Serum osteoprotegerin levels are increased in patients with advanced prostate cancer.

PURPOSE: Osteoprotegerin (OPG) is a soluble osteoclastogenesis inhibitor that regulates bone turnover. We reported recently that OPG protein expression is significantly increased in prostate cancer (CaP) cells present in bone metastases. The aim of this study was to determine serum OPG levels in patients at different stages of CaP and correlate the results with disease status. EXPERIMENTAL DESIGN: OPG levels were examined in patients with benign prostatic hyperplasia, clinically localized CaP, early recurrence of CaP, and advanced CaP and evidence of bone metastases. Serum OPG levels were measured by sandwich ELISA assays. The serum Crosslaps (sCTX) assay was used to quantify bone resorption in the advanced CaP group. RESULTS: Serum OPG levels were increased significantly in the advanced CaP group versus all other groups. There was no significant correlation between serum OPG levels and PSA levels either in the advanced CaP group or within any of three treatment subclasses of this group: no Tx, those not treated; Tx, those treated; and R, those treated with resorption blockers. Levels of OPG were negatively correlated with sCTX levels only in the advanced CaP Tx group. sCTX levels correlated with prostate-specific antigen levels in the advanced CaP Tx and R groups but not in the no-Tx group. CONCLUSIONS: Our data show that serum OPG levels are increased with advanced CaP. We hypothesize that OPG levels are related to CaP progression and suggest that further studies of the biological effects of OPG on CaP metastases are warranted.     

Elevation of cytokine levels in cachectic patients with prostate carcinoma

BACKGROUND: Approximately 60-70% of patients with advanced prostate carcinoma (CaP) suffer from cachexia, one of the most devastating conditions associated with advanced malignant disease. The pathophysiology of cachexia is multifactorial, and several cytokines, such as tumor necrosis factor alpha (TNFalpha) and interleukin 1 (IL-1), IL-6, and IL-8, may be involved. The objective of the current study was to determine whether cachexia associated with advanced CaP is accompanied by increased serum levels of TNFalpha, IL-1beta, IL-6, and IL-8. METHODS: The levels of TNFalpha, IL-1beta, IL-6, IL-8, and prostate specific antigen (PSA) were examined in serum samples from normal donors (n = 10 donors), from patients with organ-confined CaP (n = 19 patients), from patients with advanced CaP without cachexia (n = 17 patients), and from patients with cachectic CaP (n = 26 patients). DPC Immulite and Abbott IMx Total-PSA assays were used to determine cytokine and PSA levels, respectively. RESULTS: Levels of TNFalpha, IL-6, and IL-8 were elevated significantly in the group of patients with advanced, cachectic CaP compared with patients who were without cachexia. In the cachectic patients, levels of TNFalpha were correlated positively with IL-8, and there was no correlation between PSA levels and any of the cytokine levels. IL-1beta levels were below the limit of detection in all samples. CONCLUSIONS: The current results show that levels of TNFalpha, IL-6, and IL-8 were increased in CaP patients with cachexia. Increased levels of these cytokines were not correlated with PSA levels, suggesting that they are regulated by a mechanism that is independent of PSA synthesis. Additional fundamental research is needed to determine the mechanisms involved and to identify potential therapeutic targets in patients with cachexia.     

Clinical utility of serum macrophage migration inhibitory factor in men with prostate cancer as a novel biomarker of detection and disease progression

Macrophage migration inhibitory factor (MIF) has been shown to play an important role in the growth and metastasis of prostate cancer. The objective of this study was to determine whether the serum level of MIF could be used as a diagnostic biomarker for prostate cancer as well as a predictor of disease progression. A total of 369 men who underwent systematic prostate biopsy from January 2000 to April 2004 and 30 healthy controls were included in this study. The serum MIF level was measured using an enzyme linked immunosorbent assay. Associations between the serum MIF level and several clinicopathological factors were analyzed. Among 359 patients, 137 were diagnosed as having prostate cancer. The mean values of serum MIF in the control, benign and cancer-groups were 2.1, 3.5 and 10.8 ng/ml, respectively. The MIF levels of patients with prostate cancer were significantly higher than those of the other two groups. A comparison of MIF and PSA values in all patients showed a positive correlation (correlation coefficient r(2)=0.56, p<0.0001). The MIF value in patients with prostate cancer was significantly associated with clinical T stage, Gleason score and percentage of positive biopsy core (PPBC). MIF levels in patients with metastasis were significantly elevated compared with those in patients without metastasis. Among patients undergoing radical prostatectomy, the level of MIF in those with pathologically confirmed extraprostatic disease was significantly higher than that in patients with organ-confined disease. Moreover, multivariate analysis showed that MIF values, PSA values and PPBC were independent predictors for extraprostatic disease. These findings suggest that an increased serum MIF level is closely associated with the progression of prostate cancer, and thus the serum MIF level could be useful as a novel biomarker for the detection of prostate cancer as well as a predictor of disease progression.     

EphA6 promotes angiogenesis and prostate cancer metastasis and is associated with human prostate cancer progression

Metastasis is the primary cause of prostate cancer (CaP)-related death. We investigate the molecular, pathologic and clinical outcome associations of EphA6 expression and CaP metastasis. The expression profiling of Eph receptors (Ephs) and their ephrin ligands was performed in parental and metastatic CaP cell lines. Among Ephs and ephrins, only EphA6 is consistently overexpressed in metastatic CaP cells. Metastatic potential of EphA6 is assessed by RNAi in a CaP spontaneous metastasis mouse model. EphA6 knock-down in human PC-3M cells causes decreased invasion in vitro and reduced lung and lymph node metastasis in vivo. In addition, knock-down of EphA6 decreases tube formation in vitro and angiogenesis in vivo. EphA6 mRNA expression is higher in 112 CaP tumor samples compared with benign tissues from 58 benign prostate hyperplasia patients. Positive correlation was identified between EphA6 expression and vascular invasion, neural invasion, PSA level, and TNM staging in CaP cases. Further, genome-wide gene expression analysis in EphA6 knock-down cells identified a panel of differentially regulated genes including PIK3IPA, AKT1, and EIF5A2, which could contribute to EphA6-regulated cancer progression. These findings identify EphA6 as a potentially novel metastasis gene which positively correlates with CaP progression. EphA6 may be a therapeutic target in metastatic CaP.     

Elevated level of prostate specific antigen among prostate cancer patients and high prevalence in the Gangetic zone of Bihar, India

Prostate cancer (CaP) is a common reproductive cancer among men. This study was conducted to correlate the cancer incidence with Gangetic zone and to correlate the tumor marker prostate specific antigen (PSA) level in serum with different age groups and stage of malignancy. Patients suffering from CaP in the pathology unit of Mahavir Cancer Sansthan (Hospital and Research Centre), Patna, Bihar, India were studied from June 2009 to May 2010. PSA level in the serum of CaP patients was estimated by ELISA method. CaP incidence was highly recorded in Gangetic zone than the non-Gangetic zone. Maximum patients were in the 56 - 75 years age group with a marked predominance. Results of PSA examination showed that serum PSA level was not correlating with the age of patient and stage of malignancy. Significantly, elevated level of more than 10 ng/ml of PSA was recorded among the studied cancer patients. In this study, it is concluded that Gangetic zone habitat have high risk of CaP and elevated level of PSA was marked in Bihar, India.     

A novel biomarker for staging human prostate adenocarcinoma: overexpression of matriptase with concomitant loss of its inhibitor, hepatocyte growth factor activator inhibitor-1.

BACKGROUND: Matriptase, a type II transmembrane serine protease is involved in angiogenesis, degradation of extracellular matrix, and in the progression of some epithelial cancers. Here, we establish the clinical significance of matriptase and its inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1), during the progression of human prostate cancer (CaP). METHODS: The expression patterns of matriptase and HAI-1 were determined in primary cultures of normal human prostate epithelial (NHPE) cells, human CaP cells LNCaP, DU-145, CWR22Rnu1, and PC-3, and in tissue samples of 172 patients with normal prostate, benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and adenocarcinoma of different tumor grades. RESULTS: The protein and mRNA levels of matriptase were significantly higher in all carcinoma cells as compared with NHPE cells. Conversely, all CaP cells exhibited a reduced expression of HAI-1 as compared with NHPE cells. A progressive increase in the protein levels of matriptase was observed with increasing tumor grade in CaP specimens as compared with normal and BPH tissue specimens. Tissue samples of normal prostate exhibited a high constitutive protein level of HAI-1 compared with BPH and low-grade cancer with a progressive loss with increasing tumor grade. CONCLUSION: The increased expression of matriptase and loss of HAI-1 may be an important event during the progression of CaP in humans. We suggest that the ratio of these two gene products may serve as a promising biomarker for CaP progression and a potential marker for establishing the efficacy of therapeutic and chemopreventive interventions.     

Serum microRNA expression patterns that predict early treatment failure in prostate cancer patients

We aimed to identify microRNA (miRNA) expression patterns in the serum of prostate cancer (CaP) patients that predict the risk of early treatment failure following radical prostatectomy (RP). Microarray and Q-RT-PCR analyses identified 43 miRNAs as differentiating disease stages within 14 prostate cell lines and reflectedpublically available patient data. 34 of these miRNA were detectable in the serum of CaP patients. Association with time to biochemical progression was examined in a cohort of CaP patients following RP. A greater than two-fold increase in hazard of biochemical progression associated with altered expression of miR-103, miR-125b and miR-222 (p <.0008) in the serum of CaP patients. Prediction models based on penalized regression analyses showed that the levels of the miRNAs and PSA together were better at detecting false positives than models without miRNAs, for similar level of sensitivity. Analyses of publically available data revealed significant and reciprocal relationships between changes in CpG methylation and miRNA expression patterns suggesting a role for CpG methylation to regulate miRNA. Exploratory validation supported roles for miR-222 and miR-125b to predict progression risk in CaP. The current study established that expression patterns of serum-detectable miRNAs taken at the time of RP are prognostic for men who are at risk of experiencing subsequent early biochemical progression. These non-invasive approaches could be used to augment treatment decisions.     

The usefulness of serum human kallikrein 11 for discriminating between prostate cancer and benign prostatic hyperplasia

Prostate-specific antigen (PSA) is the most useful tumor marker for diagnosis and monitoring of prostate cancer (CaP). Recently, we developed a specific immunoassay for human kallikrein 11 (hK11), one of the kallikrein gene family members, and found that hK11 was highly expressed in prostatic tissue and could be detected in seminal plasma (E. P. Diamandis et al., Cancer Res., 62: 295-300, 2002). The aim of this study was to investigate whether serum hK11 levels could be used to discriminate CaP from benign prostatic hyperplasia (BPH). We analyzed for hK11, total PSA, and percentage of free PSA, 150 serum samples from men with histologically confirmed BPH (n = 64) or CaP (n = 86). Total and free PSA levels were measured by the Immulite PSA assay, and hK11 levels were measured by our previously published immunofluorometric assay. Serum hK11 levels and the hK11:total PSA ratio were both significantly lower in CaP patients than in BPH patients. In the subgroup of patients with percentage of free PSA less than 20, an additional 54% of BPH patients could have avoided biopsies by using the hK11:total PSA ratio. Receiver operating characteristic (ROC) curve analysis demonstrated that the hK11:total PSA ratio [area under the curve (AUC), 0.83] and percentage of free PSA (AUC, 0.83) were much stronger predictors of CaP than total PSA (AUC, 0.69). These preliminary data suggest that the hK11:total PSA ratio could be a useful tumor marker for CaP and could be combined with percentage of PSA to further reduce the number of unnecessary prostatic biopsies.     

Expression of the human cachexia-associated protein (HCAP) in prostate cancer and in a prostate cancer animal model of cachexia

Prostate cancer (CaP) patients with disseminated disease often suffer from severe cachexia, which contributes to mortality in advanced cancer. Human cachexia-associated protein (HCAP) was recently identified from a breast cancer library based on the available 20-amino acid sequence of proteolysis-inducing factor (PIF), which is a highly active cachectic factor isolated from mouse colon adenocarcinoma MAC16. Herein, we investigated the expression of HCAP in CaP and its potential involvement in CaP-associated cachexia. HCAP mRNA was detected in CaP cell lines, in primary CaP tissues and in its osseous metastases. In situ hybridization showed HCAP mRNA to be localized only in the epithelial cells in CaP tissues, in the metastatic foci in bone, liver and lymph node, but not in the stromal cells or in normal prostate tissues. HCAP protein was detected in 9 of 14 CaP metastases but not in normal prostate tissues from cadaveric donors or patients with organ-confined tumors. Our Western blot analysis revealed that HCAP was present in 9 of 19 urine specimens from cachectic CaP patients but not in 19 urine samples of noncachectic patients. HCAP mRNA and protein were also detected in LuCaP 35 and PC-3M xenografts from our cachectic animal models. Our results demonstrated that human CaP cells express HCAP and the expression of HCAP is associated with the progression of CaP and the development of CaP cachexia.     

Serum prostate specific antigen levels in mice bearing human prostate LNCaP tumors are determined by tumor volume and endocrine and growth factors.

The ability of prostate-specific antigen (PSA) to predict tumor volume and stage in patients with prostate cancer would be improved if factors regulating its production and clearance were better defined. A thorough understanding of the pharmacokinetics (regulation of production, metabolism, and excretion) of PSA has been precluded, however, by the absence of an in vivo animal model. The purposes of this study are to develop a murine model for evaluating PSA pharmacokinetics in vivo and to assess factors that influence PSA production in vitro. The human prostate cancer cell line, LNCaP, was chosen because it is androgen sensitive and PSA positive. Although LNCaP cells are usually nontumorigenic when inoculated s.c. in athymic mice, coinoculation of 1 x 10(6) LNCaP cells with 1 x 10(6) human bone fibroblasts reliably produces PSA-secreting carcinomas. This LNCaP model provides accurate correlation between tumor volume and serum PSA levels (r = 0.94) and demonstrates that tumor volume and androgens are codeterminants of circulating PSA levels. Following castration, serum PSA levels decrease rapidly up to 8-fold and increase up to 20-fold following androgen supplementation, without detectable castration-induced tumor cell death or concomitant changes in tumor volume. Serum PSA levels increase 0.24 ng/ml/mm3 of tumor, which is approximately 5-fold less than that estimated for humans. Most likely this reduced PSA index (PSA:tumor volume ratio) results from a 7-fold faster clearance of PSA in athymic mice than in humans; other than this shorter half-life, PSA elimination in the murine model appears similar to that in humans, with both following first-order kinetics characteristic of a two-compartment model. Interestingly, following prolonged growth (greater than 21 days) in castrate hosts, LNCaP tumors are capable of adapting to an androgen-deprived environment whereby LNCaP tumors regain the ability to secrete PSA in amounts similar to the precastrate state. In LNCaP cells, androgens increase PSA mRNA levels 4-fold in vivo and in vitro. PSA mRNA expression is also altered by various growth factors. Changes in PSA production induced by androgens and growth factors do not always parallel changes in LNCaP cell growth rate induced by these factors, suggesting that PSA production occurs independently of cell growth rate and may be influenced by various interrelated factors, including hormonal and stromal milieu. Observations from this murine model suggest that androgens and tumor volume are independent determinants of serum PSA levels and imply that decreases in circulating PSA following antiandrogen therapy may not always reflect a corresponding reduction in tumor volume.     

Free-to-total prostate-specific antigen ratios 18-24 months following external beam radiation for adenocarcinoma of the prostate

BACKGROUND AND OBJECTIVES: The purpose of this study was to evaluate free-to-total prostate-specific antigen (PSA) ratios after definitive external beam radiation therapy for men with adenocarcinoma of the prostate (CaP). METHODS: A prospective evaluation of percent free PSA in men following definitive external beam radiation therapy for CaP was compared to men with untreated CaP and men at very low risk for CaP. Statistical comparison of clinical and pathologic parameters was performed. RESULTS: There was no statistically significant difference in free-to-total PSA ratios for men with newly diagnosed CaP and men with detectable PSA who were treated with external beam radiation therapy. CONCLUSIONS: Free-to-total PSA ratios after definitive external beam radiation therapy for CaP are consistent with percent free PSA in patients with newly diagnosed CaP. This supports the theory that PSA from in situ prostate tissue following external beam radiation therapy is produced by malignant cells.     

Long-term antiepileptic treatment with histone deacetylase inhibitors may reduce the risk of prostate cancer

Various antiepileptic drugs such as valproic acid, carbamazepine, oxcarbazepine, lamotrigine and levetiracetam are known to exert histone deacetylase inhibitory (HDACi) properties, which can modify aberrantly silenced gene expression by an epigenetic mechanism. This study was initiated to examine a potential beneficial effect of these drugs on prostate cancer (PC) development. The prostate-specific antigen (PSA) levels of 106 patients under long-term treatment with antiepileptic drugs and known HDACi properties were examined. PSA represents a hallmark in the early detection of PC, and its levels may predict an invasive disease in subsequent years. For in-vitro experiments, the PC cell line LNCaP was treated with HDACi drugs; subsequently, PSA and further PC markers were assessed. When men over 50 years of age were treated with HDACi drugs they had lower age-corrected PSA levels compared with control groups, according to the following ranking: valproic acid>levetiracetam>carbamazepine/oxcarbazepine>lamotrigine. Furthermore, there was a correlation between PSA reduction and the number of HDACi drugs within the medication, lending credence to the idea that a synergistic effect might be possible. Moreover, in vitro, HDACi drugs decrease PSA on mRNA and protein levels and exhibit further oncoprotective properties.The fact that HDACi drugs exert antiproliferative effects on neoplastic cells in vitro and in vivo, which are paralleled by expression alterations of aberrantly regulated genes, underlines the potential therapeutic value of HDACi drugs. These data suggest that long-term HDACi treatment can positively influence the characteristically slow transformation of tumour precursor cells in the prostate and may thus reduce a patient's risk of developing PC.     

Clinical relevance of genetic instability in prostatic cells obtained by prostatic massage in early prostate cancer

We investigated whether genetic lesions such as loss of heterozygosity (LOH) are detected in prostatic cells obtained by prostatic massage during early diagnosis of prostate cancer (CaP) and discussed their clinical relevance. Blood and first urine voided after prostatic massage were collected in 99 patients with total prostate-specific antigen (PSA) between 4 and 10 ng ml(-1), prior to prostate biopsies. Presence of prostatic cells was confirmed by quantitative RT-PCR analysis of PSA mRNA. Genomic DNA was analysed for LOH on six chromosomal regions. One or more allelic deletions were found in prostatic fluid from 57 patients analysed, of whom 33 (58%) had CaP. Sensitivity and specificity of LOH detection and PSA free to total ratio <15% for positive biopsy were respectively 86.7 and 44% (P=0.002) for LOH, and 55 and 74% (P=0.006) for PSA ratio <15%. Analysis of LOH obtained from prostatic tumours revealed similar patterns compared to prostatic fluid cells in 86% of cases, confirming its accuracy. The presence of LOH of urinary prostatic cells obtained after prostatic massage is significantly associated with CaP on biopsy and may potentially help to identify a set of patients who are candidates for further prostate biopsies.     

Interleukin-6 induces androgen responsiveness in prostate cancer cells through up-regulation of androgen receptor expression.

Interleukin-6 (IL-6) induces prostate cancer (CaP) cell proliferation in vitro. Several lines of evidence suggest that IL-6 may promote CaP progression through induction of an androgen response. In this work, we explored whether IL-6 induces androgen responsiveness through modulation of androgen receptor (AR) expression. We found that in the absence of androgen, IL-6 increased prostate-specific antigen (PSA) mRNA levels and activated several androgen-responsive promoters, but not the non-androgen responsive promoters in LNCaP cells. Bicalutamide, an antiandrogen, abolished the IL-6 effect and IL-6 could not activate the PSA and murine mammary tumor virus reporters in AR-negative DU-145 and PC3 cells. These data indicate the IL-6 induces an androgen response in CaP cells through the AR. Pretreatment of LNCaP cells with SB202190, PD98059, or tyrphostin AG879 [p38 mitogen-activated protein kinase (MAPK), MAP/extracellular signal-regulated protein kinase kinase 1/2, and ErbB2 MAPK inhibitors, respectively) but not wortmannin (PI3-kinase inhibitor) blocked IL-6-mediated induction of the PSA promoter, which demonstrates that IL-6 activity is dependent on a MAPK pathway. Finally, IL-6 activated the AR gene promoter, resulting in increased AR mRNA and protein levels in LNCaP cells. These results demonstrate that IL-6 induces AR expression and are the first report of cytokine-mediated induction of the AR promoter. Taken together, our results suggest that IL-6 induces AR activity through both increasing AR gene expression and activating the AR in the absence of androgen in CaP cells. These results provide a mechanism through which IL-6 may contribute to the development of androgen-independent CaP.