Tumor Suppressor in Lung Cancer (TSLC)1 Suppresses Epithelial Cell Scattering and Tubulogenesis

The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases. The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases. TSLC1/IGSF4A is a tumor suppressor gene in non-small cell lung cancer (1Gomyo H. Arai Y. Tanigami A. Murakami Y. Hattori M. Hosoda F. Arai K. Aikawa Y. Tsuda H. Hirohashi S. Asakawa S. Shimizu N. Soeda E. Sakaki Y. Ohki M. Genomics. 1999; 62: 139-146Crossref PubMed Scopus (103) Google Scholar, 2Kuramochi M. Fukuhara H. Nobukuni T. Kanbe T. Maruyama T. Ghosh H.P. Pletcher M. Isomura M. Onizuka M. Kitamura T. Sekiya T. Reeves R.H. Murakami Y. Nat. Genet. 2001; 27: 427-430Crossref PubMed Scopus (398) Google Scholar), which encodes a single membrane-spanning glycoprotein belonging to the family of immunoglobulin superfamily cell adhesion molecules (3Masuda M. Yageta M. Fukuhara H. Kuramochi M. Maruyama T. Nomoto A. Murakami Y. J. Biol. Chem. 2002; 277: 31014-31019Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Because of its diverse functions in various tissues, TSLC1 has been characterized by several independent research groups. It is also known, therefore, as RA175 (4Fujita E. Soyama A. Momoi T. Exp. Cell Res. 2003; 287: 57-66Crossref PubMed Scopus (26) Google Scholar), SgIGSF (5Ito A. Jippo T. Wakayama T. Morii E. Koma Y. Onda H. Nojima H. Iseki S. Kitamura Y. Blood. 2003; 101: 2601-2608Crossref PubMed Scopus (108) Google Scholar), Necl2 (6Shingai T. Ikeda W. Kakunaga S. Morimoto K. Takekuni K. Itoh S. Satoh K. Takeuchi M. Imai T. Monden M. Takai Y. J. Biol. Chem. 2003; 278: 35421-35427Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), and SynCAM1 (7Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (636) Google Scholar). In a primary non-small cell lung cancer, we have found a 2-bp deletion in the TSLC1 that causes replacement of the C-terminal 19 amino acid residues, indicating that the cytoplasmic domain of TSLC1 is crucial for tumor suppression (2Kuramochi M. Fukuhara H. Nobukuni T. Kanbe T. Maruyama T. Ghosh H.P. Pletcher M. Isomura M. Onizuka M. Kitamura T. Sekiya T. Reeves R.H. Murakami Y. Nat. Genet. 2001; 27: 427-430Crossref PubMed Scopus (398) Google Scholar). In fact, the cytoplasmic domain was shown to be necessary for tumor suppressor activity of TSLC1 in nude mice (8Mao X. Seidlitz E. Ghosh K. Murakami Y. Ghosh H.P. Cancer Res. 2003; 63: 7979-7985PubMed Google Scholar). The cytoplasmic tail of TSLC1 contains a juxtamembrane sequence interacting with protein 4.1 binding motif (protein 4.1-BM) 4The abbreviations used are: BMbinding motifMDCKMadin-Darby canine kidneyHGFhepatocyte growth factorEMTepithelial mesenchymal transitionsPDZPSD95/Dlg/ZO-1GSTglutathione S-transferaseMAGuKmembrane-associated guanylate kinase homologDoxdoxycyclinePAKp21-activated kinasemAbmonoclonal antibodypAbpolyclonal antibodyHAhemagglutininGFPgreen fluorescent proteinAJadherens junction. and a class II PDZ binding motif (PDZ-BM) at the C terminus. We have demonstrated that protein4.1B/DAL-1, a member of the protein 4.1 family, connects TSLC1 to the actin cytoskeleton, and the interaction is involved in actin reorganization (9Yageta M. Kuramochi M. Masuda M. Fukami T. Fukuhara H. Maruyama T. Shibuya M. Murakami Y. Cancer Res. 2002; 62: 5129-5133PubMed Google Scholar). Along with others, we have reported that TSLC1 also interacts with membrane-associated guanylate kinase homologs (MAGuKs) through its class II PDZ domain (6Shingai T. Ikeda W. Kakunaga S. Morimoto K. Takekuni K. Itoh S. Satoh K. Takeuchi M. Imai T. Monden M. Takai Y. J. Biol. Chem. 2003; 278: 35421-35427Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 7Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (636) Google Scholar, 10Fukuhara H. Masuda M. Yageta M. Fukami T. Kuramochi M. Maruyama T. Kitamura T. Murakami Y. Oncogene. 2003; 22: 6160-6165Crossref PubMed Scopus (71) Google Scholar), implying that TSLC1, protein 4.1, and MAGuK form a tripartite complex. TSLC1 was originally identified based upon its ability to sup-press tumorigenicity of human lung adenocarcinoma A549 cells in nude mice (2Kuramochi M. Fukuhara H. Nobukuni T. Kanbe T. Maruyama T. Ghosh H.P. Pletcher M. Isomura M. Onizuka M. Kitamura T. Sekiya T. Reeves R.H. Murakami Y. Nat. Genet. 2001; 27: 427-430Crossref PubMed Scopus (398) Google Scholar). Restoration of TSLC1 expression in A549 cells inhibited the metastasis from the spleen to the liver (9Yageta M. Kuramochi M. Masuda M. Fukami T. Fukuhara H. Maruyama T. Shibuya M. Murakami Y. Cancer Res. 2002; 62: 5129-5133PubMed Google Scholar). Loss of TSLC1 has been observed at high frequency in advanced lung cancers as well as many other human cancers (2Kuramochi M. Fukuhara H. Nobukuni T. Kanbe T. Maruyama T. Ghosh H.P. Pletcher M. Isomura M. Onizuka M. Kitamura T. Sekiya T. Reeves R.H. Murakami Y. Nat. Genet. 2001; 27: 427-430Crossref PubMed Scopus (398) Google Scholar, 11Fukami T. Fukuhara H. Kuramochi M. Maruyama T. Isogai K. Sakamoto M. Takamoto S. Murakami Y. Int. J. Cancer. 2003; 107: 53-59Crossref PubMed Scopus (102) Google Scholar). Although these observations strongly suggest that the C-terminal motifs of TSLC1 play a crucial role in the suppression of tumor invasion and/or metastasis, as yet the precise roles of these motifs remain elusive. binding motif Madin-Darby canine kidney hepatocyte growth factor epithelial mesenchymal transitions PSD95/Dlg/ZO-1 glutathione S-transferase membrane-associated guanylate kinase homolog doxycycline p21-activated kinase monoclonal antibody polyclonal antibody hemagglutinin green fluorescent protein adherens junction. Epithelial-mesenchymal transitions (EMT), in which polarized epithelial cells become motile fibroblast-like cells, occur during critical phases of normal embryonic development in multicellular organisms (12Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5487) Google Scholar). It has been known, however, that EMT contributes to invasion and metastasis of carcinoma cells from epithelial tumors (12Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5487) Google Scholar, 13Gotzmann J. Mikula M. Eger A. Schulte-Hermann R. Foisner R. Beug H. Mikulits W. Mutat. Res. 2004; 566: 9-20Crossref PubMed Scopus (274) Google Scholar). The ‘scattering’ of Madin-Darby canine kidney (MDCK) epithelial cells by HGF is one of the well documented in vitro model systems that have been used to study the mechanisms of EMT (12Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5487) Google Scholar). When exposed to HGF, MDCK cells grown as small colonies on tissue culture plates respond by spreading, dissociating from each other, and scattering away from the colonies (12Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5487) Google Scholar). HGF induces a vast array of signaling through its receptor, c-Met, and one clear downstream target is the cytoskeleton, which is dramatically rearranged in response to HGF (12Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5487) Google Scholar). During HGF-induced EMT, the Rho family GTPases, especially Rho and Rac, function as key mediators of actin cytoskeletal dynamics (14Kamei T. Matozaki T. Sakisaka T. Kodama A. Yokoyama S. Peng Y.F. Nakano K. Takaishi K. Takai Y. Oncogene. 1999; 18: 6776-6784Crossref PubMed Scopus (176) Google Scholar). Another in vitro model involving EMT is HGF-induced MDCK tubulogenesis. A MDCK cell embedded in a collagen type I matrix forms a polarized lumen-enclosing epithelial monolayer called a cyst (15O'Brien L.E. Zegers M.M. Mostov K.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 531-537Crossref PubMed Scopus (497) Google Scholar). When treated with HGF, the cyst develops branching tubules. Although tubulogenesis is a complex morphogenic process, partial EMT involving rearrangements of the cytoskeleton and the junctional complexes between cells plays a key role at the stage where single file chains of cells are extended away from the cyst (15O'Brien L.E. Zegers M.M. Mostov K.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 531-537Crossref PubMed Scopus (497) Google Scholar). In the present study using a series of truncation mutants, we examined the effect of TSLC1 on EMT in the MDCK-HGF model systems. The results suggest that TSLC1 suppresses the HGF-induced EMT through regulating the activation of small Rho GTPases. Cells and Proliferation Assay—The MDCK Tet-Off cell line was obtained from BD Biosciences and maintained in Eagle's minimum essential medium (Sigma) supplemented with 100 μm amino 100 and 100 expression of the of doxycycline was to the Cell growth was examined by using a cell to the polyclonal the C of TSLC1 (3Masuda M. Yageta M. Fukuhara H. Kuramochi M. Maruyama T. Nomoto A. Murakami Y. J. Biol. Chem. 2002; 277: 31014-31019Abstract Full Text Full Text PDF PubMed Scopus (145) Google and the of TSLC1 (8Mao X. Seidlitz E. Ghosh K. Murakami Y. Ghosh H.P. Cancer Res. 2003; 63: 7979-7985PubMed Google A and a monoclonal antibody from and and from BD used to and for and from and used for the Rac and Rho activation a and a and TSLC1 was a TSLC1 by using (3Masuda M. Yageta M. Fukuhara H. Kuramochi M. Maruyama T. Nomoto A. Murakami Y. J. Biol. Chem. 2002; 277: 31014-31019Abstract Full Text Full Text PDF PubMed Scopus (145) Google or (3Masuda M. Yageta M. Fukuhara H. Kuramochi M. Maruyama T. Nomoto A. Murakami Y. J. Biol. Chem. 2002; 277: 31014-31019Abstract Full Text Full Text PDF PubMed Scopus (145) Google as a and a a lacking the cytoplasmic tail of TSLC1 the amino acid from to and the at was with an The truncation mutants and to amino the protein 4.1-BM and amino the PDZ-BM, These the cells with to the in medium of of cell and (9Yageta M. Kuramochi M. Masuda M. Fukami T. Fukuhara H. Maruyama T. Shibuya M. Murakami Y. Cancer Res. 2002; 62: 5129-5133PubMed Google Scholar). was with in and three-dimensional culture was to the Cells to the collagen I and plates at examined for cyst and subcellular localization of the full-length TSLC1 and the deletion induction of with medium HGF for and examined by taken with a with a the of and was with Cell at 1 in culture the the cells with medium in the or of HGF and for Cell scattering was examined by and as Cell scattering was by the of cells from colonies and the of cells in and the two-dimensional cells at a of 1 and to to for The cells and as (3Masuda M. Yageta M. Fukuhara H. Kuramochi M. Maruyama T. Nomoto A. Murakami Y. J. Biol. Chem. 2002; 277: 31014-31019Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). the cells examined with a with a and a The with a a single When for was to the of the in collagen type I was as Y. Mostov K.E. J. Cell Biol. PubMed Scopus Google Scholar). Rac and Rho of Rac and Rho was by a glutathione S-transferase using a Rac activation and a Rho activation to the cells at 1 culture and the cells with HGF for various and in an and from the with the p21-activated kinase binding domain and the binding domain on and by using was where using the or of Cell or Tet-Off cells, which the with the or TSLC1 and a expressing also has that the obtained the at a and MDCK Tet-Off cells a low of TSLC1 as in 1 and of the was with in the A cell of the showed growth with that of parental MDCK cells indicating that of TSLC1 or of the truncation mutants not cell growth in MDCK cells the 4.1-BM and the PDZ-BM for the of TSLC1 in in a have reported that TSLC1 to the lateral membrane of polarized cells on (3Masuda M. Yageta M. Fukuhara H. Kuramochi M. Maruyama T. Nomoto A. Murakami Y. J. Biol. Chem. 2002; 277: 31014-31019Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). We deletion of the cytoplasmic domain or the motifs its lateral for in the of the cells expressing the deletion mutants, the forms of TSLC1 to the lateral of the cells, as in cells expressing the TSLC1 In the three-dimensional MDCK cells form that are by a monolayer of the cells (15O'Brien L.E. Zegers M.M. Mostov K.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 531-537Crossref PubMed Scopus (497) Google Scholar). We embedded each of the in collagen I and found that of that from the one from the parental MDCK cell and demonstrated that TSLC1 was localized to the lateral membrane and whereas was in the and indicating that the truncation of the cytoplasmic domain abolished the lateral localization of TSLC1. Although a of to the lateral was also in the of the and PDZ-BM its localization from the lateral to the of the lateral membrane and the and We have also the for a of which is on the of parental MDCK (15O'Brien L.E. Zegers M.M. Mostov K.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 531-537Crossref PubMed Scopus (497) Google Scholar). In the expressing the or TSLC1, was as the of the and indicating that expression of TSLC1 or of the mutants not of cell localization of the TSLC1 and the deletion mutants in a three-dimensional in collagen type I for TSLC1 and with a and a in the and The with and are also HGF-induced tubulogenesis. from each grown with or HGF for and branching was examined by A cyst treated with HGF is in the at the of of TSLC1 in MDCK Cells HGF-induced the of TSLC1 HGF-induced tubulogenesis. In collagen I parental MDCK cells and the cells expressing TSLC1, or in a The of not either the development of from each not or tubulogenesis of parental MDCK cells and When with HGF for tubulogenesis was induced in parental MDCK and and In contrast, observed in the from the membrane When TSLC1 expression was with in cells, tubulogenesis to the with that of the parental MDCK cells Furthermore, and in a to that observed in parental MDCK and of for each cell demonstrated that the in tubulogenesis between the and cells was whereas other cell showed between the and These that TSLC1 HGF-induced tubulogenesis and that either of the cytoplasmic motifs is for the inhibitory TSLC1 HGF-induced Cell by of examined the of TSLC1 on HGF-induced cell scattering of MDCK In the of HGF, the cell small colonies on two-dimensional which from one The of not either the of each not or scattering of parental MDCK cells and a with HGF, and cells a and a culture at a and the other the HGF-treated cells not from each other and showed with the HGF When TSLC1 expression was with cells to the parental MDCK cells HGF also induced scattering of and cells and the of scattering in cells in parental MDCK cells A of cell scattering that the between the and cells was with In contrast, other cell showed between the and cells of cell scattering. Cell scattering is as the of cells the of cells in the the of The with as by the of EMT in these cells, we examined localization of adherens and for mesenchymal cells in parental and cells after a with or HGF. In the of HGF, the small colonies from each for the and at the cell-cell and but not for the mesenchymal and and In HGF-treated MDCK and cells, and and and from the cell these cells and and and that In contrast, HGF-treated cells to TSLC1 and at the cell-cell but mesenchymal and was We also the expression of these by with the results of expression of and was induced in HGF-treated MDCK and cells but not in cells The expression of was in HGF-treated MDCK and cells but not in HGF-treated cells indicating that cells retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These results suggest that TSLC1 the induction of EMT and the epithelial in HGF-treated MDCK Although the of was not in either the HGF-treated MDCK or cells by the of was not in these cells by has been reported to from the membrane to the and/or E-cadherin-based cell adhesion is (12Thiery J.P. Nat. Rev. Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5487) Google Scholar). is to have the of in TSLC1 Rac and Rho in HGF-treated MDCK Rho family are well key mediators of actin cytoskeletal rearrangements during HGF-induced cell scattering and tubulogenesis K. S. M. M. Cell Biol. 1999; PubMed Scopus Google Scholar). A study has demonstrated in MDCK cells on a tissue culture HGF transient activation of Rac and sustained activation of Rho, which is correlated with cell scattering response J.P. J. Cell Biol. PubMed Scopus Google Scholar). of and we that in response to HGF, the parental cells showed in Rac which to as well as sustained Rho activation In contrast, the HGF-treated cells prolonged Rac which was observed after but was in Rho activity These results demonstrated that the expression of TSLC1 induces the prolonged activation of Rac and the activation of Rho in MDCK It therefore, that the ability of TSLC1 to the cell scattering from its on Rac and Rho The effect of TSLC1 on Rac activity to upon its cytoplasmic domain cells showed a of Rac activation to that in parental MDCK cells however, not the sustained activation of Rho to the observed in parental MDCK cells the mechanisms of tumor and/or metastasis suppression by TSLC1, we the of TSLC1 on EMT in MDCK-HGF using a series of mutants with subcellular In a three-dimensional culture, but not in a two-dimensional culture, we found that the protein 4.1-BM and the PDZ-BM for the lateral localization of TSLC1. Because three-dimensional culture systems are to the in two-dimensional culture systems M.M. L.E. W. A. Mostov K.E. Cell Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), the protein 4.1-BM and the PDZ-BM play roles in the lateral localization of TSLC1 in human with the protein truncation of the PDZ-BM showed on subcellular localization of TSLC1. the PDZ-BM the localization from the lateral membrane to the of the MDCK The PDZ-BM of TSLC1 has been shown to with that as (6Shingai T. Ikeda W. Kakunaga S. Morimoto K. Takekuni K. Itoh S. Satoh K. Takeuchi M. Imai T. Monden M. Takai Y. J. Biol. Chem. 2003; 278: 35421-35427Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 7Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. Science. 2002; 297: 1525-1531Crossref PubMed Scopus (636) Google Scholar, 10Fukuhara H. Masuda M. Yageta M. Fukami T. Kuramochi M. Maruyama T. Kitamura T. Murakami Y. Oncogene. 2003; 22: 6160-6165Crossref PubMed Scopus (71) Google Scholar). The the that are involved in lateral of TSLC1 or of TSLC1 in the lateral In MDCK-HGF we have demonstrated that expression of TSLC1 suppresses HGF-induced tubulogenesis and cell scattering and that either the protein 4.1-BM or the PDZ-BM is necessary for The inhibitory effect of tubulogenesis to be correlated with the lateral localization of TSLC1 in HGF-induced tubulogenesis of MDCK cells through of of single file cell to and of M.M. L.E. W. A. Mostov K.E. Cell Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). A of observed in the by implying that TSLC1 its inhibitory effect at stage It has been that partial EMT during stage I and II to form and chains M.M. L.E. W. A. Mostov K.E. Cell Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). TSLC1 therefore, the induction of EMT during HGF-induced tubulogenesis. HGF-induced cell scattering of MDCK cells is also cell spreading, of cells from each other, and as HGF-treated cells showed but not from each other, that TSLC1 the in stage Furthermore, we found that cells to at cell-cell but not mesenchymal after with HGF. TSLC1 expression an epithelial of MDCK cells by EMT induced by HGF. It has been that Rac and Rho are involved in cell scattering at (14Kamei T. Matozaki T. Sakisaka T. Kodama A. Yokoyama S. Peng Y.F. Nakano K. Takaishi K. Takai Y. Oncogene. 1999; 18: 6776-6784Crossref PubMed Scopus (176) Google Scholar). from have shown that a Rac scattering K. S. M. M. Cell Biol. 1999; PubMed Scopus Google Scholar, J.P. F. Science. 278: PubMed Scopus Google and high in Rac and low in Rho are correlated with an epithelial by cell-cell adhesion J.P. F. Science. 278: PubMed Scopus Google Scholar, E. Nat. Cell Biol. 2003; PubMed Scopus Google Scholar). with these we observed high Rac and low Rho with of cell-cell adhesion in the HGF-treated It therefore, that TSLC1 HGF-induced EMT of MDCK cells by sustained activation of Rac and low activity of Rho. of Rac activity during HGF-induced MDCK cell scattering showed that a transient in Rac activity necessary to cell-cell F. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). that a transient in Rac activity is essential to of cell-cell prolonged Rac activation in the HGF-treated cells with the of cell cell scattering. The effect of TSLC1 on Rac activity to upon its cytoplasmic In addition, the protein 4.1-BM and PDZ-BM, which in the cytoplasmic are for the inhibitory of TSLC1 on tubulogenesis and cell scattering. It is therefore, that interacting with these motifs be for the prolonged Rac activation induced by TSLC1. We however, that the HGF-treated cells not the prolonged Rho activation at the observed in parental MDCK that of Rho activity caused by TSLC1 required domain of TSLC1 its cytoplasmic of TSLC1 is frequently observed in advanced non-small cell lung cancer as well as in many other human its involvement in invasion and/or metastasis (2Kuramochi M. Fukuhara H. Nobukuni T. Kanbe T. Maruyama T. Ghosh H.P. Pletcher M. Isomura M. Onizuka M. Kitamura T. Sekiya T. Reeves R.H. Murakami Y. Nat. Genet. 2001; 27: 427-430Crossref PubMed Scopus (398) Google Scholar, 11Fukami T. Fukuhara H. Kuramochi M. Maruyama T. Isogai K. Sakamoto M. Takamoto S. Murakami Y. Int. J. Cancer. 2003; 107: 53-59Crossref PubMed Scopus (102) Google Scholar). with of TSLC1 expression in non-small cell lung cancer cells dramatically suppressed metastasis from the spleen to the liver in nude mice (9Yageta M. Kuramochi M. Masuda M. Fukami T. Fukuhara H. Maruyama T. Shibuya M. Murakami Y. Cancer Res. 2002; 62: 5129-5133PubMed Google Scholar). In addition, of primary lung demonstrated that tumor cells to be A. M. K. Wakayama T. Koma Y. Iseki S. N. Y. Kitamura Y. 2003; PubMed Scopus Google Scholar, K. A. Wakayama T. Koma Y. T. Iseki S. Kitamura Y. N. Y. M. Cancer. 2003; PubMed Scopus Google Scholar, A. T. D. Murakami Y. N. M. Cancer PubMed Scopus Google Scholar). these observations suggest that the of TSLC1 is involved in EMT during tumor cell invasion and/or metastasis in various advanced human The present study in in vitro systems the that TSLC1 suppresses induction of EMT by the of Rac and Rho. on TSLC1 induces high Rac and low Rho activity in MDCK cells are It also be that we not the between TSLC1 and the for HGF, c-Met, by a not the signaling involved in the activity of TSLC1 and also a between TSLC1, EMT, and tumor metastasis. We are to and and the of the of the Cancer for with the We are to for of We for the of and S. for critical of the

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