MicroRNAs associated with metastatic prostate cancer

Objective

Metastasis is the most common cause of death of prostate cancer patients. Identification of specific metastasis biomarkers and novel therapeutic targets is considered essential for improved prognosis and management of the disease. MicroRNAs (miRNAs) form a class of non-coding small RNA molecules considered to be key regulators of gene expression. Their dysregulation has been shown to play a role in cancer onset, progression and metastasis, and miRNAs represent a promising new class of cancer biomarkers. The objective of this study was to identify down- and up-regulated miRNAs in prostate cancer that could provide potential biomarkers and/or therapeutic targets for prostate cancer metastasis.

Methods

Next generation sequencing technology was applied to identify differentially expressed miRNAs in a transplantable metastatic versus a non-metastatic prostate cancer xenograft line, both derived from one patient''s primary cancer. The xenografts were developed via subrenal capsule grafting of cancer tissue into NOD/SCID mice, a methodology that tends to preserve properties of the original cancers (e.g., tumor heterogeneity, genetic profiles).

Results

Differentially expressed known miRNAs, isomiRs and 36 novel miRNAs were identified. A number of these miRNAs (21/104) have previously been reported to show similar down- or up-regulation in prostate cancers relative to normal prostate tissue, and some of them (e.g., miR-16, miR-34a, miR-126*, miR-145, miR-205) have been linked to prostate cancer metastasis, supporting the validity of the analytical approach.

Conclusions

The use of metastatic and non-metastatic prostate cancer subrenal capsule xenografts derived from one patient''s cancer makes it likely that the differentially expressed miRNAs identified in this study include potential biomarkers and/or therapeutic targets for human prostate cancer metastasis.     

The Lipid Phenotype of Breast Cancer Cells Characterized by Raman Microspectroscopy: Towards a Stratification of Malignancy

Although molecular classification brings interesting insights into breast cancer taxonomy, its implementation in daily clinical care is questionable because of its expense and the information supplied in a single sample allocation is not sufficiently reliable. New approaches, based on a panel of small molecules derived from the global or targeted analysis of metabolic profiles of cells, have found a correlation between activation of de novo lipogenesis and poorer prognosis and shorter disease-free survival for many tumors. We hypothesized that the lipid content of breast cancer cells might be a useful indirect measure of a variety of functions coupled to breast cancer progression. Raman microspectroscopy was used to characterize metabolism of breast cancer cells with different degrees of malignancy. Raman spectra from MDA-MB-435, MDA-MB-468, MDA-MB-231, SKBR3, MCF7 and MCF10A cells were acquired with an InVia Raman microscope (Renishaw) with a backscattered configuration. We used Principal Component Analysis and Partial Least Squares Discriminant Analyses to assess the different profiling of the lipid composition of breast cancer cells. Characteristic bands related to lipid content were found at 3014, 2935, 2890 and 2845 cm⁻¹, and related to lipid and protein content at 2940 cm⁻¹. A classificatory model was generated which segregated metastatic cells and non-metastatic cells without basal-like phenotype with a sensitivity of 90% and a specificity of 82.1%. Moreover, expression of SREBP-1c and ABCA1 genes validated the assignation of the lipid phenotype of breast cancer cells. Indeed, changes in fatty acid unsaturation were related with the epithelial-to-mesenchymal transition phenotype. Raman microspectroscopy is a promising technique for characterizing and classifying the malignant phenotype of breast cancer cells on the basis of their lipid profiling. The algorithm for the discrimination of metastatic ability is a first step towards stratifying breast cancer cells using this rapid and reagent-free tool.     

Self-renewing Pten-/- TP53-/- protospheres produce metastatic adenocarcinoma cell lines with multipotent progenitor activity

Prostate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem/progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease.     

Metabolite profiling of blood plasma of patients with prostate cancer

Prostate cancer is one of the most common types of cancer in men. It is though extremely important to search for specific markers including metabolites, which concentration in blood could be a diagnostic measure. In this regard, the metabolite profiling of blood plasma was performed with two groups of people: healthy volunteers (n = 30) and patients with prostate cancer, second stage (n = 40). The profiling protocol included proteins removal from blood plasma with methanol and direct analysis of metabolite fractions by mass spectrometry. Identification of the most abundant metabolites in samples was performed using an accurate mass tag and an isotope pattern methods. Cancer-specific metabolites were revealed by statistical analysis of metabolite intensities in the mass spectra. Six different metabolites were found to be cancer-specific. Two metabolites, acylcarnitine and arachidonoyl amine, have the AUC 0.97 and 0.86, respectively, which are higher than those from PSA test, 0.59.     

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC₅₀ value of 15 μM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na⁺ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.     

ZBTB38 suppresses prostate cancer cell proliferation and migration via directly promoting DKK1 expression

Prostate cancer is still one of the most common malignancies in men all around the world. The mechanism of how prostate cancer initiates and develops is still not clear. Here in this study, we show that tumor suppressor ZBTB38 could suppress the migration and proliferation of prostate cancer cells. We find lower ZBTB38 expression in prostate cancer tissues, which also strongly predicts a poorer prognosis of prostate cancer. ZBTB38 binds DKK1 (Dickkopf WNT signaling pathway inhibitor 1) locus and promotes DKK1 expression in prostate cancer cell lines. Consistently, reduction of DKK1 expression significantly restores ZBTB38-mediated suppression of migration and proliferation of prostate cancer cell lines. Mechanistically, we find that ZBTB38 primarily binds the promoters of target genes, and differentially regulates the expression of 1818 genes. We also identify PRKDC (protein kinase, DNA-activated, catalytic subunit) as a ZBTB38-interacting protein that could repress the function of ZBTB38 in suppressing migration and proliferation of prostate cancer cells. Taken together, our results indicate that ZBTB38 could repress cell migration and proliferation in prostate cancer via promoting DKK1 expression, and also provide evidence supporting ZBTB38 as a potential prognosis marker for prostate cancer.Subject terms: Tumour-suppressor proteins, Prostate cancer     

Targeting lactate production and efflux in prostate cancer

Prostate cancer (PCa) is the most commonly diagnosed cancer in men worldwide. Screening and management of PCa remain controversial and, therefore, the discovery of novel molecular biomarkers is urgently needed. Alteration in cancer cell metabolism is a recognized hallmark of cancer, whereby cancer cells exhibit high glycolytic rates with subsequent lactate production, regardless of oxygen availability. To maintain the hyperglycolytic phenotype, cancer cells efficiently export lactate through the monocarboxylate transporters MCT1 and MCT4. The impact of inhibiting lactate production/extrusion on PCa cell survival and aggressiveness was investigated in vitro and ex vivo using primary tumor and metastatic PCa cell lines and the chicken embryo chorioallantoic membrane (CAM) model. In this study, we showed the metastatic PCa cell line (DU125) displayed higher expression levels of MCT1/4 isoforms and glycolysis-related markers than the localized prostate tumor-derived cell line (22RV1), indicating these proteins are differentially expressed throughout prostate malignant transformation. Moreover, disruption of lactate export by MCT1/4 silencing resulted in a decrease in PCa cell growth and motility. To support these results, we pharmacological inhibited lactate production (via inhibition of LDH) and release (via inhibition of MCTs) and a reduction in cancer cell growth in vitro and in vivo was observed. In summary, our data provide evidence that MCT1 and MCT4 are important players in prostate neoplastic progression and that inhibition of lactate production/export can be explored as a strategy for PCa treatment.     

Spermine and Citrate as Metabolic Biomarkers for Assessing Prostate Cancer Aggressiveness

Separating indolent from aggressive prostate cancer is an important clinical challenge for identifying patients eligible for active surveillance, thereby reducing the risk of overtreatment. The purpose of this study was to assess prostate cancer aggressiveness by metabolic profiling of prostatectomy tissue and to identify specific metabolites as biomarkers for aggressiveness. Prostate tissue samples (n = 158, 48 patients) with a high cancer content (mean: 61.8%) were obtained using a new harvesting method, and metabolic profiles of samples representing different Gleason scores (GS) were acquired by high resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS). Multivariate analysis (PLS, PLS-DA) and absolute quantification (LCModel) were used to examine the ability to predict cancer aggressiveness by comparing low grade (GS = 6, n = 30) and high grade (GS≥7, n = 81) cancer with normal adjacent tissue (n = 47). High grade cancer tissue was distinguished from low grade cancer tissue by decreased concentrations of spermine (p = 0.0044) and citrate (p = 7.73·10⁻⁴), and an increase in the clinically applied (total choline+creatine+polyamines)/citrate (CCP/C) ratio (p = 2.17·10⁻⁴). The metabolic profiles were significantly correlated to the GS obtained from each tissue sample (r = 0.71), and cancer tissue could be distinguished from normal tissue with sensitivity 86.9% and specificity 85.2%. Overall, our findings show that metabolic profiling can separate aggressive from indolent prostate cancer. This holds promise for the benefit of applying in vivo magnetic resonance spectroscopy (MRS) within clinical MR imaging investigations, and HR-MAS analysis of transrectal ultrasound-guided biopsies has a potential as an additional diagnostic tool.     

MicroRNA-100 regulates IGF1-receptor expression in metastatic pancreatic cancer cells

Abstract

Patients with pancreatic adenocarcinoma have the lowest 5 year survival rate and yearly rates of incidence are nearly equal to the mortality rates. Long term cure rates by standard therapies are disappointing owing to disseminated disease at diagnosis and chemotherapeutic resistance. New therapeutic targets are necessary to decrease the progression of pancreatic cancer and the ability to identify targets specific to metastasis would improve patient care. We evaluated the levels of microRNA of metastatic and non-metastatic cell lines. The expression levels of microRNAs and mRNAs were determined using microarray analysis to examine and compare five pancreatic cancer cell lines, two that can metastasize in vivo (S2VP10 and S2CP9) and three that do not metastasize (MiaPaCa2, Panc-1 and ASPC-1). MicroRNA analysis indicated an increase in miR-100 and a decrease in miR-138 expression in metastatic cancer cells. Microarray analysis of different expressions of mRNAs in metastatic and non-metastatic pancreatic cell lines also indicated significantly increased insulin growth factor-1 receptor (IGF1-R) expression in metastatic pancreatic cancer cell lines compared to non-metastatic pancreatic cancer cell lines. To confirm microarray analysis results, western blot and immunocytochemistry were performed. Western blot revealed that IGF1-R expression exhibited in metastatic cancer cell lines a seven-fold increase compared to non-metastatic cell lines. In addition, downstream expressions of the proteins, GRB2 and phosphorylated PI3K, also were increased in aggressive cancer cell lines. Immunocytochemistry confirmed the linkage of IGF1-R to miR-100, because cells transfected with miR-100 inhibitor showed a decrease in IGF1-R. Cells transfected with a miR-138 mimic, however, did not affect IGF1-R expression.     

miR-888 is an expressed prostatic secretions-derived microRNA that promotes prostate cell growth and migration

microRNAs (miRNAs) are a growing class of small non-coding RNAs that exhibit widespread dysregulation in prostate cancer. We profiled miRNA expression in syngeneic human prostate cancer cell lines that differed in their metastatic potential in order to determine their role in aggressive prostate cancer. miR-888 was the most differentially expressed miRNA observed in human metastatic PC3-ML cells relative to non-invasive PC3-N cells, and its levels were higher in primary prostate tumors from cancer patients, particularly those with seminal vesicle invasion. We also examined a novel miRNA-based biomarker source called expressed prostatic secretions in urine (EPS urine) for miR-888 expression and found that its levels were preferentially elevated in prostate cancer patients with high-grade disease. These expression studies indicated a correlation for miR-888 in disease progression. We next tested how miR-888 regulated cancer-related pathways in vitro using human prostate cancer cell lines. Overexpression of miR-888 increased proliferation and migration, and conversely inhibition of miR-888 activity blocked these processes. miR-888 also increased colony formation in PC3-N and LNCaP cells, supporting an oncogenic role for this miRNA in the prostate. Our data indicates that miR-888 functions to promote prostate cancer progression and can suppress protein levels of the tumor suppressor genes RBL1 and SMAD4. This miRNA holds promise as a diagnostic tool using an innovative prostatic fluid source as well as a therapeutic target for aggressive prostate cancer.     

Valproic acid inhibits the invasion of PC3 prostate cancer cells by upregulating the metastasis suppressor protein NDRG1

Valproic acid (VPA) is a clinically available histone deacetylase inhibitor with promising anticancer attributes. Recent studies have demonstrated the anticancer effects of VPA on prostate cancer cells. However, little is known about the differential effects of VPA between metastatic and non-metastatic prostate cancer cells and the relationship between the expression of metastasis suppressor proteins and VPA. In the present study, we demonstrate that inhibition of cell viability and invasion by VPA was more effective in the metastatic prostate cancer cell line PC3 than in the tumorigenic but non-metastatic prostate cell line, RWPE2. Further, we identified that the metastasis suppressor NDRG1 is upregulated in PC3 by VPA treatment. In contrast, NDRG1 was not increased in RWPE2 cells. Also, the suppressed invasion of PC3 cells by VPA treatment was relieved by NDRG1 knockdown. Taken together, we suggest that the anticancer effect of VPA on prostate cancer cells is, in part, mediated through upregulation of NDRG1. We also conclude that VPA has differential effects on the metastasis suppressor gene and invasion ability between non-metastatic and metastatic prostate cancer cells.     

Induction of Neuroendocrine Differentiation in Prostate Cancer Cells by Dovitinib (TKI-258) and its Therapeutic Implications

Prostate cancer (PCa) remains the second-leading cause of cancer-related deaths in American men with an estimated mortality of more than 26,000 in 2016 alone. Aggressive and metastatic tumors are treated with androgen deprivation therapies (ADT); however, the tumors acquire resistance and develop into lethal castration resistant prostate cancer (CRPC). With the advent of better therapeutics, the incidences of a more aggressive neuroendocrine prostate cancer (NEPC) variant continue to emerge. Although de novo occurrences of NEPC are rare, more than 25% of the therapy-resistant patients on highly potent new-generation anti-androgen therapies end up with NEPC. This, along with previous observations of an increase in the number of such NE cells in aggressive tumors, has been suggested as a mechanism of resistance development during prostate cancer progression. Dovitinib (TKI-258/CHIR-258) is a pan receptor tyrosine kinase (RTK) inhibitor that targets VEGFR, FGFR, PDGFR, and KIT. It has shown efficacy in mouse-model of PCa bone metastasis, and is presently in clinical trials for several cancers. We observed that both androgen receptor (AR) positive and AR-negative PCa cells differentiate into a NE phenotype upon treatment with Dovitinib. The NE differentiation was also observed when mice harboring PC3-xenografted tumors were systemically treated with Dovitinib. The mechanistic underpinnings of this differentiation are unclear, but seem to be supported through MAPK-, PI3K-, and Wnt-signaling pathways. Further elucidation of the differentiation process will enable the identification of alternative salvage or combination therapies to overcome the potential resistance development.     

Identification,prioritization, and evaluation of glycoproteins for aggressive prostate cancer using quantitative glycoproteomics and antibody‐based assays on tissue specimens

Prostate cancer is highly heterogeneous in nature; while the majority of cases are clinically insignificant, some cases are lethal. Currently, there are no reliable screening methods for aggressive prostate cancer. Since most established serum and urine biomarkers are glycoproteins secreted or leaked from the diseased tissue, the current study seeks to identify glycoprotein markers specific to aggressive prostate cancer using tissue specimens. With LC‐MS/MS glycoproteomic analysis, we identified 350 glycopeptides with 17 being altered in aggressive prostate cancer. ELISA assays were developed/purchased to evaluate four candidates, that is, cartilage oligomeric matrix protein (COMP), periostin, membrane primary amine oxidase (VAP‐1), and cathepsin L, in independent tissue sets. In agreement with the proteomic analysis, we found that COMP and periostin expressions were significantly increased in aggressive prostate tumors while VAP‐1 expression was significantly decreased in aggressive tumor. In addition, the expression of these proteins in prostate metastases also follows the same pattern observed in the proteomic analysis. This study provides a workflow for biomarker discovery, prioritization, and evaluation of aggressive prostate cancer markers using tissue specimens. Our data suggest that increase in COMP and periostin and decrease in VAP‐1 expression in the prostate may be associated with aggressive prostate cancer.     

Proteomic Profiling of Androgen-independent Prostate Cancer Cell Lines Reveals a Role for Protein S during the Development of High Grade and Castration-resistant Prostate Cancer

Androgen deprivation constitutes the principal therapy for advanced and metastatic prostate cancers. However, this therapeutic intervention usually results in the transition to a more aggressive androgen-independent prostate cancer. The elucidation of molecular alterations during the progression to androgen independence is an integral step toward discovering more effective targeted therapies. With respect to identifying crucial mediators of this transition, we compared the proteomes of androgen-independent (PC3, DU145, PPC1, LNCaP-SF, and 22Rv1) and androgen-dependent (LNCaP and VCaP) and/or normal prostate epithelial (RWPE) cell lines using mass spectrometry. We identified more than 100 proteins that were differentially secreted in the androgen-independent cell lines. Of these, Protein S (PROS1) was elevated in the secretomes of all of the androgen-independent prostate cancer cell lines, with no detectable secretion in normal and androgen-dependent cell lines. Using quantitative PCR, we observed significantly higher (p < 0.05) tissue expression levels of PROS1 in prostate cancer samples, further indicating its importance in prostate cancer progression. Similarly, immunohistochemistry analysis revealed elevation of PROS1 in high grade prostate cancer (Gleason grade ≥8), and further elevation in castration-resistant metastatic prostate cancer lesions. We also observed its significant (p < 0.05) elevation in high grade prostate cancer seminal plasma samples. Taken together, these results show that PROS1 is elevated in high grade and castration-resistant prostate cancer and could serve as a potential biomarker of aggressive disease.     

Caveolin-1, a metastasis-related gene that promotes cell survival in prostate cancer

Metastasis represents the ultimate target in cancer therapy as this complex biological process is the direct cause of mortality for a variety of human malignancies. The current high level of mortality from prostate cancer results in large part from the inexorable growth of overt or occult metastasis present at the time of diagnosis. Currently, there are no curative therapies for metastatic prostate cancer. To better understand the metastatic phenotype in prostate cancer, we developed a strategy to identify mRNAs that are expressed differentially in cell lines derived from primary versus metastatic mouse prostate cancer using differential display-PCR. In using this system a number of metastasis-related sequences were identified including a cDNA that encodes caveolin-1. Caveolin-1 was found to be overexpressed not only in metastatic mouse prostate cancer, but also in human metastatic disease. Recent studies have indicated that suppression of caveolin-1 expression induces androgen sensitivity in high caveolin-1, androgen-insensitive mouse prostate cancer cells derived from metastases. Conversely, overexpression of caveolin-1 leads to androgen insensitivity in low caveolin, androgen-sensitive mouse prostate cancer cells. Caveolin-1, therefore, is both a metastasis-related gene as well as a candidate androgen resistance gene for prostate cancer in man. Interestingly, recent studies also point to a potential role for caveolin-1 in the resistance of various malignancies to multiple antineoplastic agents. The linkage of caveolin-1 expression with the androgen-resistant phenotype in prostate cancer and the multidrug resistance phenotype in various solid tumors establishes a novel paradigm for understanding these clinically important and now potentially related processes in malignant progression.     

The significance of galectin-3 as a new basal cell marker in prostate cancer

Prostate cancer may originate from distinct cell types, resulting in the heterogeneity of this disease. Galectin-3 (Gal-3) and androgen receptor (AR) have been reported to play important roles in the progression of prostate cancer, and their heterogeneous expressions might be associated with different cancer subtypes. Our study found that in various prostate cancer cell lines Gal-3 expression was always opposite to AR expression and other luminal cell markers but consistent with basal cell markers including glutathione S-transferase-π and Bcl-2. This expression pattern was confirmed in human prostate cancer tissues. Our results also showed that prostate cancer cells positive with basal cell markers were more aggressive. Downregulation of Gal-3 expression resulted in increased apoptotic potential and decreased metastasis potential of prostate cancer cells. Our findings demonstrate for the first time that Gal-3 may serve as a new marker for basal characteristics of prostate cancer epithelium. This study helps us to better understand the heterogeneity of prostate cancer. The clinical significance of this study lies in the application of Gal-3 to distinguish prostate cancer subtypes and improve treatment efficacy with designed personalized therapy.     

Ubiquitin C-terminal hydrolase-L3 regulates EMT process and cancer metastasis in prostate cell lines

Ubiquitin C-terminal hydrolase-L3 (UCH-L3) is among the deubiquitinating enzymes (DUBs) that cleave ubiquitin (Ub) from Ub precursors or protein substrates. Many DUBs have been shown to participate in cancer progression in various tissues. However, the mechanism and role of UCH-L3 in carcinogenesis has largely been unknown until recently. Here we investigated the implication of UCH-L3 in prostate cancer progression. Interestingly, UCH-L3 is upregulated in normal or non-metastatic prostate cancer cells and is downregulated in metastatic prostate cancer cell lines. Notably, knockdown of UCH-L3 in normal prostate cell line RWPE1 promotes epithelial-to-mesenchymal transition (EMT), an important process for cancer cell invasion and metastasis. The induction of EMT by UCH-L3 knockdown results in an increase of cell migration and invasion. Yet, to the contrary, overexpression of UCH-L3 in highly metastatic prostate cancer cell line PC3 reverses EMT but the active site mutant UCH-L3 did not. Collectively, our findings identify UCH-L3 as a novel EMT regulator in prostate cells and highlight UCH-L3 as a potential therapeutic target for preventing metastatic prostate cancer.     

Key pathways involved in prostate cancer based on gene set enrichment analysis and meta analysis

Prostate cancer is one of the most common male malignant neoplasms; however, its causes are not completely understood. A few recent studies have used gene expression profiling of prostate cancer to identify differentially expressed genes and possible relevant pathways. However, few studies have examined the genetic mechanics of prostate cancer at the pathway level to search for such pathways. We used gene set enrichment analysis and a meta-analysis of six independent studies after standardized microarray preprocessing, which increased concordance between these gene datasets. Based on gene set enrichment analysis, there were 12 down- and 25 up-regulated mixing pathways in more than two tissue datasets, while there were two down- and two up-regulated mixing pathways in three cell datasets. Based on the meta-analysis, there were 46 and nine common pathways in the tissue and cell datasets, respectively. Three up- and 10 down-regulated crossing pathways were detected with combined gene set enrichment analysis and meta-analysis. We found that genes with small changes are difficult to detect by classic univariate statistics; they can more easily be identified by pathway analysis. After standardized microarray preprocessing, we applied gene set enrichment analysis and a meta-analysis to increase the concordance in identifying biological mechanisms involved in prostate cancer. The gene pathways that we identified could provide insight concerning the development of prostate cancer.