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[Cancer Research 64, 3855-3864, June 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

A Novel Role for Mixed-Lineage Kinase-Like Mitogen-Activated Protein Triple Kinase {alpha} in Neoplastic Cell Transformation and Tumor Development

Yong-Yeon Cho, Ann M. Bode, Hideya Mizuno, Bu Young Choi, Hong Seok Choi and Zigang Dong

Hormel Institute University of Minnesota, Austin, Minnesota


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, no member of the mixed-lineage kinase (MLK) protein family was known to function as an oncogene. Here, we demonstrate that MLK-like mitogen-activated protein triple kinase (MLTK)-{alpha}, a member of the MLK family, induced neoplastic cell transformation and tumorigenesis in athymic nude mice. Introduction of small interference RNA (siRNA)-MLTK-{alpha} into MLTK-{alpha}-overexpressing cells dramatically suppressed cell transformation. Nuclear accumulation of the pHisG-MLTK-{alpha} fusion protein was observed after epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate treatment. Phosphorylation of downstream mitogen-activated protein kinase-targeted transcription factors including c-Myc, Elk-1, c-Jun, and activating transcription factor (ATF) 2 was also differentially enhanced in MLTK-{alpha}-overexpressing cells exposed to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate stimulation compared with cells expressing mock vector or siRNA-MLTK-{alpha}. Very importantly, MLTK-{alpha}-overexpressing cells formed fibrosarcomas when injected s.c. into athymic nude mice, whereas almost no tumor formation was observed in mice that received injections of mock or siRNA-MLTK-{alpha} stably transfected cells. These results are the first to indicate that MLTK-{alpha} plays a key role in neoplastic cell transformation and cancer development.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mixed-lineage kinases (MLKs) are a family of serine/threonine protein kinases that act as mitogen-activated protein (MAP) kinase (MAPK)-kinase kinases to activate the various MAPK pathways via the MAPK kinases (MKKs; Ref. 1 ). The MLKs are divided into three subfamilies including the MLKs (MLK1–4), the dual-leucine-zipper-bearing kinases, and the zipper sterile-{alpha}-motif kinases (ZAKs). Most of the MLK family members appear to share similar structural features including a kinase domain and one or two leucine zipper regions (1) . The ZAK subfamily is made up of the most recently discovered members of the MLK family. ZAK-{alpha} and ZAK-ß are alternative splicing products and are also referred to as MLK-like MAP triple kinases (MLTK-{alpha} and MLTK-ß; Refs. 1 and 2 ) or MLK-related kinases (MRK-{alpha} and MRK-ß; Refs. 1 and 2 ). To avoid confusion, we will use the MLTK terminology for the remainder of this manuscript.

Currently no evidence suggests that any MLK family member functions as an oncogene. The potential role of MLKs in cancer remains to be determined. Similar to other MLK family members, the MLTKs regulate signaling of the c-Jun N-terminal kinases (JNKs), p38 MAPKs (1) , and MAP/extracellular signal-regulated kinase (ERK) kinases (MEKs; Ref. 3 ). MLTK-{alpha} and MLTK-ß have a high N-terminal homology with MLK2 and TAK1; and MLTK-ß also shows homology to the COOH-terminal region of TAK1 (2) . The most distinguishable features of MLTK-{alpha} are the SAM domain and one leucine zipper region. The SAM domain is an independently folding module that is usually found near the NH2 or COOH terminus in many signaling molecules, including receptor tyrosine kinases, adapter proteins, and GTPase-activating proteins (4 , 5) . Crystal structure and biochemical studies indicate that SAM domains can mediate homo- and hetero-dimerization (6) and oligomerization (7) . An accumulation of evidence suggests that MLTK-{alpha} can interact with various MAPKs, which regulate diverse cellular responses, including cell proliferation, differentiation, development, inflammation, and apoptosis.

MAPKs comprise complex phosphorylation cascades that transmit and integrate extracellular signals into the cell resulting in diverse cellular responses (8) . These pathways mediate multiple physiological processes, including cell proliferation, differentiation, and cell death as well as stress-induced responses (9, 10, 11) . To date, MLKs are known to phosphorylate and activate MKKs such as MKK4 and/or MKK7 (12, 13, 14) , which in turn activate JNKs and MKK3/6 (15) . Subdomains I–VII of the MLK kinase resemble MEK kinase (MEKK) and Raf, which is an upstream kinase of ERK1/2; whereas subdomains VIII–XI more closely resemble tyrosine kinases, such as Src and the fibroblast-growth factor receptor (1) . Importantly, a very recent paper reported that MLK3 directly phosphorylates and activates MEK1, an upstream kinase of ERKs (3) , thus confirming that MLKs can act on all known MAPK pathways. Although some signaling events of MLTK-{alpha} have been elucidated, the specific signaling pathway of MLTK-{alpha} is poorly understood.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and epidermal growth factor (EGF) are well-known tumor promotion agents used to study malignant cell transformation in cell and animal models of cancer (16) . These two agents induce activation of the transcription factor, activator protein-1 (AP-1; Refs. 17 and 18 ). When treated with TPA or EGF, JB6 Cl41 skin epidermal cells showed an induction of AP-1 transcriptional activation in promotion-sensitive (P+) phenotypes but not in promotion-resistant (P–) phenotypes (19) . Blocking AP-1 activation causes P+ cells to revert to the P– phenotype, indicating a unique requirement for AP-1 activation in TPA- or EGF-induced cell transformation (20) . In previous studies, MLTK was shown to be highly expressed in heart, skeletal muscle, ovary, small intestine, and colon as well as in HT 1080 cells, a human p53-deficient fibroblast cancer cell line (21) . These results suggested that MLTK might regulate mitogen-derived signal transduction induced by tumor promoters, such as EGF or TPA. Although TPA and EGF signaling cascades have been thoroughly studied, the involvement of MLTK-{alpha} signaling is not yet clearly understood. Moreover, downstream targets or transcription factors activated by MLTK-{alpha} are unknown.

Here, we demonstrate that ectopically expressed MLTK-{alpha} induced proliferation and malignant cell transformation in JB6 Cl41 skin epidermal cells. However, when small interference RNA (siRNA)-MLTK-{alpha} was stably expressed in cells overexpressing MLTK-{alpha}, proliferation and transformation were decreased dramatically. Furthermore, pHisG-MLTK-{alpha} fusion protein accumulated in the nucleus when cells were treated with the tumor promoters EGF or TPA. Phosphorylation of downstream MAPK-targeted transcription factors, including c-Myc, Elk-1, c-Jun, and ATF2, was increased in MLTK-{alpha}-overexpressing cells. Importantly, MLTK-{alpha}-overexpressing cells formed tumors when injected s.c. into athymic nude mice whereas, almost no tumor formation was observed in mice that received injections of cells stably transfected with the mock vector or siRNA-MLTK-{alpha}. This study is the first to report a novel function of MLTK-{alpha} in cancer development both in vitro and in vivo.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies.
Chemical reagents, including Tris, NaCl, and SDS for molecular biology and buffer preparation were purchased from Sigma-Aldrich (St. Louis, MO). Restriction enzymes and some modifying enzymes were purchased from New England BioLabs, Inc. (Beverly, MA). Superscript II RNase H– reverse transcriptase and TaqDNA polymerase were from Life Technologies, Inc. (Rockville, MD) and Qiagen, Inc. (Valencia, CA), respectively. The DNA ligation kit (version 2.0) was purchased from TAKATA Bio, Inc. (Otsu, Shiga, Japan). The pcDNA4/HisMAX plasmid used for the construction of the expression vector was from Invitrogen (Carlsbad, CA). Cell culture medium and other supplements were purchased from Life Technologies, Inc. Antibodies for immunoblotting and immunocytochemical analysis were purchased from Cell Signaling Technology, Inc. (Beverly, MA), Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), or Upstate Biotechnology, Inc. (Charlottesville, VA).

Animals.
Athymic mice (Cr:NIH(S), NIH Swiss nude, 4–6 weeks old) were purchased from the National Cancer Institute (NIH). Mice were maintained under specific pathogen-free conditions according to guidelines established by Research Animal Resources, University of Minnesota. The mice were acclimated for 2 weeks and housed in sterile filter top covered cages. Mice were maintained on a 12-h dark/light cycle and allowed food and water ad libitum.

Cell Culture and Transfections.
JB6 Cl41 skin epidermal cells were cultured with minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS) and antibiotics in a 5% CO2 incubator. The cells were maintained by splitting at 90% confluence, and media were changed every 3 days. JB6 Cl41 skin epidermal cells (5.0 x 105) in 5% FBS-MEM were seeded in 100-mm tissue culture dishes. After culturing at 37°C for 16 h, the cells were transfected with 4 µg of pHisG-MLTK-{alpha} or psi-MLTK-{alpha} using LipofectAMINE Plus reagent (Life Technologies, Inc.) following the manufacturer’s suggested protocol. The cells were selected with zeocin (Life Technologies, Inc.) for expression of pHisG-MLTK-{alpha} and with G418 for expression of psi-MLTK-{alpha}.

Construction of pHisG-MLTK-{alpha} and psi-MLTK-{alpha}.
The human MLTK-{alpha} coding fragment, including the open reading frame, was amplified by reverse transcription-PCR using the following primers: 5'-GGG GTA CCT ATG TCG TCT CTC GGT CCC TCC-3' (KpnI site underlined) and 5'-CGG GAT CCT CAT CAA AAG TTT CTC CAT CCA CG-3' (BamHI site underlined) and then introduced into the KpnI/BamHI site of pcDNA4/HisMAX, a mammalian expression vector (pHisG-MLTK-{alpha}). To construct the siRNA-MLTK{alpha} (psi-MLTK-{alpha}), the pU6pro vector [a gift kindly provided by Dr. David L. Turner (Mental Health Research Institute, University of Michigan)] was digested with XbaI and BbsI. The annealed synthetic primers were then introduced: sense, 5'-TTT GCC ACA CAA CAC ACA TGT CCT TCA AGA GAG GAC ATG TGT GTT GTG TGG TTT TT-3' (BbsI cohesive end underlined); and antisense, 5'-CTA GAA AAA CCA CAC AAC ACA CAT GTC CTC TCT TGA AGG ACA TGT GTG TTG TGT GG-3' (XbaI cohesive end underlined) following the recommended protocols.1 The recombinant plasmids of pHisG-MLTK-{alpha} and psi-MLTK-{alpha} were confirmed by agarose gel electrophoresis and DNA sequencing.

3-(4,5-Dimethylthiazol-2-yl)-5-(3-Carboxymethoxy-Phenyl)-2-(4-Sulfonyl)-2H-Tetrazolium Assay.
To estimate cell proliferation, JB6 Cl41 cells transfected with the mock vector, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} were seeded (1 x 103) in 96-well plates in 100 µl of 5% FBS-MEM in a 37°C, 5% CO2 incubator. After culturing for 12 h, 20 µl of the CellTiter 96 Aqueous One Solution (Promega, Madison, WI) were added to each well, and cells were then incubated for 1 h at 37°C, 5% CO2. To stop the reaction, 25 µl of a 10% SDS solution were added, and absorbance was measured at 492 and 690 nm.

Anchorage-Independent Cell Transformation Assay.
EGF- or TPA-induced cell transformation was investigated in mock, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cells. In brief, cells (8 x 103/ml) were exposed to EGF (0.1–10 ng/ml) or TPA (1–40 ng/ml) in 1 ml of 0.3% basal medium Eagle agar containing 10% FBS. The cultures were maintained in a 37°C, 5% CO2 incubator for 10 days (EGF) or 3–4 weeks (TPA), and the cell colonies were scored using a microscope and the Image-Pro PLUS computer software program (Media Cybernetics, Silver Spring, MD) as described by Colburn et al. (22) .

Nuclear Transport Assay.
JB6 Cl41 cells transfected with the mock vector or pHisG-MLTK-{alpha} were plated (6 x 104/well) in 12-well plates and cultured 18 h with 5% FBS-MEM in a 37°C, 5% CO2 incubator. The cells were starved with 0.1% FBS-MEM for 24 h and then treated EGF (1 ng/ml) or TPA (10 ng/ml). At then end of 24 h, the cells were washed with PBS, fixed by adding 1 ml of ice-cold methanol, and then incubated 5 min at room temperature. The cells were washed with PBS two times and incubated with the anti-HisG mouse antibody (1:500) in PBS-10% FBS for 2 h at room temperature with gentle rocking. They were then washed three times and hybridized with an antimouse IgG goat antibody (horseradish peroxidase conjugated). After 1 h, the cells were washed with PBS three times, and 0.5 ml of Sigma FAST 3,3'-diaminobenzidine peroxidase substrate was added. The cells were maintained at room temperature for 1 h in the dark, after which they were washed with PBS and observed under a microscope.

In Vivo Tumor Formation Assay.
Log-growing JB6 Cl41 cells transfected with mock, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} were harvested at 90% confluence and suspended (3 x 106) in 200 µl of MEM without FBS or antibiotics. Mice were divided into three groups of 10 or 12 mice each. Each individual cell line (3 x 106) was injected into mice (10, mock vector control; 12, pHisG-MLTK-{alpha}; and 12, pHisG-MLTK-{alpha}/si-MLTK-{alpha}) for each cell line. The cells were injected s.c. into the right flank of each mouse. The mice were weighed twice each week and monitored every day for tumor growth. When tumors appeared, they were measured by caliper daily. Tumor volume was calculated from measurements of two diameters of the individual tumor according to the formula: tumor volume (mm3) = [longer diameter x shorter diameter2 ]/2. Mice were monitored until tumors reached 1000 mm3 total volume, at which time mice were euthanized, and tumors were extracted for histochemical analysis. All experiments were performed according to guidelines approved by the University of Minnesota Institutional Animal Care and Use Committee.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MLTK-{alpha} Overexpression Increased Cell Proliferation.
To assess whether overexpression of MLTK-{alpha} affected cell proliferation, we cloned the human MLTK-{alpha} cDNA, including the open reading frame, and recombined it into the mammalian expression vector, pcDNA4/HisMAX-A. This plasmid was then introduced into JB6 Cl41 skin epidermal cells, and proliferation was assessed. Overexpression of MLTK-{alpha} was detected in pHisG-MLTK-{alpha} stably transfected cells using an anti-HisG antibody (Fig. 1A)Citation . Expression was not detectable in wild-type JB6 Cl41 cells or empty ("mock") vector stably transfected cells (Fig. 1A)Citation . 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assay results indicated that MLTK-{alpha}-overexpressing cells proliferated at a significantly higher rate compared with mock vector stably transfected control cells (*, P < 0.005; Fig. 1BCitation ). These results indicated that MLTK-{alpha} is involved in cell proliferation.



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  		Fig. 1. Cell proliferation was enhanced by ectopic expression of MLTK-{alpha}. A, JB6 Cl41 cells were transfected with pHisG-MLTK-{alpha}, selected with 200 µg/ml zeocin for 10 days, and then pooled. The pHisG-MLTK-{alpha} stably transfected cells were disrupted by sonication with NP40 buffer and then subjected to Western blotting to detect HisG-MLTK-{alpha} expression using an anti-HisG antibody as described in "Experimental Procedures." The lower band is nonspecific. B, cells stably transfected with the mock vector or pHisG-MLTK-{alpha} were seeded (1 x 103/well) in 96-well plates in 100 µl of 5% FBS-MEM, and cell proliferation was estimated using the CellTiter 96 Aqueous One Solution detection kit (Promega). Cell proliferation was estimated by absorbance (A492) at 24-h intervals up to 72 h. Each bar indicates the mean ± SD of values obtained from triplicate experiments. Significant differences were evaluated using the Student’s t test (*, P < 0.005).

 
MLTK-{alpha} Overexpression Enhanced Anchorage-Independent Tumor Promoter-Induced Cell Transformation.
EGF stimulates the proliferation of a number of epithelial cells both in vivo and in vitro (23) , induces the progression from G2 to M phase in the developing Drosophila eye (24) , is required in skin development, and is implicated in epithelial tumor formation (25) . TPA is also a well-known skin cancer promoter in vivo and in vitro, and its effects are mediated through the MAPK signaling cascades (26, 27, 28, 29) . An accumulation of recent evidence showed that signaling mediated by MLTK-{alpha} is strongly implicated in regulating MAPK cascades, ERKs, p38 kinases, and JNKs (2 , 21 , 30) . However, the specific functions of MLTK-{alpha} are not yet well understood.

To determine the role of MLTK-{alpha} in neoplastic cell transformation, we used the anchorage-independent cell transformation assay. Cells, stably transfected with the mock vector or pHisG-MLTK-{alpha}, were stimulated with EGF (0.0–1.0 ng/ml) or TPA (0–20 ng/ml) as described in "Experimental Procedures." The number of colonies formed after treatment with EGF (Fig. 2, A and B)Citation or TPA (Fig. 2, C and D)Citation was significantly (*, P < 0.01) increased in pHisG-MLTK-{alpha} stably transfected cells compared with the mock vector control. Furthermore, the colony size also appeared to be larger in the MLTK-{alpha}-overexpressing cells treated with 1.0 ng/ml EGF (Fig. 2A)Citation or 10–20 ng/ml TPA (Fig. 2C)Citation . These results strongly indicated that MLTK-{alpha} has an important function in tumor development or neoplastic cell transformation in epidermal mouse skin fibroblast cells stimulated with EGF or TPA.



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  		Fig. 2. Tumor promoter-induced cell transformation was increased in pHisG-MLTK-{alpha} stably transfected cells. Cells, stably transfected with the mock vector or pHisG-MLTK-{alpha}, were subjected to an anchorage-independent cell transformation assay (soft agar assay) in the presence of EGF (A and B) or TPA (C and D). A and B, cells (1 x 103/ml) transfected with mock vector or pHisG-MLTK-{alpha} were exposed to EGF (0, 0.1 or 1 ng/ml) in 1 ml of 0.3% basal medium Eagle agar containing 10% FBS. The cultures were maintained in a 37°C, 5% CO2 incubator for 10 days, and then colonies were counted using a microscope and the Image-Pro PLUS computer software program. Each bar indicates the mean ± SD of values obtained from triplicate experiments. Significant differences were evaluated using the Student’s t test (*, P < 0.01). C and D, cells transfected as described in A were exposed to TPA (0, 10.0 or 20.0 ng/ml) in 1 ml of 0.3% basal medium Eagle agar containing 10% FBS. The cultures were maintained in a 37°C, 5% CO2 incubator for 3 weeks, and then colonies were counted as in A and B. Each bar indicates the mean ± SD of values obtained from triplicate experiments. Significant differences were evaluated using the Student’s t test (*, P < 0.01).

 
When Stimulated with Tumor Promoters, MLTK-{alpha} Accumulated in the Nucleus.
MAPK kinase (MEK1) contains the nuclear export signal (NES) and is therefore primarily located in the cytosol, both before and after mitogenic stimulation. This indicates that at certain times, MEK enters the nucleus and then must be exported (31) . Both MLTK-{alpha} and MLTK-ß contain two putative NES-like sequences in the NH2 and COOH termini. MLTKs are mainly localized in the cytosol and are known to accumulate in the nucleus when cells are treated with leptomycin, an inhibitor of the NES receptor (2) . To examine whether MLTK-{alpha} accumulates in the nucleus after stimulation of cells with EGF or TPA, cells stably transfected with the mock vector or pHisG-MLTK-{alpha} were treated with EGF or TPA. HisG-MLTK-{alpha} was detected with a HisG-tagged primary antibody and an horseradish peroxidase-conjugated secondary antibody using 3,3'-diaminobenzidine as the peroxidase substrate. Results showed that HisG-MLTK-{alpha} accumulated in the nucleus 12 or 6 h after stimulation with 1.0 ng/ml EGF (Fig. 3A)Citation or 10.0 ng/ml TPA (Fig. 3B)Citation , respectively. In contrast, cells stably transfected with mock vector showed no significant change whether stimulated or not with EGF or TPA. These results suggested that accumulation of MLTK-{alpha} in the nucleus might be important in proliferation and cell transformation.



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  		Fig. 3. Stimulation by tumor promoters resulted in a nuclear localization of the HisG-MLTK-{alpha} fusion protein. A and B, nuclear localization induced by treatment with 1.0 ng/ml EGF (A) or 10 ng/ml TPA (B) was assessed in cells stably transfected with the mock vector or pHisG-MLTK-{alpha}. Cells (6 x 104/well) were seeded in 12-well plates and cultured 18 h in 5% FBS-MEM in a 37°C, 5% CO2 incubator. The cells were starved in 0.1% FBS-MEM for 24 h, treated with 1 ng/ml EGF (12 h) or 10 ng/ml TPA (6 h), and then fixed in ice-cold methanol. The cells were incubated with an anti-HisG mouse antibody and then detected with a horseradish peroxidase-conjugated antimouse IgG goat antibody and the Sigma FAST 3,3'-diaminobenzidine peroxidase substrate. The figures are representative of three independent experiments.

 
Expression of siRNA against MLTK-{alpha} Knocked Down MLTK-{alpha} Expression.
Double-stranded RNA is processed into siRNA in mammalian cells, and short siRNAs can cause a strong and specific suppression of the expression of a targeted gene (32) . To examine the function of MLTK-{alpha}, a siRNA against MLTK-{alpha} (psi-MLTK-{alpha}) was constructed by replacing the Green Fluorescence Protein (GFP) region of the pU6pro vector with si-MLTK-{alpha} primers (Fig. 4, A and B)Citation . The recombinant plasmids of pHisG-MLTK-{alpha} and psi-MLTK-{alpha} were confirmed by agarose gel electrophoresis (Fig. 4C)Citation and DNA sequencing. The psi-MLTK-{alpha} construct was cotransfected into pHisG-MLTK-{alpha} stably transfected cells with pcDNA3.1neo and selected with G418. Total RNA was isolated from pHisG-MLTK-{alpha} or pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cells, and mRNA levels were compared. The magnitude of gene knockdown was analyzed by reverse transcription and cycle-dependent PCR (Fig. 4D)Citation . A detectable amount of MLTK-{alpha} mRNA was detected after 24 cycles in MLTK-{alpha}-overexpressing cells compared with 27 cycles in psi-MLTK-{alpha} stably transfected cells (Fig. 4E)Citation . Densitometer analysis of MLTK-{alpha} mRNA shows that MLTK-{alpha} mRNA was decreased about 82% in psi-MLTK-{alpha} stably transfected cells compared with MTLK-{alpha}-overexpressing cells (Fig. 4F)Citation . In contrast, the internal control, ß-actin, showed no difference in density, indicating that expression of psi-MLTK-{alpha} specifically knocks down MLTK-{alpha} mRNA level (Fig. 4E)Citation .



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  		Fig. 4. MLTK-{alpha} mRNA expression was suppressed by siRNA directed against MLTK-{alpha}. For the gene knockdown experiment, the si-MLTK-{alpha} expression vector was constructed using the pU6pro vector by replacing the Green Fluorescence Protein (GFP) region with si-MLTK-{alpha} synthetic oligo primers. A, schematic diagram for the construction of the si-MLTK-{alpha} stable expression vector. B, DNA sequence of the two synthetic oligo primers. C, after recombination, the recombinant si-MLTK-{alpha} (psi-MLTK-{alpha}) was smaller than the original pU6pro vector containing the GFP region. The sequence of psi-MLTK-{alpha} was confirmed by DNA sequencing. The pHisG-MLTK-{alpha} stably transfected cells were cotransfected with pcDNA3.1neo and pU6pro control or psi-MLTK-{alpha}, selected with 400 µg/ml G418 for 10 days, and then pooled. Total RNA was isolated and reverse transcribed using an oligo dT primer. D, schematic strategy for amplification of MLTK-{alpha} cDNA by PCR. Primers 1 and 2 were designed to detect an 1800-bp spanning fragment of the si-MLTK-{alpha} priming site. E, shows the amplification of MLTK-{alpha} mRNA by reverse transcription-PCR and visualization by ethidium bromide agarose gel electrophoresis. ß-actin was used as an internal control. F, densitometry analysis of the amplified MLTK-{alpha} fragments.

 
Expression of psi-MLTK-{alpha} Blocked Proliferation and Neoplastic Transformation.
If MLTK-{alpha} is involved in proliferation and cell transformation, then psi-MLTK-{alpha} transfection into pHisG-MLTK-{alpha} stably transfected cells should return proliferation and cell transformation to control levels or below. To test this hypothesis, the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assay was used to examine proliferation in cells stably transfected with mock vector, pHisG-MLTK-{alpha} or pHisG-MLTK-{alpha}/si-MLTK-{alpha}. As before, the apparent rate of proliferation of pHisG-MLTK-{alpha} stably transfected cells was faster than that of mock control (Fig. 5A)Citation . On the other hand, pHisG-MLTK-{alpha} cells stably transfected with psi-MLTK-{alpha} showed a marked decrease in the rate of proliferation compared with either the mock vector control or pHisG-MLTK-{alpha} stably transfected cells (*, P < 0.05; Fig. 5ACitation ). This result strongly supports the hypothesis that MLTK-{alpha} plays an important role in cell proliferation.



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  		Fig. 5. Cell proliferation and transformation induced by tumor promoters in MLTK-{alpha}-overexpressing cells was reduced in si-MLTK-{alpha} stably transfected cells. A, cells stably transfected with mock vector, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} were examined for the effect on proliferation. Cells stably transfected as above were seeded (1 x 103/well) into 96-well plates with 100 µl of 5% FBS-MEM; and proliferation was estimated every 12 h with the CellTiter 96 Aqueous One Solution detection kit. Each bar indicates the mean ± SD of values obtained from triplicate experiments. Significant differences were evaluated using the Student’s t test (*, P < 0.05). B, cells stably transfected with mock vector, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} were subjected to an anchorage-independent cell transformation assay (soft agar assay) in the presence of EGF or TPA. Cells (8 x 103/ml) stably transfected with mock vector, pHisG-MLTK-{alpha} or pHisG-MLTK-{alpha}/si-MLTK-{alpha} were exposed to 1.0 ng/ml EGF or 10.0 ng/ml TPA in 1 ml of 0.3% basal medium Eagle agar containing 10% FBS. The cultures were maintained in a 37°C, 5% CO2 incubator for 10 days (EGF) or 3 weeks (TPA), and then colonies were counted using a microscope and the Image-Pro PLUS computer software program. Each bar indicates the mean ± SD of values obtained from triplicate experiments. Significant differences were evaluated using the Student’s t test (*, P < 0.01).

 
Next, we tested whether psi-MLTK-{alpha} stable expression affects anchorage-independent cell transformation. The cells transfected with mock vector, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} were mixed with soft agar, in the presence or absence of either EGF or TPA, and then cultured 10 days (EGF) or 3 weeks (TPA). Results show that the pHisG-MLTK-{alpha} stably transfected cells strongly induced a 15–18-fold increase in colony formation stimulated by EGF (*, P < 0.01) or a 17–19-fold increase in colony formation stimulated by TPA treatment (*, P < 0.01; Fig. 5BCitation ). In marked contrast, pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cells dramatically suppressed EGF- or TPA-induced colony formation to levels similar to mock vector control (*, P < 0.01; Fig. 5BCitation ). Furthermore, not only were colony numbers suppressed, but colony size also appeared much smaller (data not shown). The largest colonies were observed in the EGF- or TPA-treated pHisG-MLTK-{alpha} stably transfected cells, and the size was markedly reduced by introduction of psi-MLTK-{alpha} into pHisG-MLTK-{alpha} stably transfected cells (data not shown). No colony formation was observed in any untreated cell lines, indicating that MLTK-{alpha} plays an important role in neoplastic cell transformation in JB6 Cl41 skin epidermal cells exposed to EGF or TPA.

Signaling Pathways of MLTK-{alpha}.
MLTK-{alpha} is located at the same level as the MEKKs in the MAPK signal transduction pathways (1) . EGF signaling is mediated by Raf-1 and MEKK and differentially activates ERKs and JNKs (33) . Furthermore, both MEKK1 and MEKK2 are activated by EGF; kinase-inactive mutants of each MEKK partially inhibit EGF-stimulated JNKs activity; and kinase-inactive MEKK1, not MEKK2, strongly inhibits EGF-stimulated ERKs activity through Rac/cdc42 (34) . In addition, MLK-3 activates JNKs and p38 kinase, but not ERKs (14) , and MEK kinase activity is also regulated by EGF (34) . MAPKs have many downstream targets and some of these may be involved in the signal transduction pathway originating with MLTK-{alpha}. To identify possible targeted transcription factors downstream of the MAPKs but activated via MLTK-{alpha}, we examined c-Myc, Elk-1, c-Jun, and ATF2. A weak EGF-induced phosphorylation of c-Myc and Elk-1 was observed at 15 min in mock vector-transfected cells and increased at 30–60 min (Fig. 6A)Citation . On the other hand, a strong EGF-induced phosphorylation of both c-Myc and Elk-1 was observed at 15 min and maintained for at least 120 min in MLTK-{alpha}-overexpressing cells. The response to EGF was markedly attenuated in si-MLTK-{alpha} expressing cells (Fig. 6A)Citation . c-Jun phosphorylation was strongly induced by EGF at 15 min and maintained for at least 30 min in mock vector control cells and in cells expressing si-MLTK-{alpha}, whereas the response was maintained for only 15 min in MLTK-{alpha}-overexpressing cells (Fig. 6A)Citation . ATF2 showed very little response to EGF in any of the cells, suggesting that the signaling from JNKs or p38 kinase was directed elsewhere. These results strongly indicate that when stimulated with EGF, MLTK-{alpha} plays a key role in neoplastic cell transformation and cancer development possibly mediated by activation of c-Myc and Elk-1.



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  		Fig. 6. Overexpression or knockdown of MLTK-{alpha} expression had varied effects on the phosphorylation of MAPK-targeted transcription factors. Cells stably transfected with mock vector, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} were examined for their effect on EGF-induced (A) or TPA-induced (B) phosphorylation of c-Myc, Elk-1, c-Jun, or ATF2. The cells were seeded (1.5 x 106) in 100-mm tissue culture dishes, starved for 24 h in 0.1% FBS-MEM, and then treated or not treated with 1 ng/ml EGF (A) or 10 ng/ml TPA (B). At the indicated time after treatment, the cells were harvested and subjected to Western blotting to detect phosphorylation of c-Myc, Elk-1, c-Jun, or ATF2. Total ß-actin served as an internal control to monitor equal protein loading. All of the Western blotting experiments were performed independently two or three times using different samples. Similar results were obtained each time, and representative blots are shown.

 
TPA is also a well-known skin cancer promoter both in vivo and in vitro (27 , 29) . The signal is believed to be primarily transmitted through protein kinase C and MAPKs, resulting in the activation of AP-1 (26 , 28) . To identify the transcription factors in the MLTK-{alpha} pathway induced by TPA, we tested c-Myc, Elk-1, c-Jun, and ATF2 as for EGF described above. Phosphorylation of c-Myc in the mock vector control and pHisG-MLTK-{alpha}/si-MLTK-{alpha} appeared to be slightly increased over time, whereas pHisG-MLTK-{alpha} stably transfected cells showed a slight increase up to 60 min and then almost disappeared by 120 min (Fig. 6B)Citation . The phosphorylation pattern of Elk-1 was very similar to that of c-Myc (Fig. 6B)Citation . These results indicated that MLTK-{alpha} phosphorylation of Elk-1 and c-Myc induced by TPA was probably not mediated through ERKs. The phosphorylation of c-Jun was stronger over a longer period of time compared with the mock vector control or the pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cell line (Fig. 6B)Citation . c-Jun phosphorylation in the mock vector control and pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cell line was increased at 5 min, enhanced further at 15 min, and then suddenly dropped at 30 min after TPA treatment. However, pHisG-MLTK-{alpha} stably transfected cells exhibited an increased c-Jun phosphorylation until about 60 min after TPA treatment. Moreover, the level of c-Jun phosphorylation in pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cells appeared weaker than that of mock vector control cells (Fig. 6B)Citation . In pHisG-MLTK-{alpha} stably transfected cells, phosphorylation of ATF2 at Thr-71 was strong early and then decreased gradually over time. On the other hand, the mock vector control and pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cells showed a very weak and diffuse phosphorylation (Fig. 6B)Citation . TPA- or EGF-induced phosphorylation of c-Myc, Elk-1, ATF2, and c-Jun was determined under the exact same conditions simultaneously using the same protein samples. However, these proteins are more responsive to EGF and therefore produce a stronger phosphorylation signal. The differences in band density between Fig. 6, A and BCitation , in the nonstimulated groups is due to a difference in exposure time to the chemiluminescent (ECL) substrate. For example, cells treated with EGF produced a phosphorylation signal for c-Myc after 2–3 s, whereas TPA-stimulated phosphorylation of c-Myc required 10–15-s exposure to the ECL substrate. These results strongly suggested that c-Jun and ATF2 are critically involved in TPA induced cell transformation through MLTK-{alpha}.

Cells Ectopically Expressing MLTK-{alpha} Formed Tumors in Athymic Nude Mice.
To investigate whether MLTK-{alpha} can induce tumor development in vivo, we injected cells (3 x 106) stably transfected with mock vector, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} s.c. into the right flank of athymic nude mice. A total of 34 mice (10, mock vector control; 12, pHisG-MLTK-{alpha}; 12, pHisG-MLTK-{alpha}/si-MLTK-{alpha}) received injections of stably transfected cells according to guidelines approved by the University of Minnesota Institutional Animal Care and Use Committee. Mice showed no adverse effects to the injection, and average body weight was similar in all groups throughout the study (data not shown). The first measurable tumors (minimum 13.5 mm3) were observed in seven mice that received injections of pHisG-MLTK-{alpha} stably transfected cells on day 22 after injection, whereas no mice that received injections of either mock vector control or pHisG-MLTK-{alpha}/si-MLTK-{alpha} showed any sign of tumor development (data not shown). All 12 mice that received injections of pHisG-MLTK-{alpha} stably transfected cells had developed tumors by day 26 after injection (data not shown). On the other hand, none of the mice that received injections of either mock vector or pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cells had developed tumors. Only one mouse in each group that received injections of either mock vector or pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cells had developed a measurable tumor by day 33 (data not shown). By day 39, two additional measurable (minimum 13.5 mm3) tumors were observed in mice that received injections of pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cells. No additional tumors were found in mock vector-injected mice. Compared with tumor growth in mock vector- or pHisG-MLTK-{alpha}/si-MLTK-{alpha}-injected mice, growth was exponential in mice that received injections of pHisG-MLTK-{alpha} stably transfected cells (Fig. 7A)Citation Citation . According to Institutional Animal Care and Use Committee guidelines, the end point for maximal tumor size was 1000 mm3 total volume. Therefore the study was terminated on day 39, because all remaining mice that received injections of pHisG-MLTK-{alpha} had developed tumors of allowable maximal size. Mice were euthanized by CO2 tank, and tumors were extracted for additional analysis. To confirm whether the tumors developed from JB6 Cl41 cells, protein was extracted from tumors and analyzed by Western blotting using an anti-HisG monoclonal antibody. Results indicated that HisG-MLTK-{alpha} protein was strongly detected only in tumors from mice that received injections of pHisG-MLTK-{alpha}-overexpressing cells but not from samples in mock control or in pHisG-MLTK-{alpha}/si-MLTK-{alpha} expressing cells (Fig. 7B)Citation Citation . These results matched those acquired from JB6 Cl41 cell extracts.



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  		Fig. 7. Cell lines overexpressing pHisG-MLTK-{alpha} formed tumors in athymic nude mice. Cells stably transfected with mock vector, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} were injected (3 x 106) s.c. into the right flank of athymic nude mice, and tumor formation was monitored until tumor size reached 1000 mm3 at which time the mice were euthanized according to University of Minnesota Institutional Animal Care and Use Committee guidelines. A, tumor volume was monitored everyday and calculated according to the formula: tumor volume (mm3) = [longer diameter x shorter diameter2]/2. B, Protein was extracted from tumor tissues of mock control, pHisG-MLTK-{alpha}, or pHisG-MLTK-{alpha}/si-MLTK-{alpha} and then detected HisG-MLTK-{alpha} with anti-HisG monoclonal antibody and antimouse IgG goat antibody conjugated with horseradish peroxidase. C, three mice in each group were euthanized, and tumors were extracted for histochemical analysis. A representation sample from each group is shown. Tissues were stained with H&E. Bars indicate size (1 mm) and boxed area in the photographs is magnified x200.

 


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  		Fig. 7A. Continued.

 
Also, representative tumors from mice in each group were chosen for histochemical analysis. Results indicated that pHisG-MLTK-{alpha} stably transfected cells formed large, highly proliferating fibrosarcomas in nude mice. Moreover, mock vector and pHisG-MLTK-{alpha}/si-MLTK-{alpha} stably transfected cells also formed fibrosarcomas but comparatively much smaller at the identical time of sacrifice (Fig. 7C)Citation Citation , indicating that these tumors all originated from JB6 Cl41 cells. These are the first results providing direct in vivo evidence that MLTK-{alpha} plays a key role in neoplastic tumor formation and/or skin cancer development.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The MAPK signal transduction network comprises a very complicated and highly interactive series of phosphorylation cascades that are activated by a variety of diverse extracellular stimuli received at the surface of the cell (9, 10, 11 , 35) . The most thoroughly characterized MAPK family members include the ERKs, JNKs, and p38 kinases. These MAPKs are regulated and activated by MEKKs or MAPK kinase kinases via MEK, MKK3/7 or MKK3/6. Importantly, the phenotypes of cells affected by MAPK signal transduction are dependent on downstream-targeted proteins. For example, ATF2, c-Jun, Elk-1, and c-Myc are well-known downstream-targeted transcription factors of MAPKs, and their activation results in cell transformation and cancer development (36, 37, 38) . MLTK-{alpha} has two putative NES domains and has been shown to accumulate in the nucleus when treated with 2 ng/ml leptomycin, an inhibitor of the NES receptor (2) . We observed that tumor promoters such as EGF or TPA can induce MLTK-{alpha} accumulation in the nucleus. Our data indicate that HEK293 T cells transfected with HisG-MLTK-{alpha} and treated with anysomycin, exhibit a strong band of HisG-MLTK-{alpha} in the nuclear fraction (data not shown). These results suggested that EGF or TPA can influence the export of MLTK-{alpha} from the nucleus to the cytosol.

MLKs are MAPK kinase kinases, which all appear to regulate the JNK pathway, although several members have recently been shown to mediate the ERK1/2, p38 kinase, and ERK5 pathways (1 , 3) . The MLKs activate MAPK kinases, which in turn phosphorylate and activate specific MAPKs. Because several genes encoding these proteins were simultaneously cloned by different groups, multiple names have been used to designate the same protein. This is especially true for the newest subfamily, ZAKs. ZAK-{alpha} and ZAK-ß are alternative splicing products and are also referred to as MLTK-{alpha} and MLTK-ß (1 , 2) or MRK-{alpha} and MRK-ß (1 , 21) . MLK7 is also believed to be the same or similar to MLTK-ß (1) . The majority of the members of the MLK family, thus far identified, appear to play a role in cell cycle arrest and/or apoptosis. However, our results strongly suggest a new role for MLTK-{alpha} in cell proliferation and/or skin cancer development.

Because MLTK-{alpha} is a member of the MAPK kinase kinase family and has a SAM domain capable of interacting with several signaling molecules, we speculated that MLTK-{alpha} may have a key role in skin cancer development. Indeed, we found that ectopic or overexpression of MLTK-{alpha} induced cell transformation and cancer development in vitro and in vivo. In contrast, knockdown of MLTK-{alpha} expression using the siRNA technique suppressed cell transformation and cancer development in vitro and in vivo. These findings strongly supported the hypothesis that MLTK-{alpha} is an "onco-kinase" that regulates activity of "onco-transcription" factors such as ATF2, c-Jun, Elk-1, and c-Myc through MAPK signal cascades.

Several previous studies showed that overexpression of some members of the MLK family induce cell cycle arrest and/or apoptosis, especially in neuronal cells (39) . MLK3 is a MAPK kinase kinase that activates JNK and can induce cell death in neurons. In particular, overexpression of active MLK3 activated JNK and induced cell death in superior cervical ganglion neurons (40 , 41) . In addition, expression of ZAK in mammalian cells was shown to activate JNKs, AP-1, and nuclear factor-{kappa}B, and its overexpression induced apoptosis in Hep3 cells, a hepatoma cell line (42) . One group showed that expression of wild-type MRK-ß (ZAK-ß or MLTK-ß) induces G2 arrest, whereas dominant-negative MRK attenuated the G2 arrest caused by {gamma}-radiation (21) . Exposure of cells to {gamma}-radiation induces MRK activity, all suggesting that MRK-ß may be necessary for cell cycle checkpoint regulation in cells. In addition, expression of leucine-zipper protein kinase, another MLK family member, in keratinocytes resulted in growth arrest, indicating that this kinase plays an active part in cellular processes related to terminal differentiation of epidermal keratinocytes (43) . MLK3 was recently shown to significantly inhibit Rac1-transforming activity, suggesting that MLK3 may be a negative regulator of the growth-promoting and transforming properties of Rac1 (44) . MLK3 has recently been reported to phosphorylate and activate MEK-1 directly in vitro and to induce MEK phosphorylation on its activation sites in vivo in COS-7 cells. Surprisingly, in cells expressing active MLK3, ERK became resistant to activation by growth factors and mitogens (3) . These findings appear to further support a role for MLK3 and other MLK family members in cell cycle arrest and apoptosis.

In direct contrast to these findings, our results support a novel role of MLTK-{alpha} in cell proliferation and/or cancer development. MLTK-{alpha} has been shown to be highly expressed in heart, skeletal muscle, small intestine, colon, prostate, and ovary as well as the HT 1080 human fibroblast cancer cell line (21) . These organs or cells are very sensitive to mitogenic, stress, or hormonal stimulation (45 , 46) . MLTK-{alpha}-induced targeted transcription factors are also clearly activated by different stimulations via two possible pathways. The transcription factors c-Jun and ATF2 are closely related to skin cancer development when stimulated with tumor promoters such as TPA through MLTK-{alpha}-stimulated JNKs and/or p38 MAPK signaling pathways. In contrast, when stimulated with EGF, c-Myc and ELK-1 play a key role in skin cancer development via the MLTK-{alpha}-stimulated ERK signaling pathway. This idea is supported by reports that MLKs are known to phosphorylate and activate MKKs such as MKK4 and/or MKK7 (12, 13, 14) and MKK3/6 (15) and that MLK3 directly phosphorylates and activates MEK1, an upstream kinase of ERKs (3) . However, since the cloning of the MLTK-{alpha} cDNA in 2001 (2) , no direct evidence in humans has been found until now, showing that MLTKs are involved in human cancer development. Our results clearly show that overexpression of MLTK-{alpha} in JB6 Cl41 cells results in increased proliferation, enhanced cell transformation induced by EGF or TPA, and in vivo tumor formation. In addition and very importantly, the overexpressing MLTK-{alpha} cells stably transfected with siRNA against MLTK-{alpha} completely lost their tumorigenic capacity both in vitro and in vivo. Moreover, the xenograft model used in this study represents a new methodology for testing tumorigenic capacity using siRNA stably transfected cells in vivo. The cells were not treated with EGF or TPA before injection into athymic mice. Furthermore, in the soft agar assay, MLTK-{alpha}-overexpressing cells still formed about 6-fold more colonies compared with mock only even without EGF or TPA stimulation (data not shown). Furthermore, MLTK-{alpha}-overexpressing cells showed a very high sensitivity for EGF or TPA. Generally, JB6 Cl41 cells undergo transformation in the presence of 10–20 ng/ml EGF or 20–40 ng/ml TPA. In contrast, MLTK-{alpha}-overexpressing cells showed significant colony formation at 1 ng/ml EGF or 10 ng/ml TPA. Thus these results strongly suggest that MLTK-{alpha} may represent an important new potential preventive or therapeutic target of human cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. David L. Turner for the generous gift of the pU6pro siRNA vector. We also thank Shaylie Meyer, veterinarian technician, for carefully monitoring athymic nude mice experiments and Andria Hansen for secretarial assistance.


   FOOTNOTES
 
Grant support: The Hormel Foundation and NIH Grants CA77646 and CA81064.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Zigang Dong, Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912; Phone: (507) 437-9600; Fax: (507) 437-9606; E-mail: zgdong{at}hi.umn.edu

1 http://sitemaker.umich.edu/dlturner.vectors. Back

Received 1/21/04. Revised 3/ 8/04. Accepted 3/23/04.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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