Transcriptional Activity of Heparan Sulfate Biosynthetic Machinery is Specifically Impaired in Benign Prostate Hyperplasia and Prostate Cancer.

Heparan sulfates (HSs) are key components of mammalian cells surface and extracellular matrix. Structure and composition of HS, generated by HS-biosynthetic system through non-template-driven process, are significantly altered in cancer tissues. The aim of this study was to investigate the involvement of HS-metabolic machinery in prostate carcinogenesis. Transcriptional patterns of HS-metabolic enzymes (EXT1, EXT2, NDST1, NDST2, GLCE, 3OST1/HS3ST1, SULF1, SULF2, HPSE) were determined in normal, benign, and cancer human prostate tissues and cell lines (PNT2, LNCaP, PC3, DU145). Stability of the HS-metabolic system patterns under the pressure of external or internal stimuli was studied. Overall impairment of transcriptional activity of HS-metabolic machinery was detected in benign prostate hyperplasia, while both significant decrease in the transcriptional activity and changes in the expression patterns of HS metabolism-involved genes were observed in prostate tumors. Prostate cancer cell lines possessed specific transcriptional patterns of HS metabolism-involved genes; however, expression activity of the system was similar to that of normal prostate PNT2 cells. HS-metabolic system was able to dynamically react to different external or internal stimuli in a cell type-dependent manner. LNCaP cells were sensitive to the external stimuli (5-aza-deoxycytidin or Trichostatin A treatments; co-cultivation with human fibroblasts), whereas PC3 cells almost did not respond to the treatments. Ectopic GLCE over-expression resulted in transcriptional activation of HS-biosynthetic machinery in both cell lines, suggesting an existence of a self-regulating mechanism for the coordinated transcription of HS metabolism-involved genes. Taken together, these findings demonstrate impairment of HS-metabolic system in prostate tumors in vivo but not in prostate cancer cells in vitro, and suggest that as a potential microenvironmental biomarker for prostate cancer diagnostics and treatment.

Heterogeneity of d-glucuronyl C5-epimerase expression and epigenetic regulation in prostate cancer.

Heparansulfate proteoglycans (HSPG) play an important role in cell-cell and cell-matrix interactions and signaling, and one of the key enzymes in heparansulfate biosynthesis is d-glucuronyl C5-epimerase (GLCE). A tumor suppressor function has been demonstrated for GLCE in breast and lung carcinogenesis; however, no data are available as to the expression and regulation of the gene in prostate cancer. In this study, decreased GLCE expression was observed in 10% of benign prostate hyperplasia (BPH) tissues and 53% of prostate tumors, and increased GLCE mRNA levels were detected in 49% of BPH tissues and 21% of tumors. Statistical analysis showed a positive correlation between increased GLCE expression and Gleason score, TNM staging, and prostate-specific antigen (PSA) level in the prostate tumors (Pearson correlation coefficients GLCE/Gleason = 0.56, P < 0.05; GLCE/TNM = 0.62, P < 0.05; and GLCE/PSA = 0.88, P < 0.01), suggesting GLCE as a candidate molecular marker for advanced prostate cancer. Immunohistochemical analysis revealed an intratumoral heterogeneity of GLCE protein levels both in BPH and prostate cancer cells, resulting in a mixed population of GLCE-expressing and nonexpressing epithelial cells in vivo. A model experiment on normal (PNT2) and prostate cancer (LNCaP, PC3, DU145) cell lines in vitro showed a 1.5- to 2.5-fold difference in GLCE expression levels between the cancer cell lines and an overall decrease in GLCE expression in cancer cells. Methyl-specific polymerase chain reaction (PCR), bisulfite sequencing, and deoxy-azacytidin (aza-dC) treatment identified differential GLCE promoter methylation (LNCaP 70-72%, PC3 32-35%, DU145, and PNT2 no methylation), which seems to contribute to heterogeneous GLCE expression in prostate tumors. The obtained results reveal the complex deregulation of GLCE expression in prostatic diseases compared with normal prostate tissue and suggest that GLCE may be used as a potential model to study the functional role of intratumor cell heterogeneity in prostate cancer progression.

The TCF4/ß-catenin pathway and chromatin structure cooperate to regulate D-glucuronyl C5-epimerase expression in breast cancer.

D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/ß-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/ß-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/ß-catenin mRNA levels and activated TCF4/ß-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/ß-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and ß-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/ß-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/ß-catenin complex activity.