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

Abstract

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.

Tissue-specificity of heparan sulfate biosynthetic machinery in cancer

“…It was shown that the expression levels of exostosin proteins EXT1 and EXT2 oppositely affect the amount of glucosaminyl N-deacetylase/N-sulfotransferase NDST1 in the cell, which, in turn, greatly influences HS structure; 39 knock-out of sulfatase genes (Sulf) dynamically modulates the expression of the Hs2st and Hs6st heparan sulfate sulfotransferase genes resulting to the changes in HS sulfation and significant effects on fibroblast growth factor signaling; 40 ectopic over-expression of D-glucuronyl C5-epimerase (GLCE) results in proportional up-regulation of expression of key genes and total transcriptional activation of HS-biosynthetic machinery in prostate cancer cells. 33 The presented results support an existence of the self-regulating mechanism for the coordinated transcription of HS metabolism-involved genes. As to cancer-related changes of the HS biosynthetic machinery, the obtained data show tissue-specific changes in total expression level and transcriptional pattern of the system in different tumors.…”

“…38 An interesting aspect of the results is that the balance between key genes was maintained in all normal tissues, in spite of 1,5-fold difference in total transcriptional activities, supporting an existence of molecular mechanism tightly coordinating the expression of the HS biosynthesis-involved genes. 33 The mechanism seems include feedback loop, where any gene is able to appropriately coordinate an expression of other gene(s). It was shown that the expression levels of exostosin proteins EXT1 and EXT2 oppositely affect the amount of glucosaminyl N-deacetylase/N-sulfotransferase NDST1 in the cell, which, in turn, greatly influences HS structure; 39 knock-out of sulfatase genes (Sulf) dynamically modulates the expression of the Hs2st and Hs6st heparan sulfate sulfotransferase genes resulting to the changes in HS sulfation and significant effects on fibroblast growth factor signaling; 40 ectopic over-expression of D-glucuronyl C5-epimerase (GLCE) results in proportional up-regulation of expression of key genes and total transcriptional activation of HS-biosynthetic machinery in prostate cancer cells.…”

“…33 Briefly, total RNA was extracted from the cells using the PureLink Total RNA Purification System (Invitrogen, USA) according to the manufacturer's instructions. cDNA was synthesized from 1-2 mg of total RNA using a First Strand cDNA Synthesis kit (Fermentas, USA) and 1/10th of the product was subjected to PCR analysis.…”

“…32 Although the obtained results were not analyzed in terms of overall configuration of GAG-biosynthetic machinery in tumors, they support a common disbalance of the system in cancer. Complex analysis of transcriptional activity of HS biosynthetic system in normal prostate tissue and tumors showed significant downregulation of total transcriptional activity of HS biosynthetic system and changes in overall expression patterns in prostate cancer, 33 whereas in colon cancer only qualitative changes in expression patterns are detected. 34 All the results suggest direct involvement of HS biosynthetic machinery in carcinogenesis and show complex changes of HS metabolic enzymes expression in tumors.…”

Methylation profiling identified novel differentially methylated markers includingOPCMLandFLRT2in prostate cancer

“…34 EXT1 expression was significantly lower in benign prostatic hyperplasia (BPH) and PCa in comparison with normal prostate tissue. 35 IRX5 has been linked to human PCa through downregulation via vitamin D3 [1,25(OH)2D3] in LNCaP cells; VitD3 is a potent inhibitor of the proliferation of many different cancer cell types. Knockdown of IRX5 resulted in an increase in p21 protein expression, G2-M arrest, and apoptosis, partially mediated by p53.…”