Heparanase Enhances Syndecan-1 Shedding
When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth. When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth. Heparanase is an endo-β-d-glucuronidase that releases 5–7 kDa fragments of heparan sulfate from intact heparan sulfate chains of proteoglycans and is known to have multiple important roles in promoting tumor growth, angiogenesis, and metastasis (1Ilan N. Elkin M. Vlodavsky I. Int. J. Biochem. Cell Biol. 2006; 38: 2018-2039Crossref PubMed Scopus (462) Google Scholar). In general, it is thought that the enzymatic activity of heparanase is necessary for remodeling of the extracellular matrix and particularly the subendothelial basement membrane of endothelial cells prior to their migration during angiogenesis (1Ilan N. Elkin M. Vlodavsky I. Int. J. Biochem. Cell Biol. 2006; 38: 2018-2039Crossref PubMed Scopus (462) Google Scholar). Heparanase can also liberate a number of heparan sulfate-bound pro-angiogenic growth factors (e.g. fibroblast growth factor-2, vascular endothelial growth factor (VEGF) 2The abbreviations used are: VEGF, vascular endothelial growth factor; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline. ) from the extracellular matrix to indirectly enhance endothelial cell migration and proliferation (2Elkin M. Ilan N. Ishai-Michaeli R. Friedmann Y. Papo O. Pecker I. Vlodavsky I. FASEB J. 2001; 15: 1661-1663Crossref PubMed Scopus (285) Google Scholar, 3Vlodavsky I. Miao H.Q. Medalion B. Danagher P. Ron D. Cancer Metastasis Rev. 1996; 15: 177-186Crossref PubMed Scopus (270) Google Scholar). Fragments of heparan sulfate generated by heparanase retain biological activity and can act as potent promoters of growth factor activity (2Elkin M. Ilan N. Ishai-Michaeli R. Friedmann Y. Papo O. Pecker I. Vlodavsky I. FASEB J. 2001; 15: 1661-1663Crossref PubMed Scopus (285) Google Scholar, 4Kato M. Wang H. Kainulainen V. Fitzgerald M.L. Ledbetter S. Ornitz D.M. Bernfield M. Nat. Med. 1998; 4: 691-697Crossref PubMed Scopus (287) Google Scholar). In addition, heparanase has functions not related to its enzymatic activity. For example, VEGF expression is up-regulated 3–6-fold in several heparanase-transfected tumor cell lines. This effect does not require the active form of the enzyme and occurs via the Src pathway (5Zetser A. Bashenko Y. Edovitsky E. Levy-Adam F. Vlodavsky I. Ilan N. Cancer Res. 2006; 66: 1455-1463Crossref PubMed Scopus (216) Google Scholar). The non-enzymatic form of heparanase can also promote cell adhesion (6Goldshmidt O. Zcharia E. Cohen M. Aingorn H. Cohen I. Nadav L. Katz B.Z. Geiger B. Vlodavsky I. FASEB J. 2003; 17: 1015-1025Crossref PubMed Scopus (169) Google Scholar) and Akt signaling as well as phosphoinositol 3-kinase- and p38-dependent endothelial cell migration and invasion (7Gingis-Velitski S. Zetser A. Flugelman M.Y. Vlodavsky I. Ilan N. J. Biol. Chem. 2004; 279: 23536-23541Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Thus, heparanase clearly can impact tumor progression via both enzymatic and non-enzymatic mechanisms. Heparan sulfate binds to and regulates the activity of many effector molecules capable of initiating signaling pathways critical to cancer inception, growth, and progression (e.g. growth factors, chemokines, angiogenic factors) (8Sasisekharan R. Shriver Z. Venkataraman G. Narayanasami U. Nat. Rev. Cancer. 2002; 2: 521-528Crossref PubMed Scopus (555) Google Scholar). Heparan sulfate is prevalent in many cancers and particularly abundant in multiple myeloma, where most of the tumor cells express the proteoglycan on the cell surface (9Sanderson R.D. Borset M. Ann. Hematol. 2002; 81: 125-135Crossref PubMed Scopus (63) Google Scholar). In addition to expression on the cell surface, syndecan-1 is shed from the surface of the tumor cells as an intact ectodomain bearing the extracellular portion of the core protein with attached heparan sulfate chains. Shed syndecan-1 can become lodged and incorporated into the bone marrow extracellular matrix that supports the tumor or remain as a soluble component within the bone marrow plasma. In addition, shed syndecan-1 accumulates in the sera of myeloma patients, where high serum levels of syndecan-1 reflect a high tumor burden and predict poor prognosis (10Dhodapkar M.V. Kelly T. Theus A. Athota A.B. Barlogie B. Sanderson R.D. Br. J. Haematol. 1997; 99: 368-371Crossref PubMed Scopus (102) Google Scholar, 11Seidel C. Sundan A. Hjorth M. Turesson I. Dahl I.M. Abildgaard N. Waage A. Borset M. Blood. 2000; 95: 388-392Crossref PubMed Google Scholar). Recently, we demonstrated that both soluble syndecan-1 and heparanase can promote myeloma tumor growth and metastasis in vivo (12Yang Y. MacLeod V. Bendre M. Huang Y. Theus A.M. Miao H.Q. Kussie P. Yaccoby S. Epstein J. Suva L.J. Kelly T. Sanderson R.D. Blood. 2005; 105: 1303-1309Crossref PubMed Scopus (120) Google Scholar, 13Yang Y. Yaccoby S. Liu W. Langford J.K. Pumphrey C.Y. Theus A. Epstein J. Sanderson R.D. Blood. 2002; 100: 610-617Crossref PubMed Scopus (163) Google Scholar). These results suggest that syndecan-1 and heparanase acting together serve as master regulators of the myeloma tumor microenvironment within the bone marrow niche by binding to and regulating the activity of multiple growth-promoting factors whose actions are key to the progression of this cancer (14Sanderson R.D. Yang Y. Kelly T. MacLeod V. Dai Y. Theus A. J. Cell. Biochem. 2005; 96: 897-905Crossref PubMed Scopus (132) Google Scholar, 15Sanderson R.D. Yang Y. Suva L.J. Kelly T. Matrix Biol. 2004; 23: 341-352Crossref PubMed Scopus (174) Google Scholar). We now report the unexpected finding that heparanase acts on tumor cells to regulate both the amount and location of syndecan-1 within tumors by enhancing synthesis and shedding of the proteoglycan. Thus, in addition to its role in remodeling tumor extracellular matrix and releasing fragments of heparan sulfate, heparanase also enhances levels of the soluble, tumor-promoting form of syndecan-1. This novel function of heparanase further underscores the importance of the interplay between the syndecan-1 heparan sulfate proteoglycan and heparanase in driving aggressive tumor growth and metastasis. Cells and Transfections—CAG cells were established from a bone marrow aspirate of a myeloma patient at Arkansas Cancer Research Center as previously described (16Borset M. Hjertner O. Yaccoby S. Epstein J. Sanderson R.D. Blood. 2000; 96: 2528-2536Crossref PubMed Google Scholar). ARH-77 cells were obtained from the American Type Culture Collection (Manassas, VA). MDA-MET cells were derived from MDA-MB-231 human breast adenocarcinoma cells based on their ability to home to and grow in bone (17Bendre M.S. Gaddy-Kurten D. Mon-Foote T. Akel N.S. Skinner R.A. Nicholas R.W. Suva L.J. Cancer Res. 2002; 62: 5571-5579PubMed Google Scholar). CAG and ARH-77 cells were grown in RPMI medium supplemented with 10% fetal bovine serum. MDA-MET cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. For karyotyping of ARH-77 cells (wild-type, control-transfected, or heparanase-transfected), the cells growing in culture were exposed to colcemid for 1 h, washed with alkaline Pucks saline solution, and treated with trypsin to remove the cells from the flasks. The cells were treated in hypotonic solution (0.05 m KCl) and fixed in Carnoy’s fixative (3:1 methanol:acetic acid). Cell suspensions were dropped onto slides and trypsin G-banded. Ten G-banded metaphase cells were counted and three cells karyotyped for each cell line. For transfections, human heparanase cDNA was subcloned in the sense direction into pcDNA3 or pIRES2-enhanced green fluorescent protein vectors (Clontech, Palo Alto, CA) and stably transfected into the above-described cell lines using Lipofectin (Invitrogen) and Opti-MEM I (Invitrogen) to the cells were by growth in by for green fluorescent cells by For of vectors in the enzyme active of heparanase, were generated at or J. C. 2000; PubMed Scopus Google Scholar) using a with were using and the of of were by and Cells in culture were by washed with of PBS, in of 1 and on for were at at for and from cell lines were by a protein For heparanase of protein were onto to with heparanase H.Q. E. S. D. H. H. A. D. P. Kussie P. 2002; PubMed Scopus Google and by a For syndecan-1 were on to a membrane and with syndecan-1 and a were detected using For cell suspensions were washed in and with to human syndecan-1 or were using a For of ARH-77 the levels of a of and were using to or Cells were from washed in PBS, and at with or cells were fixed in and by of of cells were in and in the levels of shed syndecan-1 in the medium were by enzyme-linked immunosorbent using an from The was between and and were to within that For cells were on a in RPMI medium with the subsequent addition of or human The cells were for at in with addition of heparanase or of cell and the addition of heparanase, of cell suspensions were from each to remove and the at prior to of syndecan-1 levels by of the the cells were fixed with and the level of syndecan-1 was by using human syndecan-1 the effects of removal of heparan sulfate chains on syndecan-1 shedding, or heparanase-transfected cells in RPMI medium were on a and of bacterial heparitinase or was and cells at heparitinase or was each during the of and of medium at h, the medium was the cells fixed in and the level of syndecan-1 by of syndecan-1 in medium were by as described were also on ARH-77 cells using bovine heparan sulfate with or with heparanase or Cells were as described for levels of syndecan-1 at the cell surface or in the medium and addition of the heparan levels were also with or the addition for of the Src levels in sera from bearing tumors formed by CAG cells or CAG cells transfected with heparanase (12Yang Y. MacLeod V. Bendre M. Huang Y. Theus A.M. Miao H.Q. Kussie P. Yaccoby S. Epstein J. Suva L.J. Kelly T. Sanderson R.D. Blood. 2005; 105: 1303-1309Crossref PubMed Scopus (120) Google Scholar) were also by and by the level of serum a of tumor was on the and the at was with an levels of syndecan-1 in heparanase transgenic and sera from animals were to a of m and onto a membrane were with syndecan-1 CA) and using a For was from or heparanase-transfected CAG or ARH-77 cells using the expression was with the CA) using previously described F. Huang Y. S. I. S. Epstein J. Yaccoby S. J. B. E. M. G. F. R. M. J. Barlogie B. Blood. 2006; PubMed Scopus Google Scholar). of heparan sulfate proteoglycan levels was with Heparanase the and of we demonstrated that of the for human heparanase into CAG myeloma cells enhances tumor growth and metastasis in vivo compared with (12Yang Y. MacLeod V. Bendre M. Huang Y. Theus A.M. Miao H.Q. Kussie P. Yaccoby S. Epstein J. Suva L.J. Kelly T. Sanderson R.D. Blood. 2005; 105: 1303-1309Crossref PubMed Scopus (120) Google Scholar). of the heparanase-transfected and CAG cells heparanase was levels of syndecan-1 that of not this to levels of cell surface cells were and by the in in cells expressing heparanase, cell surface levels of syndecan-1 were to that of of medium cells that the level of shed syndecan-1 is in heparanase-transfected cells of heparanase-transfected MDA-MET breast cancer cells a in syndecan-1 shedding as compared with suggesting that the effect in myeloma cells is not cell the syndecan-1 shedding requires the activity of the heparanase enzyme, CAG cells expressing heparanase mutated at the active of the enzyme were generated at or that the mutated forms of heparanase are as activity that heparan sulfate-degrading activity as compared with When cells expressing mutated heparanase were both the cell surface syndecan-1 and shed syndecan-1 levels were to of cells indicating that heparanase activity is to promote synthesis and shedding of syndecan-1 in an of heparanase effects on syndecan-1 shedding, we heparanase to CAG cells growing in a accelerated shedding was and by h, the amount of shed syndecan-1 that in the medium was that of cells not exposed to heparanase at the levels of cell surface syndecan-1 on cells of This is heparanase levels are by cell and a role for heparanase in promoting syndecan-1 shedding. of Heparan of from the Cell of heparanase-transfected CAG cells growing in or growing as tumors in vivo Y. MacLeod V. Bendre M. Huang Y. Theus A.M. Miao H.Q. Kussie P. Yaccoby S. Epstein J. Suva L.J. Kelly T. Sanderson R.D. Blood. 2005; 105: 1303-1309Crossref PubMed Scopus (120) Google Scholar) that the size of syndecan-1 is as compared with this is to the effect of heparanase enzymatic activity that heparan sulfate chains. Thus, the mechanism for syndecan-1 shedding on the heparanase be related to a reduction in the amount of heparan sulfate on the proteoglycan. this the CAG cells were exposed to bacterial heparitinase to heparan sulfate chains from the cell surface. This a of syndecan-1 shedding that was as as the addition of the enzyme h, shedding in cells was elevated as compared with the cells not exposed to effect was on heparanase-transfected the addition of cells exhibited a in syndecan-1 shedding cells the in the of the between and of This shedding in the of in the level of cell surface syndecan-1 Heparanase of in a Cell further the between heparanase and syndecan-1 ARH-77 cells human cell lacking syndecan-1 were transfected with the cDNA for human This in a of syndecan-1 cell surface the cell from to This from to was also in a of ARH-77 cells using a the heparanase cDNA of this unexpected of expression of syndecan-1 by a previously cell we the cell to the of cell that ARH-77 and heparanase-transfected cells the presence of a of and to the ARH-77 cell by with cell surface and that both and heparanase-transfected cells the levels of as are detected on the ARH-77 cells Thus, the of syndecan-1 expression that occurs ARH-77 cells express heparanase is not by a in cell The on CAG cells suggests that the mechanism for of syndecan-1 expression may be related to the of heparan sulfate chains by because the ARH-77 cells not express syndecan-1 prior to their expression of heparanase, it the that other heparan sulfate proteoglycans be on cells by enzyme, syndecan-1 of the ARH-77 cells to be of expression of the extracellular proteoglycan as well as of the and For the other of cell surface heparan sulfate the and was a on the for and this level of proteoglycan by enzyme promote syndecan-1 the ARH-77 cells were treated with heparanase or When for syndecan-1 it was that the cells of to enzyme not a further bovine heparan sulfate was treated with heparanase or heparitinase and to ARH-77 When at and the addition of the heparan sulfate of the cells for syndecan-1 expression not that the of syndecan-1 expression in the ARH-77 cells is not to the heparan sulfate-degrading activity of of Heparanase in of Shed a we demonstrated that tumors formed from CAG cells expressing high levels of heparanase grow and to bone CAG tumors expressing levels of heparanase (12Yang Y. MacLeod V. Bendre M. Huang Y. Theus A.M. Miao H.Q. Kussie P. Yaccoby S. Epstein J. Suva L.J. Kelly T. Sanderson R.D. Blood. 2005; 105: 1303-1309Crossref PubMed Scopus (120) Google Scholar). the levels of syndecan-1 shed by we serum from that tumors formed from heparanase-transfected or syndecan-1 levels were in animals with tumors formed from heparanase-transfected a finding with the that animals a tumor burden animals bearing tumors from for this in tumor levels of serum syndecan-1 were to the tumor burden by the syndecan-1 by the level of serum a well established of tumor burden in this Y. Yaccoby S. Liu W. Langford J.K. Pumphrey C.Y. Theus A. Epstein J. Sanderson R.D. Blood. 2002; 100: 610-617Crossref PubMed Scopus (163) Google Scholar). for tumor a of syndecan-1 levels in the sera of animals bearing tumors expressing heparanase as compared with animals bearing tumors the elevated shedding of syndecan-1 in to heparanase expression was to tumor cells or be we sera from the for compared with sera from heparanase transgenic have levels of syndecan-1 The that human heparanase, an important of tumor the expression and shedding of a proteoglycan known to promote the growth and metastasis of tumors. This is a novel and important because it a new mechanism by which heparanase its well results that expression of heparanase enhances the synthesis and shedding of expression of heparanase can syndecan-1 expression in a cell for the effect of heparanase on syndecan-1 shedding requires the active form of the enzyme, and the regulation of syndecan-1 by heparanase to tumors growing in vivo and to that soluble syndecan-1 can promote myeloma tumor growth, angiogenesis, and metastasis, the finding that heparanase promotes syndecan-1 synthesis and shedding suggests that molecules act synergistically to and tumor the mechanism of regulation of syndecan-1 is not from the ARH-77 cells is because it heparanase has a effect on initiating syndecan-1 Heparanase can enhance VEGF expression via of Src (5Zetser A. Bashenko Y. Edovitsky E. Levy-Adam F. Vlodavsky I. Ilan N. Cancer Res. 2006; 66: 1455-1463Crossref PubMed Scopus (216) Google Scholar). in both heparanase-transfected ARH-77 and CAG the Src failed to of syndecan-1 expression a not This suggests that the mechanism to the of syndecan-1 expression is not the as the mechanism that enhances VEGF effect of heparanase on syndecan-1 expression is also by the finding that ARH-77 cells heparan sulfate proteoglycan expression and not expression in to heparanase or the addition to cells of heparan sulfate that by heparanase or heparitinase not syndecan-1 Thus, the of heparanase in syndecan-1 expression in ARH-77 cells is not heparan This may also be the with the CAG cell which in to the ARH-77 syndecan-1 prior to with the for because heparanase or heparitinase the shedding of syndecan-1 from CAG cells and we the in cells expressing is a that syndecan-1 expression shedding is This occur in cells to a level of the proteoglycan on the cell surface. is that both a of syndecan-1 expression and an in to shedding, to the effects in CAG of the mechanism that syndecan-1 the critical of the is as heparanase levels in syndecan-1 shedding This an important new mechanism by which heparanase promotes tumor on from myeloma that a high level of shed syndecan-1 in the serum is an of high tumor burden and poor prognosis (10Dhodapkar M.V. Kelly T. Theus A. Athota A.B. Barlogie B. Sanderson R.D. Br. J. Haematol. 1997; 99: 368-371Crossref PubMed Scopus (102) Google Scholar, 11Seidel C. Sundan A. Hjorth M. Turesson I. Dahl I.M. Abildgaard N. Waage A. Borset M. Blood. 2000; 95: 388-392Crossref PubMed Google Scholar) and that high heparanase enzyme activity in the bone marrow of myeloma with high tumor which is also an of poor prognosis T. Miao H.Q. Yang Y. E. Kussie P. Huang Y. MacLeod V. J. L. F. M. Barlogie B. J. Sanderson R.D. Cancer Res. 2003; Google Scholar). in vivo of myeloma, we have demonstrated that of expression of soluble syndecan-1 or heparanase promotes tumor growth, angiogenesis, and metastasis (12Yang Y. MacLeod V. Bendre M. Huang Y. Theus A.M. Miao H.Q. Kussie P. Yaccoby S. Epstein J. Suva L.J. Kelly T. Sanderson R.D. Blood. 2005; 105: 1303-1309Crossref PubMed Scopus (120) Google Scholar, 13Yang Y. Yaccoby S. Liu W. Langford J.K. Pumphrey C.Y. Theus A. Epstein J. Sanderson R.D. Blood. 2002; 100: 610-617Crossref PubMed Scopus (163) Google Scholar). finding that heparanase is at least in part, for the of syndecan-1 shedding in tumors that this may be an important mechanism by which heparanase promotes tumor growth. This is further by the that shed syndecan-1 in the is elevated in animals bearing tumors formed from cells expressing high levels of heparanase the that have elevated syndecan-1 in their serum that heparanase may also regulate syndecan-1 in pathological that are in Heparanase transgenic a number of growth and of accelerated of growth, bone and bone and to of E. S. T. Aingorn H. Elkin M. Friedmann Y. T. U. Vlodavsky I. FASEB J. 2004; PubMed Scopus Google Scholar, E. D. Edovitsky E. Aingorn H. S. Vlodavsky I. Elkin M. J. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, V. Zcharia E. O. S. T. Y. R. Vlodavsky I. I. J. Cell. 2006; PubMed Scopus Google Scholar, M.L. F. Zcharia E. S. Vlodavsky I. R. U. U. S. 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Med. 1998; 4: 691-697Crossref PubMed Scopus (287) Google Scholar). The role between heparanase and heparan sulfate in driving angiogenesis and other their key role in regulating that and other The finding that heparanase promotes syndecan-1 expression and shedding new into heparanase promotes an aggressive tumor phenotype and for heparanase as a cancer We Suva of Arkansas for for of MDA-MET with