MEKK1 Regulates Chemokine Expression in Mammary Fibroblasts: Implications for the Breast Tumor Microenvironment.

Breast tumors contain both transformed epithelial cells and non-transformed stroma cells producing secreted factors that can promote metastasis. Previously, we demonstrated that the kinase MEKK1 regulates cell migration and gene expression, and that transgene-induced breast tumor metastasis is markedly inhibited in MEKK1-deficient mice. In this report, we examined the role of MEKK1 in stroma cell gene expression and the consequent effect on breast tumor cell function. Using a heterotypic cell system to quantify the effect of stroma cells on breast tumor cell function, we discovered that MEKK1-/- fibroblasts are significantly less effective at inducing tumor cell invasion than MEKK1+/+ fibroblasts. Expression array analysis revealed that both baseline and tumor cell-induced expression of the chemokines CCL3, CCL4, and CCL5 were markedly reduced in MEKK1-/- mammary fibroblasts. By focusing on the role of MEKK1 in CCL5 regulation, we discovered that MEKK1 kinase activity promotes CCL5 expression, and inactive mutant MEKK1 strongly inhibits CCL5 transcription. CCL5 and the other MEKK1-dependent chemokines are ligands for the GPCR CCR5, and we show that the CCR5 antagonist Maraviroc strongly inhibits fibroblast-induced tumor cell migration. Finally, we report that fibroblast growth factor 5 (FGF-5) is secreted by MDA-MB 231 cells, that FGF-5 activates MEKK1 effectors ERK1/2 and NFκB in fibroblasts, and that chemical inhibition of NFκB inhibits CCL5 expression. Our results suggest that MEKK1 contributes to the formation of a breast tumor microenvironment that supports metastasis by promoting expression of stroma cell chemokine genes in response to tumor cell-induced paracrine signaling.

Stimulation of hERG1 channel activity promotes a calcium-dependent degradation of cyclin E2, but not cyclin E1, in breast cancer cells.

Cyclin E2 gene amplification, but not cyclin E1, has been recently defined as marker for poor prognosis in breast cancer, and appears to play a major role in proliferation and therapeutic resistance in several breast cancer cells. Our laboratory has previously reported that stimulation of the hERG1 potassium channel with selective activators led to down-regulation of cyclin E2 in breast cancer cells. In this work, we demonstrate that stimulation of hERG1 promotes an ubiquitin-proteasome-dependent degradation of cyclin E2 in multiple breast cancer cell lines representing Luminal A, HER2+ and Trastuzumab-resistant breast cancer cells. In addition we have also reveal that hERG1 stimulation induces an increase in intracellular calcium that is required for cyclin E2 degradation. This novel function for hERG1 activity was specific for cyclin E2, as cyclins A, B, D E1 were unaltered by the treatment. Our results reveal a novel mechanism by which hERG1 activation impacts the tumor marker cyclin E2 that is independent of cyclin E1, and suggest a potential therapeutic use for hERG1 channel activators.

Interaction with the Paxillin LD1 Motif Relieves MEKK2 Auto-inhibition.

The cell signaling molecule MEK kinase 2 (MEKK2) is a key upstream regulator of MAPK activity that regulates numerous cellular functions, but the mechanisms that control MEKK2 activity are not well understood. Recently, we reported that MEKK2 both binds and promotes ubiquitylation of the scaffold protein paxillin, and thereby modulates the composition of adhesion complexes. In this study, we have extended our examination of this interaction and report that recombinant paxillin is sufficient to induce MEKK2 auto-phosphorylation. Furthermore, we utilize siRNA-mediated paxillin expression knockdown to reveal that MEKK2 activity is reduced in paxillin-deficient cells. Finally, we show that the paxillin leucine-rich motif 1 (LD1) is sufficient to bind to the MEKK2 amino terminal region and activate MEKK2. Taken together, our results show for the first time that paxillin association promotes MEKK2 activation and reveal the existence of a novel bi-directional regulatory relationship between MEKK2 and paxillin.

MEKK2 regulates paxillin ubiquitylation and localization in MDA-MB 231 breast cancer cells.

The intracellular kinase MEKK2 (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase 2) is an upstream regulator of JNK (c-Jun N-terminal kinase), but additional functions for MEKK2 have not been well defined. Silencing MEKK2 expression in invasive breast tumour cells markedly inhibits xenograft metastasis, indicating that MEKK2 controls tumour cell function required for tumour progression. In our previous investigation of MEKK2 function, we discovered that tumour cell attachment to fibronectin recruits MEKK2 to focal adhesion complexes, and that MEKK2 knockdown is associated with stabilized focal adhesions and significant inhibition of tumour cell migration. In the present study we investigate MEKK2 function in focal adhesions and we report that MEKK2 physically associates with the LD1 motif of the focal adhesion protein paxillin. We reveal that MEKK2 induces paxillin ubiquitylation, and that this function requires both the paxillin LD1 motif and MEKK2 kinase activity. Finally, we demonstrate that MEKK2 promotes paxillin redistribution from focal adhesions into the cytoplasm, but does not promote paxillin degradation. Taken together, our results reveal a novel function for MEKK2 as a regulator of ubiquitylation-dependent paxillin redistribution in breast tumour cells.

MEKK2 regulates focal adhesion stability and motility in invasive breast cancer cells.

MEK Kinase 2 (MEKK2) is a serine/threonine kinase that functions as a MAPK kinase kinase (MAP3K) to regulate activation of Mitogen-activated Protein Kinases (MAPKs). We recently have demonstrated that ablation of MEKK2 expression in invasive breast tumor cells dramatically inhibits xenograft metastasis, but the mechanism by which MEKK2 influences metastasis-related tumor cell function is unknown. In this study, we investigate MEKK2 function and demonstrate that silencing MEKK2 expression in breast tumor cell significantly enhances cell spread area and focal adhesion stability while reducing cell migration. We show that cell attachment to the matrix proteins fibronectin or Matrigel induces MEKK2 activation and localization to focal adhesions. Further, we reveal that MEKK2 ablation enhances focal adhesion size and frequency, thereby linking MEKK2 function to focal adhesion stability. Finally, we show that MEKK2 knockdown inhibits fibronectin-induced Extracellular Signal-Regulated Kinase 5 (ERK5) signaling and Focal Adhesion Kinase (FAK) autophosphorylation. Taken together, our results strongly support a role for MEKK2 as a regulator of signaling that modulates breast tumor cell spread area and migration through control of focal adhesion stability.

An MAPK-dependent pathway induces epithelial-mesenchymal transition via Twist activation in human breast cancer cell lines.

BACKGROUND: Twist is an epithelial-mesenchymal transition (EMT) transcription factor that instigates cell invasion. Our research has shown that osteopontin (OPN) regulates the EMT factor Twist. The underlying signaling pathway is unknown. We hypothesized that OPN activates Twist to induce EMT in human breast cancer. METHODS: Potential kinases for Twist were identified using NetPhosK. Inhibitors of MEK1/2, JNK, p38 MAPK, and PI3K were applied to human breast cancer cells MDA-MB231 (OPN high). After 24 h, Twist was immunoprecipitated and incubated with phosphoserine. Expression of the Twist target protein, Bmi-1, was determined following 24-h osteopontin aptamer (APT) treatment; mutant aptamer (MuAPT) was used as the control. Scratch-wound assay was imaged 12, 24, and 48 h after APT and MuAPT treatment. RESULTS: MEK1/2 inhibition caused ≈ twofold decrease in Twist serine phosphorylation (P < .05). APT blockade of OPN in MB231 decreased Bmi1 protein twofold (P < .05). Aptamer-treated cells were significantly decreased in cell migration and wound closure in the scratch wound-assay (P < .001). CONCLUSION: We demonstrate that OPN extracellular binding to MB231 activates an autocrine MAPK intracellular signaling pathway resulting in Twist activation and promoting Bmi1 expression to further EMT initiation and cellular migration. Our results elucidate a previously undescribed role for OPN as a prime regulator of EMT in human breast cancer cells.

Osteopontin up-regulates critical epithelial-mesenchymal transition transcription factors to induce an aggressive breast cancer phenotype.

BACKGROUND: Tumor cells undergoing epithelial-mesenchymal transition (EMT) develop cellular properties leading to stroma invasion and intravasation. We have previously shown in a xenograft breast cancer model that blocking osteopontin (OPN), a secreted phosphoprotein, decreases EMT. This study examines OPN's role in EMT initiation through its regulation of EMT transcription factors (TFs) Snail, Slug, and Twist. OPN's role in Twist activation is examined through immunoprecipitation and Western blot. STUDY DESIGN: MDA-MB-231 breast cancer cells secreting high levels of OPN were treated with OPN aptamer (APT) or mutant APT. Osteopontin APT binds to and inhibits extracellular OPN. Low-OPN-secreting breast cancer cells, MCF-7, were treated with OPN, OPN+APT, or OPN+mutant APT. Twist was isolated in MDA-MB-231 with immunoprecipitation. Phospho-serine antibody detected activated Twist in Western blot. Activation of Twist was confirmed by chromatin immunoprecipitation. RESULTS: Analysis through quantitative polymerase chain reaction demonstrated APT inhibition of OPN in MDA-MB-231 cells caused a decrease in EMT-TF expression (MDA-MB-231 vs MDA-MB-231+APT: *Twist ΔΔCT: 1.0 vs 0.07; *Snail ΔΔCT: 1.0 vs 0.11; *Slug ΔΔCT: 1.0 vs 0.11; *p < 0.001). Mutant APT did not change EMT-TF expression (NS). Treatment of MCF-7 cells with OPN caused an increase in EMT-TF expression (MCF-7 vs MCF-7+OPN: Twist ΔΔCT: 1.0 vs 9.1; *Snail ΔΔCT: 1.0 vs 11.2; *Slug ΔΔCT: 1.0 vs 10.9; *p < 0.001). The EMT-TF expression in MCF-7 treated with OPN+APT did not differ significantly from MCF-7 alone. Phosphorylated Twist protein was reduced 2-fold with APT in MDA-MB-231 compared with MDA-MB-231 and MDA-MB-231+mutant APT. Twist phorphorylation induced binding to the promoter regions of Twist-regulated gene, B lymphoma Mo-MLV insertion region 1 homolog, a critical protein for EMT progression. CONCLUSIONS: This study shows that OPN is critical in EMT initiation through activation of Twist via serine phosphorylation. These unique observations indicate that OPN APT can serve a clinical role as a novel therapeutic agent by diminishing breast cancer oncogenesis.

The MEKK1 SWIM domain is a novel substrate receptor for c-Jun ubiquitylation.

MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1] is a MAP3K (MAPK kinase kinase) that regulates MAPK activation, and is the only known mammalian kinase that is also a ubiquitin ligase. MEKK1 contains a RING domain within its N-terminal regulatory region, and MEKK1 has been shown to ubiquitylate the AP-1 (activator protein 1) transcription factor protein c-Jun, but the mechanism by which MEKK1 interacts with c-Jun to induce ubiquitylation has not been defined. Proximal to the RING domain is a SWIM (SWI2/SNF2 and MuDR) domain of undetermined function. In the present study, we demonstrate that the MEKK1 SWIM domain, but not the RING domain, directly associates with the c-Jun DNA-binding domain, and that the SWIM domain is required for MEKK1-dependent c-Jun ubiquitylation. We further show that this MEKK1 SWIM-Jun interaction is specific, as SWIM domains from other proteins failed to bind c-Jun. We reveal that, although the Jun and Fos DNA-binding domains are highly conserved, the MEKK1 SWIM domain does not bind Fos. Finally, we identify the sequence unique to Jun proteins required for specific interaction with the MEKK1 SWIM domain. Therefore we propose that the MEKK1 SWIM domain represents a novel substrate-binding domain necessary for direct interaction between c-Jun and MEKK1 that promotes MEKK1-dependent c-Jun ubiquitylation.

Ablation of MEKK4 kinase activity causes neurulation and skeletal patterning defects in the mouse embryo.

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

MEKK1 regulates the AP-1 dimer repertoire via control of JunB transcription and Fra-2 protein stability.

Activator protein 1 (AP-1) transcription factor dimers are composed of Jun, Fos, and ATF member proteins, but the mechanisms that determine AP-1 composition are not clearly defined and the function of specific dimers is not well understood. MEKK1 is a mitogen-activated protein kinase (MAPK) kinase kinase and an ubiquitin ligase that regulates both the extracellular signal-regulated kinase 1/2 and the c-Jun amino-terminal kinase. Herein, we demonstrate that MEKK1 regulates the AP-1 protein repertoire. Both FGF-2 and phorbol ester-inducible urokinase-type plasminogen activator (uPA) expression requires AP-1 binding to an enhancer element in the uPA promoter, and we have previously shown that FGF-2 or PMA induction of uPA expression is strongly dependent on MEKK1. JunB mRNA is significantly increased in MEKK1-/- cells, demonstrating that MEKK1 suppresses JunB mRNA expression. Upregulation of JunB expression in MEKK1-/- cells forms an inhibitory AP-1 complex that binds to the uPA promoter and inhibits uPA transcription. MEKK1 also regulates Fra-2 protein stability by inducing Fra-2 ubiquitination and degradation. MEKK1 regulates AP-1-dependent gene expression by regulating the expression, activity and degradation of component members of the AP-1 complex. Controlling the repertoire of a transcription factor complex is a newly defined function for an MAPK kinase kinase.

Wiring diagrams of MAPK regulation by MEKK1, 2, and 3.

Mitogen-activated protein kinase (MAPK) pathways are activated by a plethora of stimuli. The literature is filled with papers describing the activation of different MAPKs by almost any stimulus or insult imaginable to cells. In this review, we use signal transduction wiring diagrams to illustrate putative upstream regulators for the MAPK kinase kinases, MEKK1, 2, and 3. Targeted gene disruption of MEKK1, 2, or 3 defined phenotypes for each MEKK associated with loss of specific MAPK regulation. Genetic analysis of MEKK function clearly defines specific components of the wiring diagram that require MEKK1, 2, or 3 for physiological responses. We propose that signal transduction network wiring diagrams are valuable tools for hypothesis building and filtering physiologically relevant phenotypic responses from less connected protein relations in the regulation of MAPK pathways.

Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock.

Sensing the osmolarity of the environment is a critical response for all organisms. Whereas bacteria will migrate away from high osmotic conditions, most eukaryotic cells are not motile and use adaptive metabolic responses for survival. The p38 MAPK pathway is a crucial mediator of survival during cellular stress. We have discovered a novel scaffold protein that binds to actin, the GTPase Rac, and the upstream kinases MEKK3 and MKK3 in the p38 MAPK phospho-relay module. RNA interference (RNAi) demonstrates that MEKK3 and the scaffold protein are required for p38 activation in response to sorbitol-induced hyperosmolarity. FRET identifies a cytoplasmic complex of the MEKK3 scaffold protein that is recruited to dynamic actin structures in response to sorbitol treatment. Through its ability to bind actin, relocalize to Rac-containing membrane ruffles and its obligate requirement for p38 activation in response to sorbitol, we have termed this protein osmosensing scaffold for MEKK3 (OSM). The Rac-OSM-MEKK3-MKK3 complex is the mammalian counterpart of the CDC42-STE50-STE11-Pbs2 complex in Saccharomyces cerevisiae that is required for the regulation of p38 activity.

MEKK1 regulates calpain-dependent proteolysis of focal adhesion proteins for rear-end detachment of migrating fibroblasts.

Herein, we define how MEKK1, a MAPK kinase kinase, regulates cell migration. MEKK1 is associated with actin fibers and focal adhesions, localizing MEKK1 to sites critical in the control of cell adhesion and migration. EGF-induced ERK1/2 activation and chemotaxis are inhibited in MEKK1-/- fibroblasts. MEKK1 deficiency causes loss of vinculin in focal adhesions of migrating cells, increased cell adhesion and impeded rear-end detachment. MEKK1 is required for activation of the cysteine protease calpain and cleavage of spectrin and talin, proteins linking focal adhesions to the cytoskeleton. Inhibition of ERK1/2 or calpain, but not of JNK, mimics MEKK1 deficiency. Therefore, MEKK1 regulates calpain-mediated substratum release of migrating fibroblasts.

MEKK1 is required for inducible urokinase-type plasminogen activator expression.

Urokinase-type plasminogen activator (uPA) regulates the remodeling of extracellular matrix and controls reparative processes such as wound healing and liver regeneration. Here we show inducible uPA expression is controlled by MEKK1, a MAPK kinase kinase that regulates the ERK1/2 and JNK pathways. MEKK1 is activated in response to growth factors and cytoskeletal changes. We have found MEKK1 to be necessary for uPA up-regulation in response to treatment with phorbol 12-myristate 13-acetate or basic fibroblast growth factor. We demonstrate that growth factor-treated MEKK1-deficient fibroblasts display greatly reduced uPA expression and activity compared with control fibroblasts. Further, we show that growth factor-induced uPA expression requires MEKK1-dependent MKK1 and JNK activity and that transfection of MEKK1 into knockout cells restores inducible uPA expression and activity. Importantly, disrupted expression of MEKK2, a related MAPK kinase kinase, had no effect on uPA activity. Therefore, we conclude that MEKK1 expression is required for PMA- or FGF-2-induced signals to control uPA expression and function.