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Background Treatment of prostate cancer (PCa) may be improved by identifying biological mechanisms of tumor growth that directly impact clinical disease progression. We investigated whether genes associated with a highly tumorigenic, drug resistant, progenitor phenotype impact PCa biology and recurrence. Methods Radical prostatectomy (RP) specimens (±disease recurrence, N = 276) were analyzed by qRT-PCR to quantify expression of genes associated with self-renewal, drug resistance, and tumorigenicity in prior studies. Associations between gene expression and PCa recurrence were confirmed by bootstrap internal validation and by external validation in independent cohorts (total N = 675) and in silico. siRNA knockdown and lentiviral overexpression were used to determine the effect of gene expression on PCa invasion, proliferation, and tumor growth. Results Four candidate genes were differentially expressed in PCa recurrence. Of these, low AXIN2 expression was internally validated in the discovery cohort. Validation in external cohorts and in silico demonstrated that low AXIN2 was independently associated with more aggressive PCa, biochemical recurrence, and metastasis-free survival after RP. Functionally, siRNA-mediated depletion of AXIN2 significantly increased invasiveness, proliferation, and tumor growth. Conversely, ectopic overexpression of AXIN2 significantly reduced invasiveness, proliferation, and tumor growth. Conclusions Low AXIN2 expression was associated with PCa recurrence after RP in our test population as well as in external validation cohorts, and its expression levels in PCa cells significantly impacted invasiveness, proliferation, and tumor growth. Given these novel roles, further study of AXIN2 in PCa may yield promising new predictive and therapeutic strategies. Prostate © 2016 Wiley Periodicals, Inc.

Primary extragonadal germ cell tumors are rare, and 60% of such cases are seminomas. While the tumors can occur in the mediastinum, thymus, retroperitoneal organs and pineal gland, seminoma originating in the prostate tissue is extremely rare. The present study reports the case of a 54-year-old male with prostate seminoma. The patient was followed up at the First Affiliated Hospital of Dalian Medical University (Dalian, Liaoning, China) from 2001 onwards. The patient received chemotherapy with cyclophosphamide following total resection of the pelvic organs, pelvic lymph node dissection, continent detenial cecum-ascending colic bladder and orchidectomy. The patient experienced considerable post-operative quality of life for >10 years and the disease did not reappear. The study indicated that extragonadal seminoma is sensitive to chemotherapy, and that radical surgery and post-operative adjuvant chemotherapy with cyclophosphamide is a reasonable and feasible treatment method.

The present study aimed to investigate whether microRNA (miR)‑185 downregulated androgen receptor expression in the LNCaP prostate carcinoma cell line. Human prostate cancer (PCa) LNCaP cells were cultured and transfected with synthetic has‑miR‑185 mimic or inhibitor. The transfected cells were subsequently evaluated with a viability assay, nuclear staining, reverse transcription quantitative polymerase chain reaction (RT‑qPCR), dual luciferase assay and western blot analysis. The results of the western blot analysis and RT‑qPCR indicated that transfection with an miR‑185 mimic markedly reduced the androgen receptor (AR) protein expression levels in LNCaP cells, whereas transfection with an miR‑185 inhibitor increased the protein expression of AR in the LNCaP cells. The results of the luciferase reporter assay demonstrated that the predicted target site in the AR 3' untranslated regions was a specific functional binding site for miR‑185, and that AR was a direct target of miR‑185. In addition, downregulation of AR by miR‑185 impaired the interaction between AR and androgen response element, and downregulated the expression of the AR target gene prostate specific antigen. Data also suggested that the downregulation of AR mediated by miR‑185, inhibited the proliferation and induced the apoptosis of the LNCaP cells. Therefore, the results of the present study suggested that miR‑185 may be a potential negative modulator of AR‑mediated signaling and may act as a tumor suppressor in prostate cancer cells.

It is known that aquaporin 9 (AQP9) in the prostate was strictly upregulated by androgen and may represent a novel therapeutic target for several cancers, but whether AQP9 plays a role in the regulation of androgen-independent prostate cancer still remains unclear. In the present study, AQP9 was determined in prostate cancer and adjacent cancer tissues; AQP9-siRNA was applied to silencing AQP9 in androgen-independent prostate cancer cell PC3 cell line. Western blot and flow cytometry analysis were employed to detect changes in related-function of control and AQP9-siRNA groups. The results showed that AQP9 is significantly induced in cancer tissues than that in adjacent cancer tissues. Moreover, knockdown of AQP9 in PC3 androgen-independent prostate cancer cell prostate cancer cells increased inhibition rates of proliferation. In addition, knockdown of AQP9 resulted in a significant decrease in the expression of the Bcl-2 and with a notable increase in the expression of Bax and cleaved caspase 3, indicated that AQP9 knockdown promoted apoptosis in prostate cancer cells. From wound healing assay and matrigel invasion, we suggested that AQP9 expression affects the motility and invasiveness of prostate cancer cells. Moreover, In order to explore the pathway may be involved in AQP9-mediated motility and invasion of prostate cancer cells, the phosphorylation of ERK1/2 was significant suppressed in AQP9 siRNA-transfected cells compared with that in control cells, suggesting that AQP9 is involved in the activation of the ERK pathway in androgen-independent prostate cancer cells.

BACKGROUND. Treatment of prostate cancer (PCa) may be improved by identifying biological mechanisms of tumor growth that directly impact clinical disease progression. We investigated whether genes associated with a highly tumorigenic, drug resistant, progenitor phenotype impact PCa biology and recurrence. METHODS. Radical prostatectomy (RP) specimens (±disease recurrence, N = 276) were analyzed by qRT-PCR to quantify expression of genes associated with self-renewal, drug resistance, and tumorigenicity in prior studies. Associations between gene expression and PCa recurrence were confirmed by bootstrap internal validation and by external validation in independent cohorts (total N = 675) and in silico. siRNA knockdown and lentiviral overexpression were used to determine the effect of gene expression on PCa invasion, proliferation, and tumor growth. RESULTS. Four candidate genes were differentially expressed in PCa recurrence. Of these, low AXIN2 expression was internally validated in the discovery cohort. Validation in external cohorts and in silico demonstrated that low AXIN2 was independently associated with more aggressive PCa, biochemical recurrence, and metastasis-free survival after RP. Functionally, siRNA-mediated depletion of AXIN2 significantly increased invasiveness, proliferation, and tumor growth. Conversely, ectopic overexpression of AXIN2 significantly reduced invasiveness, proliferation, and tumor growth. CONCLUSIONS. Low AXIN2 expression was associated with PCa recurrence after RP in our test population as well as in external validation cohorts, and its expression levels in PCa cells significantly impacted invasiveness, proliferation, and tumor growth. Given these novel roles, further study of AXIN2 in PCa may yield promising new predictive and therapeutic strategies.

Remodeling of the extracellular matrix (ECM), which is induced by lysyl oxidase (LOX), has been demonstrated to accompany tumor progression; however, the association between LOX expression levels and the malignant behavior of prostate cancer (Pca) remains unclear. The present study aimed to analyze the tumor-associated expression profile of LOX in patients with Pca and to evaluate its potential prognostic value. In the form of a retrospective study, the expression patterns of LOX and collagen I were analyzed in patients with benign prostate hyperplasia and Pca by immunohistochemical examination. The results demonstrated that, with the initiation and progression of Pca, the expression levels of LOX and collagen I were closely associated with Gleason score and tumor stage. In addition, although LOX was expressed in cancer and non-cancer tissues, the differential expression pattern observed in the ECM of Pca cells may indicate that LOX is an important molecule that affects the progression of this disease. Therefore, LOX expression level in the ECM may function as an independent predictor of Pca.

INTRODUCTION AND OBJECTIVES: Better biomarkers are needed in prostate cancer (PCa) to predict disease recurrence and guide optimal therapy. We investigated whether genes associated with a highly tumorigenic, drug resistant, progenitor phenotype impact PCa biology and clinical outcomes in localized disease. METHODS: Radical prostatectomy (RP) specimens (þ/disease recurrence) were analyzed by qRT-PCR to quantify expression of genes associated with self-renewal, drug resistance, and tumorigenicity in prior studies. Univariable and multivariable associations between gene expression and PCa recurrence were confirmed by bootstrap internal validation and by external validation in independent cohorts and in-silico. siRNA knockdown and lentiviral overexpression were used to determine the effect of gene expression on PCa proliferation, invasion and tumor growth. RESULTS: Four candidate genes were differentially expressed in PCa recurrence, and of these, low AXIN2 expression was internally validated. In external cohorts and in silico, low AXIN2 was independently associated with more aggressive PCa, biochemical recurrence, and metastasis-free survival after RP. In vitro and in vivo, low AXIN2 expression was associated with a cancer stem-like cell-surface signature, and siRNA knockdown of AXIN2 resulted in significantly greater invasiveness. Conversely, ectopic overexpression of AXIN2 significantly reduced cell proliferation and tumor growth in mice (Figure). CONCLUSIONS: Low AXIN2 expression was associated with PCa recurrence after RP in our test population as well as in external validation cohorts, and its expression levels in PCa cells significantly impacted invasiveness, proliferation and tumor growth. Based on these novel roles in PCa, AXIN2 may represent a new putative biomarker and potential therapeutic target in this disease.

Objective To investigate the role of miR-221/222 in cell proliferation and apoptosis in human prostate cancer cells, and examine the effects of miR-221/222 on caspase-10 expression. Methods Prostate cancer cells were transfected with miR-221/222 mimics or inhibitors. Cell proliferation was assessed by MTT assay. The expression levels of miR-221/222 were detected with quantitative real-time PCR. Apoptosis was induced with TNF-α/CHX treatment, and evaluated by Hoechst 33342 staining, propidium iodide (PI) flow cytometric analysis, caspase-3 activity measurement, and Western blot analysis. Luciferase activity assay, quantitative real-time PCR, and Western blot were performed to evaluate the effects of miR-221/222 on caspase-10 expression. Results Our results showed that miR-221/222 could promote the proliferation of prostate cancer cells, including LNCaP and PC3 cells. After transfection and apoptosis induction, Hoechst 33342 staining and PI flow cytometric assay showed that apoptosis was dramatically decreased in prostate cancer cells treated with miR-221/222 mimics. Moreover, caspase-3 activity was dramatically decreased, and the cleaved forms of caspase-3 were reduced, in the miR-221/222 mimic-treated group. On the contrary, miR-221/222 knockdown sensitized the prostate cancer cells to TNF-α/CHX-induced apoptosis. In addition, a negative correlation was observed between the expressions of miR-221/222 and caspase-10 in prostate cancer cells. miR-221/222 could repress the expression of caspase-10, which was confirmed by the luciferase reporter assay. Conclusion miR-221/222 promote cell proliferation and repress apoptosis, through suppressing caspase-10, in prostate cancer cells. Our results provide promising evidence for the miRNA-based therapeutic strategy of prostate cancers.

Urocortin (Ucn), a corticotropin-releasing factor (CRF)-related neuropeptide binding both CRF type 1 receptor (CRFR1) and CRFR2, has recently been found in prostate cancer. However, no report has yet been known to elucidate the roles of Ucn in prostate cancer via the two receptors. In this study, the expression of both CRFR1 and CRFR2 in the mouse prostate cancer cell line RM-1 were detected and cellular apoptosis was monitored in the presence of CRF or Ucn2, the CRFR1- and CRFR2-selective agonist respectively. CRF promoted apoptosis while Ucn2 exerted the opposite effect. CRF reduced Bcl-2 expression, induced Bax expression, and hyperpolarized the mitochondrial membrane potential to activate caspase-9. On the contrary, Ucn2 increased Bcl-2 expression and decreased Bax expression, in which phosphorylation of Akt and cyclic AMP response element-binding (CREB) was involved. Pretreatment with phosphatidylinositide 3-kinase/Akt inhibitor (LY-294002) prior to Ucn2 led to downregulation of CREB phosphorylation and hence reduced Bcl-2 expression. These effects of CRF and Ucn2 were abolished by antalarmin (Anta) and antisauvagine-30, the CRFR1- and CRFR2-selective antagonist respectively. In LNCaP cell line, similar effects on cell apoptosis by CRF and Ucn2 were observed. In summary, our results demonstrated CRFR1 and CRFR2 expression in prostate cancer and indicated the opposite apoptotic roles of the two different CRFRs. These data may contribute to uncovering the pathophysiological function of endogenous Ucn in prostate tumorigenesis and progression.

29 Background: Better biomarkers are needed in prostate cancer (PCa) to predict disease recurrence and guide optimal therapy. We investigated whether genes associated with a highly tumorigenic, drug resistant, progenitor cancer phenotype impact PCa biology and clinical outcomes in localized disease. Methods: Genes associated with self-renewal, drug resistance, and tumorigenicity were analyzed by qRT-PCR on PCa mRNA from radical prostatectomy (RP) specimens (+/- disease recurrence). Wilcoxon rank sum two-sample test, multivariable recursive partitioning, and bootstrap internal validation measured and confirmed associations with recurrence. Further validation was conducted in external cohorts and in-silico, as well as in vitro and in vivo using siRNA knockdown and lentiviral overexpression to determined the effect of gene expression on PCa proliferation, invasion and tumor growth. Results: Four candidate genes were differentially expressed in PCa recurrence, and of these, low AXIN2 expression was internally validated. Validation in external cohorts demonstrated low AXIN2 expression was associated with more aggressive prostate cancer and was independently associated with biochemical recurrence (BCR) and metastasis-free survival (MFS) after RP. In vitro, low AXIN2 expression was associated with a cancer stem-like cell-surface signature, and siRNA knockdown of AXIN2 resulted in significantly greater invasiveness. Conversely, ectopic overexpression of AXIN2 significantly reduced cell proliferation and tumor growth in mice. Conclusions: Low AXIN2 expression was associated with PCa recurrence after RP in our test population as well as in external validation cohorts. AXIN2 expression levels in PCa cells significantly impacted invasiveness, proliferation and tumor growth. AXIN2 represents a putative biomarker and potential therapeutic target in early prostate cancer.

145 Background: Sub-populations of cancer stem cells (CSC) present in prostate cancer (PCa) and other malignancies have been associated with more aggressive characteristics. We investigated whether tumor expression of genes putatively linked to the CSC phenotype is associated with PCa recurrence. Methods: Expression of mRNA from radical prostatectomy specimens in 241 patients (cases 147, control 94) was analyzed by RT-PCR. Univariate analysis and recursive partitioning measured associations between expression of 12 CSC genes and PCa recurrence. Bootstrap internal validation was used to confirm associations. External validation was performed in-silico using the Oncomine microarray data compendium, analyzing differential gene expression in relevant studies. Results: Four genes (AXIN2, p-9 to 0.07) and locally-advanced versus low grade (four of seven studies, p= 0.007 to 0.037). Lastly, low AXIN2 expression was associated with worse disease in multiple other cancers (10 of 10 studies, p=7.7x10-6 to 0.007). Conclusions: Low AXIN2 expression was internally and externally validated as predictive of PCa recurrence after radical prostatectomy. In-silico analysis demonstrates low AXIN2 expression is associated with more aggressive disease and worse clinical outcomes in prostate and other cancer cohorts. This novel finding delineates a link between the CSC phenotype and disease prognosis, suggesting AXIN2’s role as a potential biomarker.

4563 Background: Novel biomarkers are needed to predict recurrence after radical prostatectomy for localized prostate cancer. Recent data suggest that cancer stem-like cells (CSC) are present in prostate tumors, and the CSC phenotype has been associated with an aggressive cancer phenotype. We investigated whether tumor mRNA levels of genes typically expressed by CSC are associated with disease recurrence. Methods: Clinically annotated prostatectomy specimens (FFPE) were used for this nested case-control study. Samples represented men who underwent radical prostatectomy and either experienced a recurrence (PSA or clinical; n=152) or no recurrence (controls; n=124) with minimum 5-year follow-up. Men were excluded if they received neoadjuvant hormone therapy. Cases and controls were frequency matched based on D’Amico risk group. Laser capture microdissection with mRNA extraction and quantitative RT-PCR were used to quantify the expression of 12 candidate CSC-associated genes (ALDH1A1, Axin2, Bmi1, CD133, CD44a, CTNNB1/β-catenin, ITGA2/integrin α2, NANOG, Nkx3-1, Notch1, OCT 4, TACSTD2/Trop2). Univariable and multivariable analyses were conducted to measure associations with recurrence. Results: mRNA data were evaluable for 241 of 276 patients. Univariate Wilcoxon analysis showed that 4 genes (Axin2, plow/NANOGhigh; n=94, 95%CI 3.7-19.2) and 5.1 (Axin2low/NANOGvery high/CTNNB1low; n=26, 95%CI 1.7-14.8). Conclusions: We identified a novel CSC-associated 3 gene expression signature with the potential to predict recurrence after radical prostatectomy. Axin2 is known to interact with CTNNB1/β-catenin in the Wnt signaling pathway, which in turn has been shown to regulate expression of NANOG, a key protein that mediates pluripotency. In vitro experiments and an independent cohort validation study are planned based on these novel findings.

31 Background: New biomarkers are needed to better predict outcome after radical prostatectomy for clinically localized prostate cancer. Recent data suggest that subpopulations of cancer stem-like cells (CSC) are present in prostate cancer, and the CSC phenotype has been associated with high tumorigenicity, drug resistance, and disease progression. We sought to determine whether the expression of genes associated with the CSC phenotype are associated with disease recurrence. Methods: A nested case-control study was performed. Cases were men who underwent radical prostatectomy and had disease recurrence (n=147). Controls were men who underwent radical prostatectomy and did not have disease recurrence (n=59). Cases and controls were matched based on D’Amico risk stratification group. Laser capture microdissection with mRNA extraction and quantitative RT-PCR were used to measure the expression of 9 novel candidate CSC-associated genes (Axin2, Bmi1, CD133, CD44a, CTNNB1, ITGA2, Nkx3-1, Notch1, TACSTD2) in formalin-fixed, paraffin-embedded radical prostatectomy specimens. The Wilcoxon test was used to examine the association between CSC-associated gene expression and D’Amico risk stratification group. The optimal chi-square approach and recursive partitioning analysis were used to determine the association between CSC-associated gene expression and disease recurrence. Results: Complete gene expression data were evaluable for 206 out of 261 patients (79%). The median age was 64 years (range, 42-78 years). High expression of TACSTD2 (p=0.04) and CD44a (p=0.05) were associated with D’Amico low risk group. The optimal chi-square approach showed that Axin2 was associated with disease recurrence (recurrence rate: Axin2 ≤ 3.83 = 80% versus Axin2 > 3.83 = 43%, p=0.0015). Recursive partitioning analysis confirmed an association between Axin2 and disease recurrence. Conclusions: A novel finding of the current study was that a low gene expression level of Axin2 was associated with disease recurrence. High gene expression levels of TACSTD2 and CD44a were associated with D’Amico low risk group. Further analysis of the Wnt/beta-catenin pathway is ongoing in this cohort.