.

BCL2-associated athanogene 6 exon24 contributes to testosterone synthesis and male fertility in mammals.

OBJECTIVES: BCL2-associated athanogene 6 (BAG6) plays critical roles in spermatogenesis by maintaining testicular cell survival. Our previous data showed porcine BAG6 exon24-skipped transcript is highly expressed in immature testes compared with mature testes. The objective of this study is to reveal the functional significance of BAG6 exon24 in mammalian spermatogenesis. MATERIALS AND METHODS: CRISPR/Cas9 system was used to generate Bag6 exon24 knockout mice. Testes and cauda epididymal sperm were collected from mice. TMT proteomics analysis was used to discover the protein differences induced by Bag6 exon24 deletion. Testosterone enanthate was injected into mice to generate a high-testosterone mice model. H&E staining, qRT-PCR, western blotting, vector/siRNA transfection, immunofluorescence, immunoprecipitation, transmission electron microscopy, TUNEL and ELISA were performed to investigate the phenotypes and molecular basis. RESULTS: Bag6 exon24 knockout mice show sub-fertility along with partially impaired blood-testis barrier, increased apoptotic testicular cell rate and abnormal sperm morphology. Endoplasmic reticulum stress occurs in Bag6 exon24-deficient testes and sterol regulatory element-binding transcription factor 2 is activated; as a result, cytochrome P450 family 51 subfamily A member 1 expression is up-regulated, which causes a high serum testosterone level. Additionally, serine/arginine-rich splicing factor 1 down-regulates BAG6 exon24-skipped transcripts in porcine Sertoli cells by binding to 35-51 nt on BAG6 exon24 via its N-terminal RNA-recognition domain. CONCLUSIONS: Our findings reveal the critical roles of BAG6 exon24 in testosterone biosynthesis and male fertility, which provides new insights into the regulation of spermatogenesis and pathogenesis of subfertility in mammals.

Long-term outcomes of radiofrequency ablation vs. partial nephrectomy for cT1 renal cancer: A meta-analysis and systematic review.

Background: Partial nephrectomy (PN) is one of the most preferred nephron-sparing treatments for clinical T1 (cT1) renal cancer, while radiofrequency ablation (RFA) is usually used for patients who are poor surgical candidates. The long-term oncologic outcome of RFA vs. PN for cT1 renal cancer remains undetermined. This meta-analysis aims to compare the treatment efficacy and safety of RFA and PN for patients with cT1 renal cancer with long-term follow-up of at least 5 years. Method: This meta-analysis was performed following the PRISMA reporting guidelines. Literature studies that had data on the comparison of the efficacy or safety of RFA vs. PN in treating cT1 renal cancer were searched in databases including PubMed, Embase, Web of Science, and the Cochrane Library from 1 January2000 to 1 May 2022. Only long-term studies with a median or mean follow-up of at least 5 years were included. The following measures of effect were pooled: odds ratio (OR) for recurrence and major complications; hazard ratio (HR) for progression-free survival (PFS), cancer-specific survival (CSS), and overall survival (OS). Additional analyses, including sensitivity analysis, subgroup analysis, and publication bias analysis, were also performed. Results: A total of seven studies with 1,635 patients were finally included. The treatment efficacy of RFA was not different with PN in terms of cancer recurrence (OR = 1.22, 95% CI, 0.45-3.28), PFS (HR = 1.26, 95% CI, 0.75-2.11), and CSS (HR = 1.27, 95% CI, 0.41-3.95) as well as major complications (OR = 1.31, 95% CI, 0.55-3.14) (P > 0.05 for all). RFA was a potential significant risk factor for OS (HR = 1.76, 95% CI, 1.32-2.34, P < 0.001). No significant heterogeneity and publication bias were observed. Conclusion: This is the first meta-analysis that focuses on the long-term oncological outcomes of cT1 renal cancer, and the results suggest that RFA has comparable therapeutic efficacy with PN. RFA is a nephron-sparing technique with favorable oncologic efficacy and safety and a good treatment alternative for cT1 renal cancer.

CMTM5 inhibits the development of prostate cancer via the EGFR/PI3K/AKT signaling pathway.

Prostate cancer (PCa) endangers the life and health of older men. Most PCa cases develop into castration­resistant PCa (CRPC) within 2 years. At present, the molecular mechanisms of the occurrence and development of PCa and its transformation to CRPC remain unknown. The present study aimed to investigate the role of CKLF­like Marvel transmembrane domain containing family member 5 (CMTM5) in PCa and its molecular mechanism in vitro. PCa tissues and paired adjacent normal prostate tissues from 70 patients were collected to examine the expression levels of CMTM5 and EGFR via immunohistochemistry, reverse transcription­quantitative PCR and western blotting. Then, CMTM5­overexpressing DU145 cells were constructed, and CMTM5 expression in these transfected cells and vector control cells was examined via western blotting. Cell Counting Kit­8 and plate clone formation assays were used to evaluate the proliferation and colony number of CMTM5­overexpressing cells and vector control cells. Then, cell migration and invasion were assessed using wound healing assay, Transwell assay and immunofluorescence analysis with DAPI staining. The effect of CMTM5 on apoptosis and its underlying molecular mechanism were examined using western blotting and flow cytometry. The results demonstrated that CMTM5 expression in PCa tissues and cell lines was significantly downregulated, while EFGR expression was significantly upregulated. The proportion of high CMTM5 expression in PCa tissues was significantly lower compared with that in normal prostate tissues. By contrast, the proportion of high EGFR expression in PCa tissues was significantly increased compared with that in normal prostate tissues. Moreover, CMTM5 overexpression significantly inhibited cell proliferation, migration and invasion, and promoted cell apoptosis compared with vector control cells in vitro. Furthermore, the regulation of PCa by CMTM5 was associated with the downregulation of PI3K/AKT and its downstream Bcl­2 expression, as well as the upregulation of Bax expression. In conclusion, CMTM5 may be an effective tumor suppressor gene for PCa, especially for castration­resistant PCa, by downregulating EGFR and PI3K/AKT signaling pathway components.

Efficacy and safety of PD-1/PD-L1 inhibitors combined with CTLA-4 inhibitor versus chemotherapy for advanced lung cancer: A meta-analysis.

BACKGROUND: This meta-analysis was performed to compare efficacy and tolerability between antiprogrammed cell death (PD-1)/programmed cell death-ligand-1 (PD-L1) + anticytotoxic T-lymphocyte-associated protein-4 (CTLA-4) treatment and chemotherapy in advanced lung cancer. METHODS: Cochrane Library, Embase, and PubMed databases were searched for potential articles. The fixed-effect model or random-effect model was adopted for pooled analysis based on the I2 and P-value. RESULTS: Six articles with 1338 patients were identified and subjected to meta-analysis. Compared with chemotherapy, anti-PD-1/PD-L1 + anti-CTLA-4 treatment could significantly improve the overall survival (hazard ratio [HR] = 0.78, 95%confidence interval [CI]: 0.71-0.84, P = .21) and progression-free survival (HR = 0.77, 95%CI: 0.71-0.83, P = .30) of advanced lung cancer patients. Moreover, there was no obvious difference in the incidence of 3 to 4 adverse events (AEs) serious adverse reactions (HR = 1.35, 95%CI: 0.66-2.74, P < .00001) between the 2 treatment groups, but the incidence rates of AEs leading to discontinuation (HR = 2.56, 95%CI: 1.53-4.30, P < .00001) and AEs leading to death (HR = 2.10, 95%CI: 1.21-3.63, P = .20) were higher. Furthermore, no remarkable differences in objective response rate (HR = 1.31, 95%CI: 0.97-1.77, P = .02) were observed between the 2 groups. CONCLUSION: Our meta-analysis revealed that PD-1/PD-L1 inhibitors plus CTLA-4 inhibitor could markedly improve the endpoint outcomes of patients compared with chemotherapy alone, and did not significantly increase the serious adverse reactions. Thus, it can serve as a new treatment strategy for advanced lung cancer.

Chronic morphine induces cyclic adenosine monophosphate formation and hyperpolarization-activated cyclic nucleotide-gated channel expression in the spinal cord of mice.

Chronic morphine exposure persistently activates Gαi/o protein-coupled receptors and enhances adenylyl cyclase (AC) activity, which can increase cyclic adenosine monophosphate (cAMP) production. Direct binding of cAMP to the cytoplasmic site on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels increases the probability of channel opening. HCN channels play a prominent role in chronic pain the disease that shares some common mechanisms with opioid tolerance. This compensatory AC activation may be responsible for the induction of morphine-induced analgesic tolerance. We investigated spinal cAMP formation and expression of HCN2 in the spinal cord, and observed the effect of AC inhibition on the induction of morphine analgesic tolerance. We found that chronic morphine-induced antinociceptive tolerance increased spinal cAMP formation and the expression of spinal HCN2. Inhibition of spinal AC partially blocked chronic morphine-induced cAMP formation and prevented the induction of morphine-induced analgesic tolerance. Inhibition of HCN2 also showed a partial preventive effect on morphine-induced tolerance, hypothermia tolerance and also the right-shift of the dose-response curve. We conclude that repeated morphine treatment increases AC activity and cAMP formation, and also spinal HCN2 expression, blockade of AC or HCN2 can prevent the development of morphine-induced analgesic tolerance.

Identification of Four Pathological Stage-Relevant Genes in Association with Progression and Prognosis in Clear Cell Renal Cell Carcinoma by Integrated Bioinformatics Analysis.

Clear cell renal cell carcinoma (ccRCC) is a major histological subtype of renal cell carcinoma and can be clinically divided into four stages according to the TNM criteria. Identifying clinical stage-related genes is beneficial for improving the early diagnosis and prognosis of ccRCC. By using bioinformatics analysis, we aim to identify clinical stage-relevant genes that are significantly associated with the development of ccRCC. First, we analyzed the gene expression microarray data sets: GSE53757 and GSE73731. We divided these data into five groups by staging information-normal tissue and ccRCC stages I, II, III, and IV-and eventually identified 500 differentially expressed genes (DEGs). To obtain precise stage-relevant genes, we subsequently applied weighted gene coexpression network analysis (WGCNA) to the GSE73731 dataset and KIRC data from The Cancer Genome Atlas (TCGA). Two modules from each dataset were identified to be related to the tumor TNM stage. Several genes with high inner connection inside the modules were considered hub genes. The intersection results between hub genes of key modules and 500 DEGs revealed UBE2C, BUB1B, RRM2, and TPX2 as highly associated with the stage of ccRCC. In addition, the candidate genes were validated at both the RNA expression level and the protein level. Survival analysis also showed that 4 genes were significantly correlated with overall survival. In conclusion, our study affords a deeper understanding of the molecular mechanisms associated with the development of ccRCC and provides potential biomarkers for early diagnosis and individualized treatment for patients at different stages of ccRCC.

Runt-related transcription factor 2 influences cell adhesion-mediated drug resistance and cell proliferation in B-cell non-Hodgkin's lymphoma and multiple myeloma.

Several lines of evidence show that RUNX2 as a transcription factor is closely involved in carcinogenesis in a variety of human cancers. Cell adhesion-mediated drug resistance (CAM-DR) is an important part of the mechanism underlying drug resistance in hematological tumors. In this study, we investigated the biological function of RUNX2 in B-cell Non-Hodgkin's lymphoma (B-NHL) and multiple myeloma (MM). We assessed the expression of RUNX2 in suspension and adhesion model by western blot in B-NHL and MM. Adhesion assay, flow cytometry and CCK-8 were utilized to examine the role and mechanism of RUNX2 in CAM-DR and proliferation in B-NHL and MM. RUNX2 was highly expressed in adherent B-NHL and MM cells compared to suspension cells, and knockdown the expression of RUNX2 could reverse CAM-DR. Besides, RUNX2 could promote the proliferation of B-NHL and MM cells. Furthermore, RUNX2 participated the process of CAM-DR and proliferation by regulating the AKT/GSK-3ß pathway. Developing RUNX2 inhibitor may be a possible strategy for drug resistance.

Correction to: Liquid Biopsy Applications in the Clinic.

In the original publication of the article, the name of the fourth author, which previously read "Howard Chan", should read as "Howard Chang". The original article has been updated.

Liquid Biopsy Applications in the Clinic.

The global liquid biopsy industry is expected to exceed $US5 billion by 2023. One application of liquid biopsy technology is the diagnosis of disease using biomarkers found in blood, urine, stool, saliva, and other biological samples from patients. These biomarkers could be DNA, RNA, protein, or even a cell. More recently, the use of cell-free DNA from plasma is emerging as an important minimally invasive tool for clinical diagnosis. The development of technology has increased the diversity of its application. Here, we discuss how liquid biopsies have been used in the clinic, and how personalized medicine are likely to use liquid biopsies in the near future.

Glutathione S-transferase M1 and T1 null genotypes and bladder cancer risk: A meta-analysis in a single ethnic group.

AIM OF STUDY: To further evaluate the influence of glutathione S-transferase M1 (GSTM1) and glutathione S-transferase T1 (GSTT1) null genotypes on bladder cancer risk, we conducted a meta-analysis in the Chinese population. MATERIALS AND METHODS: PubMed and Chinese databases were electronically searched through April 2016. RESULTS: Nine studies were included for our meta-analysis, involving 1646 bladder cancer cases and 1938 controls. In general, our findings indicated that a significant association existed between GSTM1-null genotype and the risk of bladder cancer in the studied Chinese population (odds ratio = 1.56, 95% confidence interval: 1.36-1.79). However, no significant association between GSTT1 polymorphism and bladder cancer was found. After stratification of the subgroup analyses by source of controls and geographical areas, a substantially elevated risk was revealed between GSTM1-null genotype and bladder cancer in the population-based studies and those conducted in South China and North China. CONCLUSION: Our meta-analysis suggested that GSTM1-null genotype is associated with an increased bladder cancer risk in the Chinese individuals.

Identification of Key Genes and miRNAs in Osteosarcoma Patients with Chemoresistance by Bioinformatics Analysis.

Chemoresistance is a significant factor associated with poor outcomes of osteosarcoma patients. The present study aims to identify Chemoresistance-regulated gene signatures and microRNAs (miRNAs) in Gene Expression Omnibus (GEO) database. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) included positive regulation of transcription, DNA-templated, tryptophan metabolism, and the like. Then differentially expressed genes (DEGs) were uploaded to Search Tool for the Retrieval of Interacting Genes (STRING) to construct protein-protein interaction (PPI) networks, and 9 hub genes were screened, such as fucosyltransferase 3 (Lewis blood group) (FUT3) whose expression in chemoresistant samples was high, but with a better prognosis in osteosarcoma patients. Furthermore, the connection between DEGs and differentially expressed miRNAs (DEMs) was explored. GEO2R was utilized to screen out DEGs and DEMs. A total of 668 DEGs and 5 DEMs were extracted from GSE7437 and GSE30934 differentiating samples of poor and good chemotherapy reaction patients. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform GO and KEGG pathway enrichment analysis to identify potential pathways and functional annotations linked with osteosarcoma chemoresistance. The present study may provide a deeper understanding about regulatory genes of osteosarcoma chemoresistance and identify potential therapeutic targets for osteosarcoma.

Long noncoding RNA GHET1 promotes the development of bladder cancer.

In spite of the advances in the diagnosis and treatment of bladder cancer, the prognosis of bladder cancer remains relatively poor. As a result, it is vital to identify novel diagnostic and prognostic marker of bladder cancer. A growing volume of literature has implicated the vital role of long noncoding RNA in the development of cancer. GHET1, a recently identified lncRNA, was initially characterized in gastric cancer. However, its role in bladder cancer remains largely unknown. In this study, we demonstrated that GHET1 was upregulated in bladder cancer tissues compared to adjacent normal tissues and its over-expression correlates with tumor size, advanced tumor and lymph node status, and poor survival. GHET1 knockdown suppressed the proliferation and invasion of bladder cancer cells in vitro. In the meantime, inhibition of GHET1 reversed the epithelial-mesenchymal-transition in bladder cancer cell line. Taken together, our study suggests that the potential use of GHET1 as a prognostic marker and therapeutic target of bladder cancer.