Hyaluronan Synthase 2 (HAS2) Promotes Breast Cancer Cell Invasion by Suppression of Tissue Metalloproteinase Inhibitor 1 (TIMP-1)
2011; Elsevier BV; Volume: 286; Issue: 49 Linguagem: Inglês
10.1074/jbc.m111.278598
ISSN1083-351X
AutoresBerit Bernert, Helena Porsch, Paraskevi Heldin,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoInvasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion. Invasion and metastasis are the primary causes of breast cancer mortality, and increased knowledge about the molecular mechanisms involved in these processes is highly desirable. High levels of hyaluronan in breast tumors have been correlated with poor patient survival. The involvement of hyaluronan in the early invasive phase of a clone of breast cancer cell line MDA-MB-231 that forms bone metastases was studied using an in vivo-like basement membrane model. The metastatic to bone tumor cells exhibited a 7-fold higher hyaluronan-synthesizing capacity compared with MDA-MB-231 cells predominately due to an increased expression of hyaluronan synthase 2 (HAS2). We found that knockdown of HAS2 completely suppressed the invasive capability of these cells by the induction of tissue metalloproteinase inhibitor 1 (TIMP-1) and dephosphorylation of focal adhesion kinase. HAS2 knockdown-mediated inhibition of basement membrane remodeling was rescued by HAS2 overexpression, transfection with TIMP-1 siRNA, or addition of TIMP-1-blocking antibodies. Moreover, knockdown of HAS2 suppressed the EGF-mediated induction of the focal adhesion kinase/PI3K/Akt signaling pathway. Thus, this study provides new insights into a possible mechanism whereby HAS2 enhances breast cancer invasion. IntroductionA hallmark of the malignant phenotype is acquisition of an invasive phenotype that allows cancer cells to degrade the basement membrane before they enter the circulation via blood or lymphatic vessels. The basement membrane is a specialized form of the extracellular matrix that underlies epithelial cells and surrounds blood vessels (1Kalluri R. Nat. Rev. Cancer. 2003; 3: 422-433Crossref PubMed Scopus (1312) Google Scholar). Turnover of the basement membrane and extracellular matrix is regulated by the balance between metalloproteinases (MMPs) 2The abbreviations used are: MMPmetalloproteinaseTIMPtissue inhibitor of MMPMT1membrane type 1HAShyaluronan synthaseFAKfocal adhesion kinase. and their naturally occurring tissue inhibitors of MMP (TIMPs). Up-regulation of MMPs or lack of TIMPs has been reported to be involved in cancer cell invasion (1Kalluri R. Nat. Rev. Cancer. 2003; 3: 422-433Crossref PubMed Scopus (1312) Google Scholar, 2Hotary K. Li X.Y. Allen E. Stevens S.L. Weiss S.J. Genes Dev. 2006; 20: 2673-2686Crossref PubMed Scopus (292) Google Scholar, 3Srivastava A. Pastor-Pareja J.C. Igaki T. Pagliarini R. Xu T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2721-2726Crossref PubMed Scopus (158) Google Scholar). However, the relationship between MMPs and TIMPs is complex because both of them can be overexpressed in malignancies and be associated with tumorigenesis (4Bigelow R.L. Williams B.J. Carroll J.L. Daves L.K. Cardelli J.A. Breast Cancer Res. Treat. 2009; 117: 31-44Crossref PubMed Scopus (55) Google Scholar, 5Schrohl A.S. Holten-Andersen M.N. Peters H.A. Look M.P. Meijer-van Gelder M.E. Klijn J.G. Brünner N. Foekens J.A. Clin. Cancer Res. 2004; 10: 2289-2298Crossref PubMed Scopus (106) Google Scholar). The MMP family of proteolytic enzymes consists of >20 secreted or membrane-anchored enzymes. MMP2, MMP7, and MMP9 are secreted as latent precursor forms (pro-MMP2, pro-MMP7, and pro-MMP9) that are activated at the cell surface and cleave collagen type IV, which is the principal structural constituent of the basement membrane. Membrane type 1 (MT1) MMP catalyzes the activation of pro-MMP2, which in turn activates pro-MMP9, propagating basement membrane proteolysis (1Kalluri R. Nat. Rev. Cancer. 2003; 3: 422-433Crossref PubMed Scopus (1312) Google Scholar, 2Hotary K. Li X.Y. Allen E. Stevens S.L. Weiss S.J. Genes Dev. 2006; 20: 2673-2686Crossref PubMed Scopus (292) Google Scholar, 6Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5079) Google Scholar, 7Itoh Y. Seiki M. J. Cell. Physiol. 2006; 206: 1-8Crossref PubMed Scopus (413) Google Scholar). MMPs can localize transiently to the plasma membrane and become activated through their interactions with adhesion molecules, such as CD44 (8Yu Q. Stamenkovic I. Genes Dev. 1999; 13: 35-48Crossref PubMed Scopus (603) Google Scholar, 9Yu W.H. Woessner Jr., J.F. McNeish J.D. Stamenkovic I. Genes Dev. 2002; 16: 307-323Crossref PubMed Scopus (379) Google Scholar, 10Stamenkovic I. Semin. Cancer Biol. 2000; 10: 415-433Crossref PubMed Scopus (644) Google Scholar).Accumulating evidence has demonstrated that CD44 and its ligand hyaluronan, a core component of the extracellular matrix, contribute to the invasive behavior and progression of multiple cancers. CD44 exists in various isoforms, the standard form (CD44s) and the variant forms (CD44v1–10), all of which can bind hyaluronan (11Ponta H. Sherman L. Herrlich P.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 33-45Crossref PubMed Scopus (1819) Google Scholar, 12Toole B.P. Clin. Cancer Res. 2009; 15: 7462-7468Crossref PubMed Scopus (287) Google Scholar, 13Naor D. Wallach-Dayan S.B. Zahalka M.A. Sionov R.V. Semin. Cancer Biol. 2008; 18: 260-267Crossref PubMed Scopus (239) Google Scholar, 14Orian-Rousseau V. Eur. J. Cancer. 2010; 46: 1271-1277Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Increased hyaluronan synthesis and increased expression of CD44 have been observed in aggressive breast cancer cells (15de la Torre M. Heldin P. Bergh J. Anticancer Res. 1995; 15: 2791-2795PubMed Google Scholar, 16Corte M.D. González L.O. Junquera S. Bongera M. Allende M.T. Vizoso F.J. J. Cancer Res. Clin. Oncol. 2010; 136: 745-750Crossref PubMed Scopus (13) Google Scholar). Hyaluronan-activated CD44 signaling has been shown to enhance oncogenic events induced by growth factors and MMPs, and CD44 can form complexes with growth factor receptors, as well as with MMPs (10Stamenkovic I. Semin. Cancer Biol. 2000; 10: 415-433Crossref PubMed Scopus (644) Google Scholar, 12Toole B.P. Clin. Cancer Res. 2009; 15: 7462-7468Crossref PubMed Scopus (287) Google Scholar, 17Li L. Heldin C.H. Heldin P. J. Biol. Chem. 2006; 281: 26512-26519Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Orian-Rousseau V. Ponta H. Adv. Cancer Res. 2008; 101: 63-92Crossref PubMed Scopus (65) Google Scholar, 19Bourguignon L.Y. Semin. Cancer Biol. 2008; 18: 251-259Crossref PubMed Scopus (233) Google Scholar).The expression of hyaluronan synthases (HAS1, HAS2, and HAS3) increases during embryonic development, as well as during malignant progression, specifically in nests of cancer cells and at the invading edges of breast carcinomas (20Toole B.P. Semin. Cell Dev. Biol. 2001; 12: 79-87Crossref PubMed Scopus (433) Google Scholar, 21de la Torre M. Wells A.F. Bergh J. Lindgren A. Hum. Pathol. 1993; 24: 1294-1297Crossref PubMed Scopus (42) Google Scholar, 22Auvinen P. Tammi R. Parkkinen J. Tammi M. Agren U. Johansson R. Hirvikoski P. Eskelinen M. Kosma V.M. Am. J. Pathol. 2000; 156: 529-536Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 23Boregowda R.K. Appaiah H.N. Siddaiah M. Kumarswamy S.B. Sunila S. Thimmaiah K.N. Mortha K. Toole B. Banerjee S. J. Carcinog. 2006; 5: 2Crossref PubMed Scopus (66) Google Scholar). Of the three HAS genes, HAS2 is vital because of its involvement in epithelial-mesenchymal transition during embryonic cardiac cushion morphogenesis (24Camenisch T.D. Spicer A.P. Brehm-Gibson T. Biesterfeldt J. Augustine M.L. Calabro Jr., A. Kubalak S. Klewer S.E. McDonald J.A. J. Clin. Invest. 2000; 106: 349-360Crossref PubMed Scopus (700) Google Scholar). Notably, overexpression of HAS2 in the epithelium induces the transition of epithelial cells to a more fibroblastic, migratory phenotype and enhances anchorage-independent growth in soft agar, i.e. two of the key properties of cells undergoing malignant transformation (25Li Y. Heldin P. Br. J. Cancer. 2001; 85: 600-607Crossref PubMed Scopus (105) Google Scholar, 26Zoltan-Jones A. Huang L. Ghatak S. Toole B.P. J. Biol. Chem. 2003; 278: 45801-45810Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Moreover, studies both in vivo and in vitro have shown that ectopic expression of HAS proteins (and consequently, increased hyaluronan synthesis) promotes tumor progression, angiogenesis, and lymphangiogenesis, as well as recruitment of stromal cells (27Jacobson A. Rahmanian M. Rubin K. Heldin P. Int. J. Cancer. 2002; 102: 212-219Crossref PubMed Scopus (109) Google Scholar, 28Koyama H. Hibi T. Isogai Z. Yoneda M. Fujimori M. Amano J. Kawakubo M. Kannagi R. Kimata K. Taniguchi S. Itano N. Am. J. Pathol. 2007; 170: 1086-1099Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 29Koyama H. Kobayashi N. Harada M. Takeoka M. Kawai Y. Sano K. Fujimori M. Amano J. Ohhashi T. Kannagi R. Kimata K. Taniguchi S. Itano N. Am. J. Pathol. 2008; 172: 179-193Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In contrast, suppression of HAS2 using antisense inhibition or specific siRNA has been shown to suppress the malignant phenotype of breast cancer cells (30Udabage L. Brownlee G.R. Waltham M. Blick T. Walker E.C. Heldin P. Nilsson S.K. Thompson E.W. Brown T.J. Cancer Res. 2005; 65: 6139-6150Crossref PubMed Scopus (114) Google Scholar, 31Li Y. Li L. Brown T.J. Heldin P. Int. J. Cancer. 2007; 120: 2557-2567Crossref PubMed Scopus (125) Google Scholar). Growth factors, such as PDGF-BB and TGF-β (32Suzuki M. Asplund T. Yamashita H. Heldin C.H. Heldin P. Biochem. J. 1995; 307: 817-821Crossref PubMed Scopus (118) Google Scholar, 33Jacobson A. Brinck J. Briskin M.J. Spicer A.P. Heldin P. Biochem. J. 2000; 348: 29-35Crossref PubMed Scopus (185) Google Scholar, 34Heldin P. www.glycoforum.gr.jp/science/hyaluronan/HA33/HA33E.htmlDate: 2009Google Scholar), as well as tumor promoting agents (phorbol 12-myristate 13-acetate) (32Suzuki M. Asplund T. Yamashita H. Heldin C.H. Heldin P. Biochem. J. 1995; 307: 817-821Crossref PubMed Scopus (118) Google Scholar) and glucocorticoids (33Jacobson A. Brinck J. Briskin M.J. Spicer A.P. Heldin P. Biochem. J. 2000; 348: 29-35Crossref PubMed Scopus (185) Google Scholar, 35Zhang W. Watson C.E. Liu C. Williams K.J. Werth V.P. Biochem. J. 2000; 349: 91-97Crossref PubMed Scopus (63) Google Scholar), modulate expression of the HAS genes, especially the HAS2 isoform. Furthermore, hyaluronan levels are modulated by the supply of UDP-sugar substrates that are produced during glycolysis (36Jokela T.A. Makkonen K.M. Oikari S. Kärnä R. Koli E. Hart G.W. Tammi R.H. Carlberg C. Tammi M.I. J. Biol. Chem. 2011; 286: 33632-33640Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Notably, aberrant hyaluronan production seen in hyperglycemia has been associated with higher HAS2 mRNA expression (37Jones S. Jones S. Phillips A.O. Kidney Int. 2001; 59: 1739-1749Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 38Wang A. Hascall V.C. J. Biol. Chem. 2004; 279: 10279-10285Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Hyaluronan is degraded by hyaluronidases, the most important being HYAL1 and HYAL2 (39Stern R. Semin. Cancer Biol. 2008; 18: 275-280Crossref PubMed Scopus (203) Google Scholar).In this study, we explored the possibility that hyaluronan plays an important role during the initial steps of breast cancer invasion through the basement membrane. We compared the biological properties of wild-type MDA-MB-231 breast cancer cells with those of a clone of this line that forms bone metastases (MDA-MB-231-BM) with regard to hyaluronan-synthesizing capacity, CD44 expression, and interference of MMPs. Our data indicate that the abundant expression of HAS2 by MDA-MB-231-BM cells confers an invasive phenotype by suppression of TIMP-1 expression, presumably increasing MMP activity and consequently basement membrane degradation.DISCUSSIONIn this study, we have demonstrated that the invasive MDA-MB-231-BM cells synthesized high amounts of hyaluronan through up-regulation of the HAS2 gene compared with MDA-MB-231 cells. Suppression of HAS2 decreased the invasive phenotype of MDA-MB-231-BM cells in conjunction with increased expression of TIMP-1; the phenotype was reverted after overexpressing HAS2 (Fig. 4), silencing the TIMP-1 gene, or addition of TIMP-1-blocking antibodies (Fig. 6A).Our findings are consistent with the observation that coumarin-like 4-methylumbelliferone, an inhibitor of hyaluronan synthesis that exhibits both anti-inflammatory and antitumor properties, increases TIMP-1 expression in aortic smooth muscle cell cultures (52Vigetti D. Rizzi M. Viola M. Karousou E. Genasetti A. Clerici M. Bartolini B. Hascall V.C. De Luca G. Passi A. Glycobiology. 2009; 19: 537-546Crossref PubMed Scopus (76) Google Scholar). The reverse correlation between HAS2 and TIMP-1 is most likely a property of hyaluronan endogenously produced by HAS2 because exogenously added hyaluronan in osteoclast cultures (53Pivetta E. Scapolan M. Wassermann B. Steffan A. Colombatti A. Spessotto P. J. Cell. Physiol. 2011; 226: 769-779Crossref PubMed Scopus (22) Google Scholar) and mature hematopoietic cells (54Schraufstatter I. Serobyan N. DiScipio R. Feofanova N. Orlovskaya I. Khaldoyanidi S.K. J. Stem Cells. 2009; 4: 191-202PubMed Google Scholar) increases TIMP-1 expression. Our present and previous results (17Li L. Heldin C.H. Heldin P. J. Biol. Chem. 2006; 281: 26512-26519Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 31Li Y. Li L. Brown T.J. Heldin P. Int. J. Cancer. 2007; 120: 2557-2567Crossref PubMed Scopus (125) Google Scholar), as well as those of others (55Ghatak S. Misra S. Toole B.P. J. Biol. Chem. 2005; 280: 8875-8883Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) support the conclusion that the functional properties of endogenously synthesized hyaluronan cannot always be mimicked by exogenously added hyaluronan.It is possible that HAS2 has a hyaluronan CD44-independent signaling property because the HAS2 isoform appears to be a trait of breast cancer cells metastasizing to bone, whereas this phenotype is unrelated to the other HASs, hyaluronidases, and CD44 (Fig. 1). Increased understanding of the regulation of the HAS2 gene and protein in various pathological contexts is desirable. Specific inhibition of the HAS2 gene and hyaluronan synthesis occurs through glucocorticoids (33Jacobson A. Brinck J. Briskin M.J. Spicer A.P. Heldin P. Biochem. J. 2000; 348: 29-35Crossref PubMed Scopus (185) Google Scholar, 35Zhang W. Watson C.E. Liu C. Williams K.J. Werth V.P. Biochem. J. 2000; 349: 91-97Crossref PubMed Scopus (63) Google Scholar) that induce a rapid and sustained decrease in both gene transcription and HAS2 mRNA stability. Moreover, changes in hyaluronan synthesis can be affected by post-translational modifications of the HAS2 protein through phosphorylation (32Suzuki M. Asplund T. Yamashita H. Heldin C.H. Heldin P. Biochem. J. 1995; 307: 817-821Crossref PubMed Scopus (118) Google Scholar) and ubiquitination (56Karousou E. Kamiryo M. Skandalis S.S. Ruusala A. Asteriou T. Passi A. Yamashita H. Hellman U. Heldin C.H. Heldin P. J. Biol. Chem. 2010; 285: 23647-23654Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Accumulating evidence supports the notion that induction of HAS2 often accounts for the increased amount of hyaluronan synthesis seen in many tumors, which correlates with poor outcome. Interestingly, HAS2 is one of the 0.3% of genes that were up-regulated through tumor-stromal interactions during pancreatic cell invasion (57Sato N. Maehara N. Goggins M. Cancer Res. 2004; 64: 6950-6956Crossref PubMed Scopus (131) Google Scholar). Furthermore, HAS2 has been implicated in chromosomal copy number alterations associated with periodic fever syndrome (58Olsson M. Meadows J.R. Truvé K. Rosengren Pielberg G. Puppo F. Mauceli E. Quilez J. Tonomura N. Zanna G. Docampo M.J. Bassols A. Avery A.C. Karlsson E.K. Thomas A. Kastner D.L. Bongcam-Rudloff E. Webster M.T. Sanchez A. Hedhammar A. Remmers E.F. Andersson L. Ferrer L. Tintle L. Lindblad-Toh K. PLoS Genet. 2011; 7e1001332Crossref PubMed Scopus (102) Google Scholar). In addition, HAS2 chromosomal rearrangements and their deregulated mRNA expression are oncogenic events in lipoblastomas (59Hibbard M.K. Kozakewich H.P. Dal Cin P. Sciot R. Tan X. Xiao S. Fletcher J.A. Cancer Res. 2000; 60: 4869-4872PubMed Google Scholar) and breast cancer (60Unger K. Wienberg J. Riches A. Hieber L. Walch A. Brown A. O'Brien P.C. Briscoe C. Gray L. Rodriguez E. Jackl G. Knijnenburg J. Tallini G. Ferguson-Smith M. Zitzelsberger H. Endocr. Relat. Cancer. 2010; 17: 87-98Crossref PubMed Scopus (33) Google Scholar). Thus, transcriptional up-regulation of HAS2 might reflect early molecular events in inflammation and cancer. Analysis of the promoter region of the HAS2 gene indicated transcriptional regulation in response to growth factors, cytokines, and protein kinase C activators through putative Sp1, CREB1, STAT, and NF-κB transcription initiation sites, all of which are fine-balanced to assure hyaluronan production in response to external stimuli (61Tammi R.H. Passi A.G. Rilla K. Karousou E. Vigetti D. Makkonen K. Tammi M.I. FEBS J. 2011; 278: 1419-1428Crossref PubMed Scopus (170) Google Scholar).To elucidate the mechanism whereby decreased HAS2 expression and increased TIMP-1 expression could mediate decreased invasiveness, we investigated the activation status of several signaling pathways. No significant differences were noted in cells expressing HAS2 or not with regard to activation of ERK, JNK, and the Smad2 pathway; no activation of Akt was seen in any of the cells. However, we found that HAS2 expression is necessary for EGF-mediated activation of the FAK/PI3K/Akt pathway because this pathway was drastically attenuated upon knockdown of HAS2. Interestingly, knockdown of HAS2 correlated with a decreased activation of FAK, which could be rescued by siRNA-mediated knockdown of TIMP-1.The TIMP-1-mediated inhibition of matrix remodeling, e.g. in conjunction with cancer invasion and inflammation, has been associated with the ability of TIMP-1 to suppress the activities of MMPs. However, TIMP-1 has also been shown to have MMP-independent functions, i.e. an anti-apoptotic activity in several cell types, including hematopoietic cells (62Lambert E. Boudot C. Kadri Z. Soula-Rothhut M. Sowa M.L. Mayeux P. Hornebeck W. Haye B. Petitfrere E. Biochem. J. 2003; 372: 767-774Crossref PubMed Scopus (77) Google Scholar) and normal breast epithelial cells via activation of FAK, which binds and activates PI3K, resulting in phosphorylation of Akt and then Bad, known to be crucial for cell survival (63Liu X.W. Bernardo M.M. Fridman R. Kim H.R. J. Biol. Chem. 2003; 278: 40364-40372Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 64Liu X.W. Taube M.E. Jung K.K. Dong Z. Lee Y.J. Roshy S. Sloane B.F. Fridman R. Kim H.R. Cancer Res. 2005; 65: 898-906Crossref PubMed Scopus (51) Google Scholar, 65Jung K.K. Liu X.W. Chirco R. Fridman R. Kim H.R. EMBO J. 2006; 25: 3934-3942Crossref PubMed Scopus (229) Google Scholar). However, there was no difference in the apoptotic rate of cells expressing HAS2 or not as determined by annexin V staining (data not shown). TIMP-1 was shown to inhibit microvascular endothelial cell migration by both MMP-dependent (induction of VE-cadherin) and MMP-independent (inhibition of FAK activity) mechanisms (48Akahane T. Akahane M. Shah A. Connor C.M. Thorgeirsson U.P. Exp. Cell Res. 2004; 301: 158-167Crossref PubMed Scopus (85) Google Scholar). Whether the suppression of MDA-MB-231-BM cell invasion by TIMP-1 induction following HAS2 knockdown is exerted via MMP-dependent or MMP-independent mechanisms or both remains to be determined. The mechanism whereby TIMP-1 affects FAK phosphorylation remains to be elucidated. However, our finding of a correlation between TIMP-1-induced loss of invasive activity and FAK dephosphorylation is consistent with previous data that demonstrated an important role for FAK in regulation of cell motility (66Schlaepfer D.D. Mitra S.K. Curr. Opin. Genet. Dev. 2004; 14: 92-101Crossref PubMed Scopus (353) Google Scholar). Notably, a correlation between down-regulation of FAK and up-regulation of TIMP-1 was recently seen in prostate cancer cells (67Liu K.C. Huang A.C. Wu P.P. Lin H.Y. Chueh F.S. Yang J.S. Lu C.C. Chiang J.H. Meng M. Chung J.G. Oncol. Rep. 2011; 26: 177-184PubMed Google Scholar).Bone metastases are common in breast carcinomas. Based on the "seed and soil" theory of Paget (68Paget S. Lancet. 1889; 1: 571-573Abstract Scopus (2238) Google Scholar), bone tissue most likely provides a suitable milieu for breast cancer cells to grow. Hyaluronan and CD44 are abundantly found in breast tumor and bone marrow matrices especially during osteoclastic bone metastases (40Heldin P. de la Torre M. Ytterberg D. Bergh J. Oncol. Rep. 1996; 3: 1011-1016PubMed Google Scholar, 69Draffin J.E. McFarlane S. Hill A. Johnston P.G. Waugh D.J. Cancer Res. 2004; 64: 5702-5711Crossref PubMed Scopus (213) Google Scholar). It is possible that high hyaluronan synthesis by breast cancer cells not only plays an important function in determining their early invasive phenotype but could also promote interactions with CD44-expressing cells in the bone tissue microenvironment and create a physiological "niche" in which breast cancer cells thrive.An increased understanding of the regulation of the HAS2 gene and protein in various pathological contexts is desirable. Specific inhibition of the HAS2 gene and hyaluronan synthesis occurs through glucocorticoids (33Jacobson A. Brinck J. Briskin M.J. Spicer A.P. Heldin P. Biochem. J. 2000; 348: 29-35Crossref PubMed Scopus (185) Google Scholar, 35Zhang W. Watson C.E. Liu C. Williams K.J. Werth V.P. Biochem. J. 2000; 349: 91-97Crossref PubMed Scopus (63) Google Scholar), which induce a rapid and sustained decrease in both gene transcription and HAS2 mRNA stability. Moreover, changes in hyaluronan synthesis can be affected by post-translational modifications of HAS2 protein through phosphorylation (32Suzuki M. Asplund T. Yamashita H. Heldin C.H. Heldin P. Biochem. J. 1995; 307: 817-821Crossref PubMed Scopus (118) Google Scholar) and ubiquitination (56Karousou E. Kamiryo M. Skandalis S.S. Ruusala A. Asteriou T. Passi A. Yamashita H. Hellman U. Heldin C.H. Heldin P. J. Biol. Chem. 2010; 285: 23647-23654Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In addition to our study, these previous studies provide a rationale for inhibition of hyaluronan synthesis in tumor treatment. IntroductionA hallmark of the malignant phenotype is acquisition of an invasive phenotype that allows cancer cells to degrade the basement membrane before they enter the circulation via blood or lymphatic vessels. The basement membrane is a specialized form of the extracellular matrix that underlies epithelial cells and surrounds blood vessels (1Kalluri R. Nat. Rev. Cancer. 2003; 3: 422-433Crossref PubMed Scopus (1312) Google Scholar). Turnover of the basement membrane and extracellular matrix is regulated by the balance between metalloproteinases (MMPs) 2The abbreviations used are: MMPmetalloproteinaseTIMPtissue inhibitor of MMPMT1membrane type 1HAShyaluronan synthaseFAKfocal adhesion kinase. and their naturally occurring tissue inhibitors of MMP (TIMPs). Up-regulation of MMPs or lack of TIMPs has been reported to be involved in cancer cell invasion (1Kalluri R. Nat. Rev. Cancer. 2003; 3: 422-433Crossref PubMed Scopus (1312) Google Scholar, 2Hotary K. Li X.Y. Allen E. Stevens S.L. Weiss S.J. Genes Dev. 2006; 20: 2673-2686Crossref PubMed Scopus (292) Google Scholar, 3Srivastava A. Pastor-Pareja J.C. Igaki T. Pagliarini R. Xu T. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2721-2726Crossref PubMed Scopus (158) Google Scholar). However, the relationship between MMPs and TIMPs is complex because both of them can be overexpressed in malignancies and be associated with tumorigenesis (4Bigelow R.L. Williams B.J. Carroll J.L. Daves L.K. Cardelli J.A. Breast Cancer Res. Treat. 2009; 117: 31-44Crossref PubMed Scopus (55) Google Scholar, 5Schrohl A.S. Holten-Andersen M.N. Peters H.A. Look M.P. Meijer-van Gelder M.E. Klijn J.G. Brünner N. Foekens J.A. Clin. Cancer Res. 2004; 10: 2289-2298Crossref PubMed Scopus (106) Google Scholar). The MMP family of proteolytic enzymes consists of >20 secreted or membrane-anchored enzymes. MMP2, MMP7, and MMP9 are secreted as latent precursor forms (pro-MMP2, pro-MMP7, and pro-MMP9) that are activated at the cell surface and cleave collagen type IV, which is the principal structural constituent of the basement membrane. Membrane type 1 (MT1) MMP catalyzes the activation of pro-MMP2, which in turn activates pro-MMP9, propagating basement membrane proteolysis (1Kalluri R. Nat. Rev. Cancer. 2003; 3: 422-433Crossref PubMed Scopus (1312) Google Scholar, 2Hotary K. Li X.Y. Allen E. Stevens S.L. Weiss S.J. Genes Dev. 2006; 20: 2673-2686Crossref PubMed Scopus (292) Google Scholar, 6Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5079) Google Scholar, 7Itoh Y. Seiki M. J. Cell. Physiol. 2006; 206: 1-8Crossref PubMed Scopus (413) Google Scholar). MMPs can localize transiently to the plasma membrane and become activated through their interactions with adhesion molecules, such as CD44 (8Yu Q. Stamenkovic I. Genes Dev. 1999; 13: 35-48Crossref PubMed Scopus (603) Google Scholar, 9Yu W.H. Woessner Jr., J.F. McNeish J.D. Stamenkovic I. Genes Dev. 2002; 16: 307-323Crossref PubMed Scopus (379) Google Scholar, 10Stamenkovic I. Semin. Cancer Biol. 2000; 10: 415-433Crossref PubMed Scopus (644) Google Scholar).Accumulating evidence has demonstrated that CD44 and its ligand hyaluronan, a core component of the extracellular matrix, contribute to the invasive behavior and progression of multiple cancers. CD44 exists in various isoforms, the standard form (CD44s) and the variant forms (CD44v1–10), all of which can bind hyaluronan (11Ponta H. Sherman L. Herrlich P.A. Nat. Rev. Mol. Cell Biol. 2003; 4: 33-45Crossref PubMed Scopus (1819) Google Scholar, 12Toole B.P. Clin. Cancer Res. 2009; 15: 7462-7468Crossref PubMed Scopus (287) Google Scholar, 13Naor D. Wallach-Dayan S.B. Zahalka M.A. Sionov R.V. Semin. Cancer Biol. 2008; 18: 260-267Crossref PubMed Scopus (239) Google Scholar, 14Orian-Rousseau V. Eur. J. Cancer. 2010; 46: 1271-1277Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Increased hyaluronan synthesis and increased expression of CD44 have been observed in aggressive breast cancer cells (15de la Torre M. Heldin P. Bergh J. Anticancer Res. 1995; 15: 2791-2795PubMed Google Scholar, 16Corte M.D. González L.O. Junquera S. Bongera M. Allende M.T. Vizoso F.J. J. Cancer Res. Clin. Oncol. 2010; 136: 745-750Crossref PubMed Scopus (13) Google Scholar). Hyaluronan-activated CD44 signaling has been shown to enhance oncogenic events induced by growth factors and MMPs, and CD44 can form complexes with growth factor receptors, as well as with MMPs (10Stamenkovic I. Semin. Cancer Biol. 2000; 10: 415-433Crossref PubMed Scopus (644) Google Scholar, 12Toole B.P. Clin. Cancer Res. 2009; 15: 7462-7468Crossref PubMed Scopus (287) Google Scholar, 17Li L. Heldin C.H. Heldin P. J. Biol. Chem. 2006; 281: 26512-26519Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 18Orian-Rousseau V. Ponta H. Adv. Cancer Res. 2008; 101: 63-92Crossref PubMed Scopus (65) Google Scholar, 19Bourguignon L.Y. Semin. Cancer Biol. 2008; 18: 251-259Crossref PubMed Scopus (233) Google Scholar).The expression of hyaluronan synthases (HAS1, HAS2, and HAS3) increases during embryonic development, as well as during malignant progression, specifically in nests of cancer cells and at the invading edges of breast carcinomas (20Toole B.P. Semin. Cell Dev. Biol. 2001; 12: 79-87Crossref PubMed Scopus (433) Google Scholar, 21de la Torre M. Wells A.F. Bergh J. Lindgren A. Hum. Pathol. 1993; 24: 1294-1297Crossref PubMed Scopus (42) Google Scholar, 22Auvinen P. Tammi R. Parkkinen J. Tammi M. Agren U. Johansson R. Hirvikoski P. Eskelinen M. Kosma V.M. Am. J. Pathol. 2000; 156: 529-536Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 23Boregowda R.K. Appaiah H.N. Siddaiah M. Kumarswamy S.B. Sunila S. Thimmaiah K.N. Mortha K. Toole B. Banerjee S. J. Carcinog. 2006; 5: 2Crossref PubMed Scopus (66) Google Scholar). Of the three HAS genes, HAS2 is vital because of its involvement in epithelial-mesenchymal transition during embryonic cardiac cushion morphogenesis (24Camenisch T.D. Spicer A.P. Brehm-Gibson T. Biesterfeldt J. Augustine M.L. Calabro Jr., A. Kubalak S. Klewer S.E. McDonald J.A. J. Clin. Invest. 2000; 106: 349-360Crossref PubMed Scopus (700) Google Scholar). Notably, overexpression of HAS2 in the epithelium induces the transition of epithelial cells to a more fibroblastic, migratory phenotype and enhances anchorage-independent growth in soft agar, i.e. two of the key properties of cells undergoing malignant transformation (25Li Y. Heldin P. Br. J. Cancer. 2001; 85: 600-607Crossref PubMed Scopus (105) Google Scholar, 26Zoltan-Jones A. Huang L. Ghatak S. Toole B.P. J. Biol. Chem. 2003; 278: 45801-45810Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Moreover, studies both in vivo and in vitro have shown that ectopic expression of HAS proteins (and consequently, increased hyaluronan synthesis) promotes tumor progression, angiogenesis, and lymphangiogenesis, as well as recruitment of stromal cells (27Jacobson A. Rahmanian M. Rubin K. Heldin P. Int. J. Cancer. 2002; 102: 212-219Crossref PubMed Scopus (109) Google Scholar, 28Koyama H. Hibi T. Isogai Z. Yoneda M. Fujimori M. Amano J. Kawakubo M. Kannagi R. Kimata K. Taniguchi S. Itano N. Am. J. Pathol. 2007; 170: 1086-1099Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 29Koyama H. Kobayashi N. Harada M. Takeoka M. Kawai Y. Sano K. Fujimori M. Amano J. Ohhashi T. Kannagi R. Kimata K. Taniguchi S. Itano N. Am. J. Pathol. 2008; 172: 179-193Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In contrast, suppression of HAS2 using antisense inhibition or specific siRNA has been shown to suppress the malignant phenotype of breast cancer cells (30Udabage L. Brownlee G.R. Waltham M. Blick T. Walker E.C. Heldin P. Nilsson S.K. Thompson E.W. Brown T.J. Cancer Res. 2005; 65: 6139-6150Crossref PubMed Scopus (114) Google Scholar, 31Li Y. Li L. Brown T.J. Heldin P. Int. J. Cancer. 2007; 120: 2557-2567Crossref PubMed Scopus (125) Google Scholar). Growth factors, such as PDGF-BB and TGF-β (32Suzuki M. Asplund T. Yamashita H. Heldin C.H. Heldin P. Biochem. J. 1995; 307: 817-821Crossref PubMed Scopus (118) Google Scholar, 33Jacobson A. Brinck J. Briskin M.J. Spicer A.P. Heldin P. Biochem. J. 2000; 348: 29-35Crossref PubMed Scopus (185) Google Scholar, 34Heldin P. www.glycoforum.gr.jp/science/hyaluronan/HA33/HA33E.htmlDate: 2009Google Scholar), as well as tumor promoting agents (phorbol 12-myristate 13-acetate) (32Suzuki M. Asplund T. Yamashita H. Heldin C.H. Heldin P. Biochem. J. 1995; 307: 817-821Crossref PubMed Scopus (118) Google Scholar) and glucocorticoids (33Jacobson A. Brinck J. Briskin M.J. Spicer A.P. Heldin P. Biochem. J. 2000; 348: 29-35Crossref PubMed Scopus (185) Google Scholar, 35Zhang W. Watson C.E. Liu C. Williams K.J. Werth V.P. Biochem. J. 2000; 349: 91-97Crossref PubMed Scopus (63) Google Scholar), modulate expression of the HAS genes, especially the HAS2 isoform. Furthermore, hyaluronan levels are modulated by the supply of UDP-sugar substrates that are produced during glycolysis (36Jokela T.A. Makkonen K.M. Oikari S. Kärnä R. Koli E. Hart G.W. Tammi R.H. Carlberg C. Tammi M.I. J. Biol. Chem. 2011; 286: 33632-33640Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Notably, aberrant hyaluronan production seen in hyperglycemia has been associated with higher HAS2 mRNA expression (37Jones S. Jones S. Phillips A.O. Kidney Int. 2001; 59: 1739-1749Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 38Wang A. Hascall V.C. J. Biol. Chem. 2004; 279: 10279-10285Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Hyaluronan is degraded by hyaluronidases, the most important being HYAL1 and HYAL2 (39Stern R. Semin. Cancer Biol. 2008; 18: 275-280Crossref PubMed Scopus (203) Google Scholar).In this study, we explored the possibility that hyaluronan plays an important role during the initial steps of breast cancer invasion through the basement membrane. We compared the biological properties of wild-type MDA-MB-231 breast cancer cells with those of a clone of this line that forms bone metastases (MDA-MB-231-BM) with regard to hyaluronan-synthesizing capacity, CD44 expression, and interference of MMPs. Our data indicate that the abundant expression of HAS2 by MDA-MB-231-BM cells confers an invasive phenotype by suppression of TIMP-1 expression, presumably increasing MMP activity and consequently basement membrane degradation.
Referência(s)