Hyaluronan Constitutively Regulates Activation of Multiple Receptor Tyrosine Kinases in Epithelial and Carcinoma Cells

Hyaluronan (HA) is enriched in the pericellular matrices of many malignant human tumors, and manipulations of HA interactions have strong effects on tumor progression in animal models. Increased HA production stimulates ERBB2 activation, leading to increased cell survival activities and several malignant cell properties. On the other hand, inhibition of constitutive HA-tumor cell interactions in malignant cells inhibits these properties. We have now investigated the role of HA in activation of several additional receptor tyrosine kinases (RTKs), i.e. IGF1R-β, PDGFR-β, EGFR and c-MET, in colon, prostate, and breast carcinoma cells. In each case we show that antagonists of endogenous HA interactions inhibit their tyrosine phosphorylation, i.e. activation. On the other hand, we show that these RTKs are activated in phenotypically normal or relatively benign tumor cells by experimentally increasing HA production. We also investigated the role of HA in constitutive versus ligand-induced activation of RTKs. In HCA7 colon and C4-2 prostate carcinoma cells, ERBB2 is constitutively activated in a ligand-independent manner, whereas IGF1R-β and PDGFR-β require ligand interaction for activation. We show that both constitutive activation of ERBB2 and ligand-mediated activation of IGF1R-β and PDGFR-β are reversed by co-treatment of the cells with a HA antagonist. We conclude that HA serves a general function in RTK activation. Hyaluronan (HA) is enriched in the pericellular matrices of many malignant human tumors, and manipulations of HA interactions have strong effects on tumor progression in animal models. Increased HA production stimulates ERBB2 activation, leading to increased cell survival activities and several malignant cell properties. On the other hand, inhibition of constitutive HA-tumor cell interactions in malignant cells inhibits these properties. We have now investigated the role of HA in activation of several additional receptor tyrosine kinases (RTKs), i.e. IGF1R-β, PDGFR-β, EGFR and c-MET, in colon, prostate, and breast carcinoma cells. In each case we show that antagonists of endogenous HA interactions inhibit their tyrosine phosphorylation, i.e. activation. On the other hand, we show that these RTKs are activated in phenotypically normal or relatively benign tumor cells by experimentally increasing HA production. We also investigated the role of HA in constitutive versus ligand-induced activation of RTKs. In HCA7 colon and C4-2 prostate carcinoma cells, ERBB2 is constitutively activated in a ligand-independent manner, whereas IGF1R-β and PDGFR-β require ligand interaction for activation. We show that both constitutive activation of ERBB2 and ligand-mediated activation of IGF1R-β and PDGFR-β are reversed by co-treatment of the cells with a HA antagonist. We conclude that HA serves a general function in RTK activation. Aberrant activities of several types of RTKs 3The abbreviations used are: RTK, receptor tyrosine kinase; HA, hyaluronan; o-HA, HA oligomer(s); PI3K, phosphoinositide 3-kinase; EGFR, EGF receptor; IGF1R, insulin growth factor 1 receptor; PDGFR, PDGF receptor; DMEM, Dulbecco's modified Eagle's medium; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. 3The abbreviations used are: RTK, receptor tyrosine kinase; HA, hyaluronan; o-HA, HA oligomer(s); PI3K, phosphoinositide 3-kinase; EGFR, EGF receptor; IGF1R, insulin growth factor 1 receptor; PDGFR, PDGF receptor; DMEM, Dulbecco's modified Eagle's medium; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. have been implicated in the genesis of a significant proportion of human cancers, including breast, colon, and prostate carcinomas. Increased RTK activity can arise from gene amplification, activating mutations, or altered regulation, e.g. by cross-talk between RTKs, integrins, and other receptors or by autocrine and paracrine stimulation by various regulatory factors. These changes lead in turn to enhanced tumor cell growth, motility, survival, and resistance to therapies (1Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3073) Google Scholar, 2Skorski T. Nat. Rev. Cancer. 2002; 2: 351-360Crossref PubMed Scopus (111) Google Scholar, 3Gschwind A. Fischer O.M. Ullrich A. Nat. Rev. Cancer. 2004; 4: 361-370Crossref PubMed Scopus (962) Google Scholar, 4Krause D.S. Van Etten R.A. N. Engl. J. Med. 2005; 353: 172-187Crossref PubMed Scopus (1149) Google Scholar). HA is a very large, anionic polysaccharide that is localized at the cell surface and in the extracellular matrix of numerous tissues, wherein it plays an important structural role under homeostatic conditions. However, when cells proliferate or migrate, e.g. in embryonic processes, tissue remodeling, inflammation, and diseases such as cancer and atherosclerosis, HA-induced signaling is activated (5Toole B.P. Wight T.N. Tammi M. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 6Yasuda M. Nakano K. Yasumoto K. Tanaka Y. Histol. Histopathol. 2002; 17: 945-950PubMed Google Scholar, 7Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1603) Google Scholar, 8Mummert M.E. Immunol. Res. 2005; 31: 189-206Crossref PubMed Scopus (106) Google Scholar, 9Hascall V.C. Majors A.K. De La Motte C.A. Evanko S.P. Wang A. Drazba J.A. Strong S.A. Wight T.N. Biochim. Biophys. Acta. 2004; 1673: 3-12Crossref PubMed Scopus (226) Google Scholar, 10Adamia S. Maxwell C.A. Pilarski L.M. Curr. Drug Targets Cardiovasc. Haematol. Disord. 2005; 5: 3-14Crossref PubMed Scopus (137) Google Scholar). High levels of HA in tumors are a prognostic factor in several malignancies (5Toole B.P. Wight T.N. Tammi M. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar), and manipulations of HA production or interaction with cell surface receptors strongly influence tumor growth and metastasis (7Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1603) Google Scholar, 10Adamia S. Maxwell C.A. Pilarski L.M. Curr. Drug Targets Cardiovasc. Haematol. Disord. 2005; 5: 3-14Crossref PubMed Scopus (137) Google Scholar). CD44 is a major cell surface receptor for HA and also plays an important role in tumor progression (11Turley E.A. Noble P.W. Bourguignon L.Y. J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 12Naor D. Nedvetzki S. Golan I. Melnik L. Faitelson Y. Crit. Rev. Clin. Lab. Sci. 2002; 39: 527-579Crossref PubMed Scopus (436) Google Scholar). We and others have shown that interaction of HA with CD44 induces formation of complexes containing CD44 and ERBB2 or EGFR in a variety of tumor cells (13Bourguignon L.Y. Zhu H. Zhou B. Diedrich F. Singleton P.A. Hung M.C. J. Biol. Chem. 2001; 276: 48679-48692Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 14Tsatas D. Kanagasundaram V. Kaye A. Novak U. J. Clin. Neurosci. 2002; 9: 282-288Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 15Wobus M. Rangwala R. Sheyn I. Hennigan R. Coila B. Lower E.E. Yassin R.S. Sherman L.S. Appl. Immunohistochem. Mol. Morphol. 2002; 10: 34-39Crossref PubMed Scopus (53) Google Scholar, 16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). We have shown that HA constitutively regulates formation of a signaling complex containing phosphorylated ERBB2, CD44, ezrin, PI3K, and the chaperone molecules HSP90 and CDC37 (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 17Misra S. Ghatak S. Toole B.P. J. Biol. Chem. 2005; 280: 20310-20315Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). In this work we have used various antagonists of interactions of HA with CD44. These antagonists include HA oligomers that compete for endogenous HA interactions, soluble HA-binding proteins that also act as competitive inhibitors, and RNA interference silencing of CD44. Each of these antagonists inhibits ERBB2 activation and formation of the ERBB2-containing complex in malignant colon and breast cancer cells, whereas experimentally increased HA production induces activation and assembly of this complex (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). These manipulations of HA lead to alterations in cell survival pathways, anchorage-independent growth, invasiveness, epithelial-mesenchymal transition, drug sensitivity, and tumor progression in animal models (7Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1603) Google Scholar). In this study, we show that HA has a general effect on RTK activation. In malignant colon, prostate, and breast carcinoma cells, HA antagonists inhibit activation of multiple RTKs, including ERBB2, EGFR, IGF1R-β, PDGFR-β, and c-MET, as well as assembly of signaling complexes containing these activated RTKs. Increased HA production, however, induces RTK activation and signaling complex assembly in phenotypically normal epithelial cells. We find that these manipulations of HA affect both ligand-dependent and constitutive RTK activation. Therefore, we conclude that HA plays an intrinsic role in RTK-induced oncogenic pathways. Materials—HA oligomers were prepared as described previously (18Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2002; 277: 38013-38020Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Mouse monoclonal antibody against human CD44 (HCAM; 1:2000), rabbit polyclonal antibodies against PDGFR-β (P-20), and PI3K/p110α (H-201;1:2000) were from Santa Cruz Biotechnology (Santa Cruz, CA). Rat monoclonal anti-CD44 (1:2000) was from EMD Biosciences (La Jolla, CA). Rabbit polyclonal antibodies against ERBB2 (1:3000), phospho-ERBB2 (Y1248; 1:3000), phospho-MET (Y1234, Y1235; 1:3000) and phospho-PDGFR-β (Tyr716) (1:1000), and mouse monoclonal antibody against phospho-IGF-1R (Tyr-1131; clone JY202) were from Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase-linked sheep anti-rabbit and goat anti-mouse antibodies were purchased from Amersham Biosciences. Galardin (GM6001) and its negative control, adenine, biotin, and triiodothyronine were from EMD Biosciences. DMEM (high glucose), RPMI 1640, Ham's F-12, penicillin/streptomycin, glutamine, Versene (1:5000), insulin, insulin-transferrin-selenium-G supplement, and human recombinant EGF were from Invitrogen. Recombinant human PDGF-BB was purchased from BD Biosciences, (Bedford, MA). IGF1 was purchased from Sigma. All other reagents were of analytical grade and purchased from Sigma. Cell Culture—LNCaP and C4-2 human prostate carcinoma cells were from Dr. C. Vokel-Johnson, Medical University of South Carolina, Charleston, SC. LNCaP cells were grown in RPMI 1640 medium, 200 mm pyruvate, 0.15 g/ml sodium bicarbonate, 0.45 g/ml glucose, 10 mm HEPES buffer, pH 7.4, 5 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 10% fetal bovine serum. C4-2 cells were grown in Ham's F12/DMEM (20:80 v/v), 1× insulin-transferrin-selenium-G supplement, 13.6 ng/ml triiodothyronine, 25 ng/ml biotin, 2.5 ng/ml adenine, 100 units/ml penicillin, 100 μg/ml streptomycin, 1 mm glutamine, and 10% fetal bovine serum. PC-3 and DU145 cells (ATCC, Manassas, VA) were grown in RPMI 1640 medium, Glutamax 1 plus 10% fetal bovine serum. MCF-7/Adr drug-resistant human mammary carcinoma cells (K. Cowan, University of Nebraska) and MCF-7 cells (C. Sonnenschein, Tufts University, Boston, MA) were grown in the same medium as PC-3 and DU145 cells. HCA7 (Colony 29) human colon carcinoma cells (European Collection of Cell Cultures) were maintained in DMEM, 10% fetal bovine serum, 2 mm glutamine, 11 mg/ml pyruvate. HT29 cells (ATCC) were grown in McCoy's 5 a medium, 1.5 mm l-glutamine, 2.2 g/liter sodium bicarbonate, 10% fetal bovine serum. IEC6 rat intestinal epithelial cells (ATCC) were grown in DMEM with 4 mm l-Glutamine, 4.5 g/liter glucose, 1.5 g/L Na bicarbonate, 100 units/liter bovine insulin, 10% fetal bovine serum. HIEC-6 human intestinal cells (J.-F. Beaulieu, University of Sherbrook, Quebec, Canada) were maintained in DMEM (high glucose), 4% fetal bovine serum, 20 mm HEPES buffer, pH 7.4, 50 units/ml penicillin, 50 μg/ml streptomycin, 10 μg/ml insulin, 5 ng/ml human recombinant EGF (19Perreault N. Jean-Francois B. Exp. Cell Res. 1996; 224: 354-364Crossref PubMed Scopus (132) Google Scholar) and used between 15th and 17th passage in this study. Cell lines were grown at 37 °C in 5% CO2 and passed every 3–4 days. RNA Silencing and HAS2 Adenovirus—siRNA for human CD44 and the recombinant HAS2 adenovirus were produced and employed as described previously (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Preparation of Cell Lysates—After treatment, cells were washed twice in cold phosphate-buffered saline and then harvested in Versene with gentle pipetting at 37 °C for 15 min, followed by washing twice in cold phosphate-buffered saline. The cell pellet was treated with lysis buffer containing 1% Nonidet P-40, 0.5 mm EGTA, 5 mm sodium orthovanadate, 10% (v/v) glycerol, 100 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 50 mm HEPES, pH 7.5. The suspension was vortexed for three cycles of 15 s at maximum speed followed by cooling on ice. The lysate was centrifuged at 12,000 × g for 15 min at 4 °C in an Eppendorf 5415R centrifuge. An aliquot of the extract was diluted with 0.5% SDS and protein concentration measured. The remaining supernatant was frozen and stored at –80 °C. Immunoprecipitations—All procedures were done at 4 °C unless otherwise mentioned. Cell lysates (500 μl; 1 μg of protein/μl) were mixed with 5 μg of antibody against ERBB2, EGFR, c-MET, IGF1R-β, or PDGFR-β and incubated for 2 h while rotating on a wheel. The immune complexes were captured by incubating with 80 μl of 1:1 (v/v) protein A-Sepharose 4B suspension and incubated for another 1 h. After collecting by brief centrifugation, the Sepharose 4B beads were washed three times in ice-cold lysis buffer, three times with lithium chloride buffer (5 mm LiCl, 0.1 mm sodium orthovanadate, 0.1 m Tris-HCl, pH 7.4), and three times with buffer containing 150 mm NaCl, 5 mm EDTA, 0.1 mm sodium orthovanadate, 10 mm Tris-HCl, pH 7.4. Finally, the immune complexes were recovered from the beads in 500 μl of SDS-containing denaturing buffer and heated to 65 °C for 5 min. SDS-PAGE—Prestained molecular weight standards or 10–30 μg of denatured protein per lane were loaded onto NuPAGE Novex BisTris gels, electrophoresed in a Novex minicell apparatus (Invitrogen) at 200 V for 50 min, transferred to nitrocellulose membranes at V for min. The membranes were with 5% in 20 buffer for 1 washed in buffer, with antibody diluted in 5% bovine or 5% then treated with peroxidase-linked anti-mouse or anti-rabbit antibodies at in 5% for 1 h at were by and protein with of the was by Hyaluronan was in cell h by an A. V. Toole B.P. PubMed Google Scholar). and of RTKs on HA in we have shown that antagonists of interaction inhibit ERBB2 activation tyrosine in colon and breast carcinoma cells (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 17Misra S. Ghatak S. Toole B.P. J. Biol. Chem. 2005; 280: 20310-20315Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). We have now investigated the effects of HA antagonists on activation of several RTKs, ERBB2, EGFR, IGF1R-β, c-MET, and PDGFR-β, in several types of malignant colon, breast, and prostate carcinoma cells. The cells used were relatively levels of tyrosine of the RTKs shown in when incubated in However, it can that these RTKs are activated to in the various cells used In each case it was shown that i.e. HA oligomers or CD44 (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), inhibit activation of RTKs to various from to and significant effects on RTK were effects of in the HA we several We that the oligomers to 100 °C for 10 min effect on their to inhibit ERBB2 activation lane 4 versus lane We treated the oligomers with the oligomers to that are by CD44 J. V.C. Tammi M. R. J. Biol. Chem. Full Text Full Text PDF PubMed Google was used as a the oligomers inhibit ERBB2 activation lane 5 versus lane We have also shown previously that and HA the effects of HA oligomers on various signaling (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, S. Misra S. Toole B.P. J. Biol. Chem. 2002; 277: 38013-38020Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). We then ERBB2, IGF1R-β, and PDGFR-β are constitutively activated versus ligand-dependent in three of these cell i.e. HCA7 colon, C4-2 prostate and MCF-7 breast carcinoma cells. We that ERBB2 is the same in the and of in these cell of IGF1R-β and PDGFR-β was cells were treated in the of with the i.e. IGF1 or the RTK was activation was reversed to the of for and for phospho-PDGFR-β by co-treatment of cells with HA oligomers also both constitutive activation of ERBB2 and ligand-dependent activation of IGF1R-β and PDGFR-β are on endogenous HA interactions in these cells. The of inhibition of RTK activation in the described to several factors. inhibition of CD44 by the from 65 to was to in of with HA oligomers on the of endogenous HA to CD44, as S. Ghatak S. Toole B.P. J. Biol. Chem. 2005; 280: 20310-20315Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). of RTKs by HA and others of on previously that formation of a signaling complex containing activated ERBB2, CD44, PI3K, ezrin, and the HSP90 and is on endogenous interaction of HA with CD44 and that this complex is by of the cells with antagonists (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). We have now shown that complexes with other RTKs, i.e. EGFR, IGF1R-β, PDGFR-β, and We RTKs from lysates of HCA7 colon, HT29 colon, or C4-2 prostate carcinoma cells and that CD44, the of PI3K, and CDC37 with each of these RTKs. of the cells with HA oligomers or CD44 a in of activated RTK, CD44, and in these complexes of three in RTK of In the case of the IGF1R-β were of for activated IGF1R-β, for CD44, for and for were for the other RTK However, the of RTKs in each of the were by the HA These that the antagonists of CD44, and from the complexes as well as of the RTKs. the to in of the antagonists or of of RTKs to HA regulation, as have shown that HA oligomers can of CD44 T. H. H. H. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. H. R. T. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (106) Google Scholar), by matrix T. Y. H. T. Y. T. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, H. N. K. H. K. M. Y. M. Res. 2004; PubMed Scopus Google Scholar). the that the of CD44 in RTK complexes to CD44 we the of CD44 in cell lysates of cells with HA oligomers or a matrix We significant effect with HA oligomers or matrix on the or of CD44. CD44 is a major for its interaction with ERBB2 with HA We conclude that endogenous interaction assembly of several complexes in malignant cells and that antagonists to of these that the ERBB2-containing complex a (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). We the to the various RTK complexes or Increased HA of RTKs in to inhibition of ERBB2 activation by we have also shown that increased HA production induces ERBB2 activation in MCF-7 breast carcinoma cells, levels of constitutive ERBB2 activation when (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). we show that increased HA production stimulates activation of multiple RTKs in phenotypically normal human and rat IEC6 intestinal epithelial cells, as well as in MCF-7 cells. We treated these cells with a recombinant HAS2 in HA production in these cells under the used in activation of ERBB2, EGFR, IGF1R-β, c-MET, or PDGFR-β was In RTK activation was reversed to the same or levels by co-treatment with HA oligomers or CD44 also for RNA interference silencing of CD44 with interactions, while HA oligomers with HA interactions J. V.C. Tammi M. R. J. Biol. Chem. Full Text Full Text PDF PubMed Google Scholar). we have used these reagents to with interactions and signaling However, it is that also HA signaling by the in HA production by recombinant HAS2 we their effects on HA production in the human cells. we that the CD44 HA oligomers reversed the in HA production as well as the of have effect on CD44 were with rat IEC6 cells these oligomers compete for endogenous interaction of HA with CD44 also affect HA production. The this effect of HA oligomers is under and (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 17Misra S. Ghatak S. Toole B.P. J. Biol. Chem. 2005; 280: 20310-20315Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) that interactions constitutively activation of several RTKs, ERBB2, EGFR, PDGFR-β, IGF1R-β, and c-MET, as well as formation of signaling complexes in malignant carcinoma cells. These interactions lead to numerous for malignant cell (7Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1603) Google Scholar). that these changes were by alterations in HA levels and In we that of HA the effects of endogenous HA on ERBB2 activation or signaling (16Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, S. Misra S. Toole B.P. J. Biol. Chem. 2002; 277: 38013-38020Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), whereas others have that HA induces ERBB2 activation (13Bourguignon L.Y. Zhu H. Zhou B. Diedrich F. Singleton P.A. Hung M.C. J. Biol. Chem. 2001; 276: 48679-48692Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). a that HA formation of a complex in to inhibition of activation L. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These the by and produced HA with cells (7Toole B.P. Nat. Rev. Cancer. 2004; 4: 528-539Crossref PubMed Scopus (1603) Google Scholar). The effects also to the cells used in these i.e. normal versus cancer cells. however, the that HA, as to tumor HA, inhibit tumor progression J. J.A. Res. 2005; PubMed Scopus Google Scholar), the that levels of HA are prognostic for tumor progression (5Toole B.P. Wight T.N. Tammi M. J. Biol. Chem. 2002; 277: 4593-4596Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). that of HA an important factor in tumor and that the between HA and B. J.A. S. L. Kaye Novak U. 2002; Google Scholar, N. T. F. S. R. M. K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, L.Y. Singleton P.A. Diedrich F. R. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Res. 2005; PubMed Scopus Google Scholar, J. Full Text Full Text PDF PubMed Scopus Google Scholar). work is to the HA-induced signaling is activated and and the of HA signaling in malignant cell with

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