Tissue Transglutaminase Is an Essential Participant in the Epidermal Growth Factor-stimulated Signaling Pathway Leading to Cancer Cell Migration and Invasion

Abstract

Epidermal growth factor (EGF) exerts pleiotropic effects during oncogenesis, including the stimulation of cell migration and invasiveness. Although a number of traditional signaling proteins (e.g. Ras and Rho GTPases) have been implicated in EGF-stimulated cancer cell migration, less is known about the identity of those proteins functioning further downstream in this growth factor pathway. Here we have used HeLa carcinoma cells as a model system for investigating the role of tissue transglutaminase (TGase), a protein that has been linked to oncogenesis, in EGF-stimulated cancer cell migration and invasion. Treatment of HeLa cells with EGF resulted in TGase activation and its accumulation at their leading edges, whereas knocking down TGase expression, or treating cells with a TGase inhibitor, blocked EGF-stimulated cell migration and invasion. We show that EGF signaling through Ras and c-Jun N-terminal kinase is responsible for targeting TGase to the leading edges of cells and activating it. The requirement for EGF to properly localize and activate TGase can be circumvented by the expression of oncogenic Ras (G12V), whose ability to stimulate migration is also dependent on TGase. We further show that, in the highly aggressive breast cancer cell line MDAMB231, where EGF stimulation is unnecessary for migration and invasive activity, TGase is already at the leading edge and activated. These findings demonstrate that TGase plays a key role in cancer cell motility and invasiveness and represents a previously unappreciated participant in the EGF pathway that stimulates these processes in cancer cells. Epidermal growth factor (EGF) exerts pleiotropic effects during oncogenesis, including the stimulation of cell migration and invasiveness. Although a number of traditional signaling proteins (e.g. Ras and Rho GTPases) have been implicated in EGF-stimulated cancer cell migration, less is known about the identity of those proteins functioning further downstream in this growth factor pathway. Here we have used HeLa carcinoma cells as a model system for investigating the role of tissue transglutaminase (TGase), a protein that has been linked to oncogenesis, in EGF-stimulated cancer cell migration and invasion. Treatment of HeLa cells with EGF resulted in TGase activation and its accumulation at their leading edges, whereas knocking down TGase expression, or treating cells with a TGase inhibitor, blocked EGF-stimulated cell migration and invasion. We show that EGF signaling through Ras and c-Jun N-terminal kinase is responsible for targeting TGase to the leading edges of cells and activating it. The requirement for EGF to properly localize and activate TGase can be circumvented by the expression of oncogenic Ras (G12V), whose ability to stimulate migration is also dependent on TGase. We further show that, in the highly aggressive breast cancer cell line MDAMB231, where EGF stimulation is unnecessary for migration and invasive activity, TGase is already at the leading edge and activated. These findings demonstrate that TGase plays a key role in cancer cell motility and invasiveness and represents a previously unappreciated participant in the EGF pathway that stimulates these processes in cancer cells. The EGF 2The abbreviations used are: EGFepidermal growth factorBPA5-(biotinamido)pentylamineERKextracellular-signal-regulated protein kinaseMAPKmitogen-activated protein kinaseMEKMAPK/ERK kinaseMDCmonodansylcadaverinePI3Kphosphatidylinositol 3-kinaseRAretinoic acidTGasetissue transglutaminaseJNKc-Jun N-terminal kinaseRNAiRNA interferencesiRNAsmall interference RNAHAhemagglutininDAPI4′,6-diamidino-2-phenylindoleFBSfetal bovine serum. receptor is a cell surface receptor tyrosine kinase that is expressed in a variety of normal cell lineages. Upon binding ligand, the EGF receptor becomes activated and initiates a highly regulated series of signaling events, which direct changes in gene transcription and various cellular responses that are vital for proper development and tissue homeostasis (1.Citri A. Yarden Y. Nat. Rev. Mol. Cell Biol. 2006; 7: 505-516Crossref PubMed Scopus (1639) Google Scholar, 2.Henson E.S. Gibson S.B. Cell. Signal. 2006; 18: 2089-2097Crossref PubMed Scopus (238) Google Scholar, 3.Katz M. Amit I. Yarden Y. Biochim. Biophys. Acta. 2007; 1773: 1161-1176Crossref PubMed Scopus (343) Google Scholar). In addition to its physiological functions, the EGF receptor has also been intimately associated with oncogenesis. Enhanced EGF receptor signaling, as a result of receptor overexpression and/or an autocrine stimulation of the receptor, is a hallmark of a variety of human tumors (2.Henson E.S. Gibson S.B. Cell. Signal. 2006; 18: 2089-2097Crossref PubMed Scopus (238) Google Scholar, 4.Laskin J.J. Sandler A.B. Cancer Treat. Rev. 2004; 30: 1-17Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 5.Zandi R. Larsen A.B. Andersen P. Stockhausen M.T. Poulsen H.S. Cell. Signal. 2007; 19: 2013-2023Crossref PubMed Scopus (240) Google Scholar). Moreover, high levels of EGF receptor expression detected in brain, breast, pancreatic, and lung tumors are well correlated with poor prognosis, and therapeutic strategies targeting the EGF receptor itself or signaling events initiated by the receptor are currently being used as cancer treatments (4.Laskin J.J. Sandler A.B. Cancer Treat. Rev. 2004; 30: 1-17Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 6.Bublil E.M. Yarden Y. Curr. Opin. Cell Biol. 2007; 19: 124-134Crossref PubMed Scopus (327) Google Scholar, 7.Li G. Wong A.J. Expert Rev. Vaccines. 2008; 7: 977-985Crossref PubMed Scopus (59) Google Scholar). These findings, coupled with the fact that ectopically expressing the EGF receptor in normal cell types is sufficient to induce ligand-dependent cellular transformation (8.Moscatello D.K. Ramirez G. Wong A.J. Cancer Res. 1997; 57: 1419-1424PubMed Google Scholar), strongly implicate EGF receptor signaling in cancer development. Thus, it is that a of to be EGF receptor activation to growth factor protein kinase protein kinase kinase tissue transglutaminase c-Jun N-terminal kinase interference interference bovine serum. cancer cells to EGF can or a of with the oncogenic or These the stimulation of cell the responses by and cell motility and invasive (8.Moscatello D.K. Ramirez G. Wong A.J. Cancer Res. 1997; 57: 1419-1424PubMed Google Scholar, I. M. M. D.K. Wong A.J. PubMed Scopus Google Scholar, M. Amit I. A. G. M. R. P. A. G. Yarden Y. Nat. Cell Biol. 2007; PubMed Scopus Google Scholar). has been to the that the ability of cancer cells to and/or in to cells that these are to have an aggressive the of EGF in cancer it is that be the signaling events that are responsible for the development of highly aggressive cell motility a that a highly activation and of signaling cells with of the and Nat. Rev. 2007; 7: PubMed Scopus Google Scholar). The of this is the of a cellular where at the or leading edge of the cell by and cell at the of the signaling that have been implicated in the migration of cancer cells of the Ras of and Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar, M. G. M. Cell 2007; PubMed Scopus (59) Google Scholar). protein kinase and of the protein kinase which are known for cell growth and M. Amit I. Yarden Y. Biochim. Biophys. Acta. 2007; 1773: 1161-1176Crossref PubMed Scopus (343) Google Scholar), of signaling that are also for their in cancer cell motility Cell 2004; PubMed Scopus Google Scholar). with the ability of EGF to stimulate the or to the EGF-stimulated migration of cancer as well as lung carcinoma cells Cancer Res. Google Scholar, Cell Res. PubMed Scopus Google Scholar). the breast carcinoma cell line with the these cells to the of EGF I. Cancer Res. 2006; PubMed Scopus Google Scholar). Although these findings the of Ras and and the and as in the cancer cell to be those proteins that are further downstream this growth factor at the of the leading edge and/or the of is a protein that an ability to and as well as an that in the of proteins or a protein and a Nat. Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). this protein an to is that the overexpression and/or activation of TGase have been in highly aggressive and human brain, breast, and tumors M.T. G. Cancer Res. 2008; PubMed Scopus Google Scholar, G. I. 2006; PubMed Scopus Google Scholar, A. P. A.B. G. Cancer Res. 2008; PubMed Scopus Google Scholar, M. 2007; PubMed Scopus Google Scholar). In ectopically expressing TGase in the breast cancer cell line sufficient to a to these cells with the J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the invasive and of breast carcinoma cells be the TGase associated with these cells by knocking down TGase expression or by the cells with the TGase J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. 2007; PubMed Scopus Google Scholar, Cancer Res. 2006; PubMed Scopus Google Scholar). these TGase and of cancer we the that it be a role in the that to cancer cell migration and the human carcinoma HeLa cell line as a model for these We that treating HeLa cells with EGF TGase to at the leading edges of as well as activated its and or the of TGase with blocked the ability of EGF to HeLa cell migration and that TGase is in fact an participant in the pathway responsible for these We further that EGF can through Ras and to localize TGase to the leading edges of cells and to activate it. these for the that cell is coupled to its ability to activate and in demonstrate that TGase plays an role in the ability of EGF to stimulate cancer cell migration and invasive Cell the and TGase and the and and whereas the inhibitor, and The TGase and whereas the that the activated of and c-Jun Cell The EGF receptor and the receptor HeLa and cell in bovine whereas HeLa cells expressing or Ras and in the growth with The the various of TGase cells whereas the and TGase cells the cell with or in the and of or in the and of the The cells with or with cell and the protein of cell by and the proteins to The with the various in and The detected with by to of the cells with with and blocked in bovine the cells with the for with and with or for an used to and used to The cells and the or on a cells or cells with or TGase to at which with for The for an and the cell a The to cells and with or the cells and by These on the in migration In these the cells the and with a TGase and HeLa cells or HeLa cells that been with or TGase and in at in the of a with EGF to the and the for The cells that on the of the with with and The and the These as previously J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J.J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). of HeLa cells in or of HeLa cells with or TGase and in of the with and a cells for of the and the of cells cells that have to cells that have These and the of cell for The of this to the TGase and cancer M.T. G. Cancer Res. 2008; PubMed Scopus Google Scholar, G. I. 2006; PubMed Scopus Google Scholar, A. P. A.B. G. Cancer Res. 2008; PubMed Scopus Google Scholar, M. 2007; PubMed Scopus Google Scholar, J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. 2007; PubMed Scopus Google an role for this protein in the pathway that stimulates cancer cell migration and invasive The human carcinoma HeLa cell line used to this these cells the EGF receptor and TGase and the as well as an model system for cancer cell migration and M. Amit I. A. G. M. R. P. A. G. Yarden Y. Nat. Cell Biol. 2007; PubMed Scopus Google Scholar, Y. Nat. Cell Biol. 2006; PubMed Scopus Google Scholar). that treating HeLa cells with EGF for of in the activation of the EGF receptor, with the receptor being activated of EGF stimulation and to levels by the effects of the of the EGF receptor on downstream signaling events, that the of and used to the activation levels of these signaling The in demonstrate that EGF stimulates the of the the and the the of activation for these signaling through of growth factor whereas the activation of and and the activation levels of the EGF We EGF TGase HeLa cells in in the or of EGF or and the and of TGase in the cells to EGF the of TGase with as by the of its expression the cell for activity, as by the of a in detected in the cells with the cells is also that EGF to be to of the known and of TGase Biol. Full Text PDF PubMed Google Scholar, M. PubMed Scopus Google Scholar, A.B. Cancer Res. Google Scholar), at the and of TGase and We EGF also the of TGase. on HeLa cells that TGase expressed the with its levels of expression being the of the the of TGase the cells of the EGF a in the cellular of that of the cells an accumulation of TGase at their of the and the of TGase detected the of the cells EGF stimulation to be to of cell and of the these cell a to the leading edge in cells Nat. Rev. 2007; 7: PubMed Scopus Google Scholar), we the that TGase be the leading edges of HeLa cells to a to induce cell and the leading edges of we or on of HeLa cells with or of the cell by with a the EGF-stimulated HeLa the to leading edges as by the of cell the and the accumulation of the surface of these cell the the cells with an that TGase in fact detected the leading edges of EGF-stimulated HeLa cells at the of with the that HeLa cells to EGF strongly cell motility as by the of the that TGase be an in cell is to the leading edges of cells. of on of HeLa cells or with of the on these a TGase and The are of cells edge of the The of the is and TGase is with a of cell the or the and and by represents a that edges of a that in a of cells to the The of for is The the of the at the of the We to the of TGase in EGF-stimulated cell migration the in HeLa cells with the TGase EGF to stimulate their ability to with these knocking down TGase expression blocked EGF-stimulated HeLa cell migration further implicate TGase as an of EGF-stimulated cell migration, in with TGase can cells to J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. 2007; PubMed Scopus Google Scholar, A. G. A.B. R. Cancer Res. 2006; PubMed Scopus Google Scholar), we to that the of EGF to stimulate cell migration, where TGase expression and/or its in be to a cell Cell on HeLa cells that the used for the migration in with the of TGase HeLa cells cell whereas the cells to the of the cells to We TGase as a of HeLa cell migration or its effects for the cells to a that used or bovine to stimulate cell that, the cell with of cell migration, the cells with the those cells with the of cell migration with that with the to the to a as with EGF for with for In we with EGF the cells to on the ability of of or to stimulate cell migration these that TGase is a role in cell migration is a of the pathway that stimulates the motility of these cancer cells. cell invasiveness on cell migration Nat. Rev. 2007; 7: PubMed Scopus Google Scholar). Thus, we to TGase also to the ability of EGF to invasiveness. We this by of the in that HeLa cells to EGF a in the number of cells that the the of in the cells. HeLa cells with on the of cell with the the invasive by Moreover, knocking down TGase expression by the EGF-stimulated invasiveness of the cells by as as These findings that, in addition to being for EGF-stimulated cell migration, TGase is also for EGF-stimulated invasiveness. that the EGF-stimulated motility and invasive of HeLa cells are coupled to the ability of this growth factor to TGase activation and its accumulation at the leading edges of we to EGF the and of TGase. We on TGase activation for its at the leading edges of cells. this we expressed of or of TGase that and/or in HeLa cells to TGase and the of cells with has been for cell J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. G. A.B. R. Cancer Res. 2006; PubMed Scopus Google Scholar, 2007; PubMed Scopus Google Scholar), ectopically expressed TGase in HeLa cells the and the ability to the of cell proteins on these that, being of and activity, the ectopically expressed TGase in cells. their with the TGase be detected in the of the the that expressed TGase in HeLa cells in the of EGF stimulation is to cell an to TGase to the leading edges of cells be linked to the of this HeLa cell expressing or a of TGase to in the or of and the of cell migration and the cells in the of EGF of The of in the and of cells that with a TGase and to a The cells in the that ectopically expressed the TGase protein by the with an by with The of these show that activating TGase by its overexpression in cells the of EGF and stimulate further to the that the activation and of TGase are for its ability to cell motility The to previously and/or of TGase 2007; PubMed Scopus Google also be to the leading edges of cells in an of as the TGase in Although the expression levels of the the and the TGase that of these in HeLa cells less that with we that of the TGase to EGF stimulation and at the leading edges of cells The that the of TGase is for its to the leading edge is also with the in the of the of cells that been with the show that the on the ability of EGF to localize TGase to the of cells. these findings that the and the of TGase are sufficient for its EGF-stimulated We to the EGF-stimulated signaling pathway responsible for the of TGase to the leading edges of cells. as well as the and to on the that these signaling proteins to cell migration responses in a variety of cancer cell types Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Cell 2004; PubMed Scopus Google Scholar). The in show that, where EGF stimulates cell migration of it and to a the of these signaling proteins in TGase to the of we that blocked their of HeLa cells with EGF in the or of the inhibitor, the inhibitor, or the and of cells the that the the of and the cell with that the of and c-Jun The in show that the EGF-stimulated and by and the of HeLa the cellular of TGase by the cell with an The in and the in show that the cells with or the ability of EGF to TGase to the leading edges of that is to this the the cells with the inhibitor, resulted in a in the of TGase EGF The of the at the EGF-stimulated accumulation of TGase at cell to be on of as in the in cell signaling as well as the inhibitor, also and to be at the EGF-stimulated of TGase. The of for a direct and the of TGase in which we show that the ability of the to the of TGase the leading edges of EGF-stimulated cells be to the fact that as a of cell In we show that, activation can the EGF-stimulated migration of HeLa cells as as the inhibitor, with the of TGase to the cell surface in to growth factor stimulation the requirement for TGase in the EGF-stimulated migration of HeLa cells it of to the signaling pathway that is responsible for the growth activation of TGase. We for and to or of these signaling for the ability of EGF to activate TGase. that of these EGF-stimulated TGase expression, that in the of TGase that the of or of the signaling is to changes in the expression of TGase. The cell used for the for the of proteins as a the of EGF-stimulated treating the cells with and by with it by the a of in EGF-stimulated activity, that it the levels of TGase in cells. These findings the of for the EGF-stimulated of TGase to the leading edges of as well as for the activation of its findings to this that the of TGase and its accumulation the leading edges of cells are linked to the ability of EGF to stimulate HeLa cell motility and invasive we to TGase activation and its for the migration responses by proteins that have been implicated in or that in human cancer cell Thus, we the of TGase in the of an oncogenic of Ras HeLa as well as the TGase and the highly by the human breast cancer cell line J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. 2007; PubMed Scopus Google Scholar). Ras to be a that it as a downstream of the EGF receptor, and is the in human M. M. Nat. Rev. PubMed Scopus Google Scholar). HeLa cells expressing the or a of Ras (G12V), and the expression of the Ras protein by the We Ras expression TGase activation by the levels of in cell HeLa cells to Ras cells. The in that the detected in cells expressing the of Ras to the cells the and Moreover, the of TGase in the Ras cells to that the cells with the that the EGF receptor the activation of TGase in HeLa cells is through its ability to to of also to the levels of TGase expression and activation in the highly aggressive and invasive breast cancer cells. to findings by and J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. 2007; PubMed Scopus Google Scholar), a of the TGase expression levels in HeLa and cells that TGase expression is in cells of the associated with TGase expressed in as by the of that TGase is activated in this cancer cell line the of cells with EGF to a further in TGase activity, that TGase is already activated in these cancer cells. The of TGase HeLa cells expressing or the Ras as well as in cells to various by the cells with a TGase of the cell are in the of the cells where TGase expression is HeLa cells expressing the Ras and the cells show a accumulation of TGase at their In we that of HeLa cells expressing Ras and of the cells of TGase Moreover, the cellular of TGase HeLa cells expressing the of as well as in the cells to the TGase inhibitor, and their cells to the and further that the ability of TGase to be to the surface of cells its levels of TGase activity, and its to be to leading edges, are by Ras HeLa cells and we these cell by as in the Ras HeLa cells and the cells to in of or Moreover, we that the migration of of these cell to that TGase is for the cell migration responses by the expression of a activated of Ras in HeLa and as by the highly aggressive breast cancer cell line further by the fact that knocking down TGase expression in the HeLa cells or in cells also blocked the ability of these cells to and on these findings, we that TGase and the accumulation of TGase at the leading edges of cancer cells as of cell including those tumors where their aggressive is dependent on EGF the responsible for the aggressive of human cancer cells is for of cancer also for the development of for cancer of the hallmark of transformation is the ability of growth EGF to stimulate cell migration and invasiveness M. Amit I. A. G. M. R. P. A. G. Yarden Y. Nat. Cell Biol. 2007; PubMed Scopus Google Scholar, J.J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. Nat. Rev. PubMed Scopus Google Scholar). We have used the carcinoma HeLa cell line as a model to in these EGF-stimulated Here we show that TGase is protein that plays an role in the ability of EGF to stimulate HeLa cell migration and invasive The that this the findings that treating HeLa cells with EGF to the activation of the and of as well as the accumulation of TGase the leading edges of cells. We that by knocking down TGase expression in HeLa cells or by a that at the of EGF of cell migration or invasion. ectopically expressed TGase in HeLa cells and it is to the of EGF and a cell migration Thus, TGase activation is sufficient for the aggressive by HeLa cells. the EGF-stimulated of TGase to the leading edges of cells is to be an with TGase in the ability of this growth factor to cell migration and invasive a of EGF the activation and of TGase to the leading edges of cells. on where we that the of TGase expression and the activation of its and in various cell with and/or EGF dependent on J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), we that a role in the of TGase in HeLa cells. with the ability of EGF to stimulate TGase activation in HeLa cells. we that signaling to a of the is for the activation of TGase as well as its to the leading and for EGF-stimulated cell The role of in EGF signaling to TGase to be highly of signaling including of the and at TGase or its to the leading edges of cells. is that the pathway is responsible for of these of TGase the that the accumulation of TGase at the leading edges of cells and the EGF-stimulated activation of are coupled be that TGase is in an through of with a binding in the that that TGase to the cell surface also this that EGF through Ras to to and TGase as cells expressing oncogenic Ras can the for EGF stimulation to TGase to the leading edges of cells and activate its and Moreover, the requirement for EGF stimulation can be circumvented in where TGase activation and accumulation at their leading edges, and these cells are highly and invasive in the of growth factor We EGF signaling through Ras and TGase to the leading edges of we it is the of a is it TGase plays an role in EGF-stimulated cell migration and invasiveness. have also been to for proteins to at the leading edges of cells their to and signaling Nat. Rev. 2007; 7: PubMed Scopus Google Scholar, M. G. M. Cell 2007; PubMed Scopus (59) Google Scholar, Y. Nat. Cell Biol. 2006; PubMed Scopus Google Scholar, Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar, Cell 2004; PubMed Scopus Google Scholar). The EGF-stimulated of TGase to the leading edges of HeLa cells it to a protein or a of protein that to be in for cell motility to Although a number of signaling and proteins have been in as TGase Nat. Rev. Mol. Cell Biol. PubMed Scopus Google Scholar), we have been to implicate of these in the cell migration in cancer cells. we have a TGase a role in cell migration, the that it can a with as by the of these proteins HeLa cells as well as human breast cancer cell and M. is about these findings is that TGase is of a with a of TGase in which the has been to is to be with Although we have that can as a of TGase in as by the of of of and TGase are in the of we have that is by in HeLa or cells Moreover, the fact that the of TGase with to as a it to an it be to TGase the an Thus, these findings to that the of TGase be in binding to TGase to as a for the of proteins that or effects on in cells. be at further this of have linked TGase to human cancer TGase overexpression and/or its activation have been in a of the aggressive brain, breast, and tumors and cell M.T. G. Cancer Res. 2008; PubMed Scopus Google Scholar, G. I. 2006; PubMed Scopus Google Scholar, A. P. A.B. G. Cancer Res. 2008; PubMed Scopus Google Scholar, A. R. Cancer Res. 2006; PubMed Scopus Google Scholar). Moreover, the TGase in of these cancer cell (e.g. by knocking down TGase expression or the cells with TGase has been and in to cancer cells to as well as their aggressive M.T. G. Cancer Res. 2008; PubMed Scopus Google Scholar, J.J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, A. 2007; PubMed Scopus Google Scholar, Cancer Res. 2006; PubMed Scopus Google Scholar). Although is to further the the signaling and TGase in types of findings TGase as a for therapeutic those human whose aggressive is coupled to EGF receptor expression and We for