Antiproliferative effect of D-glucuronyl C5-epimerase in human breast cancer cells

D-glucuronyl C5-epimerase (GLCE) is one of the key enzymes responsible for biosynthesis of the carbohydrate part of heparan sulfate proteoglycans (HSPGs) - complex protein-carbohydrate molecules localized on the cell surface and in extracellular matrix (ECM). HSPGs interact with numerous ligands, including many growth factors, cytokines, receptors and extracellular matrix molecules and mediate cell signaling events controlling migration, proliferation and differentiation [1‐4]. Abnormal expression or deregulated function of these proteoglycans crucially affect cell-cell and cell-matrix interactions and promote different pathologies including malignant transformation [5, 6].

In many cases, the structure of the heparan sulfate (HS) polysaccharide chains is a major determinant of HSPGs function [7]. Changes in expression of heparan sulfates, as well as of enzymes involved in their biosynthesis and degradation, contribute to different steps of tumour progression [8] and study of the heparan sulfate biosynthesis system has a critical importance since its defect affects all HSPGs synthesised by the cell [5].

One of the key enzymes of HS biosynthesis is D-glucuronyl C5-epimerase that is responsible for epimerization of D-glucuronyl residue (D-GlcUA) into L-iduronyl residue (L-IdoUA) in HS carbohydrate chains [9]. Up to date, there are not so many data concerning mammalian D-glucuronyl C5-epimerase and no data on human GLCE. The gene was cloned from bovine lung [10], mouse liver [11] and mouse mastocytoma cells [12]; knockdown of a murine glucuronyl C5-epimerase gene resulted in neonatal lethality of experimental animals [13]. It was shown that the gene is involved in the embryonic development of Danio rerio [14] and its expression is regulated via beta-catenin-TCF4 transactivation pathway [15].

Some indirect data also support an importance of GLCE in cell physiology - a presence of flexible IdoUA residues in HS is necessary for the interaction of heparan sulfates with FGF2 and subsequent cell signaling [16] and for the interaction of hepatocyte growth factor/scatter factor with its signaling receptor MET [17].

Our previous data on significant down-regulation of GLCE expression in human breast tumours suggested a possible involvement of the gene in carcinogenesis [18, 19].

We hypothesized that GLCE expression could be involved in regulation of breast cancer cell proliferation through the changed structure/composition of cell surface heparan sulfates and tumour microenvironment. To test this hypothesis, we ectopically expressed D-glucuronyl C5-epimerase in MCF7 breast cancer cells at the physiological level and analyzed a proliferative activity and viability of the epimerase-expressing cells as well as possible molecular mechanisms of the functional effect of GLCE in vitro.

The primary aim of this study was to investigate a possible interrelation between D-glucuronyl C5-epimerase expression and proliferative activity of human breast cancer cells. The obtained results support the hypothesis that GLCE re-expression in human breast cancer cells MCF7 indeed suppresses their proliferation in vitro. It is not something unexpected - tumour-suppressor function was shown for another HS-synthesizing enzyme EXT1, which is responsible for the initial step of heparan sulfate (HS) chain elongation [20]. It was shown that epigenetic inactivation of EXT1, leading to an abrogation of HS biosynthesis, is common for leukemia and non-melanoma skin cancer, and reintroduction of EXT1 into HS-deficient leukemia cells HL-60 suppressed colony formation and growth of tumour xenografts [20]. Ext1 mutation in mouse embryonic fibroblasts resulted in substantially reduced HS chains length, reduced ability to attach to collagen I and impaired FGF2 signaling that also could be rescued by reintroduction of Ext1[21]. In our target cell line MCF7, EXT1 is actively expressed and HS chains are present in the cells [20] but GLCE expression is almost abrogated. It suggests a decreased IdoUA content in heparan sulfate chains that is critically important for interaction of the HS with growth factors and appropriate cell signaling [16, 17]. The changes in HS structure could influence, in turn, a gene expression profile of the cells and various molecular mechanisms involved in the regulation of cell proliferation.

In our in vitro experimental system, re-expression of D-glucuronyl C5-epimerase in breast cancer cells MCF7 significantly (3-8 fold) increased expression level of the breast tumour suppressor genes BRCA1 [22] and SYK (Spleen Tyrosine Kinase) [23] which are known as important inhibitors of breast cancer cell growth and metastasis. Other cancer-related genes influenced by the ectopic expression of GLCE were transcription factors p53 and E2F1 that are key molecules of the INK4A-Rb-E2f and ARF-MDM2-p53 pathways and pivotal regulators of cell proliferation and viability. Recent data reveal that there is extensive crosstalk between the pathways and in particular between the transcription factors E2F1 and p53 which cooperate in inhibition of tumourigenesis by activation of many pro-apoptotic genes and inducing cell death [24]. From the other side, E2F-containing protein complexes mediate p53-induced growth arrest and senescence indicating the contradictory role of E2F1 in tumourigenesis that is context dependent and tissue specific [24].

In this study, the ectopic expression of human D-glucuronyl C5-epimerase in MCF7 cells increased the expression of both p53 and E2F1 but the expression of almost all tested apoptosis-involved genes were unaffected and viability of the epimerase-expressing MCF7 cells were even slightly higher that the control ones (Fig.3). It suggests that GLCE-activated p53 and E2F1 expression more likely affects molecular pathways promoting growth arrest and senescence rather than apoptotic functions of both p53 and E2F1, like their potentially crucial regulator SIRT1 [24]. The hypothesis is also supported by an increased expression of the anti-apoptotic genes BCL2 [25], TNF and transcriptional factor NF-kappaB which protects breast cancer cells from apoptosis [26].

Taken together, these data suggest that up-regulation of the expression of the tumour-suppressor genes (р53, BRCA1, SYK) and transcription factor E2F1 could be responsible for the antimitotic effect of D-glucuronyl C5-epimerase in human breast cancer cells in vitro.

Another important observation was that ectopic D-glucuronyl C5-epimerase expression led to significant change of the expression of genes involved in angiogenesis (IL8, IFNB1, TNF) and invasion/metastasis (SYK, NME1, S100A4, TGFβ). Among them, there are both microenvironment-dependent regulators of tumour initiation, progression and metastasis like TGFβ that has diverse and often conflicting roles in tumour progression [27] and tumour- and metastasis-suppressor gene SYK [23], metastasis suppressor gene NME1 [28] and metastasis-associated gene S100A4 (MTS1, metastasin) [29].

Interestingly, all those changes have a clearly anti-metastatic trend - re-expression of GLCE in breast cancer cells MCF7 up-regulates the expression of suppressor genes SYK and NME1 and down-regulates the expression of metastasis marker S1004A and TGFβ. It is not something completely unexpected since the process of metastasis is dependent on adhesion properties of cells and their interaction with tumour microenvironment, which are determined in significant extent by the structure and composition of heparan sulfates at ECM and cell surface. From the obtained data, we can hypothesize that the expression of D-glucuronyl C5-epimerase in breast cancer cells not only inhibits their proliferation in vitro but could suppress tumour growth and metastasis in vivo through the influence on cell microenvironment and tumour angiogenesis, which could be one of the future perspectives to study.

In summary, the obtained results showed that D-glucuronyl C5-epimerase significantly suppress proliferative activity of breast cancer cells in vitro through the activation of tumour suppressor genes р53, BRCA1, SYK and E2F1 and change of a balance of pro- and apoptotic factors BCL2, NFKB1 and TNF. The ability of D-glucuronyl C5-epimerase to affect simultaneously several different key genes of the cell cycle regulation (р53, E2F1, BRCA1), angiogenesis (IL8, IFNB1, TNF, TGFB1) and metastasis (SYK, NME1, S100A4, TGFb) supports the idea on the involvement of the gene in regulation of breast cancer cell proliferation.