Hyaluronan-CD44 Interaction with IQGAP1 Promotes Cdc42 and ERK Signaling, Leading to Actin Binding, Elk-1/Estrogen Receptor Transcriptional Activation, and Ovarian Cancer Progression
2005; Elsevier BV; Volume: 280; Issue: 12 Linguagem: Inglês
10.1074/jbc.m411985200
ISSN1083-351X
AutoresLilly Bourguignon, Eli Gilad, Kori Rothman, Karine Peyrollier,
Tópico(s)Fibroblast Growth Factor Research
ResumoIn this study, we have examined the interaction of hyaluronan (HA)-CD44 with IQGAP1 (one of the binding partners for the Rho GTPase Cdc42) in SK-OV-3.ipl human ovarian tumor cells. Immunological and biochemical analyses indicated that IQGAP1 (molecular mass of ∼190 kDa) is expressed in SK-OV-3.ipl cells and that IQGAP1 interacts directly with Cdc42 in a GTP-dependent manner. Both IQGAP1 and Cdc42 were physically linked to CD44 in SK-OV-3.ipl cells following HA stimulation. Furthermore, the HA-CD44-induced Cdc42-IQGAP1 complex regulated cytoskeletal function via a close association with F-actin that led to ovarian tumor cell migration. In addition, the binding of HA to CD44 promoted the association of ERK2 with the IQGAP1 molecule, which stimulated both ERK2 phosphorylation and kinase activity. The activated ERK2 then increased the phosphorylation of both Elk-1 and estrogen receptor-α (ERα), resulting in Elk-1- and estrogen-responsive element-mediated transcriptional up-regulation. Down-regulation of IQGAP1 (by treating cells with IQGAP1-specific small interfering RNAs) not only blocked IQGAP1 association with CD44, Cdc42, F-actin, and ERK2 but also abrogated HA-CD44-induced cytoskeletal function, ERK2 signaling (e.g. ERK2 phosphorylation/activity, ERK2-mediated Elk-1/ERα phosphorylation, and Elk-1/ERα-specific transcriptional activation), and tumor cell migration. Taken together, these findings indicate that HA-CD44 interaction with IQGAP1 serves as a signal integrator by modulating Cdc42 cytoskeletal function, mediating Elk-1-specific transcriptional activation, and coordinating "cross-talk" between a membrane receptor (CD44) and a nuclear hormone receptor (ERα) signaling pathway during ovarian cancer progression. In this study, we have examined the interaction of hyaluronan (HA)-CD44 with IQGAP1 (one of the binding partners for the Rho GTPase Cdc42) in SK-OV-3.ipl human ovarian tumor cells. Immunological and biochemical analyses indicated that IQGAP1 (molecular mass of ∼190 kDa) is expressed in SK-OV-3.ipl cells and that IQGAP1 interacts directly with Cdc42 in a GTP-dependent manner. Both IQGAP1 and Cdc42 were physically linked to CD44 in SK-OV-3.ipl cells following HA stimulation. Furthermore, the HA-CD44-induced Cdc42-IQGAP1 complex regulated cytoskeletal function via a close association with F-actin that led to ovarian tumor cell migration. In addition, the binding of HA to CD44 promoted the association of ERK2 with the IQGAP1 molecule, which stimulated both ERK2 phosphorylation and kinase activity. The activated ERK2 then increased the phosphorylation of both Elk-1 and estrogen receptor-α (ERα), resulting in Elk-1- and estrogen-responsive element-mediated transcriptional up-regulation. Down-regulation of IQGAP1 (by treating cells with IQGAP1-specific small interfering RNAs) not only blocked IQGAP1 association with CD44, Cdc42, F-actin, and ERK2 but also abrogated HA-CD44-induced cytoskeletal function, ERK2 signaling (e.g. ERK2 phosphorylation/activity, ERK2-mediated Elk-1/ERα phosphorylation, and Elk-1/ERα-specific transcriptional activation), and tumor cell migration. Taken together, these findings indicate that HA-CD44 interaction with IQGAP1 serves as a signal integrator by modulating Cdc42 cytoskeletal function, mediating Elk-1-specific transcriptional activation, and coordinating "cross-talk" between a membrane receptor (CD44) and a nuclear hormone receptor (ERα) signaling pathway during ovarian cancer progression. Ovarian cancer cells are characterized by their ability to freely invade the peritoneal cavity, which is consistent with the well known aggressiveness and high morbidity rate of ovarian tumors (1Greenlee R.T. Murray T. Bolden S. Wingo P.A. CA-Cancer J. Clin. 2000; 50: 7-33Crossref PubMed Scopus (3963) Google Scholar, 2Katso R.M. Manek S. O'Bryne K. Playford M.P. Le Meuth V. Ganesan T.S. Cancer Metastasis Rev. 1997; 16: 81-107Crossref PubMed Scopus (23) Google Scholar, 3Auersperg N. Wong A.S. Choi K.C. Kang S.K. Leung P.C. Endocr. Rev. 2001; 22: 255-288Crossref PubMed Scopus (896) Google Scholar). A number of studies have aimed at identifying specific molecule(s) expressed in ovarian carcinomas that correlate with tumor cell invasive behaviors. Among such candidate molecules is CD44 (a major hyaluronan (HA) 1The abbreviations used are: HA, hyaluronan; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; ER; estrogen receptor; ERE, estrogen-responsive element; siRNA, small interfering RNA; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PBS, phosphate-buffered saline; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.1The abbreviations used are: HA, hyaluronan; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; ER; estrogen receptor; ERE, estrogen-responsive element; siRNA, small interfering RNA; GTPγS, guanosine 5′-O-(3-thiotriphosphate); PBS, phosphate-buffered saline; GST, glutathione S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase. receptor) (4Underhill C. J. Cell Sci. 1992; 103: 293-298Crossref PubMed Google Scholar), which belongs to a family of multifunctional transmembrane glycoproteins expressed in ovarian tumor cells and carcinoma tissues (5Bourguignon L.Y.W. Zhu H.B. Chu A. Zhang L. Hung M.C. J. Biol. Chem. 1997; 272: 27913-27918Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 6Bourguignon L.Y.W. Zhu H. Shao L. Chen Y.W. J. Biol. Chem. 2001; 276: 7327-7336Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 7Bourguignon L.Y.W. 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 (164) Google Scholar, 8Cannistra S.A. Kansas G.S. Niloff J. DeFranzo B. Kim Y. Ottensmeier C. Cancer Res. 1993; 53: 3830-3838PubMed Google Scholar, 9Cannistra S.A. DeFranzo B. Niloff J. Ottensmeier C. Clin. Cancer Res. 1995; 1: 333-342PubMed Google Scholar). CD44 has been found to interact with HA at the N terminus of its extracellular domain (10Turley E.A. Nobel P.W. Bourguignon L.Y.W. J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar, 11Liao H.X. Lee D.M. Levesque M.C. Haynes B.F. J. Immunol. 1995; 155: 3938-3945PubMed Google Scholar, 12Yang B. Yang B.L. Savani R.C. Turley E.A. EMBO J. 1994; 13: 286-296Crossref PubMed Scopus (335) Google Scholar). Ovarian cancer cells express CD44 isoforms that cause very strong cell adhesion to HA-enriched peritoneal mesothelium (8Cannistra S.A. Kansas G.S. Niloff J. DeFranzo B. Kim Y. Ottensmeier C. Cancer Res. 1993; 53: 3830-3838PubMed Google Scholar, 9Cannistra S.A. DeFranzo B. Niloff J. Ottensmeier C. Clin. Cancer Res. 1995; 1: 333-342PubMed Google Scholar, 13Gardner M.J. Jones L.M. Catterall J.B. Turner G.A. Cancer Lett. 1995; 91: 229-234Crossref PubMed Scopus (103) Google Scholar, 14Yeo T.K. Nagy J.A. Yeo K.T. Dvorak H.F. Toole B.P. Am. J. Pathol. 1996; 148: 1733-1740PubMed Google Scholar). A significant reduction in tumor implants has been found to occur in nude mice 5 weeks after intraperitoneal injection of ovarian cancer cells incubated with anti-CD44 antibody compared with injected cells pretreated with antibodies against other cell-surface proteins (8Cannistra S.A. Kansas G.S. Niloff J. DeFranzo B. Kim Y. Ottensmeier C. Cancer Res. 1993; 53: 3830-3838PubMed Google Scholar, 9Cannistra S.A. DeFranzo B. Niloff J. Ottensmeier C. Clin. Cancer Res. 1995; 1: 333-342PubMed Google Scholar). These findings suggest that CD44 interaction with HA may be one of the important requirements for the peritoneal spread of ovarian cancer. However, the cellular and molecular mechanisms controlling the ability of CD44-positive ovarian tumor cells to undergo cancer progression at HA-enriched extracellular matrix within the peritoneal cavity remain poorly understood. The binding of HA to CD44 isoforms triggers direct "cross-talk" between two different tyrosine kinase (e.g. p185HER2 tyrosine kinase (5Bourguignon L.Y.W. Zhu H.B. Chu A. Zhang L. Hung M.C. J. Biol. Chem. 1997; 272: 27913-27918Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and c-Src kinase (6Bourguignon L.Y.W. Zhu H. Shao L. Chen Y.W. J. Biol. Chem. 2001; 276: 7327-7336Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar))-linked signaling pathways (cell growth versus cell migration, respectively) in ovarian tumor cells. Recent studies have shown that HA-CD44 interaction in caveolin/cholesterol-enriched lipid raft microdomains plays an important role in promoting membrane-cytoskeleton association and stimulating several intracellular signaling pathways (e.g. Ca2+ mobilization, cellular pH changes, Rho signaling, phosphatidylinositol 3-kinase/Akt activation), leading to the onset of cytoskeletal function and ovarian tumor cell-specific behaviors (e.g. cell survival, growth, migration, and invasion) (15Singleton P.A. Bourguignon L.Y.W. Exp. Cell Res. 2004; 295: 102-118Crossref PubMed Scopus (88) Google Scholar, 16Bourguignon L.Y.W. Singleton P. Diedrich F. Stern R. Gilad E. J. Biol. Chem. 2004; 279: 26991-27007Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 17Bourguignon L.Y.W. Singleton P. Zhu H. Diedrich F. J. Biol. Chem. 2003; 278: 29420-29434Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). These findings clearly demonstrate that CD44 plays a pivotal role in activating oncogenic signaling, leading to ovarian tumor cell function. Members of the Rho subclass of the Ras superfamily (small molecular mass GTPases, e.g. RhoA, Rac1, and Cdc42) are known to be associated with changes in the membrane-linked cytoskeleton (18Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5193) Google Scholar). For example, activation of RhoA, Rac1, and Cdc42 has been found to produce specific structural changes in the plasma membrane-cytoskeleton associated with membrane ruffling, lamellipodia, filopodia, and stress fiber formation (18Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5193) Google Scholar). The rationale for our present focus on Rho GTPase-related signaling is based on previous reports suggesting that CD44-associated cytoskeletal proteins (e.g. ankyrin and ERM) and certain tumor cell-specific phenotypes are dependent on Rho GTPase activation (19Bourguignon L.Y.W. Zhu H. Shao L. Zhu D. Chen Y.W. Cell Motil. Cytoskeleton. 1999; 43: 269-287Crossref PubMed Scopus (144) Google Scholar, 20Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. Tsukita S. J. Cell Biol. 1996; 135: 36-51Crossref Scopus (508) Google Scholar, 21Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (331) Google Scholar). Furthermore, overexpression of Rho GTPases in human tumors often correlates with a poor cancer prognosis (22Fritz G. Just I. Kaina B. Int. J. Cancer. 1999; 81: 682-687Crossref PubMed Scopus (575) Google Scholar, 23Suwa H. Ohshio G. Imamura T. Watanabe G. Arii S. Imamura M. Narumiya S. Hiai H. Fukumoto M. Br. J. Cancer. 1998; 77: 147-152Crossref PubMed Scopus (251) Google Scholar). Consequently, coordinated Rho GTPase signaling is considered to be a possible mechanism underlying cell growth and migration, both of which are obvious prerequisites for metastasis (24Bourguignon L.Y.W. Zhu D. Zhu H. Front. Biosci. 1998; 3: 637-649Crossref PubMed Scopus (106) Google Scholar, 25Bourguignon L.Y.W. J. Mammary Gland Biol. Neoplasia. 2000; 6: 287-297Crossref Scopus (143) Google Scholar). In the search for additional cellular targets of HA-CD44-activated Rho GTPases that correlate with metastatic behavior in ovarian tumor cells, a molecule identified as IQGAP1 (a 190-kDa protein, one of the downstream effectors of Cdc42) has been detected (26Weissbach L. Settleman J. Kalady M.F. Snijders A.J. Murrthy A.E. Yan Y.-X. Bernards A. J. Biol. Chem. 1994; 269: 20517-20521Abstract Full Text PDF PubMed Google Scholar, 27Briggs M.W. Sacks D.B. FEBS Lett. 2003; 542: 7-11Crossref PubMed Scopus (119) Google Scholar). IQGAP1 inhibits the intrinsic GTPase activity of Cdc42, thereby significantly increasing the cellular levels of active Cdc42 (28Brill S. Li S. Lyman C.W. Church D.M. Wasmuth J.J. Weissbach L. Bernards A. Snijders A.J. Mol. Cell. Biol. 1996; 16: 4869-4878Crossref PubMed Scopus (221) Google Scholar, 29Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (325) Google Scholar, 30Ho Y.D. Joyal J.L. Li Z. Sack D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 31Swart-Mataraza J.M. Li Z. Sack D.B. J. Biol. Chem. 2002; 277: 24753-24763Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 32Zhang B. Wang Z.X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). This molecule contains numerous functional domains and motifs found in many signal transduction proteins and oncoproteins. These structural domains include four putative calmodulin-binding (IQ) motifs, a calponin homology domain, a polyproline-binding (WW) domain, and a Ras GTPase-activating protein-related domain (26Weissbach L. Settleman J. Kalady M.F. Snijders A.J. Murrthy A.E. Yan Y.-X. Bernards A. J. Biol. Chem. 1994; 269: 20517-20521Abstract Full Text PDF PubMed Google Scholar, 27Briggs M.W. Sacks D.B. FEBS Lett. 2003; 542: 7-11Crossref PubMed Scopus (119) Google Scholar). Some of these motifs are involved in IQGAP1 interaction with specific proteins, such as Cdc42 (28Brill S. Li S. Lyman C.W. Church D.M. Wasmuth J.J. Weissbach L. Bernards A. Snijders A.J. Mol. Cell. Biol. 1996; 16: 4869-4878Crossref PubMed Scopus (221) Google Scholar, 29Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (325) Google Scholar, 30Ho Y.D. Joyal J.L. Li Z. Sack D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 31Swart-Mataraza J.M. Li Z. Sack D.B. J. Biol. Chem. 2002; 277: 24753-24763Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 32Zhang B. Wang Z.X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 33Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (209) Google Scholar, 34Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), Rac (33Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (209) Google Scholar, 34Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), actin (30Ho Y.D. Joyal J.L. Li Z. Sack D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 35Mateer S.C. McDaniel A.E. Nicolas V. Habermacher G.M. Lin M.J. Cromer D.A. King M.E. Bloom G.S. J. Biol. Chem. 2002; 277: 12324-12333Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 36Erickson J.W. Cerione R.A. Hart M.J. J. Biol. Chem. 1997; 272: 24443-24447Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), calmodulin (29Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (325) Google Scholar, 30Ho Y.D. Joyal J.L. Li Z. Sack D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), ERK2 (37Roy M. Li Z. Sacks D.B. J. Biol. Chem. 2004; 279: 17329-17337Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), E-cadherin (38Li Z. Kim S.H. Higgins J.M. Brenner M.B. Sacks D.B. J. Biol. Chem. 1999; 274: 37885-37892Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), and β-catenin (39Briggs M.W. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 7453-7465Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The establishment of physical linkage between IQGAP1 and its binding partners has been shown to generate several important biological activities. For example, Cdc42-IQGAP1 binding mediates oncogenic signaling events that result in the activation of the actin cytoskeleton (28Brill S. Li S. Lyman C.W. Church D.M. Wasmuth J.J. Weissbach L. Bernards A. Snijders A.J. Mol. Cell. Biol. 1996; 16: 4869-4878Crossref PubMed Scopus (221) Google Scholar, 29Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (325) Google Scholar, 30Ho Y.D. Joyal J.L. Li Z. Sack D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 31Swart-Mataraza J.M. Li Z. Sack D.B. J. Biol. Chem. 2002; 277: 24753-24763Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 32Zhang B. Wang Z.X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 33Bashour A.M. Fullerton A.T. Hart M.J. Bloom G.S. J. Cell Biol. 1997; 137: 1555-1566Crossref PubMed Scopus (209) Google Scholar, 34Kuroda S. Fukata M. Kobayashi K. Nakafuku M. Nomura N. Iwamatsu A. Kaibuchi K. J. Biol. Chem. 1996; 271: 23363-23367Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 35Mateer S.C. McDaniel A.E. Nicolas V. Habermacher G.M. Lin M.J. Cromer D.A. King M.E. Bloom G.S. J. Biol. Chem. 2002; 277: 12324-12333Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 36Erickson J.W. Cerione R.A. Hart M.J. J. Biol. Chem. 1997; 272: 24443-24447Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and tumor cell migration/invasion (40Mataraza J.M. Briggs M.W. Li Z. Entwistle A. Ridley A.J. Sacks D.B. J. Biol. Chem. 2003; 278: 41237-41245Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). IQGAP1 binding to β-catenin promotes both the rearrangement of the cadherin cell adhesion complex (41Kuroda S. Fukata M. Nakagawa M. Fujii K. Nakamura T. Ookubo T. Izawa I. Nagase T. Nomura N. Tani H. Shoji I. Matsuura Y. Yonehara S. Kaibuchi K. Science. 1998; 281: 832-835Crossref PubMed Scopus (427) Google Scholar, 42Fukata M. Kuroda S. Nakagawa M. Kawajiri A. Itoh N. Shoji I. Matsuura Y. Yonehara S. Fujisawa H. Kikuchi A. Kaibuchi K. J. Biol. Chem. 1999; 274: 26044-26050Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) and transcriptional activation (39Briggs M.W. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 7453-7465Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Moreover, IQGAP1-ERK2 association modulates the Ras/MAPK pathways (in particular, ERK signaling) (37Roy M. Li Z. Sacks D.B. J. Biol. Chem. 2004; 279: 17329-17337Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). These findings suggest that IQGAP1 not only serves as a scaffolding protein (mediating multiprotein complex assembly), but also directly participates in cytoskeleton activation and signaling coordination. ERKs, members of the MAPK family, are known to be activated by receptor tyrosine kinases, cytokine receptors, and G-protein-coupled receptors (43Chen Z. Gibson T.B. Robinson F. Silvestro L. Pearson G. Xu B. Wright A. Vanderbilt C. Cobb M.H. Chem. Rev. 2001; 101: 2449-2476Crossref PubMed Scopus (779) Google Scholar, 44Scaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1400) Google Scholar). In particular, ERK phosphorylation of certain target proteins is directly involved in transcriptional activation by coordinating extracellular cues and intracellular signals (43Chen Z. Gibson T.B. Robinson F. Silvestro L. Pearson G. Xu B. Wright A. Vanderbilt C. Cobb M.H. Chem. Rev. 2001; 101: 2449-2476Crossref PubMed Scopus (779) Google Scholar, 44Scaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1400) Google Scholar). For example, during epidermal growth factor (EGF) stimulation or integrin signaling, ERK activation is coupled with the phosphorylation of specific target molecules, including transcription factors (e.g. the ternary complex factor Elk-1) (45Lenormand P. Sardet C. Pages G. L'Allemain G. Brunet A. Pouyssegur J. J. Cell Biol. 1993; 122: 1079-1088Crossref PubMed Scopus (581) Google Scholar, 46Aplin A.E. Stewart S.A. Assoian R.K. Juliano R.L. J. Cell Biol. 2001; 153: 273-281Crossref PubMed Scopus (218) Google Scholar) and the nuclear hormone receptor (e.g. estrogen receptor) (47Bunone G. Briand P.A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (845) Google Scholar, 48Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1705) Google Scholar), resulting in cyclin D1 expression and cell cycle progression (45Lenormand P. Sardet C. Pages G. L'Allemain G. Brunet A. Pouyssegur J. J. Cell Biol. 1993; 122: 1079-1088Crossref PubMed Scopus (581) Google Scholar, 46Aplin A.E. Stewart S.A. Assoian R.K. Juliano R.L. J. Cell Biol. 2001; 153: 273-281Crossref PubMed Scopus (218) Google Scholar, 47Bunone G. Briand P.A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (845) Google Scholar, 48Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1705) Google Scholar, 49Castro-Rivera E. Samudio I. Safe S. J. Biol. Chem. 2001; 276: 30853-30861Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Furthermore, growth factors have been found to activate ERK and to augment estradiol-mediated activation of nuclear hormone receptors/estrogen receptors during malignant transformation (47Bunone G. Briand P.A. Miksicek R.J. Picard D. EMBO J. 1996; 15: 2174-2183Crossref PubMed Scopus (845) Google Scholar, 48Kato S. Endoh H. Masuhiro Y. Kitamoto T. Uchiyama S. Sasaki H. Masushige S. Gotoh Y. Nishida E. Kawashima H. Metzger D. Chambon P. Science. 1995; 270: 1491-1494Crossref PubMed Scopus (1705) Google Scholar, 49Castro-Rivera E. Samudio I. Safe S. J. Biol. Chem. 2001; 276: 30853-30861Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Previous studies indicate that HA interaction with the hyaluronan receptor RHAMM regulates ERK activity (50Zhang S. Chang M.C.Y. Zylka D. Turley S. Harrison R. Turley E.A. J. Biol. Chem. 1998; 273: 11342-11348Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Most importantly, HA-activated ERK signaling participates in the up-regulation of certain transcription factors (e.g. NF-κB and AP-1) (50Zhang S. Chang M.C.Y. Zylka D. Turley S. Harrison R. Turley E.A. J. Biol. Chem. 1998; 273: 11342-11348Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 51Haslinger B. Mandl-Weber S. Sellmayer A. Sitter T. Cell Tissue Res. 2001; 305: 79-86Crossref PubMed Scopus (40) Google Scholar) and cell growth and cell migration (50Zhang S. Chang M.C.Y. Zylka D. Turley S. Harrison R. Turley E.A. J. Biol. Chem. 1998; 273: 11342-11348Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Although ERK activation is closely associated with HA and RHAMM signaling, the regulatory mechanisms that modulate ERK-mediated transcriptional activation and cellular functions in HA-CD44-stimulated ovarian cancer progression are not well understood. In this study, we have described a new HA-CD44-mediated oncogenic mechanism occurring in ovarian tumor cells. Specifically, we have focused on CD44 interactions with IQGAP1 during HA signaling in SK-OV-3.ipl ovarian tumor cells. Our results indicate that HA-CD44-stimulated IQGAP1 binds to both Cdc42 and ERK (in particular, ERK2). These interactions enhance F-actin binding and promote ERK activation, leading to the phosphorylation of Elk-1 and estrogen receptor (ER)-α, Elk-1-specific transcriptional up-regulation, and estrogen-responsive element (ERE) reporter gene activation, as well as ovarian tumor cell migration. Down-regulation of IQGAP1 (by treating cells with IQGAP1-specific small interfering RNAs (siRNAs)) effectively blocks HA-CD44-stimulated cytoskeletal function, ERK signaling, and ovarian tumor cell behaviors. These findings suggest that HA-CD44-activated IQGAP1 plays an essential role in regulating Cdc42-cytoskeleton interaction and in modulating ERK activity required for Elk-1 and nuclear hormone receptor (ERα) transcriptional up-regulation and ovarian cancer progression. Cell Culture—The SK-OV-3.ipl cell line was established from ascites that developed in a nu/nu mouse given an intraperitoneal injection of the SK-OV-3 human ovarian carcinoma cell line (obtained from American Type Culture Collection) as described previously (5Bourguignon L.Y.W. Zhu H.B. Chu A. Zhang L. Hung M.C. J. Biol. Chem. 1997; 272: 27913-27918Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 6Bourguignon L.Y.W. Zhu H. Shao L. Chen Y.W. J. Biol. Chem. 2001; 276: 7327-7336Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 7Bourguignon L.Y.W. 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 (164) Google Scholar). Cells were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 supplemented with 10% fetal bovine serum. Cells were routinely serum-starved (and therefore deprived of serum HA) before adding HA. Antibodies and Reagents—Rat anti-CD44 monoclonal antibody (clone 020, isotype IgG2b; obtained from CMB-TECH, Inc., San Francisco, CA) recognizes a common determinant of the HA-binding region of CD44 isoforms, including CD44s, CD44E, and CD44 variant species. This rat anti-CD44 antibody was routinely used for HA-related blocking experiments. Both mouse anti-IQGAP1 and rabbit anti-phospho-ERα antibodies were purchased from Upstate Cell Signaling Solutions (Lake Placid, NY). A number of immunoreagents, including rabbit anti-Elk-1, mouse anti-phospho-Elk-1, and goat anti-actin antibodies, were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Other immunoreagent such as mouse anti-Cdc42, rabbit anti-ERK, rabbit anti-ERK2, and rabbit anti-phospho-ERK antibodies were purchased from BD Biosciences, Oncogene Research Products (San Diego, CA), and Cell Signaling Technology (Beverly, MA). Mouse anti-ERα antibody and recombinant ERα were manufactured by Lab Vision Corp. (Freemont, CA) and Calbiochem, respectively. 17β-[3H]Estradiol (specific radioactivity of 52 Ci/mmol) was purchased from Amersham Biosciences. Water-soluble β-estradiol was obtained from Sigma. High molecular mass Healon HA polymers (∼500,000-Da polymers) were prepared by gel filtration chromatography using a Sephacryl S1000 column. The purity of the high molecular mass HA polymers used in our experiments was further verified by anion exchange high performance liquid chromatography. HA polymers were also digested by PH20 hyaluronidase. Both intact HA and PH20 hyaluronidase-treated HA fragments were used for analyzing their effects on biological activities as described under "Results." Measurement of Cdc42 Activation—SK-OV-3.ipl cells (∼5.0 × 106 cells) were resuspended in buffer containing 118 mm KCl, 5 mm NaCl, 0.4 mm CaCl2, 1 mm EGTA, 1.2 mm magnesium acetate, 1.2 mm KH2PO4, 25 mm Tris-HCl (pH 7.4), and 20 mg/ml bovine serum albumin. An aliquot of the cell suspension was added to the electroporation cuvette and incubated at 4 °C for 5-10 min, followed by the addition of [35S]GTPγS (12.5 μCi). Subsequently, cells were electroporated at 25 microfarads and 2.0 kV/cm, followed by incubation either with HA (50 μg/ml) in the presence or absence of rat anti-CD44 antibody (50 μg/ml) or with PH20 hyaluronidase-treated HA fragments (50 μg/ml) or without any HA treatment at 37 °C for 10 min. Subsequently, [35S]GTPγS-labeled cells were washed with phosphate-buffered saline (PBS; pH 7.4) and solubilized in 1.0% Nonidet P-40 with 1 mm GTP, 25 mm magnesium acetate, and protease inhibitors in PBS (pH 7.4). Nonidet P-40-solubilized cells were then incubated with mouse anti-Cdc42 IgG (5 μg/ml) plus goat anti-mouse antibody-conjugated beads. The amount of [35S]GTPγS bound to Cdc42 associated with anti-Cdc42 antibody-conjugated Immunobeads was measured using a γ-counter. The values expressed in Table I represent an average of triplicate determinations of five experiments with S.D. < 5%.Table IDetection of Cdc42 activation in SK-OV-3.ipl cellsCellsAmount of [35S]GTPγS bound to Cdc42% of controlIncubated without HA (control)100Incubated with HA285Incubated with anti-CD44 IgG + HA98Incubated with PH20 hyaluronidase-treated HA fragments95 Open table in a new tab Preparations of IQGAP1 siRNA—The siRNA sequence targeting human IQGAP1 (from the mRNA sequence, GenBank™/EBI accession number AJ251595) corresponds to the coding region relative to the first nucleotide of the start codon. Target sequences were selected using the software developed by Ambion Inc. As recommended by Ambion Inc., IQGAP1-specific targeted regions were selected beginning 50-100 nucleotides downstream from the start codon. Sequences with close to 50% G/C content were chosen. Specifically, the IQGAP1 target sequence 5′-AAAGTTCTACGGGAAGTAATT-3′ and scrambled sequence 5′-AATAGAGGCAAGGGGTTACG-3′ were used. The IQGAP1-specific target sequence was then aligned with the human genome data base in
Referência(s)