Yunhua Mao

Overexpression of Cdc20 in clinically localized prostate cancer: Relation to high Gleason score and biochemical recurrence after laparoscopic radical prostatectomy

OBJECTIVES This study was aimed to explore Cdc20 expression and its correlation with clinicopathological characteristics and biochemical recurrence (BCR) after laparoscopic radical prostatectomy (LRP) in clinically localized prostate cancer (PCa). METHODS Cdc20 expression was examined by immunohistochemistry in 166 cases, including 60 cases of benign hyperplasia of prostate (BPH) patients treated by transurethral resection and 106 cases of consecutive PCa patients treated by LRP without neoadjuvant therapy in a single Chinese institution. The correlation with clinicopathological features and the predictive value for BCR were statistically analyzed. RESULTS Cdc20 expression was detected in 52 (86.7%) BPH and 97 (91.5%) PCa samples, which was statistically insignificant (P= 0.675). The rate of patients with high expression of Cdc20 was 21.7% in BPH and 37.7% in PCa (P= 0.033). A correlation was revealed between Cdc20 expression and postoperative Gleason scores (P= 0.046), positive surgical margin (P< 0.001). BCR-free survival was significantly lower in patients with high Cdc20 expression than those with low Cdc20 expression (P= 0.018). Univariate analysis indicated pTstage, post operative Gleason score, seminal vesicle invasion, lymph node invasion, surgical margin and Cdc20 expression significantly influenced BCR. Multivariate analysis revealed that postoperative Gleason score, seminal vesicle invasion, lymph node invasion, surgical margin and Cdc20 expression were independent predictors for BCR. After stratified by Gleason score and surgical margin status, Cdc20 expression and lymph node invasion remained significant in Cox regression analysis. CONCLUSIONS Overexpression of Cdc20 may serve as an independent predictor for BCR in patients of clinically localized PCa undergoing LRP without neoadjuvant therapy.

Gold-chrysophanol nanoparticles suppress human prostate cancer progression through inactivating AKT expression and inducing apoptosis and ROS generation in vitro and in vivo

Controlled releasing of regulations remains the most convenient method to deliver various drugs. In the present study, we precipitated gold nanoparticles with chrysophanol. The gold-chrysophanol into poly (DL-lactide-co-glycolide) nanoparticles was loaded and the biological activity of chrysophanol nanoparticles on human LNCap prostate cancer cells, was tested to acquire the sustained releasing property. The circular dichroism spectroscopy indicated that chrysophanol nanoparticles effectively resulted in conformational alterations in DNA and regulated different proteins associated with cell cycle arrest. The reactive oxygen species (ROS), apoptosis, cell cycle, DNA damage, Cyto-c and caspase-3 activity were analyzed, and the expression levels of different anti- and pro-apoptotic were studied using immunoblotting analysis. The cytotoxicity assay suggested that chrysophanol nanoparticles preferentially killed prostate cancer cells in comparison to the normal cells. Chrysophanol nanoparticles reduced histone deacetylases (HDACs) to suppress cell proliferation and induce apoptosis by arresting the cell cycle in sub-G phase. In addition, the cell cycle-related proteins, including p27, CHK1, cyclin D1, CDK1, p-AMP-activated protein kinase (AMPK) and p-protein kinase B (AKT), were regulated by chrysophanol nanoparticles to prevent human prostate cancer cell progression. Chrysophanol nanoparticles induced apoptosis in LNCap cells by promoting p53/ROS crosstalk to prevent proliferation. Pharmacokinetic study in mice indicated that chrysophanol nanoparticle injection showed high bioavailability compared to the free chrysophanol. Also, in vivo study revealed that chrysophanol nanoparticles obviously reduced tumor volume and weight. In conclusion, the data above suggested that chrysophanol nanoparticles might be effective to prevent human prostate cancer progression.

Calpain-2 triggers prostate cancer metastasis via enhancing CRMP4 promoter methylation through NF-κB/DNMT1 signaling pathway

Metastasis is the major cause of cancer‐specific death in patients with prostate cancer (PCa). We previously reported that collapsing response mediator protein‐4 (CRMP4) is a PCa metastasis‐suppressor gene and the hypermethylation in CRMP4 promoter is responsible for the transcription repression in metastatic PCa. However, the underlying mechanisms remain unknown. In this study, we aimed to investigate the role of calpain‐2 in CRMP4 promoter hypermethylation and its functional modulation in PCa metastasis.

Identification of endonuclease domain-containing 1 as a novel tumor suppressor in prostate cancer

BackgroundEndonuclease domain containing 1 (ENDOD1) is implicated in tumorigenesis and aggressiveness of multiple tumors. In this study, we aimed to investigate the role of ENDOD1 in prostate cancer (PCa).MethodsImmunohistochemistry were performed in 30 cases of benign prostatic hyperplasia (BPH) and 50 cases of PCa to identify its association with clinicopathological characteristics. Real-time PCR and western blot were used to detect ENDOD1 mRNA and protein expression in normal prostatic epithelial and PCa cell lines. MTT assays were employed to determine the effect of cell proliferation. Flow cytometry was used to explore the cell cycle distribution and apoptotic effects. Transwell migration and invasion assays were done to evaluate changes in the ability of cell migration and invasion.ResultsImmunoreactivity scores of ENDOD1 showed no statistical difference between BPH and low-grade PCa, whereas lower immunostaining scores were observed in high-grade compared with low-grade PCa. Real-time PCR data indicated that ENDOD1 mRNA expression was markedly increased in LNCaP and 22Rv1 cells and decreased in PC3 and DU145 cells compared to the normal epithelial cells RWPE1. Western blot showed that androgen-sensitive LNCaP cells had the highest protein expression level of ENDOD1, whereas castration-resistant PCa cell lines PC3 and DU145 had significantly lower protein levels. Meanwhile, overexpression of ENDOD1 suppressed cell proliferation, induced G0/G1 cell cycle arrest and inhibited cell migration and invasion. Conversely, siRNA-mediated silencing of ENDOD1 promoted cell proliferation, migration and invasion. No apoptotic effects occurred upon manipulation of ENDOD1 expression.ConclusionOur results indicate that ENDOD1 is a novel tumor suppressor in PCa, which may be employed as a new drug target of preventing progression to metastatic castration-resistant prostate cancer.

Association of doublecortin-like kinase 1 with tumor aggressiveness and poor biochemical recurrence-free survival in prostate cancer

Background Doublecortin-like kinase 1 (DCLK1) has been proven to be involved in numerous tumors, while its role in prostate cancer (PCa) is still unclear. This study aimed at investigating the expression pattern and prognostic value of DCLK1 in PCa. Patients and methods Real-time polymerase chain reaction and Western blot were employed to determine DCLK1 mRNA and protein levels in 25 paired fresh samples of PCa and benign prostatic hyperplasia (BPH) as well as in PCa cell lines. Immunohistochemistry (IHC) was also performed in 125 PCa and 65 BPH tissues to assess DCLK1 expression. Then, the association of DCLK1 expression with clinicopathological parameters and biochemical recurrence (BCR) after radical prostatectomy was statistically analyzed. In addition, the role of DCLK1 in PCa cell proliferation, migration, and invasion was evaluated by using MTT and transwell assays. Results The mRNA and protein levels of DCLK1 were markedly higher in the fresh samples of PCa than that in BPH. Consistently, IHC revealed increased expression of DCLK1 in PCa paraffin-embedded tissues compared with BPH. Moreover, increased DCLK1 expression was significantly associated with postoperative Gleason grading (P=0.012), pathological T stage (P=0.001), seminal vesicle invasion (P=0.026), and lymph node involvement (P=0.017), respectively. The Kaplan–Meier curve analysis demonstrated that high DCLK1 expression was associated with lower postoperative BCR-free survival (bRFS). Furthermore, multivariate Cox analysis showed that postoperative Gleason grading (P=0.018), pathological T stage (P<0.001), seminal vesicle invasion (P=0.012), lymph node involvement (P=0.014), and DCLK1 expression (P=0.014) were independent predictors of BCR. In vitro, the overexpression and knockdown of DCLK1 in PCa cell lines indicated that DCLK1 could promote cell proliferation, migration, and invasion. Conclusion Increased DCLK1 expression is associated with PCa aggressiveness and may independently predict poor bRFS in patients with PCa.

MP99-04 TRUNCATED-CRMP4 BY CALPAIN-2 SUPPRESSES CRMP4 TO PROMOTE METASTASIS OF PROSTATE CANCER VIA PROMOTER METHYLATION THROUGH E2F1/NF-?B/DNMT1 SIGNALING

exploited this link to identify actionable targets by performing a shRNA genomic screen in obese and lean mice targeting the entire kinome. Our functional screen identified multiple kinases, which appear to be essential for obesity-driven PC growth including kinases previously implicated in PC and others not previously studied such as Right Open Reading Frame Kinase 2 (RIOK2). METHODS: LAPC-4 cells were inoculated with an shRNA library of ~5000 lentivirus targeting 513 kinases. 5x106 cells (~1,000 cells per shRNA) were grafted to chronically obese mice. Tumors were established to ~200 mm3 and a portion collected for reference. Remaining mice were randomized to continue on ad lib WD or 25% CR diet. Genome-integrated shRNA inserts were amplified using nested barcoded primers and sequenced using Illumina Hi-Seq 2000 and quantified. A virtual screen based on a RIOK2 homology model generated using MODELLER based on two RIOK2 crystal structures. Global gene expression analysis of RNA from scramble control and RIOK2 knockdown with two shRNAs in 22RV1 cells was conducted with Affymetrix U133A Plus Array. RESULTS: RIOK2 expression correlates with Gleason grade in radical prostatectomy tissue and RIOK2 kinase activity is elevated in metastatic vs localized PCs. ENCODE ChIP-seq data shows Androgen Receptor and Myc bind to the RIOK2 promoter and regulate expression. Targeting RIOK2, via newly identified small molecule inhibitors reduces cell viability and soft agar colony growth. Gene set enrichment analysis of RIOK2 depleted PC cells showed reduction of cell cycle, adipogenesis, EMT and cancer stem cell genes. RIOK2 also regulates Neuropeptide Y2 Receptor (NYP2R), which is part of the NPY obesogenic signaling axis that correlates with obesity and worse PC outcomes. CONCLUSIONS: Our in vivo screen highlighted RIOK2 as an actionable PC target in obese hosts. Targeting RIOK2, pharmacologically with our lead compounds or genetically, drastically reduces PC cell viability. RIOK2 may regulate NPY2R expression, which when coupled with elevated NPY in both obese hosts and PCs can amplify NPY protumorigenic signaling.

Erratum to: Identification of endonuclease domain-containing 1 as a novel tumor suppressor in prostate cancer

Background: Endonuclease domain containing 1 (ENDOD1) is implicated in tumorigenesis and aggressiveness of multiple tumors. In this study, we aimed to investigate the role of ENDOD1 in prostate cancer (PCa). Methods: Immunohistochemistry were performed in 30 cases of benign prostatic hyperplasia (BPH) and 50 cases of PCa to identify its association with clinicopathological characteristics. Real-time PCR and western blot were used to detect ENDOD1 mRNA and protein expression in normal prostatic epithelial and PCa cell lines. MTT assays were employed to determine the effect of cell proliferation. Flow cytometry was used to explore the cell cycle distribution and apoptotic effects. Transwell migration and invasion assays were done to evaluate changes in the ability of cell migration and invasion. Results: Immunoreactivity scores of ENDOD1 showed no statistical difference between BPH and low-grade PCa, whereas lower immunostaining scores were observed in high-grade compared with low-grade PCa. Real-time PCR data indicated that ENDOD1 mRNA expression was markedly increased in LNCaP and 22Rv1 cells and decreased in PC3 and DU145 cells compared to the normal epithelial cells RWPE1. Western blot showed that androgen-sensitive LNCaP cells had the highest protein expression level of ENDOD1, whereas castration-resistant PCa cell lines PC3 and DU145 had significantly lower protein levels. Meanwhile, overexpression of ENDOD1 suppressed cell proliferation, induced G0/G1 cell cycle arrest and inhibited cell migration and invasion. Conversely, siRNA-mediated silencing of ENDOD1 promoted cell proliferation, migration and invasion. No apoptotic effects occurred upon manipulation of ENDOD1 expression. Conclusion: Our results indicate that ENDOD1 is a novel tumor suppressor in PCa, which may be employed as a new drug target of preventing progression to metastatic castration-resistant prostate cancer.