Abstract
Many cancers have constitutively activated NFkappaB, 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 NFkappaB, 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 NFkappaB 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 NFkappaB activity. We show that stable cell lines overexpressing MEKK3 not only had elevated levels of NFkappaB 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 NFkappaB 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 NFkappaB 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.
Highlights
Many cancers have constitutively activated NFB, the elevation of which contributes to cancer cell resistance to chemotherapeutic agent-induced apoptosis
mitogen-activated protein kinase/extracellular-regulated kinase kinase kinase-3 (MEKK3) Cooperates with IL-1 and tumor necrosis factor (TNF) to Activate NFB— MEKK3 expression plasmids were cotransfected into U373 cells with a p(B)3/luciferase reporter gene
To further confirm that MEKK3 plays an active role in NFB activation, a dominant-negative (DN)-MEKK3 expression vector was cotransfected with p(B)3/luciferase reporter gene and its ability to block NFB activation by IL-1 examined
Summary
Chemicals and Reagents—Anti-MEKK3 antibody and its secondary antibody were purchased from Upstate Biotechnology Inc. Cell extracts from resistant cells were analyzed by Western blotting to assess the levels of HAMEKK3 expression. Frozen breast tumors, their matched normal tissues, and ovarian tumors were obtained from our institution tumor tissue banks. U373 and U373-MEKK3 cells (2 ϫ 106) were incubated with indicated concentrations of TRAIL for 7 h. The cells were washed, collected, and lysed in lysis buffer (20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 1 mM dithiothreitol, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 2 g/ml leupeptin, 2 g/ml aprotinin, and 0.5 g/ml benzamidine) on ice for 30 min. The interpretations of the p values were calculated from the Graph Pad Quickcalc, which is an on-line version (www.graphpadcom/quickcalc/ttest.cfm) of the calculators for scientists
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