Overexpression of MEKK3 Confers Resistance to Apoptosis through Activation of NFκB

Abstract

Many cancers have constitutively activated NFκB, the elevation of which contributes to cancer cell resistance to chemotherapeutic agent-induced apoptosis. Although mitogen-activated protein kinase/extracellular-regulated kinase kinase kinase-3 (MEKK3) has been shown to participate in the activation of NFκB, its relations to apoptosis and cancer are unclear. In this study, we established cell model systems to examine whether stable expression of MEKK3 could lead to increased NFκB activity and confer resistance to apoptosis. In addition, we investigated in breast and ovarian cancers whether MEKK3 expression may be altered and correlated with aberrant NFκB activity. We show that stable cell lines overexpressing MEKK3 not only had elevated levels of NFκB binding activity but also were more responsive to cytokine stimulation. These stable cells showed 2–4-fold higher basal expression of Bcl-2 and xIAP than the parental cells. Consistent with this increased expression of cell survival genes, MEKK3 stable cells showed reduced activation of caspases 3 and 8 and poly(ADP-ribose) polymerase cleavage and dramatically increased resistance to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin, daunorubicin, camptothecin, and paclitaxel. Intriguingly, analysis of human breast and ovarian cancers showed that a significant fraction of these samples have elevated MEKK3 protein levels with corresponding increases in NFκB binding activities. Thus, our results established that elevated expression of MEKK3 appears to be a frequent occurrence in breast and ovarian cancers and that overexpression of MEKK3 in cells leads to increased NFκB activity and increased expression of cell survival factors and ultimately contributes to their resistance to apoptosis. As such, MEKK3 may serve as a therapeutic target to control cancer cell resistance to cytokine- or drug-induced apoptosis. Many cancers have constitutively activated NFκB, the elevation of which contributes to cancer cell resistance to chemotherapeutic agent-induced apoptosis. Although mitogen-activated protein kinase/extracellular-regulated kinase kinase kinase-3 (MEKK3) has been shown to participate in the activation of NFκB, its relations to apoptosis and cancer are unclear. In this study, we established cell model systems to examine whether stable expression of MEKK3 could lead to increased NFκB activity and confer resistance to apoptosis. In addition, we investigated in breast and ovarian cancers whether MEKK3 expression may be altered and correlated with aberrant NFκB activity. We show that stable cell lines overexpressing MEKK3 not only had elevated levels of NFκB binding activity but also were more responsive to cytokine stimulation. These stable cells showed 2–4-fold higher basal expression of Bcl-2 and xIAP than the parental cells. Consistent with this increased expression of cell survival genes, MEKK3 stable cells showed reduced activation of caspases 3 and 8 and poly(ADP-ribose) polymerase cleavage and dramatically increased resistance to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin, daunorubicin, camptothecin, and paclitaxel. Intriguingly, analysis of human breast and ovarian cancers showed that a significant fraction of these samples have elevated MEKK3 protein levels with corresponding increases in NFκB binding activities. Thus, our results established that elevated expression of MEKK3 appears to be a frequent occurrence in breast and ovarian cancers and that overexpression of MEKK3 in cells leads to increased NFκB activity and increased expression of cell survival factors and ultimately contributes to their resistance to apoptosis. As such, MEKK3 may serve as a therapeutic target to control cancer cell resistance to cytokine- or drug-induced apoptosis. The role of mitogen-activated protein kinases (MAPKs) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; NFκB, nuclear factor κB; IκB, inhibitor of NFκB; MEKK, MAPK/extracellular signal-regulated kinase kinase kinase; IL, interleukin; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; PARP, poly(ADP-ribose) polymerase; HA, hemagglutinin; EMSA, electrophoretic mobility shift assay; HIV, human immunodeficiency virus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DN, dominant-negative. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; NFκB, nuclear factor κB; IκB, inhibitor of NFκB; MEKK, MAPK/extracellular signal-regulated kinase kinase kinase; IL, interleukin; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; PARP, poly(ADP-ribose) polymerase; HA, hemagglutinin; EMSA, electrophoretic mobility shift assay; HIV, human immunodeficiency virus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DN, dominant-negative. in the growth and survival of many cell types has been well documented (1Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4307) Google Scholar). MAPKs and stress-activated protein kinases function as mediators that regulate the cellular responses to environmental stress. They serve as key integration points along the signal transduction cascade that not only link diverse extracellular stimuli to subsequent signaling molecules but also amplify the initiating signals to ultimately activate the effector molecules and induce cell proliferation, differentiation, and survival (1Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4307) Google Scholar, 2Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3393) Google Scholar). The MAPK pathway participates in determining whether a cell re-enters the cell cycle or undergoes cell cycle arrest, senescence, or apoptosis (3Reddy K.B. Nabha S.M. Atanaskova N. Cancer Metastasis Rev. 2003; 22: 395-403Crossref PubMed Scopus (452) Google Scholar). The MAPK/extracellular signal-regulated kinase kinase kinases (MEKKs) are upstream regulators of the three-module cascade of MAPKs (1Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4307) Google Scholar). MEKKs consists of a small gene family, MEKK1–6, that belongs to the serine/threonine protein kinases (1Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4307) Google Scholar). Upon phosphorylation, MEKKs are activated and can subsequently phosphorylate and activate downstream signaling molecules, including c-Jun N-terminal kinase and p38 (2Johnson G.L. Lapadat R. Science. 2002; 298: 1911-1912Crossref PubMed Scopus (3393) Google Scholar). Transient overexpression of MEKK1–3, transforming growth factor-β-activating kinase, and NFκB-activating kinase has been shown to activate NFκB (4Zhao Q. Lee F.S. J. Biol. Chem. 1999; 274: 8355-8358Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 5Tojima Y. Fujimoto A. Delhase M. Chen Y. Hatakeyama S. Nakayama K. Kaneko Y. Nimura Y. Motoyama N. Ikeda K. Karin M. Nakanishi M. Nature. 2000; 404: 778-782Crossref PubMed Scopus (311) Google Scholar). However, except for MEKK3, genetic studies have raised uncertainties regarding their involvement in the activation of NFκB (6Yang J. Boerm M. McCarty M. Bucana C. Fidler I.J. Zhuang Y. Su B. Nat. Genet. 2000; 24: 309-313Crossref PubMed Scopus (190) Google Scholar). In contrast, mouse embryonic fibroblasts deficient in MEKK3 were unable to activate NFκB in response to stimulation by IL-1 and TNF, indicating the functional importance of MEKK3 in the proinflammatory cytokine-mediated activation of NFκB (7Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (349) Google Scholar). In addition to its importance in immunity and inflammation (8Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4550) Google Scholar), NFκB has been implicated as a major regulator that controls cancer cell survival, proliferation, and metastasis (9Huang S. DeGuzman A. Bucana C.D. Fidler I.J. Clin. Cancer Res. 2000; 6: 2573-2581PubMed Google Scholar, 10Lin A. Karin M. Semin. Cancer Biol. 2003; 13: 107-114Crossref PubMed Scopus (345) Google Scholar). The transcription factor NFκB is composed primarily of heterodimeric complexes of the Rel family proteins p65 and p50. In normal resting cells, NFκB is physically confined to the cytoplasm through interaction with its protein inhibitor IκB (11Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2422) Google Scholar, 12Gilmore T.D. Cancer Treat Res. 2003; 115: 241-265Crossref PubMed Google Scholar). Cytokines such as IL-1, tumor necrosis factor (TNF), and epidermal growth factor bind to their membrane receptors and trigger a cascade of signaling events that ultimately leads to the activation of IκB kinase, which phosphorylates IκB at two serine residues (13Rothwarf D.M. Karin M. Sci. STKE. 1999; 1999: 1-16Google Scholar, 14Orlowski R.Z. Baldwin Jr., A.S. Trends Mol. Med. 2002; 8: 385-389Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). Phosphorylated IκB is then rapidly ubiquitinated and degraded through the 26 S proteosome pathway (15Senftleben U. Karin M. Crit. Care Med. 2002; 30: S18-S26Crossref Scopus (260) Google Scholar). As a result, NFκB is released from its inhibitor and translocates to the nucleus where it binds to regulatory DNA elements. NFκB regulates the expression of many genes essential for cell growth and differentiation, such as cytokines, growth factors and their receptors, and adhesion molecules (9Huang S. DeGuzman A. Bucana C.D. Fidler I.J. Clin. Cancer Res. 2000; 6: 2573-2581PubMed Google Scholar, 11Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2422) Google Scholar, 12Gilmore T.D. Cancer Treat Res. 2003; 115: 241-265Crossref PubMed Google Scholar, 16Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3389) Google Scholar, 17Boone D.L. Lee E.G. Libby S. Gibson P.J. Chien M. Chan F. Madonia M. Burkett P.R. Ma A. Inflamm. Bowel Dis. 2002; 8: 201-212Crossref PubMed Scopus (49) Google Scholar). It also up-regulates the expression of genes that actively participate in controlling cell survival (Bcl-2, Bcl-xL, survivin, and IAP family) (11Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Crossref PubMed Scopus (2422) Google Scholar, 16Pahl H.L. Oncogene. 1999; 18: 6853-6866Crossref PubMed Scopus (3389) Google Scholar), angiogenesis (IL-8, basic fibroblast growth factor, and vascular endothelial growth factor) (9Huang S. DeGuzman A. Bucana C.D. Fidler I.J. Clin. 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In addition to expression of cellular factors also a major role in activation of NFκB in cancer cells A. 2002; 16: PubMed Scopus Google Scholar). cancer IL-1, overexpression of epidermal growth factor has been shown to to the activation of NFκB in breast Chen L. L. Z. Oncogene. 2003; 22: PubMed Scopus Google Scholar, Lin J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Sci. U. S. A. 2000; PubMed Scopus Google Scholar). Although studies showed that of MEKK3 can NFκB activation A. Mol. 2002; PubMed Scopus Google Scholar, B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), has been that correlated aberrant MEKK3 expression with In this we investigated whether stable overexpression of MEKK3 to lead to higher NFκB activity and increased expression of survival genes and to confer increased resistance to apoptosis. In addition, we whether is altered expression of MEKK3 in human breast and ovarian which to the aberrant NFκB activity. We show that stable expression of MEKK3 in cells not only elevated levels of activated NFκB but also induced the expression of cell survival proteins and dramatically increased their resistance to apoptosis. we for the that breast and ovarian cancers have elevated expression of MEKK3 and show elevated MEKK3 expression and increased NFκB activity. and and its were from from c-Jun N-terminal kinase, and 8 were from and were from from and from and kinase were from and were from and IL-1 from the Cancer camptothecin, and were from and Cancer and cells and their MEKK3 stable cell lines were in essential embryonic cells and its stable cell were in of the cells were in with at in MEKK3 stable cell parental cells were with MEKK3 expression and for resistance to small of and and were from cells were by to the levels of breast their normal and ovarian were from our tumor and for cell the cells were with and The cells were then with and and were at in a for at and the protein in the were as of cell were with protein The were then at with of in a of a of protein and for The were then with of by two with of the were at for in The proteins in the were and to The were with and with the by which with the for NFκB the of and J. Immunol. 1998; Google Scholar). nuclear were used or at EMSA, of nuclear with and from the J. Immunol. 1998; Google Scholar). DNA binding by with or complexes were the Transient and cells were in and with of of the in of a (7Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (349) Google Scholar). the cells were with or with IL-1 or The cells were and the cell were in for activity a were the of and J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The cells were in in and with of for The cells were then with of in for at of in and at The at a with the as a in the is of and reduced cell of apoptosis by the of cleavage of J. Immunol. 1998; Google Scholar). and cells were with of for The cells were then and in and for The cell were by at for in the were in and The were then with that the and the of the of and chemotherapeutic the parental and MEKK3 stable cells were by the The of the were from the which is of the for MEKK3 with IL-1 and to MEKK3 expression were cells with a in activity with a at of MEKK3 MEKK3 in NFκB IL-1 and increased gene expression a stimulation of cells with IL-1 and in and increases in in activity that MEKK3 may with and signaling molecules to activate that MEKK3 role in NFκB a expression with gene and its to NFκB activation by IL-1 As shown in expression of not only reduced the basal activity but also activation of gene these results MEKK3 in the of NFκB and MEKK3 as a in the cytokine signaling events that lead to the activation of of MEKK3 in NFκB results a MEKK3 levels and the of cells to activate NFκB in response to stimulation. examine this and cells were with expression and for resistance to and stable cell These stable cell lines elevated levels of MEKK3 as by the and were used to examine the of higher MEKK3 expression the activation of stable cell lines showed elevated NFκB binding stimulation with IL-1 in higher levels of NFκB activity than their parental cells Consistent with the DNA binding of a also showed higher basal activity as well as response to IL-1 and in cells than in parental cells The of activity by MEKK3 and IL-1 is by the expression of a IκB, in with MEKK3 expression or in stable cells overexpressing MEKK3 Thus, the results from the MEKK3 stable cell lines are in with the studies and that MEKK3 can with and signals to activate MEKK3 to whether MEKK3 a role in cell survival through its to regulate NFκB and cells and their parental cells were with of TRAIL, and the cell were by As shown in parental cell lines were to TRAIL, in cell TRAIL, of the cells and than of cells were In contrast, MEKK3 stable cell lines were to TRAIL, where in cell survival at These results that overexpression of MEKK3 in and cells increased their resistance to MEKK3 of the binding to its apoptosis through the activation and of 3 and 8 to their 3 in the PARP, ultimately in apoptosis L. C. M. M. K. PubMed Scopus Google Scholar, S. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. L. R. A. C. J. 1998; Google Scholar). whether the reduced cell survival in parental cells to activation of the and cells were with for and the cell were by for the cleavage of 3 and 8 and As with TRAIL, the parental cells showed a activation of 3 and 8 as well as cleavage of to the TRAIL, a significant of 3 and 8 were and a fraction of In contrast, stable cells were to activation of caspases and Thus, these the cell survival and with parental cells, MEKK3 stable cells are to apoptosis. MEKK3 of the for the increased resistance of stable cells to TRAIL, we the expression of a cell survival Bcl-2 2003; 8: PubMed Scopus Google Scholar), and in and cells. We that increased resistance to apoptosis be to increased expression of these genes, which are of NFκB R.Z. Baldwin Jr., A.S. Trends Mol. 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Oncogene. 2002; PubMed Scopus Google Scholar). and stable cells were with TRAIL, and the levels of Bcl-2 and xIAP were by As shown in basal expression for Bcl-2 and xIAP expression were elevated in cells with the parental cells. with not their expression in cell The expression of Bcl-2 and xIAP and cells not to protein as by the these cell were for in the levels of the two cell indicating that increased cell survival in MEKK3 stable cells of activity. of MEKK3 to whether overexpression of MEKK3 in stable cells may also their to chemotherapeutic and cells were with of doxorubicin, daunorubicin, camptothecin, and and the cell survival by Although parental and MEKK3 stable cells were by chemotherapeutic in a the stable cells showed resistance than the parental cells at doxorubicin, only of the parental cells of the stable cells a higher of of the parental cells were of the stable cells The for these chemotherapeutic are in our results show that in cells that have elevated expression of MEKK3, is a corresponding in their resistance the chemotherapeutic resistance of cells to and chemotherapeutic agent-induced the that results in of cell the that results in of cell in a MEKK3 and NFκB in Breast and types of cancer have been to have constitutively elevated NFκB binding activity (1Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4307) Google Scholar, K.B. Nabha S.M. Atanaskova N. Cancer Metastasis Rev. 2003; 22: 395-403Crossref PubMed Scopus (452) Google Scholar), and many of are to apoptosis by 2003; PubMed Scopus Google Scholar, N. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The results of our studies with the MEKK3 stable cells the that overexpression of MEKK3 in cancer cells may be a that to these cancer cell whether constitutively elevated NFκB activity in cancers in be to MEKK3 we the expression of MEKK3 in human breast and with their normal breast from the cell from these samples were with we that of the breast have or MEKK3, have elevated MEKK3 In contrast, MEKK3 not or at levels in normal control of the with showed for and of the samples not from normal breast and breast with or MEKK3 expression were and NFκB binding were by As shown in breast that levels of MEKK3 have NFκB binding with MEKK3 levels have NFκB binding with of normal control breast whether this increased MEKK3 expression and NFκB binding activity in breast cancers may be to types of we MEKK3 and NFκB levels in ovarian results showed that MEKK3 at levels in ovarian cancer samples and at levels in the two samples of NFκB binding activity also MEKK3 levels and NFκB binding MEKK3 expression is NFκB binding activity also our results show that elevated MEKK3 expression is at in breast and ovarian is a MEKK3 protein levels and NFκB binding in these These are with the that MEKK3 regulatory role in the activation of NFκB in breast and ovarian In the study, we to the of MEKK3 in the activation of NFκB and to a for the resistance of cancer cells to cytokine- or chemotherapeutic agent-induced apoptosis. In addition, we whether in MEKK3 expression in human cancers that may be correlated with elevated NFκB activity. We established and model cell systems that protein kinase MEKK3 and showed that elevated MEKK3 in these cells to higher NFκB and increased expression of genes and ultimately resistance to and chemotherapeutic drug-induced apoptosis. Intriguingly, we that a significant of breast and ovarian cancers have elevated expression of MEKK3 with NFκB activity. our results a role of MEKK3 in the of NFκB activity in cancer cells. It has been that in mouse fibroblasts deficient for MEKK3, activation of NFκB by IL-1 and that MEKK3 role in the signaling pathway to the activation of NFκB (7Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (349) Google Scholar). is in this in which of MEKK3 or expression in a activation or of NFκB, These results also of MEKK3 in the activation of It is that stimulation of cells with in activation of NFκB, functional MEKK3 and signaling molecules activated by the such downstream of IL-1 and signaling pathway is which has also been to participate in the activation of NFκB Baldwin Jr., A.S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Res. 2002; 22: Google Scholar, C. M. C. 2003; PubMed Scopus Google Scholar). Consistent with this we higher activation of the gene MEKK3 and expression were cells than with J. Huang and W. Thus, it is that MEKK3 and as downstream of IL-1 and TNF, can function to regulate NFκB Although this is these studies not by signaling the of MEKK3 in the of NFκB and to examine the cellular of elevated MEKK3 we established stable cell lines in and cells that Consistent with stable expression of MEKK3 in these cells to NFκB activity. Thus, these studies showed MEKK3 levels and NFκB activities. higher levels of MEKK3 and NFκB in these stable cells are to their to or survival is of We had that mouse fibroblasts are to TNF, fibroblasts deficient in MEKK3 are to cell (7Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (349) Google Scholar). MEKK3 a and we that MEKK3 stable cells be more to such were parental and stable cells were with to induce apoptosis. Although parental and cells were by TRAIL, and cells are as by the and cleavage of and 3 and Although the to resistance in MEKK3 stable cells is to be it is that levels of MEKK3 in stable cells to increased activation of NFκB, which in up-regulates the expression of cell survival and proteins that ultimately the cells from apoptosis. Consistent with this we increased expression of cell survival Bcl-2 and in stable cells, which could from apoptosis. The role of MEKK3 in cell survival also in the studies with chemotherapeutic doxorubicin, daunorubicin, camptothecin, and used to cancer I.J. Nat. Med. 2000; 6: PubMed Scopus Google Scholar). MEKK3 stable cells higher than the parental cells, with and the In addition to cell survival, MAPK signaling pathway also contributes in cellular including cell proliferation, cell and through activation of downstream transcription factors (3Reddy K.B. Nabha S.M. Atanaskova N. Cancer Metastasis Rev. 2003; 22: 395-403Crossref PubMed Scopus (452) Google Scholar, F. Lee 2003; PubMed Scopus Google Scholar). As such, is MAPK expression and the of (3Reddy K.B. Nabha S.M. Atanaskova N. Cancer Metastasis Rev. 2003; 22: 395-403Crossref PubMed Scopus (452) Google Scholar). Although our results showed MEKK3 stable cells increased resistance to cytokine- and drug-induced we not whether also more tumor cell such as cell proliferation, and However, it is that of constitutively activated MAPK to in that expression of activated MAPK can to the of B. C. H. S. M. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of MAPK activated the cell survival pathway in and (3Reddy K.B. Nabha S.M. Atanaskova N. Cancer Metastasis Rev. 2003; 22: 395-403Crossref PubMed Scopus (452) Google Scholar, 2003; PubMed Scopus Google Scholar). In study, activated extracellular signal-regulated kinase and NFκB were to apoptosis N. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). overexpression of MEKK3 is to cell is However, our that of the human breast and ovarian cancers have elevated MEKK3 levels is many of the that have elevated expression of MEKK3 also have increased NFκB DNA binding activity. It is to that the constitutively activated NFκB in these at in be to elevated MEKK3 activity. studies this and examine the MEKK3 levels and NFκB in breast and ovarian cancers as well as types of In overexpression of MEKK3, or in stable cells, cytokine-mediated activation of In the stable cells, increased MEKK3 expression is correlated with increased expression of cell survival genes Bcl-2 and xIAP and increased resistance to and chemotherapeutic agent-induced apoptosis. we that a significant fraction of breast and ovarian cancers have expression of MEKK3 with elevated levels of NFκB activity. Although it is at this whether the NFκB activity in cancer cells can be to the MEKK3 it that the levels of NFκB activation and the expression of genes in cancer cell survival may be by of MEKK3 activity. We J. and B. Su for the and MEKK3 expression and A. for the breast cancer We also W. for