Evidence of Heavy Methylation in the Galectin 3 Promoter in Early Stages of Prostate Adenocarcinoma: Development and Validation of a Methylated Marker for Early Diagnosis of Prostate Cancer

Galectins, soluble intracellular and extracellular β-galactoside-binding proteins, are known to be involved in the progression and metastasis of various cancers, including prostate adenocarcinoma, but the detailed mechanism of their biological roles remains elusive. In the prostate cancer cell lines PC-3 and DU-145, galectin 3 (gal3) is present at normal levels, whereas in LNCaP, its expression is silenced. In LNCaP, the gal3 promoter was heavily methylated, whereas PC-3 or DU-145 cells showed negligible or no methylation in the gal3 promoter indicating a negative correlation between gal3 promoter methylation and its expression. On immunohistochemical analysis of normal and tumor prostate tissues, gal3 was found expressed both in nucleus and cytoplasm of benign prostatic hyperplasia, high-grade prostatic intraepithelial neoplasia, and stage I. The expression of the gal3 was found drastically downregulated in advanced stages and, interestingly, mostly in the cytoplasm. On methylation analysis, the gal3 promoter in stage II prostate adenocarcinoma (PCa) was found heavily methylated, whereas in stages III and IV, it was only lightly methylated. However, in stage I PCa, both heavy and light methylations were observed in the gal3 promoter. In normal and benign prostatic hyperplasia tissues, the gal3 promoter was almost unmethylated. The differential cytosine methylation in the gal3 promoter in stages I to IV PCa enabled us to develop and validate a methylation-specific polymerase chain reaction-based sensitive assay specific for stages I and II PCa. These stages are considered the critical stages for successful intervention, thus underscoring the significance of this diagnostic assay.     

CCR2 expression correlates with prostate cancer progression

Although the primary role of chemokines and their receptors is controlling the trafficking of leukocytes during inflammatory responses, they also play pleoitropic roles in cancer development. There is emerging evidence that cancer cells produce chemokines that induce tumor cell proliferation or chemotaxis in various cancer types. We have previously reported that MCP-1 acts as a paracrine and autocrine factor for prostate cancer (PCa) growth and invasion. As the cellular effects of MCP-1 are mediated by CC chemokine receptor 2 (CCR2), we hypothesized that CCR2 may contribute PCa progression. Accordingly, we first determined CCR2 mRNA and protein expression in various cancer cell lines, including PCa and other cancer types. All cells expressed CCR2 mRNA and protein, but in PCa, more aggressive cancer cells such as C4-2B, DU145, and PC3 expressed a higher amount of CCR2 compared with the less aggressive cancer cells such as LNCaP or non-neoplastic PrEC and RWPE-1 cells. Further, we found a positive correlation between CCR2 expression and PCa progression by analyzing an ONCOMINE gene array database. We confirmed that CCR2 mRNA was highly expressed in PCa metastatic tissues compared with the localized PCa or benign prostate tissues by real-time RT-PCR. Finally, CCR2 protein expression was examined by immunohistochemical staining on tissue microarray specimens from 96 PCa patients and 31 benign tissue controls. We found that CCR2 expression correlated with Gleason score and clinical pathologic stages, whereas lower levels of CCR2 were expressed in normal prostate tissues. These results suggest that CCR2 may contribute to PCa development.     

Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells

One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cells through cell cycle inhibition or induction of apoptosis without affecting normal cells. In this study, employing normal human prostate epithelial cells (PrEC), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate cancer cells (LNCaP, DU145, and PC-3), we evaluated the growth-inhibitory and apoptotic effects of tocotrienol-rich fraction (TRF) extracted from palm oil. TRF treatment to PrEC and PZ-HPV-7 resulted in almost identical growth-inhibitory responses of low magnitude. In sharp contrast, TRF treatment resulted in significant decreases in cell viability and colony formation in all three prostate cancer cell lines. The IC(50) values after 24h TRF treatment in LNCaP, PC-3, and DU145 cells were in the order 16.5, 17.5, and 22.0 microg/ml. TRF treatment resulted in significant apoptosis in all the cell lines as evident from (i) DNA fragmentation, (ii) fluorescence microscopy, and (iii) cell death detection ELISA, whereas the PrEC and PZ-HPV-7 cells did not undergo apoptosis, but showed modestly decreased cell viability only at a high dose of 80 microg/ml. In cell cycle analysis, TRF (10-40 microg/ml) resulted in a dose-dependent G0/G1 phase arrest and sub G1 accumulation in all three cancer cell lines but not in PZ-HPV-7 cells. These results suggest that the palm oil derivative TRF is capable of selectively inhibiting cellular proliferation and accelerating apoptotic events in prostate cancer cells. TRF offers significant promise as a chemopreventive and/or therapeutic agent against prostate cancer.     

A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer

DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.     

Insensitivity to transforming growth factor-beta results from promoter methylation of cognate receptors in human prostate cancer cells (LNCaP)

Prostate cancers often develop insensitivity to TGF-beta to gain a growth advantage. In this study, we explored the status of promoter methylation of TGF-beta receptors (TbetaRs) in a prostate cancer cell line, LNCaP, which is insensitive to TGF-beta. Sensitivity to TGF-beta was restored in cells treated with 5-Aza-2'-deoxycytidine (5-Aza), as indicated by an increase in the expression of phosphorylated Smad-2, type I (TbetaRI), and type II (TbetaRII) TGF-beta receptors, and a reduced rate of proliferation. The same treatment did not significantly affect a benign prostate cell line, RWPE-1, which is sensitive to TGF-beta. Mapping of methylation sites was performed by screening 82 potential CpG methylation sites in the promoter of TbetaRI and 33 sites in TbetaRII using methylation-specific PCR and sequence analysis. There were six methylation sites (-365, -356, -348, -251, -244, -231) in the promoter of TbetaRI. The -244 site was located in an activator protein (AP)-2 box. There were three methylated sites (-140, +27, +32) in the TbetaRII promoter and the -140 site was located in one of the Sp1 boxes. Chromatin immunoprecipitation analysis demonstrated DNA binding activity of AP-2 in the TbetaRI promoter and of Sp1 in the TbetaRII promoter after treatment with 5-Aza. To test whether promoter methylation is present in clinical specimens, we analyzed human prostate specimens that showed negative staining for either TbetaRI or TbetaRII in a tissue microarray system. DNA samples were isolated from the microarray after laser capture microdissection. Methylation-specific PCR was performed for TbetaRI (six sites) and TbetaRII (three sites) promoters as identified in LNCaP cells. A significant number of clinical prostate cancer specimens lacked expression of either TbetaRI and/or TbetaRII, especially those with high Gleason's scores. In those specimens showing a loss of TbetaR expression, a promoter methylation pattern similar to that of LNCaP cells was a frequent event. These results demonstrate that insensitivity to TGF-beta in some prostate cancer cells is due to promoter methylation in TbetaRs.     

A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer

DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG’s predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.     

Androgen dependent regulation of protein kinase A subunits in prostate cancer cells

Neuroendocrine (NE) cells may play a role in prostate cancer progression. Both androgen deprivation and cAMP are well known inducers of NE differentiation (NED) in the prostate. Gene-expression profiling of LNCaP cells, incubated in androgen stripped medium, showed that the Cbeta isoform of PKA is up-regulated during NE differentiation. Furthermore, by using semi-quantitative RT-PCR and immunoblotting analysis, we observed that the Cbeta splice variants are differentially regulated during this process. Whereas the Cbeta2 splice variant is down-regulated in growth arrested LNCaP cells, the Cbeta1, Cbeta3 and Cbeta4 variants, as well as the RIIbeta subunit of PKA, are induced in NE-like LNCaP cells. The opposite effect of Cbeta expression could be mimicked by androgen stimulation, implying the Cbeta gene of PKA as a putative new target gene for the androgen receptor in prostate cancer. Moreover, to investigate expression of PKA subunits during prostate cancer progression, we did immunoblotting of several prostatic cell lines and normal and tumor tissue from prostate cancer patients. Interestingly, multiple Cbeta subunits were also observed in human prostate specimens, and the Cbeta2 variant was up-regulated in tumor cells. In conclusion, it seems that the Cbeta isoforms play different roles in proliferation and differentiation and could therefore be potential markers for prostate cancer progression.     

Frequent 14-3-3 sigma promoter methylation in benign and malignant prostate lesions

14-3-3Sigma is a putative tumor suppressor gene involved in cell cycle regulation and apoptosis following DNA damage. 14-3-3Sigma loss of expression has been reported is several human cancers, including prostate adenocarcinoma and precursor lesions, and promoter hypermethylation has been proposed as the mechanism underlying gene silencing. Here, we investigate the frequency and extent of 14-3-3sigma promoter methylation in benign and cancerous prostate tissues. We examined tumor tissue from 121 patients with prostate carcinoma (PCa), 39 paired high-grade prostatic intraepithelial neoplasias (HGPIN), 29 patients with benign prostate hyperplasia (BPH), as well as four prostate cancer cell lines using quantitative methylation-specific PCR (QMSP). The percentage of methylated alleles (PMA) was calculated and correlated with clinical and pathological parameters. RT-PCR was performed in the cell lines to assess 14-3-3sigma mRNA expression. PCa, HGPIN, BPH, and cancer cell lines showed ubiquitous 14-3-3sigma promoter methylation. However, the PMA of HGPIN was significantly lower than that of PCa or BPH (P < 0.0001), while PCa and BPH did not significantly differ. The PMA did not correlate with any clinicopathological parameter. All prostate cancer cell lines expressed 14-3-3sigmamRNA. 14-3-3Sigma promoter methylation is a frequent event in prostate tissues and cancer cell lines. Furthermore, there is a progressive accumulation of neoplastic cells with 14-3-3sigma methylated alleles from HGPIN to PCa, suggesting a role for this epigenetic event in prostate carcinogenesis. However, other mechanisms besides promoter methylation might be required for effective 14-3-3sigma downregulation.     

Differential expression of CD44 during human prostate epithelial cell differentiation.

CD44 is a polymorphic transmembrane glycoprotein that binds hyaluronan and growth factors. Multiple isoforms of the protein can be generated by alternative splicing but little is known about the expression and function of these isoforms in normal development and differentiation. We have investigated the expression of CD44 during normal prostate epithelial cell differentiation. A conditionally immortalized prostate epithelial cell line, Pre2.8, was used as a model system. These cells proliferate at 33C but at 39C stop dividing and undergo changes consistent with early stages of cell differentiation. During the differentiation of these cells, the expression of the CD44 isoform v3-v10 was upregulated. Two layers of epithelial cells can clearly be distinguished in the human prostate, a basal layer expressing keratins 5/14 and a luminal layer expressing keratins 8/18. In prostate tissue the v3-v10 isoform was found predominantly in basal cells but also in keratin 14-negative, keratin 19-positive cells intermediate between the two layers. CD44 v3-v10 was also expressed in other keratin 14-negative prostate tissues, the ejaculatory ducts and prostatic urethra. Therefore, CD44 v3-v10 may be important as a cell surface marker for differentiating cells in the prostate epithelium.     

Expression and secretion of N-acylethanolamine-hydrolysing acid amidase in human prostate cancer cells

N-acylethanolamines (NAEs) are a class of bioactive lipid molecules in animal tissues, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. Enzymatic hydrolysis of NAEs is considered to be an important step to regulate their endogenous levels. Lysosomal NAE-hydrolysing acid amidase (NAAA) as well as fatty acid amide hydrolase (FAAH) is responsible for this reaction. Here, we report relatively high expression of NAAA in human prostate cancer cells (PC-3, DU-145 and LNCaP) and prostate epithelial cells (PrEC), with the highest mRNA level in LNCaP cells. FAAH and the NAE-forming enzyme N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) were also detected in these cells. NAAA activity in LNCaP cells could be distinguished from coexisting FAAH activity, based on their different pH dependency profiles and specific inhibition of FAAH activity by URB597. These results showed that both the enzymes were functionally active. We also found that NAAA was partly secreted from LNCaP cells, which underlined possible usefulness of this enzyme as a biomarker of prostate cancer.     

Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca²⁺ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca²⁺-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca²⁺. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.     

Epigenetic inactivation of PCDH10 in human prostate cancer cell lines

PCDH10 (protocadherin-10), a novel tumour suppressor gene, is down-regulated in several human cancers due to hypermethylation of promoter CGIs (CpG islands). Here, we investigated the expression of PCDH10 in different normal adult tissues and in a panel of prostate cancer cell lines. PCDH10 was widely expressed in normal tissues with higher levels in the prostate. The expression of PCDH10 was markedly reduced or silenced in prostate cancer cell lines compared with normal adult prostate tissue. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Furthermore, the DNA demethylating agent 5'-azacytidin restored PCDH10 expression by suppressing PCDH10 promoter methylation in prostate cancer cell lines. Treatment with Trichostatin A alone had no significant effect on the expression of PCDH10 but enhanced the effect of 5'-azacytidin. In conclusion, we found that the decreased PCDH10 expression in prostate cancer cells was associated with the aberrant methylation of PCDH10 promoter CGI. Our results may contribute to the understanding of the role of PCDH10 inactivation in the progression of prostate cancers.