Jun Pang

Profiling protein markers associated with lymph node metastasis in prostate cancer by DIGE-based proteomics analysis.

Current predictive tools and imaging modalities are not accurate enough for preoperative diagnosis of lymph node metastatic prostate cancer (LNM PCa). Proteomic analysis is introduced to screen potential biomarkers for early detection of LNM PCa. In our initial study, protein samples from localized and LNM PCa as well as benign prostatic hyperplasia tissues were analyzed using two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) coupled with MALDI-TOF/TOF MS. We identified 58 proteins that were differentially expressed in the LNM PCa group relative to the localized PCa group. Six of these proteins, e-FABP5, MCCC2, PPA2, Ezrin, SLP2, and SM22, are functionally relevant to cancer metastasis. Expression of these proteins was therefore further validated in tissue samples from the original cohort and also from a larger, independent cohort of patients using real time PCR, Western blotting, and immunohistochemistry staining. In addition, the serum levels of e-FABP5 were also examined by ELISA. Relative to localized PCa tissues, LNM PCa tissues had increased expression of e-FABP5, MCCC2, PPA2, Ezrin, and SLP2 and decreased expression of SM22. Patients with LNM PCa had significantly higher levels of serum e-FABP5. This study presents evidence that increased expression of e-FABP5, MCCC2, PPA2, Ezrin, and SLP2 and decreased expression of SM22 are useful diagnostic markers for the existence of LNM PCa.

ERG Rearrangement for Predicting Subsequent Cancer Diagnosis in High-Grade Prostatic Intraepithelial Neoplasia and Lymph Node Metastasis

Purpose: We aimed to analyze whether ERG rearrangement in biopsies could be used to assess subsequent cancer diagnosis in high-grade prostatic intraepithelial neoplasia (HGPIN) and the risk of lymph node metastasis in early prostate cancer. Experimental Design: Samples from 523 patients (361 with early prostate cancer and 162 with HGPIN) were collected prospectively. On the basis of the cutoff value established previously, the 162 patients with HGPIN were stratified to two groups: one with an ERG rearrangements rate ≥1.6% (n = 59) and the other with an ERG rearrangements rate <1.6% (n = 103). For the 361 prostate cancer cases undergoing radical prostatectomy, 143 had pelvic lymph node dissection (node-positive, n = 56 and node-negative, n = 87). All ERG rearrangement FISH data were validated with ERG immunohistochemistry. Results: A total of 56 (of 59, 94.9%) HGPIN cases with an ERG rearrangements rate ≥1.6% and 5 (of 103, 4.9%) HGPIN cases with an ERG rearrangements rate <1.6% were diagnosed with prostate cancer during repeat biopsy follow-ups (P < 0.001). There were significant differences in ERG rearrangement rates between lymph node–positive and -negative prostate cancer (P < 0.001). The optimal cutoff value to predict lymph node metastasis by ERG rearrangement was established, being 2.6% with a sensitivity at 80.4% [95% confidence interval (CI), 67.6–89.8] and a specificity at 85.1% (95% CI, 75.8–91.8). ERG protein expression by immunohistochemistry was highly concordant with ERG rearrangement by FISH. Conclusions: The presence of ERG rearrangement in HGPIN lesions detected on initial biopsy warrants repeat biopsies and measuring ERG rearrangement could be used for assessing the risk of lymph node metastasis in early prostate cancer. Clin Cancer Res; 18(15); 4163–72. ©2012 AACR.

Toll-like receptor 9 agonists up-regulates the expression of cyclooxygenase-2 via activation of NF-κB in prostate cancer cells

CpG-oligonucleotides (CpG-ODNs), mimicking bacterial DNA, have recently been shown to stimulate prostate cancer invasion in vitro via Toll-like receptor 9 (TLR9). Since cyclooxygenase 2 (COX-2), frequently overexpressed in multiple tumor types including prostate cancer, is a causal factor for tumor development, invasion and metastasis, an interesting question is raised whether TLR9 regulates COX-2 expression in prostate cancer cells. To address this question, herein we examined COX-2 expression in PC-3 cells stimulated with different doses and time courses of CpG-ODNs. The regulatory role of NF-κB in TLR9-mediated COX-2 expression was also investigated. CpG-ODN was found to up-regulate the expression of COX-2 in PC-3 cells in a dose- and time-dependent manner, but have little impact on COX-1 expression. Moreover, CpG-ODN also promoted nuclear translocation and activation of NF-κB, which appeared to be required for COX-2 induction by CpG-ODN. Overall, TLR9 up-regulates COX-2 expression in prostate cancer cells, at least partially through the activation of NF-κB, which may be implicated in tumor invasion and metastasis.

Detection of TMPRSS2-ETS Fusions by a Multiprobe Fluorescence in Situ Hybridization Assay for the Early Diagnosis of Prostate Cancer : A Pilot Study

Fusion of the prostate-specific and androgen-regulated transmembrane-serine protease gene (TMPRSS2) with the erythroblast transformation-specific (ETS) family members is the most common genetic alteration in prostate cancer. However, the biological and clinical role of TMPRSS2-ETS fusions in prostate cancer, especially in problematic prostate needle core biopsies, has not been rigorously evaluated. We randomly collected 85 specimens including 50 archival prostate cancer tissue blocks, 15 normal prostate specimens, and 20 benign prostatic hyperplasia specimens for TMPRSS2-ETS fusion analyses. Moreover, the fusion status in an additional 20 patients with initial negative biopsies who progressed to biopsy-positive prostate cancer at subsequent follow-ups was also characterized. Fluorescently labeled probes specific for ERG-related rearrangements involving the TMPRSS2-ERG fusion as well as TMPRSS2-ETV1 and TMPRSS2-ETV4 were used to assess samples for gene rearrangements indicative of malignancy under a design of sequential trial. Rearrangements involving TMPRSS2-ETS fusions were detected in 90.0% of the 50 postoperative prostate cancer samples. The positive rate for the rearrangements in the initial prostate cancer-negative biopsies of 20 patients who eventually progressed to prostate cancer was 60.0% (12/20). Our preliminary study demonstrates that the clinical utility of TMPRSS2-ETS fusion detection as a biomarker and ancillary diagnostic tool for the early diagnosis of prostate cancer is promising, given this approach shows significant high sensitivity and specificity in detection.

Synthesis, characterization and osteoconductivity properties of bone fillers based on alendronate-loaded poly(ε-caprolactone)/hydroxyapatite microspheres

A superior drug controlled release system capable of achieving efficient osteogenesis is in imperative demand because of limited bone substitute tissue for the treatment of bone defect. In the present study, we investigated the potential of using poly(ε-caprolactone)–hydroxyapatite (PCL–HA) composite microspheres as an injectable bone repair vehicle by controlled release of alendronate (AL), a medicine that belongs to the bisphosphonates family. The PCL/HA–AL microspheres were prepared with solid/oil/water emulsion technique, which included two processes: (1) AL was loaded on the hydroxyapatite nanoparticles; (2) the HA–AL complex was built in the PCL matrix. The spherical PCL/HA–AL microspheres were characterized with its significantly improved encapsulation efficiency of hydrophilic AL and better sustained release. Human bone mesenchymal stem cells (hMSCs) were cultured on the surface of these microspheres and exhibited high proliferative profile. Specifically, in osteogenic medium, hMSCs on the surface of PCL/HA–AL microspheres displayed superior osteogenic differentiation which was verified by alkaline phosphatase activity assay. In conclusion, by presenting strong osteogenic commitment of hMSCs in vitro, the PCL/HA–AL microspheres have the potential to be used as an injectable vehicle for local therapy of bone defect.

Dendritic cells transduced with a PSMA-encoding adenovirus and cocultured with autologous cytokine-induced lymphocytes induce a specific and strong immune response against prostate cancer cells

OBJECTIVE The lack of curative therapies for advanced prostate cancer (PCa) has prompted a search for novel treatments such as immunotherapy. In this study, we analyzed whether dendritic cells (DCs) from healthy donors transduced with a PSMA-encoding adenovirus (Ad-PSMA) and cocultured with autologous cytokine-induced killer cells (CIKs) can induce a strong specific immune response against PCa cells in vitro. MATERIALS AND METHODS Ad-PSMA was constructed by DNA recombination. DCs and CIKs were prepared by cytokines induction from peripheral blood mononuclear cells, and flow cytometry was used to measure the phenotypes of DCs and CIKs. DCs were transduced with Ad-PSMA and then cocultured with autologous CIKs. The cytotoxicity of the cocultured cells against specific target LNCaP cells and control targets DU145 and PC3 cells was analyzed by a 4-h LDH release assay. RESULTS DCs were transduced with Ad-PSMA with transfection efficiency of 70% and the transduction did not alter typical morphology of mature DCs. The PSMA protein was effectively expressed in DCs, which were transfected with Ad-PSMA. Ad-PSMA-transduced DCs stimulated CIKs strongly to lyse about 75% of PSMA-expressing PCa cells. Furthermore, the cocultivation of Ad-PSMA-transduced DCs with CIKs could significantly increase the production of interferon-gamma after restimulated with PSMA peptide mixtures. CONCLUSIONS The data demonstrate that DCs, which were transduced with a PSMA-expressing adenovirus and cocultured with autologous CIKs, induce a PSMA-specific, strong immune response against PCa cells. Therefore, this approach may have a potential for an adoptive immunotherapy for patients with advanced PCa.

Single‐port transvesical laparoscopic radical prostatectomy for organ‐confined prostate cancer: technique and outcomes

To report a novel technique for performing single‐port transvesical laparoscopic radical prostatectomy (STLRP) and to evaluate the oncological and functional outcomes in 16 patients with organ‐confined prostate cancer.

Prospective Study of CRMP4 Promoter Methylation in Prostate Biopsies as a Predictor For Lymph Node Metastases.

Background For patients with prostate cancer (PCa), the presence of pelvic lymph node metastasis (LNM) is a strong predictor of poor outcome. However, the approaches with promising sensitivity and specificity to detect LNM are still lacking. We investigated the value of collapsin response mediator protein 4 (CRMP4) promoter methylation in biopsies as a predictor for LNM. Methods CRMP4 promoter methylation at two previously identified CpG sites was determined in 80 case-matched biopsy samples (the training set) using bisulfite pyrosequencing. The predictive cutoff value was independently validated using cohort I of 339 PCa patients (Southern China) and cohort II of 328 case patients (Germany, across China). Mann-Whitney U test, the receiver operating characteristic curve, McNemars test, and logistic regression were used to assess data. All statistical tests were two-sided. Results In the training set, CRMP4 promoter methylation (≥15.0% methylated) was statistically significantly associated with LNM (P < 001). Successful validations were achieved in both cohorts I and II (sensitivity = 92.3%, 95% confidence interval [CI] = 79.3 to 97.9, and sensitivity = 92.2%, 95% CI = 81.1 to 97.8, respectively; specificity = 92.7%, 95% CI = 80.2 to 99.1, and specificity = 91.3%, 95% CI = 87.4 to 94.4, respectively). The sensitivity of CRMP4 promoter methylation is superior to conventional MRI (cohort I: 92.3% vs 26.2%, P < 001; cohort II: 92.2% vs 33.3%, P < 001). CRMP4 promoter methylation is an independent predictor of LNM (cohort I: hazard ratio [HR] = 8.35, 95% CI = 5.64 to 12.35, P < 001; cohort II: HR = 12.46, 95% CI = 5.82 to 26.70, P < 001) in a multivariable analysis model. Conclusion CRMP4 promoter methylation in diagnostic biopsies could be a robust biomarker for LNM in PCa.

Investigation of optimal prostate biopsy schemes for Chinese patients with different clinical characteristics.

Purpose: To investigate the optimal schemes of prostate biopsy according to prostate volume (PV), age and transrectal ultrasound (TRUS) status in Chinese men. Methods: 923 consecutive patients who underwent initial TRUS-guided systematic 12-core prostate biopsy (12PBx) were enrolled in this study. The 12PBx was obtained by overlapping of conventional sextant, lateral base, mid-gland of peripheral zone and apex. Each sample was individually marked and inked before fixation. Patients were divided into 8 subgroups on the basis of independent risk factors investigated using logistic regression model. Subsequently, 12PBx was defined as self-control for the analysis of biopsy schemes (6-, 8- and 10PBx) on individual core basis. The prostate cancer detection rates (CDRs) of 6-, 8-, 10- and 12PBx were compared for each individual subgroup. Results: The 12PBx detected 253 (27.4%) cases of prostate cancer (PCa), of which 67.2, 47.1 and 61.3% were located in the base, mid-gland and apex, respectively. Multivariate analysis indicated that age, TRUS status and PV were independent risk factors for PCa detection. CDR increased with increasing biopsy cores. However, for patients with age ≥65 years, positive TRUS and PV <38.5 cm3, CDR of 8PBx (30.6%) was similar to 10PBx (32.2%) and 12PBx (32.2%); for patients with age ≥65 years, negative TRUS and PV <38.5 cm3 or ones with age ≥65 years, positive TRUS and PV ≥38.5 cm3, 10PBx was as effective as 12PBx in detecting PCa (27.8, 27.5 vs. 28.9, 29.3%, respectively). Conclusion: Age, TRUS status and PV were independent risk factors for PCa detection. Traditional sextant biopsy is not recommended. 8-, 10-, or 12PBx as an individual biopsy scheme might be adopted according to these risk factors for Chinese patients.

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.