IQGAP1 Stimulates Proliferation and Enhances Tumorigenesis of Human Breast Epithelial Cells
2007; Elsevier BV; Volume: 283; Issue: 2 Linguagem: Inglês
10.1074/jbc.m708466200
ISSN1083-351X
AutoresLorraine Jadeski, Jennifer Mataraza, Ha‐Won Jeong, Zhigang Li, David B. Sacks,
Tópico(s)Microtubule and mitosis dynamics
ResumoThe scaffold protein IQGAP1 integrates signaling pathways and participates in diverse cellular activities. IQGAP1 is overexpressed in a number of human solid neoplasms, but its functional role in tumorigenesis has not been previously evaluated. Here we report that IQGAP1 contributes to neoplastic transformation of human breast epithelial cells. The amount of IQGAP1 in breast carcinoma is greater than that in normal tissue, with highly metastatic breast epithelial cells expressing the highest levels. Overexpression of IQGAP1 enhances proliferation of MCF-7 breast epithelial cells. Reduction of endogenous IQGAP1 by RNA interference impairs both serum-dependent and anchorage-independent growth of MCF-7 cells. Consistent with these in vitro observations, immortalized MCF-7 cells overexpressing IQGAP1 form invasive tumors in immunocompromised mice, whereas tumors derived from MCF-7 cells with stable knockdown of IQGAP1 are smaller and less invasive. In vitro analysis with selected IQGAP1 mutant constructs and a chemical inhibitor suggests that actin, Cdc42/Rac1, and the mitogen-activated protein kinase pathway contribute to the mechanism by which IQGAP1 increases cell invasion. Collectively, our data reveal that IQGAP1 enhances mammary tumorigenesis, suggesting that it may be a target for therapeutic intervention. The scaffold protein IQGAP1 integrates signaling pathways and participates in diverse cellular activities. IQGAP1 is overexpressed in a number of human solid neoplasms, but its functional role in tumorigenesis has not been previously evaluated. Here we report that IQGAP1 contributes to neoplastic transformation of human breast epithelial cells. The amount of IQGAP1 in breast carcinoma is greater than that in normal tissue, with highly metastatic breast epithelial cells expressing the highest levels. Overexpression of IQGAP1 enhances proliferation of MCF-7 breast epithelial cells. Reduction of endogenous IQGAP1 by RNA interference impairs both serum-dependent and anchorage-independent growth of MCF-7 cells. Consistent with these in vitro observations, immortalized MCF-7 cells overexpressing IQGAP1 form invasive tumors in immunocompromised mice, whereas tumors derived from MCF-7 cells with stable knockdown of IQGAP1 are smaller and less invasive. In vitro analysis with selected IQGAP1 mutant constructs and a chemical inhibitor suggests that actin, Cdc42/Rac1, and the mitogen-activated protein kinase pathway contribute to the mechanism by which IQGAP1 increases cell invasion. Collectively, our data reveal that IQGAP1 enhances mammary tumorigenesis, suggesting that it may be a target for therapeutic intervention. Tumor progression that culminates in clinically relevant metastatic lesions is the end point of a complex sequence of interrelated cellular events. After the initial transforming event, tumor cell proliferation, invasion, and migration, as well as vascularization of the tumor mass, occur. A thorough understanding of the molecular mechanisms that regulate tumor progression will provide the biological foundation for improving the efficacy of current therapeutic interventions (1Fidler I.J. Semin. Cancer Biol. 2002; 12: 89-96Crossref PubMed Scopus (352) Google Scholar, 2Fidler I.J. Nat. Rev. Cancer. 2003; 3: 453-458Crossref PubMed Scopus (3555) Google Scholar, 3Gupta G.P. Massague J. Cell. 2006; 127: 679-695Abstract Full Text Full Text PDF PubMed Scopus (3299) Google Scholar). IQGAP1 is a 189-kDa scaffolding protein that contains multiple protein-interacting domains (for reviews see Refs. 4Briggs M.W. Sacks D.B. FEBS Lett. 2003; 542: 7-11Crossref PubMed Scopus (119) Google Scholar, 5Briggs M.W. Sacks D.B. EMBO Rep. 2003; 4: 571-574Crossref PubMed Scopus (250) Google Scholar, 6Mateer S.C. Wang N. Bloom G.S. Cell Motil. Cytoskeleton. 2003; 55: 147-155Crossref PubMed Scopus (78) Google Scholar, 7Brown M.D. Sacks D.B. Trends Cell Biol. 2006; 16: 242-249Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). These include a calponin homology domain, a polyproline-binding domain, four calmodulin-binding motifs, and a Ras-GAP-related domain. The motifs present in IQGAP1 are involved in the interaction of IQGAP1 with specific proteins, such as actin, calmodulin, members of the Rho GTPase family (i.e. Rac1 and Cdc42), Rap1, E-cadherin, β-catenin, members of the mitogen-activated protein kinase (MAPK) 4The abbreviations used are:MAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseMEKMAPK/ERK kinaseDMEMDulbecco's modified Eagle's mediumWASPWiskott Aldrich syndrome proteinGBDGTPase-binding domainPAKp21-activated kinaseCRIBCdc42-Rac1-interative binding domainMTT3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromideGSTglutathione S-transferaseFBSfetal bovine serum. pathway, and adenomatous polyposis coli (7Brown M.D. Sacks D.B. Trends Cell Biol. 2006; 16: 242-249Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 8Jeong H.W. Li Z. Brown M.D. Sacks D.B. J. Biol. Chem. 2007; 282: 20752-20762Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). By interacting with these proteins, IQGAP1 regulates multiple fundamental cellular activities including cytoskeletal organization, cell-cell adhesion, cell migration, transcription, and signal transduction. For example, binding of IQGAP1 to β-catenin both disrupts the E-cadherin-catenin complex, inhibiting epithelial cell-cell adhesion (9Kuroda 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 (429) Google Scholar), and increases β-catenin-mediated transcriptional activation (10Briggs M.W. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 7453-7465Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). IQGAP1 increases active Cdc42 in mammalian cells, resulting in formation of actin filopodia and microspikes (11Swart-Mataraza J.M. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 24753-24763Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), and promotion of cell migration and invasion (12Mataraza 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 (177) Google Scholar). These morphological and functional changes are not observed with a mutant IQGAP1 construct with impeded Cdc42-mediated signaling (11Swart-Mataraza J.M. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 24753-24763Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 12Mataraza 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 (177) Google Scholar). More recently, functional significance of IQGAP1 in MAPK signaling was demonstrated; IQGAP1 modulates epidermal growth factor-mediated activation of extracellular signal-regulated kinase (ERK) and MAPK/ERK kinase (MEK) (13Roy M. Li Z. Sacks D.B. J. Biol. Chem. 2004; 279: 17329-17337Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 14Roy M. Li Z. Sacks D.B. Mol. Cell. Biol. 2005; 25: 9740-9752Google Scholar). In addition, IQGAP1 is required for epidermal growth factor to increase B-Raf activity (15Ren J.G. Li Z. Sacks D.B. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 10465-10469Crossref PubMed Scopus (117) Google Scholar). These findings suggest that IQGAP1 serves as a scaffolding protein that mediates multiprotein complex assembly and participates in cytoskeletal activation and coordination of signaling (5Briggs M.W. Sacks D.B. EMBO Rep. 2003; 4: 571-574Crossref PubMed Scopus (250) Google Scholar, 7Brown M.D. Sacks D.B. Trends Cell Biol. 2006; 16: 242-249Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase Dulbecco's modified Eagle's medium Wiskott Aldrich syndrome protein GTPase-binding domain p21-activated kinase Cdc42-Rac1-interative binding domain 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide glutathione S-transferase fetal bovine serum. Accumulating evidence implicates IQGAP1 in tumorigenesis and tumor progression. Many of the identified IQGAP1-binding partners contribute to malignant transformation and/or tumor progression, and several cellular functions effected as a consequence of IQGAP1 binding are important in tumor biology (5Briggs M.W. Sacks D.B. EMBO Rep. 2003; 4: 571-574Crossref PubMed Scopus (250) Google Scholar, 7Brown M.D. Sacks D.B. Trends Cell Biol. 2006; 16: 242-249Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Furthermore, genomic studies suggest IQGAP1 involvement in tumorigenesis; the IQGAP1 gene is amplified in diffuse gastric cancer cell lines (16Sugimoto N. Imoto I. Fukuda Y. Kurihara N. Kuroda S. Tanigami A. Kaibuchi K. Kamiyama R. Inazawa J. J. Hum. Genet. 2001; 46: 21-25Crossref PubMed Scopus (53) Google Scholar) and is up-regulated in lung (17Sun W. Zhang K. Zhang X. Lei W. Xiao T. Ma J. Guo S. Shao S. Zhang H. Liu Y. Yuan J. Hu Z. Ma Y. Feng X. Hu S. Zhou J. Cheng S. Gao Y. Cancer Lett. 2004; 212: 83-93Crossref PubMed Scopus (79) Google Scholar) and colon (18Bertucci F. Salas S. Eysteries S. Nasser V. Finetti P. Ginestier C. Charafe-Jauffret E. Loriod B. Bachelart L. Montfort J. Victorero G. Viret F. Ollendorff V. Fert V. Giovaninni M. Delpero J.R. Nguyen C. Viens P. Monges G. Birnbaum D. Houlgatte R. Oncogene. 2004; 23: 1377-1391Crossref PubMed Scopus (289) Google Scholar) carcinoma relative to noncancerous control tissue. At the post-transcriptional level, IQGAP1 mRNA was increased in an oligonucleotide array screen of gene expression in melanoma-derived pulmonary metastases compared with that in poorly metastatic tumor cells (19Clark E.A. Golub T.R. Lander E.S. Hynes R.O. Nature. 2000; 406: 532-535Crossref PubMed Scopus (1308) Google Scholar). Protein analyses substantiate involvement of IQGAP1 in tumorigenesis. For example, IQGAP1 protein is overexpressed in several human neoplasms, including gastric (16Sugimoto N. Imoto I. Fukuda Y. Kurihara N. Kuroda S. Tanigami A. Kaibuchi K. Kamiyama R. Inazawa J. J. Hum. Genet. 2001; 46: 21-25Crossref PubMed Scopus (53) Google Scholar), colorectal (20Nabeshima K. Shimao Y. Inoue T. Koono M. Cancer Lett. 2002; 176: 101-109Crossref PubMed Scopus (114) Google Scholar), lung (21Miyoshi T. Shirakusa T. Ishikawa Y. Iwasaki A. Shiraishi T. Makimoto Y. Iwasaki H. Nabeshima K. Pathol. Int. 2005; 55: 419-424Crossref PubMed Scopus (36) Google Scholar), ovary (22Dong P. Nabeshima K. Nishimura N. Kawakami T. Hachisuga T. Kawarabayashi T. Iwasaki H. Cancer Lett. 2006; 243: 120-127Crossref PubMed Scopus (58) Google Scholar), and liver (23Balenci L. Clarke I.D. Dirks P.B. Assard N. Ducray F. Jouvet A. Belin M.F. Honnorat J. Baudier J. Cancer Res. 2006; 66: 9074-9082Crossref PubMed Scopus (43) Google Scholar). In addition, the subcellular location of IQGAP1 is altered in neoplasia. The IQGAP1 overexpression in colorectal carcinoma is most apparent at the invasive front and in advanced carcinomas with the highest invasive potential (20Nabeshima K. Shimao Y. Inoue T. Koono M. Cancer Lett. 2002; 176: 101-109Crossref PubMed Scopus (114) Google Scholar). Similarly, immunohistochemical analyses of gastric carcinomas suggest that subcellular location of IQGAP1 varies depending on the degree of differentiation of the tumor. In poorly and well differentiated diffuse- and intestinal-type tumors, IQGAP1 is localized at the cell membrane and in the cytoplasm, respectively (24Takemoto H. Doki Y. Shiozaki H. Imamura H. Utsunomiya T. Miyata H. Yano M. Inoue M. Fujiwara Y. Monden M. Int. J. Cancer. 2001; 91: 783-788Crossref PubMed Scopus (60) Google Scholar). The localization of IQGAP1 has recently been shown to have prognostic information. In ovarian carcinoma, overexpression and a diffuse expression pattern of IQGAP1 were shown to be independent predictors of highly aggressive tumors (22Dong P. Nabeshima K. Nishimura N. Kawakami T. Hachisuga T. Kawarabayashi T. Iwasaki H. Cancer Lett. 2006; 243: 120-127Crossref PubMed Scopus (58) Google Scholar). The relevance to tumor biology of the known cellular targets of IQGAP1, combined with accumulating clinical and experimental evidence, suggest a positive relationship between IQGAP1 expression and tumorigenesis. Notwithstanding these data, it is not known whether the changes in IQGAP1 observed in these studies contribute to the tumor pathogenesis nor whether the alterations in IQGAP1 are a cause or consequence of the neoplastic transformation. To investigate these issues, we chose to directly examine the role of IQGAP1 in tumorigenesis. Analysis was performed with cultured cells in vitro and with an in vivo tumor model system. Female immunocompromised mice (i.e. NOD.CB17-Prkdcscid/JC3H/HeJ) were injected subcutaneously with one of three MCF-7-derived cell lines, which express varying amounts of IQGAP1. Differences between groups in in vitro and in vivo proliferation and tumorigenic growth were monitored. Plasmids—Myc-tagged wild type human IQGAP1 in a pcDNA3 vector was used (25Hart M.J. Callow M.G. Souza B. Polakis P. EMBO J. 1996; 15: 2997-3005Crossref PubMed Scopus (329) Google Scholar, 26Ho Y.D. Joyal J.L. Li Z. Sacks D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). The construction of IQGAP1ΔGRD, IQGAP1G75Q and IQGAP1ΔMK24 has been described previously (27Sokol S.Y. Li Z. Sacks D.B. J. Biol. Chem. 2001; 276: 48425-48430Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 28Mataraza J.M. Li Z. Jeong H.W. Brown M.D. Sacks D.B. Cell Signal. 2007; 19: 1857-1865Crossref PubMed Scopus (32) Google Scholar, 29Mataraza J.M. Briggs M.W. Li Z. Frank R. Sacks D.B. Biochem. Biophys. Res. Commun. 2003; 305: 315-321Crossref PubMed Scopus (42) Google Scholar). Myc-tagged forms of the dominant negative constructs N17Cdc42 and N17Rac1 (30Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3735) Google Scholar) were kindly provided by Alan Hall (University College London). Preparation of Fusion Proteins—GST-WASP-GBD (glutathione S-transferase Wiskott Aldrich Syndrome protein GTPase-binding domain) and GST-PAK-CRIB (p21-activated kinase Cdc42-Rac1-interactive binding domain) were expressed in Escherichia coli and isolated with glutathione-Sepharose as previously described (12Mataraza 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 (177) Google Scholar, 26Ho Y.D. Joyal J.L. Li Z. Sacks D.B. J. Biol. Chem. 1999; 274: 464-470Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 31Kim S.H. Li Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Cell Culture and Transfection—The following human breast epithelial cell lines were used: T47D, ZR-75-1, MDA-MB-231, MDA-MB-361 (generously provided by Andrea Richardson, Brigham and Women's Hospital), MCF-7, Hs578T, and MDA-MB-435s (purchased from ATCC). The cells were cultured and transfected essentially as previously described (11Swart-Mataraza J.M. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 24753-24763Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 32Li 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). Briefly, the cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. Where indicated, the cells were transiently transfected with 10 μg of pcDNA3 (empty vector), Myc-IQGAP1, Myc-IQGAP1ΔGRD, Myc-IQGAP1G75Q, or Myc-IQGAP1ΔMK24 using FuGENE 6. In addition, MCF-7 human breast epithelial cells were stably transfected with empty pcDNA3 vector (MCF/V) or pcDNA3-Myc-IQGAP1 (MCF/I); IQGAP1 protein expression is 3-fold higher in MCF/I relative to MCF/V cells (10Briggs M.W. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 7453-7465Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 11Swart-Mataraza J.M. Li Z. Sacks D.B. J. Biol. Chem. 2002; 277: 24753-24763Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Stable knockdown of IQGAP1 was obtained by integrating into the genome of MCF-7 cells, a specific siRNA targeted against IQGAP1 (12Mataraza 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 (177) Google Scholar). IQGAP1 protein expression in these cells (termed MCF-siIQ8 cells) is reduced by 80% (12Mataraza 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 (177) Google Scholar). Western Blotting—The cells were washed three times in serum-free medium and lysed in buffer A (50 mm Tris, pH 7.4, 140 mm NaCl, and 1% (v/v) Triton X-100). Equal amounts of protein lysate were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane essentially as previously described (33Joyal J.L. Annan R.S. Ho Y.D. Huddleston M.E. Carr S.A. Hart M.J. Sacks D.B. J. Biol. Chem. 1997; 272: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 34Joyal J.L. Sacks D.B. J. Biol. Chem. 1994; 269: 30039-30048Abstract Full Text PDF PubMed Google Scholar, 35Sacks D.B. McDonald J.M. Arch. Biochem. Biophys. 1992; 299: 275-280Crossref PubMed Scopus (17) Google Scholar). Immunoblots were probed with anti-IQGAP1 monoclonal antibody and anti-tubulin antibody (Sigma). The complexes were visualized with horseradish peroxidase-conjugated secondary antibody and developed by ECL. Frozen human breast tissue was thawed and homogenized in buffer A containing 1 mm EGTA and protease inhibitors. Equal amounts of protein were resolved by Western blotting. Animals—Female NOD.CB17-Prkdcscid/JC3H/HeJ mice (6-8 weeks old) were obtained from Jackson Laboratory (Bar Harbor, ME). Upon arrival at the vivarium, the animals were immediately randomized to treatment groups (i.e. MCF/V, MCF/I, and MCF-siIQ8 cell lines). The experimental procedures began after a 1-week acclimatization period. Throughout the investigation, the animals had free access to food (standard mouse chow) and water and were maintained on a 12-h light/dark cycle. The mice were housed in cages fitted with a high efficiency particulate air filter lid; the cages were contained in a chamber receiving independent, filtered air. All of the manipulations on mice were performed in a laminar flow hood using sterile procedures. The cages, bedding, food, water, nestlets, and water bottles were autoclaved prior to contact with mice. The water was acidified or supplemented with antibiotics on a rotating 4-day schedule. The animals were treated in accordance with guidelines set out by the Canadian Council on Animal Care. Cell Proliferation Assays—Incorporation of [3H]thymidine and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) dye method were used to assess cell proliferation. The cells were made quiescent by culturing them for 24 h in DMEM containing 0.5% (v/v) FBS. DNA synthesis was measured by labeling 2 × 105 cells with 1 μCi/ml [3H]thymidine for 18 h. The cells were washed with ice-cold 0.5% trichloroacetic acid and lysed with 0.25 m NaOH. Six hundred microliters of lysate was collected, and radioactivity assessed by liquid scintillation spectrometry. All of the assays were performed in quadruplicate. For the MTT assay, MCF/V and MCF/I cells were transfected with 1 μg of empty pcDNA vector, N17Cdc42, or N17Rac1. After 36 h, 5 × 103 viable cells were plated in 96-well, flat-bottom tissue culture plates. After incubating for 24, 48, and 72 h at 37 °C in 5% CO2, 20 μl of MTT labeling reagent was added, and the cells were incubated for another 4 h. Dimethyl sulfoxide (100 μl) was added to each well, and the samples were incubated overnight. The absorbances were determined at 550 nm using a Victor3 Multilabel Counter (PerkinElmer Life Sciences). Soft Agar Colony Formation Assay—Colony growth assays were performed essentially as previously described (36Vadlamudi R.K. Adam L. Wang R.A. Mandal M. Nguyen D. Sahin A. Chernoff J. Hung M.C. Kumar R. J. Biol. Chem. 2000; 275: 36238-36244Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 37Cox A.D. Der C.J. Methods Enzymol. 1994; 238: 277-294Crossref PubMed Scopus (87) Google Scholar). Briefly, a 5-ml agar solution (0.6% Difco agar in DMEM supplemented with 10% FBS) was layered onto 15-mm tissue culture plates. The cells (1 × 104; MCF/V, MCF/I and MCF-siIQ8) were then suspended in soft agar (0.36% Bactoagar solution in DMEM) and layered onto prepared 0.6% Difco agar plates. After a 14-day incubation at 37 °C in 5% CO2, colonies (>0.2 mm) were counted using a light microscope. All of the assays were performed in quadruplicate. In Vivo Assay for Primary Tumor Growth and Angiogenesis—The mice were anesthetized using an intraperitoneal injection of a hypnorm/midazolam mixture. Specifically, 1 part hypnorm (fentanyl citrate, 0.315 mg; fluanison, 10 mg/ml) and 1 part midazolam (5 mg/ml) were suspended in 2 parts of sterile water; the dose was 0.1 ml/10 g of body weight. Incision sites were cleaned using a three-step surgical preparation: isopropyl alcohol, tincture of iodine, and savlon. Hypromellose eye lubricant prevented ocular drying and damage during anesthesia. In the inguinal region, the mice received subcutaneous implants of 1 × 105 tumor cells (i.e. MCF/V, MCF/I, or MCF-siIQ8; 10 animals/group) suspended in Matrigel (Collaborative Research, Bedford, MA) (3.5 mg of Matrigel in 0.5 ml of DMEM), and on the contralateral side as controls, they received the equivalent amount of Matrigel alone. A small incision was made in the dorsal scapular region, and a slow release (60 day) 17β-estradiol pellet (Innovative Research of America, Sarasota, FL) was inserted subcutaneously. The incisions were then closed using a small drop of Vetbond surgical glue. During recovery from anesthesia, the mice were placed in a warm cage; they were monitored carefully and received subcutaneous injections of warmed saline (0.9%; 1 ml). As an indication of general health, the mice were observed and weighed regularly during the course of the experiment. Primary tumor growth was monitored at least once a week. Sixty days after tumor implantation, the mice were sacrificed using an overdose of pentobarbital. Final tumor volumes were determined by measuring the maximum and minimum tumor diameters using digital calipers; volume was calculated using the equation: tumor volume = 0.52a2b, where a and b represent the minimum and maximum tumor diameters, respectively (38Baguley B.C. Calveley S.B. Crowe K.K. Fray L.M. O'Rourke S.A. Smith G.P. Eur. J. Cancer Clin. Oncol. 1989; 25: 263-269Abstract Full Text PDF PubMed Scopus (71) Google Scholar). Matrigel implants were removed, fixed in 4% paraformaldehyde, processed for paraffin embedding, sectioned, and stained with Masson's trichrome. In vivo angiogenesis was analyzed as previously described (39Jadeski L.C. Lala P.K. Am. J. Pathol. 1999; 155: 1381-1390Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Histological sections were scanned (40× objective and 10× ocular) for areas containing tumor associated blood vessels (researcher blinded to experimental condition). These areas were systematically imaged (160× magnification), and individual vessel counts for each field were documented to identify fields of maximum blood vessel density. Fields of maximum blood vessel density were statistically analyzed for between group differences; the data are expressed as the averages of three fields of maximum blood vessel density. Active Cdc42 and Rac1 Assay—Measurement of active Cdc42 was performed essentially as previously described (12Mataraza 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 (177) Google Scholar, 31Kim S.H. Li Z. Sacks D.B. J. Biol. Chem. 2000; 275: 36999-37005Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Briefly, the cells were washed and lysed in 500 μl of buffer (20 mm Hepes, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 20 mm NaF, 20 μm GTP, 1 mm MgCl2, and protease inhibitors). After clarification by centrifugation and incubation with glutathione-Sepharose, equal amounts of lysate were incubated with 40 μg of GST-WASP-GBD. The complexes were collected with glutathione-Sepharose and resolved by SDS-PAGE and Western blotting. The blots were probed with anti-Cdc42 antibodies (BD Biosciences) and detected by ECL. In addition, 50 μg of protein lysate was examined directly by blotting as whole cell lysate. Measurement of active Rac1 was performed by as outlined above for Cdc42, except GST-PAK-CRIB was used to isolate the GTP-bound protein, and the blots were probed with anti-Rac1 antibodies (BD Biosciences) (12Mataraza 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 (177) Google Scholar, 40Brown M.D. Bry L. Li Z. Sacks D.B. J. Biol. Chem. 2007; 282: 30265-30272Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Invasion Assays—Matrigel invasion assays were performed using 24-well BD BioCoat Matrigel Invasion Chambers (Becton Dickinson Labware, Bedford, MA) according to the manufacturer's instructions. HEK-293H cells were transiently transfected with 2 μg of a plasmid that expresses wild type IQGAP1, IQGAP1G75Q, IQGAP1MK24, or pcDNA3 (as a control). After 36 h, the cells were trypsinized and counted with a hemacytometer. For the invasion assay, 2 × 105 cells were resuspended in 500 μl of medium containing 0.5% FBS and placed in the upper compartment of the chamber. The lower compartment was filled with 750 μl of medium containing 10% FBS. After allowing cells to invade for 40 h, the cells on the upper surface of the Transwell were carefully removed with a cotton swab, and the membrane was fixed and stained with DiffQuick (Dade Behring). Counting was done in five randomly selected fields (magnification, 10×) within each well. In experiments with MEK inhibitor, 2 × 105 MCF/V or MCF/I cells were resuspended in 500 μl of medium containing 1% FBS with 10 μm U0126 (Promega) or 0.1% Me2SO as a vehicle. The cells were placed in the upper compartment. The lower compartment was filled with 750 μl of growth medium containing 10% FBS. After 2 days, the number of cells that invaded were stained and counted as described above. Data Analysis—The data were analyzed using SigmaStat for Windows version 3.0.1a, and treatment groups (i.e. tumor cell lines: MCF/V, MCF/I, and MCF-siIQ8) were compared using one-way analysis of variance. A probability of 0.05 was used in determining statistical significance. Expression of IQGAP1 among Different Human Breast Cancer Cell Lines and Patient Tissue—IQGAP1 is overexpressed in several human neoplasms (16Sugimoto N. Imoto I. Fukuda Y. Kurihara N. Kuroda S. Tanigami A. Kaibuchi K. Kamiyama R. Inazawa J. J. Hum. Genet. 2001; 46: 21-25Crossref PubMed Scopus (53) Google Scholar, 20Nabeshima K. Shimao Y. Inoue T. Koono M. Cancer Lett. 2002; 176: 101-109Crossref PubMed Scopus (114) Google Scholar, 22Dong P. Nabeshima K. Nishimura N. Kawakami T. Hachisuga T. Kawarabayashi T. Iwasaki H. Cancer Lett. 2006; 243: 120-127Crossref PubMed Scopus (58) Google Scholar), but no published studies have examined breast carcinoma. Therefore, we determined the relative amounts of IQGAP1 among human breast epithelial cell lines. These cell lines can be divided into those that have estrogen receptors and progesterone receptors, namely MCF-7, T47D, ZR-75-1, and MDA-MB-361, and those lacking the receptors, namely MDA-MB-231, MDA-MB-435s, and Hs578T (41Lacroix M. Leclercq G. Breast Cancer Res. Treat. 2004; 83: 249-289Crossref PubMed Scopus (610) Google Scholar). Equal amounts of protein lysate from the cells were processed by Western blotting. Probing blots for tubulin verifies that equivalent amounts of protein are present in each sample (Fig. 1A). Analysis reveals that the amount of IQGAP1 in MCF-7, T47D, and ZR-75-1 cells is approximately equal (Fig. 1, A and B). Substantially less IQGAP1 is observed in MDA-MB-361, the other receptor-positive cell line. The reason for this finding is not known but may be because MDA-MB-361 is an adenocarcinoma (41Lacroix M. Leclercq G. Breast Cancer Res. Treat. 2004; 83: 249-289Crossref PubMed Scopus (610) Google Scholar). The highest IQGAP1 levels are detected in MDA-MB-231 cells, which have 2.7-fold more IQGAP1 than MCF-7 cells (Fig. 1, A and B). Importantly, these cells exhibit the highest invasive potential of 30 breast cell lines measured by modified Boyden chamber assays (42Neve R.M. Chin K. Fridlyand J. Yeh J. Baehner F.L. Fevr T. Clark L. Bayani N. Coppe J.P. Tong F. Speed T. Spellman P.T. DeVries S. Lapuk A. Wang N.J. Kuo W.L. Stilwell J.L. Pinkel D. Albertson D.G. Waldman F.M. McCormick F. Dickson R.B. Johnson M.D. Lippman M. Ethier S. Gazdar A. Gray J.W. Cancer Cell. 2006; 10: 515-527Abstract Full Text Full Text PDF PubMed Scopus (2398) Google Scholar). The amounts of IQGAP1 in MDA-MB-435S and Hs578T cells are not different to those in MCF-7 cells. Several factors may account for this observation. Cluster analysis of MDA-MB-435S cells reveals that these cluster with melanoma cells and may not be of breast origin (43Ross D.T. Scherf U. Eisen M.B. Perou C.M. Rees C. Spellman P. Iyer V. Jeffrey S.S. Van de Rijn M. Waltham M. Pergamenschikov A. Lee J.C. Lashkari D. Shalon D. Myers T.G. Weinstein J.N. Botstein D. Brown P.O. Nat. Genet. 200
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