Early prostate cancer (PCa) is generally treatable and associated with good prognosis. After a variable time, PCa evolves into a highly metastatic and treatment-refractory disease: castration-resistant PCa (CRPC). Currently, few prognostic factors are available to predict the emergence of CRPC, and no curative option is available. Epigenetic gene regulation has been shown to trigger PCa metastasis and androgen-independence. Most epigenetic studies have focused on DNA and histone methyltransferases. While DNA methylation leads to gene silencing, histone methylation can trigger gene activation or inactivation, depending on the target amino acid residues and the extent of methylation (me1, me2, or me3). Interestingly, some histone modifiers are essential for PCa tumor-initiating cell (TIC) self-renewal. TICs are considered the seeds responsible for metastatic spreading and androgen-independence. Histone Lysine Demethylases (KDMs) are a novel class of epigenetic enzymes which can remove both repressive and activating histone marks. KDMs are currently grouped into 7 major classes, each one targeting a specific methylation site. Since their discovery, KDM expression has been found to be deregulated in several neoplasms. In PCa, KDMs may act as either tumor suppressors or oncogenes, depending on their gene regulatory function. For example, KDM1A and KDM4C are essential for PCa androgen-dependent proliferation, while PHF8 is involved in PCa migration and invasion. Interestingly, the possibility of pharmacologically targeting KDMs has been demonstrated. In the present paper, we summarize the emerging role of KDMs in regulating the metastatic potential and androgen-dependence of PCa. In addition, we speculate on the possible interaction between KDMs and other epigenetic effectors relevant for PCa TICs. Finally, we explore the role of KDMs as novel prognostic factors and therapeutic targets. We believe that studies on histone demethylation may add a novel perspective in our efforts to prevent and cure advanced PCa.
Prostate cancer; Epigenetics; Tumor-initiating cells; Histone demethylase; Androgen receptor
The skeleton is the most common metastatic organ in patients with prostate cancer (PCa). Non-invasive biomarkers that can facilitate the detection and monitoring of bone metastases are highly desirable. We designed this study to assess the expression patterns of serum miR-141 in patients with bone-metastatic PCa. Serum samples were collected to measure the miR-141 level in 56 patients, including six with benign prostatic hyperplasia (BPH), 20 with localized PCa and 30 with bone-metastatic PCa (10 with hormone-naive PCa, 10 with hormone-sensitive PCa and 10 with hormone-refractory PCa). A bone scan was performed for each patient with PCa to assess the number of bone lesions. The quantification of serum miR-141 levels was assayed by specific TaqMan qRT-PCR. The results showed that serum miR-141 levels were elevated in patients with bone metastasis (P<0.001). There was no statistically significant difference in the serum miR-141 levels between patients with BPH and patients with localized PCa. Using Kendall's bivariate correlation test, both the Gleason score and the number of bone-metastatic lesions were found to correlate with serum miR-141 levels (P=0.012 and P<0.001, respectively). The serum miR-141 level was found to be positively correlated with alkaline phosphatase (ALP) level in patients with skeletal metastasis, using Pearson's bivariate correlation test. No relationship was found between the serum miR-141 level and the serum prostate-specific antigen (PSA) level. We concluded that serum miR-141 levels are elevated in patients with bone-metastatic PCa and that patients with higher levels of serum miR-141 developed more bone lesions. Furthermore, serum miR-141 levels are correlated with serum ALP levels but not serum PSA levels.
alkaline phosphatase (ALP); biological markers; bones; metastasis; microRNAs; miR-141; prostate-specific antigen (PSA); prostatic neoplasms; serum
Background: Emerging evidence has suggested that Notch signaling pathway may be involved in the development, progression and metastasis of prostate cancer (PCa). In the present study, we investigated the expression levels of Jagged-1 and Notch-1 in human prostate tumors and their associations with PCa progression and metastasis. Methods: Immunohistochemistry (IHC) for Jagged-1 and Notch-1 was performed on tissue microarray (TMA) slides containing 286 formalin-fixed and paraffin-embedded (FFPE) tissue specimens with various prostatic pathologies, including benign changes, high grade prostatic intraepithelial neoplasia (HGPIN), low- and high-grade PCas as well as metastatic PCa. Results: Cytoplasmic and membranous IHC scores for Jagged-1 in both metastatic PCa and high grade PCa were significantly higher than those in low grade PCa and in benign prostatic tissues. Similarly, cytoplasmic IHC scores of Notch-1 in both metastatic PCa and high grade PCa were significantly elevated compared with those observed in low grade PCa and in benign prostatic tissues. A statistically significant correlation was identified between the expression of Jagged-1 and Notch-1 in human prostatic tissues. Furthermore, significantly more highly expressed Jagged-1 in membrane was observed in Caucasian patients with high-grade or metastatic PCa (vs. African Americans) and in PCa patients with positive surgical margins (vs. negative surgical margins). Conclusion: Our results provide strong evidence that up-regulation of Jagged1-Notch1 signaling plays a role in PCa progression and metastasis and suggest that Jagged-1 and Notch-1 may be useful markers in distinguishing indolent and aggressive PCas.
Prostate cancer (PCa); cancer metastasis; Jagged-1; Notch-1; tissue microarray (TMA); immunohistochemistry (IHC)
Prostate cancer (PCa) is characterized by deregulated expression of several tumor suppressor or oncogenic miRNAs. The objective of this study was the identification and characterization of miR-let-7c as a potential tumor suppressor in PCa.
Levels of expression of miR-let-7c were examined in human PCa cell lines and tissues using qRT-PCR and in situ hybridization. Let-7c was overexpressed or suppressed to assess the effects on the growth of human PCa cell lines. Lentiviral-mediated re-expression of let-7c was utilized to assess the effects on human PCa xenografts.
We identified miR-let-7c as a potential tumor suppressor in PCa. Expression of let-7c is downregulated in castration-resistant prostate cancer (CRPC) cells. Overexpression of let-7c decreased while downregulation of let-7c increased cell proliferation, clonogenicity and anchorage-independent growth of PCa cells in vitro. Suppression of let-7c expression enhanced the ability of androgen-sensitive PCa cells to grow in androgen-deprived conditions in vitro. Reconstitution of Let-7c by lentiviral-mediated intratumoral delivery significantly reduced tumor burden in xenografts of human PCa cells. Furthermore, let-7c expression is downregulated in clinical PCa specimens compared to their matched benign tissues, while the expression of Lin28, a master regulator of let-7 miRNA processing, is upregulated in clinical PCa specimens.
These results demonstrate that microRNA let-7c is downregulated in PCa and functions as a tumor suppressor, and is a potential therapeutic target for PCa.
Prostate cancer (PCa) is the most prevalent cancer, a significant contributor to morbidity and a leading cause of cancer-related death in men in Western industrialized countries. In contrast to genetic changes that vary among individual cases, somatic epigenetic alterations are early and highly consistent events. Epigenetics encompasses several different phenomena, such as DNA methylation, histone modifications, RNA interference, and genomic imprinting. Epigenetic processes regulate gene expression and can change malignancy-associated phenotypes such as growth, migration, invasion, or angiogenesis. Methylations of certain genes are associated with PCa progression. Compared to normal prostate tissues, several hypermethylated genes have also been identified in benign prostate hyperplasia, which suggests a role for aberrant methylation in this growth dysfunction. Global and gene-specific DNA methylation could be affected by environmental and dietary factors. Among other epigenetic changes, aberrant DNA methylation might have a great potential as diagnostic or prognostic marker for PCa and could be tested in tumor tissues and various body fluids (e.g., serum, urine). The DNA methylation markers are simple in nature, have high sensitivity, and could be detected either quantitatively or qualitatively. Availability of genome-wide screening methodologies also allows the identification of epigenetic signatures in high throughput population studies. Unlike irreversible genetic changes, epigenetic alterations are reversible and could be used for PCa targeted therapies.
Epigenetics; Genome; Methylation; Prostate cancer.
Although prostate cancer (PCa) is the second leading cause of cancer death among men worldwide, not all men diagnosed with PCa will die from the disease. A critical challenge, therefore, is to distinguish indolent PCa from more advanced forms to guide appropriate treatment decisions. We used Enhanced Reduced Representation Bisulfite Sequencing, a genome-wide high-coverage single-base resolution DNA methylation method to profile seven localized PCa samples, seven matched benign prostate tissues, and six aggressive castration-resistant prostate cancer (CRPC) samples. We integrated these data with RNA-seq and whole-genome DNA-seq data to comprehensively characterize the PCa methylome, detect changes associated with disease progression, and identify novel candidate prognostic biomarkers. Our analyses revealed the correlation of cytosine guanine dinucleotide island (CGI)-specific hypermethylation with disease severity and association of certain breakpoints (deletion, tandem duplications, and interchromosomal translocations) with DNA methylation. Furthermore, integrative analysis of methylation and single-nucleotide polymorphisms (SNPs) uncovered widespread allele-specific methylation (ASM) for the first time in PCa. We found that most DNA methylation changes occurred in the context of ASM, suggesting that variations in tumor epigenetic landscape of individuals are partly mediated by genetic differences, which may affect PCa disease progression. We further selected a panel of 13 CGIs demonstrating increased DNA methylation with disease progression and validated this panel in an independent cohort of 20 benign prostate tissues, 16 PCa, and 8 aggressive CRPCs. These results warrant clinical evaluation in larger cohorts to help distinguish indolent PCa from advanced disease.
Androgen deprivation is still the standard systemic therapy for metastatic prostate cancer (PCa), but patients invariably relapse with a more aggressive form of PCa termed hormone refractory, androgen independent, or castration resistant PCa (CRPC). Significantly, the androgen receptor (AR) is expressed at high levels in most cases of CRPC, and these tumors resume their expression of multiple AR-regulated genes, indicating that AR transcriptional activity becomes reactivated at this stage of the disease. The molecular basis for this AR reactivation remains unclear, but possible mechanisms include increased AR expression, AR mutations that enhance activation by weak androgens and AR antagonists, increased expression of transcriptional coactivator proteins, and activation of signal transduction pathways that can enhance AR responses to low levels of androgens. Recent data indicate that CRPC cells may also carry out intracellular synthesis of testosterone and DHT from weak adrenal androgens and may be able to synthesize androgens from cholesterol. These mechanisms that appear to contribute to AR reactivation after castration are further outlined in this review.
androgen receptor; prostate cancer; testosterone; androgen; androgen deprivation therapy; AR antagonist
Recent technological advancements in gene expression analysis have led to the discovery of a promising new group of prostate cancer (PCa) biomarkers that have the potential to influence diagnosis and the prediction of disease severity. The accumulation of deleterious changes in gene expression is a fundamental mechanism of prostate carcinogenesis. Aberrant gene expression can arise from changes in epigenetic regulation or mutation in the genome affecting either key regulatory elements or gene sequences themselves. At the epigenetic level, a myriad of abnormal histone modifications and changes in DNA methylation are found in PCa patients. In addition, many mutations in the genome have been associated with higher PCa risk. Finally, over- or underexpression of key genes involved in cell cycle regulation, apoptosis, cell adhesion and regulation of transcription has been observed. An interesting group of biomarkers are emerging from these studies which may prove more predictive than the standard prostate specific antigen (PSA) serum test. In this review, we discuss recent results in the field of gene expression analysis in PCa including the most promising biomarkers in the areas of epigenetics, genomics and the transcriptome, some of which are currently under investigation as clinical tests for early detection and better prognostic prediction of PCa.
Prostate cancer; biomarker; epigenetics; methylation; acetylation; ncRNA; genomics; SNP; transcriptomics; miRNA; lncRNA
Although androgens are depleted in castration resistant prostate cancer (CRPC), metastases still express nuclear androgen receptor (AR) and androgen regulated genes. We recently reported that C-terminal truncated constitutively active AR splice variants contribute to CRPC development. Since specific antibodies detecting all C-terminal truncated AR variants are not available, our aim was to develop an approach to assess the prevalence and function of AR variants in prostate cancer (PCa).
Using 2 antibodies against different regions of AR protein (N- or C-terminus), we successfully showed the existence of AR variant in the LuCaP 86.2 xenograft. To evaluate the prevalence of AR variants in human PCa tissue, we used this method on tissue microarrays including 50 primary PCa and 162 metastatic CRPC tissues. RT-PCR was used to confirm AR variants. We observed a significant decrease in nuclear C-terminal AR staining in CRPC but no difference between N- and C-terminal AR nuclear staining in primary PCa. The expression of the AR regulated proteins PSA and PSMA were marginally affected by the decrease in C-terminal staining in CRPC samples. These data suggest that there is an increase in the prevalence of AR variants in CRPC based on our ability to differentiate nuclear AR expression using N- and C-terminal AR antibodies. These findings were validated using RT-PCR. Importantly, the loss of C-terminal immunoreactivity and the identification of AR variants were different depending on the site of metastasis in the same patient.
We successfully developed a novel immunohistochemical approach which was used to ascertain the prevalence of AR variants in a large number of primary PCa and metastatic CRPC. Our results showed a snapshot of overall high frequency of C-terminal truncated AR splice variants and site specific AR loss in CRPC, which could have utility in stratifying patients for AR targeted therapeutics.
Patients with advanced prostate cancer (PCa) are initially susceptible to androgen withdrawal (AW), but ultimately develop resistance to this therapy (castration-resistant PCa, CRPC). Here we show that AW can promote CRPC development by increasing the levels of the receptor tyrosine kinase (RTK) ErbB3 in androgen-dependent PCa, resulting in AW-resistant cell cycle progression and increased androgen receptor (AR) transcriptional activity. CRPC cell lines and human prostate cancer tissue overexpressed ErbB3, whereas downregulation of ErbB3 prevented CRPC cell growth. Investigation of the mechanism by which AW augments ErbB3, using normal prostate derived pRNS-1-1 cells, and androgen-dependent PCa lines LNCaP, PC346C and CWR22 mouse xenografts, revealed that the AR suppresses ErbB3 protein levels, while AW relieves this suppression, demonstrating for the first time negative regulation of ErbB3 by AR. We show that AR activation promotes ErbB3 degradation in androgen-dependent cells, and that this effect is mediated by AR-dependent transcriptional upregulation of Nrdp1, an E3 ubiquitin ligase that targets ErbB3 for degradation but whose role in PCa has not been previously examined. Therefore, AW decreases Nrdp1 expression, promoting ErbB3 protein accumulation, and leading to AR-independent proliferation. However, in CRPC sublines of LNCaP and CWR22 which strongly overexpress the AR, ErbB3 levels remain elevated due to constitutive suppression of Nrdp1, which prevents AR regulation of Nrdp1. Our observations point to a model of CRPC development where progression of PCa to castration-resistance is associated with the inability of AR to transcriptionally regulate Nrdp1, and predict that inhibition of ErbB3 during AW may impair CRPC development.
EGFR; HER2; HER3; Androgen Receptor; FLRF; RNF41
DNA methylation is an important epigenetic mechanism in prostate cancer (PCa) progression. Given the role of even-skipped homeobox 1 (EVX1) in the regulation of multiple genes during embryogenesis, we postulated that EVX1 methylation is altered in PCa progression.
Bisulphite sequencing and quantitative MethyLight were used to assess methylation in human prostate epithelial cells, four PCa cell lines, liver, lung, spleen, kidney, 35 paired tumour and tumour-associated benign tissues, and 11 normal prostate tissues. Prostate cancer cell lines were treated with 5-azacytidine (AzaC) or trichostatin A (TSA), and expression of EVX1 transcript and variants was assessed by qPCR. Hypermethylation was compared with clinicopathological features in a validation set of 58 patients using microarray.
Even-skipped homeobox 1 hypermethylation was observed in all four PCa cell lines and 57% of tumours. High-grade tumours exhibited increased methylation compared with intermediate-grade tumours. Even-skipped homeobox 1 expression was induced in PCa cell lines after treatment with AzaC or TSA. In the validation set, 83% of tumours were hypermethylated and hypermethylation was associated with worse recurrence-free survival.
In this first evaluation of EVX1 methylation in human cancer, EVX1 is one of the most commonly hypermethylated genes observed in PCa and predicted treatment failure in moderate risk patients.
EVX1; methylation; prostate cancer; PSA; prognosis
DNA methylation plays an important role in carcinogenesis and is being recognized as a promising diagnostic and prognostic biomarker for a variety of malignancies including Prostate cancer (PCa). The human kallikrein-related peptidases (KLKs) have emerged as an important family of cancer biomarkers, with KLK3, encoding for Prostate Specific Antigen, being most recognized. However, few studies have examined the epigenetic regulation of KLKs and its implications to PCa. To assess the biological effect of DNA methylation on KLK6 and KLK10 expression, we treated PC3 and 22RV1 PCa cells with a demethylating drug, 5-aza-2′deoxycytidine, and observed increased expression of both KLKs, establishing that DNA methylation plays a role in regulating gene expression. Subsequently, we have quantified KLK6 and KLK10 DNA methylation levels in two independent cohorts of PCa patients operated by radical prostatectomy between 2007–2011 (Cohort I, n = 150) and 1998–2001 (Cohort II, n = 124). In Cohort I, DNA methylation levels of both KLKs were significantly higher in cancerous tissue vs. normal. Further, we evaluated the relationship between DNA methylation and clinicopathological parameters. KLK6 DNA methylation was significantly associated with pathological stage only in Cohort I while KLK10 DNA methylation was significantly associated with pathological stage in both cohorts. In Cohort II, low KLK10 DNA methylation was associated with biochemical recurrence in univariate and multivariate analyses. A similar trend for KLK6 DNA methylation was observed. The results suggest that KLK6 and KLK10 DNA methylation distinguishes organ confined from locally invasive PCa and may have prognostic value.
biomarkers; epigenetics; kallikrein-related peptidases; prostate cancer; quantitative DNA methylation analysis
Prostate cancer (PCa) is the most common malignancy, and the third leading cancer-related cause of death among men of the Western world. Upon PCa progression into metastatic disease, androgen deprivation therapy is applied as the first-line treatment, and has been shown to be effective in most patients, leading to a decrease in serum prostate-specific antigen and relief of disease-related symptoms. However, advanced PCa almost inevitably progresses to a castration-resistant state, and is currently regarded as incurable. The large body of evidence indicates that PCa cells remain dependent on androgen receptor (AR) signaling even in an androgen-deprived environment. As such, development of drugs that target AR and AR signaling pathways have become one of the major milestones in treatment of castration-resistant PCa (CRPC). Nevertheless, currently available therapies that target AR signaling are still regarded as palliative and more potent therapies are in great need. Over the past few years, a wide range of novel therapies has entered clinical trial for treatment of CRPC, including androgen synthesis inhibitors (abiraterone acetate), chemotherapeutic agents (docetaxel and cabazitaxel), and immunotherapies (sipuleucel-T). In this context, enzalutamide (previously referred to as MDV3100) is a novel second generation antiandrogen that has been demonstrated to significantly improve survival in men with metastatic CRPC in several clinical trials. In this paper we summarize recently completed and ongoing clinical trials of enzalutamide, and briefly discuss the efficacy of the novel antiandrogen therapy and its limitations for treatment of CRPC.
castration resistant prostate cancer; drug resistance; clinical trials
Using a one-stage kinetic chromogenic assay, we studied the procoagulant activity (PCA) of prostatic tissue in an experimental model of prostate cancer in the rat. PCA was present in homogenates of rat prostate glands containing either benign or malignant tumours. The procoagulant activated factor X directly and was provisionally characterised as a tissue factor-factor VIIa complex. There was no significant differences in PCA between control rats and rats exposed to carcinogens that did not develop tumour. Levels in rats that developed tumours were significantly higher (P < 0.01) than all other groups and there was a positive correlation between tumour weight and PCA (r = 0.85, P < 0.001). Furthermore, prostatic PCA levels were higher in the metastasis (P < 0.02). We conclude that PCA reflects the malignant phenotype in this animals, the PCA of the primary tumour was compared with that of the corresponding secondary deposit and levels were higher in the metastasis (P < 0.02). We conclude that PCA reflects the malignant phenotype in this model of experimental prostate cancer and suggest that this parameter is worth evaluating as a potential tumour marker in the human disease.
Neuroendocrine prostate cancer (NEPC), also referred to as anaplastic prostate cancer, is a lethal tumor that most commonly arises in late stages of prostate adenocarcinoma (PCA) with predilection to metastasize to visceral organs. In the current study, we explore for evidence that Aurora kinase A (AURKA) and N-myc (MYCN) gene abnormalities are harbingers of treatment-related NEPC (t-NEPC). We studied primary prostate tissue from 15 hormone naïve PCAs, 51 castration-resistant prostate cancers, and 15 metastatic tumors from 72 patients at different stages of disease progression to t-NEPC, some with multiple specimens. Histologic evaluation, immunohistochemistry, and fluorescence in situ hybridization were performed and correlated with clinical variables. AURKA amplification was identified in overall 65% of PCAs (hormone naïve and treated) from patients that developed t-NEPC and in 86% of metastases. Concurrent amplification of MYCN was present in 70% of primary PCAs, 69% of treated PCAs, and 83% of metastases. In contrast, in an unselected PCA cohort, AURKA and MYCN amplifications were identified in only 5% of 169 cases. When metastatic t-NEPC was compared to primary PCA from the same patients, there was 100% concordance of ERG rearrangement, 100% concordance of AURKA amplification, and 60% concordance of MYCN amplification. In tumors with mixed features, there was also 100% concordance of ERG rearrangement and 94% concordance of AURKA and MYCN co-amplification between areas of NEPC and adenocarcinoma. AURKA and MYCN amplifications may be prognostic and predictive biomarkers, as they are harbingers of tumors at risk of progressing to t-NEPC after hormonal therapy.
The androgen receptor (AR) plays a key role in progression to incurable androgen-ablation resistant prostate cancer (PCA). We have identified three novel AR splice variants lacking the ligand binding domain (designated as AR3, AR4 and AR5) in hormone insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly upregulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells while specific knock-down of AR3 expression (without altering AR level) in hormone resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells. Furthermore, AR3 may play a distinct yet essential role in ablation-independent growth through regulating a unique set of genes including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation-independence during PCA progression and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available anti-androgen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA.
Heat shock protein 27 (Hsp-27) encoded by gene HSPB1 is a critical regulator of the behavioral phenotype of human prostate cancer (PCa) cells, enhanced expression being associated with highly aggressive disease and poor clinical outcome. In contrast, the protein is not expressed in PCas of low malignant potential. To gain insight into the mechanism regulating its expression, we tested the hypothesis that differential methylation of CpG islands within HSPB1 controls transcription and subsequent translation of the gene.
We studied prostate epithelial cell lines and tissue biopsies, including 59 BPH and 415 PCas, of which 367 were a cohort of men with up to 20 years of follow-up. Methylation across the gene (DNA methylation (DNAme)) was assayed by pyrosequencing. Hsp-27 expression was assessed by western blot and immunohistochemistry.
In cancer tissues, methylation increased in a 3′ direction (P<0.0001) whereas in benign hyperplasia methylation was constantly below 5%, a cutoff giving a specificity of 100% and sensitivity of 50%. Although methylation of the promoter region was significantly discriminating between benign and malignant prostatic epithelia, it compared poorly with methylation of the first intron. The prognostic value of HSPB1 DNAme was confirmed by both univariate (hazard ratio 1.77 per 50% increment, P=0.02) and multivariate models. Interaction between HSPB1 methylation and Gleason score revealed high DNAme to be a reliable prognostic marker of poor outcome in men with low Gleason score (P=0.014).
Our data indicate CpG methylation of the first HSPB1 intron to be an important biomarker that identifies aggressive PCas otherwise regarded as low risk by current clinical criteria but that, biologically, require immediate active management.
HSPB1; Hsp-27; methylation; biomarker
Histone deacetetylases (HDACs) are a group of corepressors of transcriptional activators and their levels of expression are potentially dysregulated in prostate cancer. Certain inhibitors of histone deacetylases show anti-tumor activity in prostate cancer cell lines. Here, we systemically studied the expression of HDACs in human prostate cancer and the suppression of prostate cancer growth and invasion by HDAC inhibitor SAHA. HDAC1-5 showed increased expression using a combination of DNA microarray, in-situ hybridization, and immunohistochemistry in benign and malignant human prostate tissue as well as RT-PCR and Western blot analysis on various PCa cell lines. Importantly, HDAC inhibitor SAHA suppressed, in particular, prostate cancer cell growth and invasion determined using cell proliferation and Matrigel invasion assays. The findings of this study show that the expression of HDACs and their associated corepressors are increased in prostate cancer in humans and HDAC inhibitor SAHA could serve as a potential therapeutic agent in prostate cancer in addition to anti-androgens.
Prostate cancer; hormone receptor; androgen; corepressor; microarray; in situ hybridization; histone deacetylase
Bone morphogenetic protein (BMP) signaling is important in prostate development and prostate cancer (PCa) progression. However, because of the multiple effects of different BMPs, no final conclusions have been made as to the role of BMPs in PCa. In our studies, we have focused on BMP-7 because it is involved in prostate morphogenesis, and its expression is regulated by androgens. The objective of our study was to determine BMP-7 expression in PCa metastases and investigate the effects of BMP-7 on PCa cells. Our results show that BMP-7 is expressed in metastatic PCa and its levels are increased in castration-resistant PCa versus androgen-dependent PCa, whereas the expression of BMP-7 is decreased in primary PCa versus normal prostate. Our in vitro results show that BMP-7 inhibits proliferation of androgen-sensitive LNCaP cells, stimulates androgen receptor signaling, increases the expression of differentiation-associated genes, and decreases the levels of some wingless-regulated transcripts. Interestingly, these effects were not detected in C4-2 castration-resistant PCa cells. In vivo expression of BMP-7 in castration-resistant C4-2 cells did not alter proliferation when these cells were grown subcutaneously, but their growth was inhibited in the bone environment. In summary, our results show that BMP-7 is expressed at the highest level in advanced castration-resistant PCa cells and that the inhibitory effects of BMP-7 are dependent on the differentiation status of PCa cells and the tumor microenvironment. Further studies are needed to identify changes in BMP-7 signaling that lead to the loss of its control of proliferation during PCa progression.
Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.
High plasma osteopontin (OPN) has been linked to tumour hypoxia, metastasis, and poor prognosis. This study aims to assess whether plasma osteopontin was a biomarker of increasing progression within prostate cancer (PCa) prognostic groups and whether it reflected treatment response to local and systemic therapies.
Baseline OPN was determined in men with localised (n=199), locally recurrent (n=9) and castrate-resistant, metastatic PCa (CRPC-MET; n=37). Receiver-operating curves (ROC) were generated to describe the accuracy of OPN for distinguishing between localised risk groups or localised vs metastatic disease. We also measured OPN pre- and posttreatment, following radical prostatectomy, external beam radiotherapy (EBRT), androgen deprivation (AD) or taxane-based chemotherapy.
The CRPC-MET patients had increased baseline values (mean 219; 56–513 ng ml−1; P<0.0001) compared with the localised, non-metastatic group (mean 72; 12–438 ng ml−1). The area under the ROC to differentiate localised vs metastatic disease was improved when OPN was added to prostate-specific antigen (PSA) (0.943–0.969). Osteopontin neither distinguished high-risk PCa from other localised PCa nor correlated with serum PSA at baseline. Osteopontin levels reduced in low-risk patients after radical prostatectomy (P=0.005) and in CRPC-MET patients after chemotherapy (P=0.027), but not after EBRT or AD.
Plasma OPN is as good as PSA at predicting treatment response in CRPC-MET patients after chemotherapy. Our data do not support the use of plasma OPN as a biomarker of increasing tumour burden within localised PCa.
osteopontin; biomarkers; radiotherapy; chemotherapy; surgery; prostate cancer
The platelet-derived growth factor receptor (PDGFR) is a receptor tyrosine kinase overexpressed in a subset of solid tumors and therefore is the target of drugs inhibiting this function such as imatinib mesylate (Gleevec). Thus far, drug therapy has played a limited role in the treatment of localized prostate cancer (PCa). This study characterizes PDGFR-β expression in a wide spectrum of PCa samples to provide empirical data as part of a rational treatment strategy. A survey of five published prostate expression array studies, including 100 clinically localized PCa, did not identify tumors with increased PDGFR-β expression level. Protein expression of PDGFR-β, as determined by immunohistochemistry, revealed 5% of clinically localized PCa and 16% of metastatic PCa cases to show moderate or strong expression. To develop a strategy to detect patients most likely to profit from Gleevec treatment, we analyzed cDNA expression array data from 10,000 transcripts for PDGFR-β expression and divided tumors in groups based on PDGFR-β expression level. Performing a supervised analysis to identify potential comarkers of PDGFR-β in PCa, we identified a set of genes whose expression was associated with PDGFR-β status including early growth response 1 (Egr1), an upstream effector of PDGF (4.2-fold upregulation), α-methylacyl-CoA racemase, as well as v-Maf and neuroblastoma suppressor of tumorigenicity (both with a 2.2-fold downregulation). Taken together, this study suggests that only a small subset of PCas may be amenable to tyrosine kinase inhibitors specific for PDGFR.
Platelet-derived growth factor receptor (PDGFR); prostate cancer; imatinib mesylate; tissue microarray; cDNA expression
An association between the metabolic syndrome and reduced testosterone levels has been identified, and a specific inverse relationship between insulin and testosterone levels suggests that an important metabolic crosstalk exists between these two hormonal axes; however, the mechanisms by which insulin and androgens may be reciprocally regulated are not well described. Androgen-dependant gene pathways regulate the growth and maintenance of both normal and malignant prostate tissue, and androgen-deprivation therapy (ADT) in patients exploits this dependence when used to treat recurrent and metastatic prostate cancer resulting in tumour regression. A major systemic side effect of ADT includes induction of key features of the metabolic syndrome and the consistent feature of hyperinsulinaemia. Recent studies have specifically identified a correlation between elevated insulin and high-grade PCa and more rapid progression to castrate resistant disease. This paper examines the relationship between insulin and androgens in the context of prostate cancer progression. Prostate cancer patients present a promising cohort for the exploration of insulin stabilising agents as adjunct treatments for hormone deprivation or enhancers of chemosensitivity for treatment of advanced prostate cancer.
Prostate cancer (pca) is the most common non-skin cancer diagnosed in North America, and it affects 1 in 6 men. Patients with recurrent or metastatic pca will inevitably develop castration-resistant disease after an initial period of hormone responsiveness. The standard first-line treatment for men with castration-resistant pca (crpc) is docetaxel, but further treatment options are limited. This review summarizes the research being conducted in crpc, with specific regard to immunotherapy and to novel targeted therapies directed against the androgen axis, vascular endothelial growth factor, chaperone proteins, the phosphoinositide 3 kinase/Akt/phosphatase and tensin homolog/mammalian target of rapamycin pathway, and endothelin-1.
Castration-resistant prostate cancer; novel therapy; targeted therapy
Androgen Receptor (AR) signaling is critically important during the development and progression of prostate cancer (PCa). The AR signaling is also important in the development of castrate resistant prostate cancer (CRPC) where AR is functional even after androgen deprivation therapy (ADT); however, little is known regarding the transcriptional and functional regulation of AR in PCa. Moreover, treatment options for primary PCa for preventing the occurrence of CRPC is limited; therefore, novel strategy for direct inactivation of AR is urgently needed. In this study, we found loss of miR-34a, which targets AR, in PCa tissue specimens, especially in patients with higher Gleason grade tumors, consistent with increased expression of AR. Forced overexpression of miR-34a in PCa cell lines led to decreased expression of AR and prostate specific antigen (PSA) as well as the expression of Notch-1, another important target of miR-34a. Most importantly, BR-DIM intervention in PCa patients prior to radical prostatectomy showed re-expression of miR-34a, which was consistent with decreased expression of AR, PSA and Notch-1 in PCa tissue specimens. Moreover, BR-DIM intervention led to nuclear exclusion both in PCa cell lines and in tumor tissues. PCa cells treated with BR-DIM and 5-aza-dC resulted in the demethylation of miR-34a promoter concomitant with inhibition of AR and PSA expression in LNCaP and C4-2B cells. These results suggest, for the first time, epigenetic silencing of miR -34a in PCa, which could be reversed by BR-DIM treatment and, thus BR-DIM could be useful for the inactivation of AR in the treatment of PCa.
BR-DIM; miR-34a; androgen receptor (AR); PSA; methylation