IGFBP5, as a Prognostic Indicator Promotes Tumor Progression and Correlates with Immune Microenvironment in Glioma.

Background: Insulin-like growth factor binding protein 5 (IGFBP5) is highly expressed in multiple human cancers, including glioma. Despite this, it remains unclear what role it plays in glioma. The aim of the present study was to analyze whether IGFBP5 could be used as a predictor of prognosis and immune infiltration in glioma. Methods: Glioma patients' clinical information was collected from the Cancer Genome Atlas (TCGA), the Chinese Glioma Genome Atlas (CGGA), Rembrandt, and Gravendeel databases. The diagnostic and prognostic roles of IGFBP5 were assessed by the Kaplan-Meier survival curves, diagnostic receiver operating characteristic (ROC) curves, nomogram model, Cox regression analysis and Enrichment analysis by R software. Moreover, the correlation between IGFBP5 expression and immune cell infiltration, and immune checkpoint genes was conducted. Immunohistochemistry staining, CCK8, colony formation, scratch and transwell assays and western blot were used to interrogate the expression and function of IGFBP5 in glioma. Results: IGFBP5 levels were obviously increased in glioma with higher malignancy and predicted poor outcomes by Univariate and multivariate Cox analysis. The biological function analysis revealed that IGFBP5 correlated closely with immune signatures. Moreover, IGFBP5 expression was associated with tumor infiltration of B cells, T cells, macrophages, and NK cells. IGFBP5 affected glioma cell proliferation, migration, and invasion probably involved in the epithelial-to-mesenchymal transition (EMT) and Hippo-YAP signaling pathway. Further study showed that IGFBP5 induced the expression of PD-L1 and CXCR4. Conclusions: IGFBP5 as an oncogene is a useful biomarker of prognosis and correlates with progression and immune infiltration in glioma.

Mesothelin-targeted CAR-NK Cells Derived From Induced Pluripotent Stem Cells Have a High Efficacy in Killing Triple-negative Breast Cancer Cells as Shown in Several Preclinical Models.

The emergence of immunotherapy has introduced a promising, novel approach to cancer treatment. While multiple chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable clinical efficacy against leukemia, their effect on solid tumors has been limited. One potential option for treating solid tumors is the engineering of natural killer (NK) cells with CARs. Mesothelin (MSLN), a tumor differentiation antigen, is expressed on triple-negative breast cancer (TNBC) cells, making it a potential target for CAR-NK therapy in the treatment of TNBC. We first constructed induced pluripotent stem cells with stable anti-MSLN-CAR expression and subsequently differentiated these cells into mesothelin-targeted CAR-NK (MSLN-NK) cells. We then assessed the effects of MSLN-NK cells on TNBC cells both in vitro (using the MDA-MB-231 cell line), in vivo (in a CDX mouse model), and ex vivo (using patient-specific primary cells and patient-specific organoids), in which MSLN surface expression was confirmed. Our CDX study results indicated that MSLN-NK cells effectively killed MDA-MB-231 (MD231) cells in vitro, reduced tumor growth in the CDX mouse model of TNBC, and lysed patient-specific primary cells and patient-specific organoids derived from the tumor samples of TNBC patients. Our data demonstrated that MSLN-NK cells had high efficacy on killing TNBC cells in in vitro, in vivo, and ex vivo. Therefore, MSLN-NK could be a promising treatment option for TNBC patients.

Identification and comprehensive analysis of epithelial-mesenchymal transition related target genes of miR-222-3p in breast cancer.

Background: Epithelial-mesenchymal transition (EMT) is a crucial mechanism that microRNA-222-3p (miR-222-3p) promotes breast cancer (BC) progression. Our study aimed to identify EMT-associated target genes (ETGs) of miR-222-3p for further analysis of their roles in BC based on bioinformatics tools. Methods: Based on bioinformatics analysis, we identified 10 core ETGs of miR-222-3p. Then, we performed a comprehensive analysis of 10 ETGs and miR-222-3p, including pathway enrichment analysis of ETGs, differential expression, clinical significance, correlation with immune cell infiltration, immune checkpoint genes (ICGs) expression, tumor mutational burden (TMB), microsatellite instability (MSI), stemness, drug sensitivity, and genetic alteration. Results: The expression of miR222-3p in basal-like BC was significantly higher than in other subtypes of BC and the normal adjacent tissue. Pathway analysis suggested that the ETGs might regulate the EMT process via the PI3K-Akt and HIF-1 signaling pathway. Six of the 10 core ETGs of miR-222-3p identified were down-expressed in BC, which were EGFR, IL6, NRP1, NTRK2, LAMC2, and PIK3R1, and SERPINE1, MUC1, MMP11, and BIRC5 were up-expressed in BC, which also showed potential diagnostic values in BC. Prognosis analysis revealed that higher NTRK2 and PIK3R1 expressions were related to a better prognosis, and higher BIRC5 and miR-222-3p expressions were related to a worse prognosis. Most ETGs and miR-222-3p were positively correlated with various infiltration of various immune cells and ICGs expression. Lower TMB scores were correlated with higher expression of MUC1 and NTRK2, and higher BIRC5 was related to a higher TMB score. Lower expression of MUC1, NTRK2, and PIK3R1 were associated with higher MSI scores. Higher expression of ETGs was associated with lower mRNAsi scores, except BIRC5 and miR-222-3p conversely. Most ETGs and miR-222-3p expression were negatively correlated with the drug IC50 values. The analysis of the genetic alteration of the ETGs suggested that amplification was the main genetic alteration of eight ETGs except for NTRK2 and PIK3R1. Conclusion: MiR-222-3p might be a specific biomarker of basal-like BC. We successfully identify 10 core ETGs of miR-222-3p, some might be useful diagnostic and prognostic biomarkers. The comprehensive analysis of 10 ETGs and miR-222-3p indicated that they might be involved in the development of BC, which might be novel therapeutic targets for the treatment of BC.

A Novel Immune-Related Signature to Predict Prognosis and Immune Infiltration of Cervical Cancer.

BACKGROUND Cervical cancer is one of the most common malignances among women globally. This study aimed to construct a novel immune-related signature to predict the prognosis and immune infiltration of cervical cancer. MATERIAL AND METHODS Transcriptomic profiles and corresponding clinical information of cervical cancer patients were obtained from The Cancer Genome Atlas (TCGA) database and GEO database. The hub immune-related genes were screened and selected using Cox regression analysis and LASSO regression analysis. A novel signature was established based on the expression levels and corresponding coefficients of the selected hub immune-related genes. Kaplan-Meier survival curve and ROC curve illustrated the prognostic value of this novel signature in cervical cancer. The predictive accuracy and stability of this novel signature were confirmed in the validation cohort, internal testing set and external testing set. Then, a nomogram was constructed to predict individual survival probability of cervical cancer patient. The association between the risk scores of novel signature and immune infiltration was investigated through single-sample gene set enrichment analysis (ssGSEA). RESULTS Ten hub immune-related genes (TFRC, SPP1, CAMP, CSF2, TUBB3, ZAP70, CHIT1, LEPR, DLL4, and DES) were selected to construct a novel signature. The risk score of this novel signature could be an independent prognostic factor in cervical cancer, which divided patients into high-risk and low-risk groups. The patients in high-risk groups showed significantly worse overall survival rates than those in low-risk groups in all training and validation cohorts (all P<0.05). A nomogram model was constructed based on the risk score of the novel signature and other clinical characteristics, which achieved the highest clinical net benefit across the entire range of reasonable threshold probabilities (concordance index=0.813). Furthermore, gene enrichment analysis revealed that the novel signature was closely related with immunology. The novel signature was negatively correlated with the infiltration of most immune cell types, especially T cell subsets (P<0.001). CONCLUSIONS The novel signature could comprehensively predict the prognosis and immune infiltration of cervical cancer. It may provide new insights for the precise treatment in cervical cancer.

Overexpressed VDAC1 in breast cancer as a novel prognostic biomarker and correlates with immune infiltrates.

BACKGROUND: More and more evidence suggests that cancer is a mitochondrial metabolic disease recently and mitochondria dysfunction is critical to tumorigenesis. As a gatekeeper of mitochondria, the voltage-dependent anion channel 1 (VDAC1) is associated with the development of breast cancer (BC). However, its potential mechanism and clinical significance remain unclear; thus, in this research, we aimed to explore it. METHODS: VDAC1 expression in BC tissues and normal tissues was obtained from The Cancer Genome Atlas (TCGA) and validated by datasets from the gene expression omnibus (GEO) database. Then, the relationships between VDAC1 expression and clinicopathological features were analyzed. Receiver operating characteristics (ROC) curves were used to identify the diagnostic value of VDAC1. The prognostic value was evaluated by Kaplan-Meier survival curves and Cox regression analysis. VDAC1 with its co-expression genes were subjected to enrichment analysis to explore potential mechanisms in BC and the protein-protein interaction (PPI) network was constructed. At last, the association between VDAC1 expression and infiltration levels of immune cell infiltration by various methods, as well as their corresponding markers, was analyzed. We also analyzed the correction between VDAC1 expression and eight immune checkpoint genes and the tumor immune dysfunction and exclusion (TIDE) scores of each BC sample in TCGA were calculated and the differences between high and low VDAC1 expression groups were analyzed. RESULTS: VDAC1 expression was remarkably elevated in BC (p < 0.001), and high expression of VDAC1 was associated with the positive expression of ER (p = 0.004), PR (p = 0.033), and HER2 (p = 0.001). ROC analysis suggested that VDAC1 had diagnosed value in BC. The Kaplan-Meier analysis suggested that higher expression of VDAC1 was associated with shorter overall survival (OS), and further Cox regression analysis revealed that VDAC1 was an independent factor of unfavorable prognosis in BC patients. Enrichment analysis of VDAC1 and its co-expression suggested that VDAC1 was related to the regulation of mitochondrial energy metabolism and protein modification, and the HIF-1 singing pathway might be the potential mechanism in BC. Notably, we found that VDAC1 expression was infiltration levels of most types of immune cells, as well as the expression of marker genes of immune cells. The ICGs PDCD1, CTLA4, LAG3, SIGLEC15, and TIGIT were negatively corrected with VDAC1 expression in BC. TIDE scores between the low and high expression groups showed no difference. CONCLUSION: Overexpressed VDAC1 in BC could be severed as a novel biomarker for diagnosis and VDAC1 was an independent factor for adverse prognosis prediction. Our study revealed that VDAC1 might inhibit tumor immunity and might be a novel therapeutic target in BC.

ARSD is responsible for carcinoma and amyloidosis of breast epithelial cells.

Breast cancer (BC) and Alzheimer's disease (AD) have pronounced female-to-male disparities and both are the major causes of death in elderly women. Intriguingly, there is an inverse incidence between BC and AD. In our previous study, we found that the expression of ARSD, a female-biased gene on chromosome Xp22.3 that encodes arylsulfatase D, is significantly downregulated in triple-negative breast cancer (TNBC) cells and tissue samples, and that ectopic ARSD overexpression could inhibit proliferation and migration of BC cells. However, the exact mechanism remains unclear. In this study, ARSD-overexpressing MDA-MB-231 cell strains were established. RNA-Seq and qRT-PCR validation were performed followed by GO and KEGG analyses. Transcriptome sequencing unveiled that Alzheimer's/Parkinson's/prion diseases were enriched in ARSD overexpressing BC cells. Besides, the top enriched pathways included lipoprotein/cholesterol metabolism, molecular chaperone and misfolding protein binding, mitochondrial respiration, dysfunction of lysosomes, etc. In which, a battery of genes, e.g., SERF1A, APOE, CD36 etc., were upregulated, while a series of genes, e.g., NDUFA11, NDUFS3, NDUFV1, etc. were downregulated, which were closely related to amyloidosis. The amyloidosis of BC cells and nerval cells caused by ARSD overexpression was verified with western blotting, immunohistochemical and Congo red staining. Collectively, downregulated ARSD may be closely associated with BC, and upregulated ARSD may cause amyloidosis of BC cells. Our findings suggest that ARSD deserves to be considered a new promising target for treating TNBC or for AD.

The EZH2- H3K27me3-DNMT1 complex orchestrates epigenetic silencing of the wwc1 gene, a Hippo/YAP pathway upstream effector, in breast cancer epithelial cells.

It is well known that epithelial-mesenchymal transition (EMT) can confer cancer cells with invasive and migratory capabilities associated with distant metastasis. As a key upstream factor in the Hippo pathway, Kibra (wwc1 gene) has been shown to suppress EMT in breast cancer cells, and we have found that its expression is reduced or lost completely in both human breast cancer cell lines and clinical tissue samples, particularly in triple negative breast cancer (TNBC). Unfortunately, the molecular mechanisms underlying this progression-associated event remain to be elucidated. Epigenetic gene silencing is one of the most common causes of suppressed expression of tumor suppressor genes. Furthermore, recent studies have demonstrated that EZH2 can recruit DNA methyltransferases, resulting in DNA methylation and subsequent gene silencing in certain circumstances. Thus, we hypothesized that there may exist a link between EZH2 and DNA methylation in association with wwc1 silencing in breast cancer. To test this hypothesis, we performed bisulfite sequencing, shRNA, co-IP, ChIP, MeDIP and ChIP-qPCR. As expected, RG108 or 5-Aza treatment improved the wwc1 gene transcription and Kibra protein expression. Both bisulfite sequencing and MeDIP demonstrated higher CpG methylation of the wwc1 promoter the TNBC cells (MDA-MB-231) than in luminal breast cancer cells (MCF7). It is noteworthy that ChIP and co-IP assays showed that EZH2, H3K27me3 and DNMT1 are enriched at the wwc1 promoter, and there exist physiologically relevant protein-protein interactions between them. We also found that EZH2 knockdown leads to a partial increase in Kibra expression and a considerable reduction in H3K27 and DNMT1 trimethylation. Moreover, ChIP-qPCR revealed more DNA fragments containing the wwc1 promoter in MDA-MB-231 than in MCF7 cells after immunoprecipitation with EZH2, DNMT1 and H3K27me3 antibodies. Collectively, our results reveal crosstalk between H3K27me3 inhibition catalyzed by EZH2 and CpG island methylation mediated by DNMT1 within the wwc1 promoter, which synergistically silence wwc1 gene expression in TNBC. Based on these results, we conclude that EZH2 shows promise as a potential anti-tumor target.

[Effect of intra-testicular testosterone withdrawal on the expression of alpha-catenin in the adult rat testis].

OBJECTIVE: To study the expression of alpha-catenin in the rat testis after intra-testicular testosterone withdrawal induced by injection of testosterone undecanoate (TU). METHODS: Ten adult male SD rats received vehicle (n = 5 ) or TU (19 mg/kg every 15 days, n = 5) for 130 days. Paraffin-embedded testicular sections were used for immunohistochemistry against a polyclonal anti-alpha-catenin antibody. RESULTS: In the control, alpha-catenin was expressed in the acrosome of spermatids and the cytoplasm of Leydig cells and peritubular myoid cells. In the TU-treated rat testis, Leydig cells were atrophied and the expression of alpha-catenin was markedly decreased or absent, but there was no evident change in the immunostaining of spermatids or myoid cells. CONCLUSION: Intra-testicular testosterone withdrawal-induced looser arrangement or sloughing of spermatogenic cells is not related to the adhesion molecule alpha-catenin. Alpha-catenin may be used as a cell identification marker for Leydig cells.

Molecular phylogeny of Sinocyclocheilus (Cypriniformes: Cyprinidae) inferred from mitochondrial DNA sequences.

More than 10 species within the freshwater fish genus Sinoncyclocheilus adapt to caves and show different degrees of degeneration of eyes and pigmentation. Therefore, this genus can be useful for studying evolutionary developmental mechanisms, role of natural selection and adaptation in cave animals. To better understand these processes, it is indispensable to have background knowledge about phylogenetic relationships of surface and cave species within this genus. To investigate phylogenetic relationships among species within this genus, we determined nucleotide sequences of complete mitochondrial cytochrome b gene (1140 bp) and partial ND4 gene (1032 bp) of 31 recognized ingroup species and one outgroup species Barbodes laticeps. Phylogenetic trees were reconstructed using maximum parsimony, Bayesian, and maximum likelihood analyses. Our phylogenetic results showed that all species except for two surface species S. jii and S. macrolepis clustered as five major monophyletic clades (I, II, III, IV, and V) with strong supports. S. jii was the most basal species in all analyses, but the position of S. macrolepis was not resolved. The cave species were polyphyletic and occurred in these five major clades. Our results indicate that adaptation to cave environments has occurred multiple times during the evolutionary history of Sinocyclocheilus. The branching orders among the clades I, II, III, and IV were not resolved, and this might be due to early rapid radiation in Sinocyclocheilus. All species distributed in Yunnan except for S. rhinocerous and S. hyalinus formed a strongly supported monophyletic group (clade V), probably reflecting their common origins. This result suggested that the diversification of Sinocyclocheilus in Yunnan may correlate with the uplifting of Yunnan Plateau.