Visium spatial transcriptomics reveals intratumor heterogeneity and profiles of Gleason score progression in prostate cancer.

Prostate cancer (PCa) frequently presents as a multifocal disease within a single gland. Herein, the transcriptome-wide profiles of glandular epithelial (GE) cells of four PCa tissues with various Gleason scores (GSs) are analyzed with Visium spatial transcriptomics (ST). The genetic classifications across PCa section sites generally matched the spatial patterns of histological structures with different GSs. Average inferred copy number variation (inferCNV) values gradually increased during GS development. Developing trajectories during GS upgrading were assessed, and differentially expressed genes (DEGs) during GS progression were analyzed which exhibited heterogeneity among individual patients with PCa. Several crucial genes, such as NANS, PABPC1L, PILRB, PPFIA2, and SESN3, were associated with GS upgrading. Enrichment analysis showed that biological functions, such as cadherin binding, Golgi vesicle transport, protein folding, and cell adhesion molecules were related to GS progression. In conclusion, this study provides insight into ST-based transcriptome-wide expression patterns during GS progression.

RNA-Sequencing Analysis Indicates That N-Cadherin Promotes Prostate Cancer Progression by the Epigenetic Modification of Key Genes.

N-cadherin (cadherin-2 [CDH2]) is widely known as the promoter of prostate cancer (PCa) invasion and castration resistance. However, the biological mechanism of N-cadherin in PCa progression is unclear. In this study, we overexpressed N-cadherin in LNCaP cells and downregulated N-cadherin in PC3 cells by lentiviral transduction. Then, differentially expressed genes (DEGs) and dysregulated biological functions were investigated through RNA sequencing (RNA-seq) analyses. We found 13 long noncoding RNA (lncRNA) transcripts, 72 messenger RNA (mRNA) transcripts, and 3 integrated genes were dysregulated by N-cadherin. In the disease enrichment, bone cancer, and neurodegenerative and nervous system diseases were associated with N-cadherin in the circular RNA (circRNA; PC3 versus [vs.,/] LNCaP [PC3/LNCaP] comparison) and DEG analysis (LNCaP_oe_CDH2 vs. LNCaP_oe_NC [LNCaP_oe_CDH2/NC] comparison). Epigenetic reprogramming, such as nucleic acid binding, and chromatin and histone modifications, was enriched in Gene Ontology (GO) analysis (DEGs in LNCaP_oe_CDH2/NC and PC3_sh_NC/CDH2, and host genes of circRNA in PC3/LNCaP). Transcriptional misregulation in cancer, post-translational protein modification, gene expression, and generic transcription pathways were dysregulated in the pathway enrichment analysis (host genes of circRNA in PC3/LNCaP, and DEGs in LNCaP_oe_CDH2/NC and PC3_sh_NC/CDH2). Verifying DEGs through TCGA-PRAD dataset revealed six oncogenes (ARHGEF1, GRAMD1A, GTF2H4, MAPK8IP3, POLD1, and PTBP1) that were commonly upregulated by N-cadherin and in advanced PCa stages. In summary, we identified several oncogenes and biological functions associated with N-cadherin expression in PCa cells. N-cadherin may trigger epigenetic reprogramming in PCa cells to promote tumor progression.

Histone lysine methylation patterns in prostate cancer microenvironment infiltration: Integrated bioinformatic analysis and histological validation.

Background: Epigenetic reprogramming through dysregulated histone lysine methylation (HLM) plays a crucial role in prostate cancer (PCa) progression. This study aimed to comprehensively evaluate HLM modification patterns in PCa microenvironment infiltration. Materials and methods: Ninety-one HLM regulators in The Cancer Genome Atlas (TCGA) dataset were analyzed using bioinformatics. Differentially expressed genes (DEGs) and survival analyses were performed using TCGA-PRAD clinicopathologic and follow-up information. Consensus clustering analysis divided patients into subgroups. Gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the DEGs. Tumor mutation burden (TMB) and tumor microenvironment (TME) cell infiltration were evaluated in different HLM clusters. Quantitative real-time PCR (qPCR) analysis assessed HLM regulators in clinical PCa tissues. Results: The tumor vs. normal (TN), Gleason score (GS) > 7 vs. GS < 7, pathological T stage (pT) = 2 vs. pT = 3, and TP53 mutation vs. wild-type comparisons using TCGA-PRAD dataset revealed 3 intersecting HLM regulators (EZH2, NSD2, and KMT5C) that were consistently upregulated in advanced PCa (GS > 7, pT3, HR > 1, and TP53 mutation) (P < 0.05) and verified in clinical PCa tissues. Consensus clustering analysis revealed three distinct HLM modification patterns (HLMclusters). However, no significant differences in recurrence-free survival (RFS) rates were found among the groups (P > 0.05). We screened 189 HLM phenotype-related genes that overlapped in the pairwise comparisons of HLMclusters and P < 0.01 in the Cox regression analysis. Three distinct subgroups (geneClusters) were revealed based on the 189 genes, in which cluster A involved the most advanced PCa (PSA > 10, T3-4, GS8-10, and biochemical recurrence) and the poorest RFS. The HLM score (HLMscore) was calculated by principal component analysis (PCA) of HLM phenotype-related genes that have positive predictive value for RFS (P < 0.001) and immune therapy responses (in the CTLA4-positive and -negative responses accompanied by a PD1-negative response). Conclusion: We comprehensively evaluated HLM regulators in the PCa microenvironment using TCGA-PRAD, revealing a nonnegligible role of HLM patterns in PCa complexity and heterogeneity. Elucidating the effects of HLM regulators in PCa may enhance prognostics, aggressiveness assessments, and immunotherapy strategies.

Construction of a risk prediction model using m6A RNA methylation regulators in prostate cancer: comprehensive bioinformatic analysis and histological validation.

BACKGROUND: Epigenetic reprogramming reportedly has a crucial role in prostate cancer (PCa) progression. RNA modification is a hot topic in epigenetics, and N6-methyladenosine (m6A) accounts for approximately 60% of RNA chemical modifications. The aim of this study was to evaluate the m6A modification patterns in PCa patients and construct a risk prediction model using m6A RNA regulators. MATERIALS AND METHODS: Analyses were based on the levels of 25 m6A regulators in The Cancer Genome Atlas (TCGA). Differentially expressed gene (DEG) and survival analyses were performed according to TCGA-PRAD clinicopathologic and follow-up information. To detect the influences of m6A regulators and their DEGs, consensus clustering analysis was performed, and tumor mutational burden (TMB) estimation and tumor microenvironment (TME) cell infiltration were assessed. mRNA levels of representative genes were verified using clinical PCa data. RESULTS: Diverse expression patterns of m6A regulators between tumor and normal (TN) tissues were detected regarding Gleason score (GS), pathological T stage (pT), TP53 mutation, and survival comparisons, with HNRNPA2B1 and IGFBP3 being intersecting genes. HNRNPA2B1 was upregulated in advanced stages (GS > 7, pT3, HR > 1, and TP53 mutation), as verified using clinical PCa tissue. Three distinct m6A modification patterns were identified through consensus clustering analysis, but no significant difference was found among these groups in recurrence-free survival (RFS) analysis. Six DEGs of m6A clusters (m6Aclusters) were screened through univariate Cox regression analysis. MMAB and PAIAP2 were intersecting genes for the five clinical factors. MMAB, which was upregulated in PCa compared with TN, was verified using clinical PCa samples. Three distinct subgroups were established according to the 6 DEGs. Cluster A involved the most advanced stages and had the poorest RFS. The m6A score (m6Ascore) was calculated based on the 6 genes, and the low m6Ascore group showed poor RFS with a negative association with infiltration for 16 of 23 immune-related cells. CONCLUSION: We screened DEGs of m6Aclusters and identified 6 genes (BAIAP2, TEX264, MMAB, JAGN1, TIMM8AP1, and IMP3), with which we constructed a highly predictive model with prognostic value by dividing TCGA-PRAD into three distinct subgroups and performing m6Ascore analysis. This study helps to elucidate the integral effects of m6A modification patterns on PCa progression.

The role of N-cadherin/c-Jun/NDRG1 axis in the progression of prostate cancer.

The dysregulation of androgen receptor (AR) signaling is a critical event in the progression of prostate cancer (PCa) and hormone therapy consisting of androgen deprivation (ADT) or AR inhibition is therefore used to treat advanced cases. It is known that N-cadherin becomes upregulated following ADT and can directly induce PCa transformation to the castration-resistant stage (CRPC). However, the relationship between AR and N-cadherin is unclear and may promote better understanding of CRPC pathogenesis and progression. Here, we demonstrate a new axis of N-cadherin/c-Jun/N-myc downstream regulated gene 1 (NDRG1) that N-cadherin promotes c-Jun expression and suppresses NDRG1 to promote invasion and migration of PCa cells through epithelial to mesenchymal transition (EMT). Targeting N-cadherin in combination with enzalutamide (ENZ) treatment synergistically suppressed PC3 cell proliferation in vivo and in vitro. Further studies showed that compared to lower Gleason score (GS) (GS < 7) cases, high GS (GS > 7) cases exhibited elevated N-cadherin expression and reduced NDRG1 expression, corroborating our in vitro observations. We further demonstrate that c-Jun, AR, and DNA methyltransferase-1 (DNMT1) form a complex in the 12-O-tetradecanoyl phorbol-13-acetate (TPA) response elements (TREs) region of the NDRG1 promoter, which suppresses NDRG1 transcription through DNA hypermethylation. In conclusion, we demonstrate an underlying mechanism for how N-cadherin collaborates with AR and NDRG1 to promote CRPC progression. Controlling N-cadherin/c-Jun/NDRG1 axis may help to overcome resistance to commonly used hormone therapy to improve long-term patient outcomes.

Upregulated Talin1 synergistically boosts ß-estradiol-induced proliferation and pro-angiogenesis of eutopic and ectopic endometrial stromal cells in adenomyosis.

Adenomyosis (ADS) is an estrogen-dependent gynecological disease with unspecified etiopathogenesis. Local hyperestrogenism may serve a key role in contributing to the origin of ADS. Talin1 is mostly identified to be overexpressed and involved in the progression of numerous human carcinomas through mediating cell proliferation, adhesion and motility. Whether Talin1 exerts an oncogenic role in the pathogenesis of ADS and puts an extra impact on the efficacy of estrogen, no relevant data are available yet. Here we demonstrated that the adenomyotic eutopic and ectopic endometrial stromal cells (ADS_Eu_ESC and ADS_Ec_ESC) treated with ß-estradiol (ß-E2) presented stronger proliferative and pro-angiogenetic capacities, accompanied by increased expression of PCNA, Ki67, VEGFB and ANGPTL4 proteins. Meanwhile, these promoting effects were partially abrogated by Fulvestrant (ICI 182780, an estrogen-receptor antagonist). Aberrantly upregulation of Talin1 mRNA and protein level was observed in ADS endometrial specimens and stromal cells. Through performing functional experiments in vitro, we further determined that merely overexpression of Talin1 (OV-Talin1) also enhanced ADS stromal cell proliferation and pro-angiogenesis, while the most pronounced facilitating effects were found in the co-intervention group of OV-Talin1 plus ß-E2 treatment. Results from the xenograft nude mice model showed that the hypodermic endometrial lesions from co-intervention group had the highest mean weight and volume, compared with that of individual OV-Talin1 or ß-E2 treatment. The expression levels of PCNA, Ki67, VEGFB and ANGPTL4 in the lesions were correspondingly elevated the most in the co-intervention group. Our findings unveiled that overexpressed Talin1 might cooperate withß-E2 in stimulating ADS endometrial stromal cell proliferation and neovascularization, synergistically promoting the growth and survival of ectopic lesions. These results may be beneficial to provide a new insight for clarifying the pathogenesis of ADS.

Elevated Circular RNA PVT1 Promotes Eutopic Endometrial Cell Proliferation and Invasion of Adenomyosis via miR-145/Talin1 Axis.

Several theories on the origin of adenomyosis (ADS) have been proposed, of which the most widely accepted is the fundamental pathogenic role of uterine eutopic endometrium. Emerging evidence suggests that circular RNAs participate in the multiple tumorgenesis. The vital importance of circular RNA PVT1 (circPVT1) in the pathological progress like malignancies has been well documented. Nevertheless, its underlying correlation with ADS remains elusive yet. The purpose of this study was to investigate the expression pattern, regulatory effect, and internal mechanism of circPVT1 in ADS. qRT-PCR was performed to detect the relative mRNA expression of circPVT1, miR-145, and Talin1 in ADS endometrial tissue and cells. The protein level of Talin1 was measured by Western blot and immunochemistry. Immunofluorescence was used to identify the primary endometrial epithelial and stromal cells. circPVT1 knockdown in vitro was achieved by transfecting with specific lentivirus vector CCK-8, and colony formation assays were utilized to assess cell proliferation; meanwhile, the transwell assay was employed for evaluating cell invasion ability. By conducting bioinformatics, dual-luciferase reporter assay, or RNA immunoprecipitation (RIP) experiment, the interaction between miR-145 and circPVT1 or Talin1 was verified. Rescue experiments further determined the regulatory effect of circPVT1/miR-145/Talin1 axis. We found both circPVT1 and Talin1 were markedly upregulated in ADS endometrial tissue and cells, whereas miR-145 was decreased. Elevated expression of circPVT1 was closely related to the severity of dysmenorrhea, menorrhagia, and uterine enlargement of patients with ADS. Knockdown of circPVT1 inhibited adenomyotic epithelial and stromal cell proliferation and invasion. Further mechanistic experiments revealed that circPVT1 negatively regulated miR-145 through serving as a molecular sponge. And the facilitating effect of circPVT1 was partially reversed by miR-145. Talin1 was demonstrated to be a down target of miR-145 and indirectly affected by circPVT1. Our findings unveiled that enhanced circPVT1 may be involved in the pathogenesis of ADS via stimulating endometrial cell proliferation and invasion. The establishment of circPVT1/miR-145/Talin1 pathway might present a novel therapeutic insight for ADS.

Talin1 Induces Epithelial-Mesenchymal Transition to Facilitate Endometrial Cell Migration and Invasion in Adenomyosis Under the Regulation of microRNA-145-5p.

Adenomyosis (ADS) is a commonly encountered benign gynecological disorder. Epithelial-mesenchymal transition (EMT) may serve a pivotal role in the pathogenesis of ADS. Talin1 has been identified to be implicated in multiple human carcinomas, probably through inducing EMT process. However, available data on the precise molecular mechanism of Talin1 in the pathogenesis of ADS remain extremely scanty. In the present study, we aim to investigate the clinical roles of Talin1 and its effects on uterine endometrial cell migration, invasion, and EMT in ADS. Relative mRNA expression of Talin1, microRNA-145-5p (miR-145-5p), and EMT-related markers was determined by qRT-PCR. Immunohistochemistry and immunofluorescence were performed to examine the distribution of Talin1 in ADS endometrium. Protein levels of Talin1, EMT-related markers, and wnt/ß-catenin pathway were measured by western blot. Wound healing assay and transwell assay were utilized for evaluating cell migration and invasion respectively. Dual-luciferase reporter assay was performed to verify the relationship between Talin1 and miR-145-5p. We found Talin1 was markedly overexpressed in ADS endometrial tissue and cells, whereas miR-145-5p was downregulated. Elevated Talin1 mRNA level might be closely related to some clinicopathological features of ADS. Through functional experiments, we demonstrated that overexpression of Talin1 induced EMT and enhanced migration and invasion ability of ADS eutopic and ectopic endometrial epithelial cells (ADS_Eu_EEC and ADS_Ec_EEC) in vitro through activating the canonical wnt/ß-catenin pathway. From a mechanistic perspective, Talin1 was inversely regulated by miR-145-5p as a direct target. Our findings unveiled that under the regulation of miR-145-5p, Talin1 might promote endometrial cell migration and invasion through inducing EMT, presenting a novel insight for elucidating the pathogenesis of ADS.

MLL5α activates AR/NDRG1 signaling to suppress prostate cancer progression.

Prostate cancer (PCa) is one of the most prevalent malignancies in men. However, the molecular mechanism controlling the transformation of androgen-dependent PCa (ADPC) to castration-resistant PCa (CRPC) is largely unknown. Androgen receptor (AR) signaling has been reported to play a key role in this process; thus, searching for the novel AR co-activator is important for identifying the mechanism underlying PCa progression. In this study, we focused on the function of mixed lineage leukemia-5α (MLL5α), an epigenetic regulator that exhibits aberrant expression in PCa. MLL5α was the primary expressed form of MLL5 protein in PCa cells and it significantly suppressed proliferation, invasion, and migration in PCa cell lines. Upon stimulation with dihydrotestosterone (DHT), knockdown of MLL5α significantly suppressed N-myc downstream regulated gene 1 (NDRG1) and Kallikrein-related peptidase 3 (KLK3) expression. MLL5α directly bound with AR on the androgen response elements (AREs) and recruited H3K4me3 to the promoters of NDRG1 and KLK3. Downregulation of NDRG1 partially restored the cell invasion and migration suppressed by MLL5α. As evaluated by the proliferation of PCa cells, overexpression of MLL5α synergistically promoted sensitivity to enzalutamide (ENZ) treatment. In PCa patients, MLL5α expression was lower in the high Gleason score (GS) (GS > 7) group than in the low GS (GS < 7) group. In conclusion, suppression of AR/NDRG1 signaling via androgen deprivation therapy (ADT) may be a potential mechanism of CRPC progression. MLL5α significantly suppressed PCa progression by promoting AR/NDRG1 signaling, indicating that regulating MLL5α expression may be a potential treatment approach for patients with advanced PCa.

Inhibition of autophagy enhances the anticancer effect of enzalutamide on bladder cancer.

BACKGROUND: Emerging preclinical evidence suggests a critical role for androgen-mediated androgen receptor (AR) signaling in bladder cancer progression. However, researchers have not determined whether autophagy modulates the efficacy of an enzalutamide (ENZ) treatment in subjects with advanced bladder cancer. In this study, we investigated the synergistic effect of ENZ and autophagy inhibitors on bladder cancer. METHODS: ENZ was used as an anti-AR drug, and chloroquine (CQ), 3-methyladenine (3-MA), and bafilomycin A1 (BAF) were used as autophagy inhibitors. J82, T24, and UMUC3 cell lines were used as models of bladder cancer. A bifluorescence autophagy system with the mRFP-GFP-LC3 plasmid was used to evaluate autophagy flux. Protein and mRNA levels were detected using Western blotting and qPCR, respectively. A Cell Counting Kit-8 (CCK-8) assay, colony assay, and flow cytometry analysis were used to evaluate cell proliferation and apoptosis. Four-week-old BALB/c athymic nude mice were used in the in vivo assay. RESULTS: Based on the results obtained using the bifluorescence autophagy system, ENZ (10-20 µM) significantly facilitated the accumulation of autophagosomes and autolysosomes in the cytoplasm of J82 and T24 cells. Additionally, ENZ significantly increased the expression of autophagy-related genes (AMP-dependent protein kinase (AMPK), autophagy-related gene 5 (ATG5), microtubule-associated protein light chain 3B (LC3B), and UNC-51-like kinase 1 (ULK1)) and proteins (microtubule-associated protein 1 light chain 3-II/I (LC3-II/I), ATG5, and phosphorylated AMP-dependent protein kinase α (p-AMPKα)). The administration of ENZ monotherapy (10-20 µM) to J82 and T24 cells failed to alter proliferation and apoptosis. Concurrent treatment with ENZ and autophagy inhibitors distinctly triggered apoptosis and inhibited proliferation. Genetic inhibition of autophagy by specifically blocking ATG5 with siRNA also increased ENZ-induced apoptosis in J82 and T24 cells. In vivo, concurrent treatment with ENZ (25 mg/kg/day) and CQ (10 mg/kg/day) improved the therapeutic sensitivity by decreasing tumor growth and apoptosis. Additionally, overexpression of AR suppressed ENZ-induced autophagy-related genes (LC3-II/I, ATG5, and p-AMPKα) in T24 cells, and CQ exerted synergistic effects with ENZ to suppressed AR-responsive genes expression (KLK2 and KLK3) in bladder cancer. In UMUC3 cells, ENZ monotherapy directly induced anticancer effects, and concurrent treatment with ENZ and CQ also had a synergistic effect on proliferation and apoptosis. CONCLUSIONS: Autophagy may be a potential mechanism underlying ENZ-resistant bladder cancer. Blockade of autophagy significantly increased ENZ-induced apoptosis in bladder cancer. Thus, concurrent treatment with autophagy inhibitors and ENZ may be a novel therapeutic strategy for bladder cancer.

Dose, duration and strain of bacillus Calmette-Guerin in the treatment of nonmuscle invasive bladder cancer: Meta-analysis of randomized clinical trials.

BACKGROUND: Intravesical bacillus Calmette-Guerin (BCG) instillation is widely used as an adjuvant therapy after transurethral resection of bladder tumor (TURBT) in patients with intermediate- and high-risk nonmuscle invasive bladder cancer (NMIBC). However, the effective dose, duration, and strain of BCG have not yet been clearly determined. We aimed to elucidate the relationship between dose, duration, and strain of BCG and clinical outcomes in NMIBC patients treated with TURBT. METHODS: We conducted a literature search in Embase, Scopus, and PubMed databases for all relevant articles published up to October 2016 in accordance with the Preferred Reporting Items for Systematic Review and Meta-analysis guidelines. The relative risks of clinical outcomes, including recurrence, progression, cancer-specific mortality, and all-cause mortality according to dose (standard vs low), duration (induction vs maintenance), and strain of BCG were presented as the pooled risk ratio (RR) and 95% confidence interval (CI). RESULTS: Nineteen studies meeting the inclusion criteria were finally selected in this meta-analysis. The risk of recurrence was significantly highly observed in case of low-dose BCG (RR, 1.17; 95% CI 1.06-1.30) and induction BCG (RR, 1.33; 95% CI 1.17-1.50) only group without heterogeneity among the included studies. Although there were no significant differences between dose or duration and other clinical outcomes. On direct comparison in each study comparing BCG strains, the Tice stain showed a relatively high probability of recurrence compared with the Connaught (RR, 1.29; 95% CI 1.01-1.64) and RIVM (RR, 2.04, 95% CI 1.28-3.25) strains. Funnel plot testing revealed no significant publication bias. CONCLUSION: The use of standard dose and maintenance BCG instillation may be effective to reduce recurrence rate after TURBT for NMIBC. Further large scale, well-designed, and prospective studies, with stratification of the patients into risk group at randomization, will be required to determine the optimal guideline of BCG use to improve clinical outcomes in NMIBC.