Cancer Cell Biology
TAK1-mediated stress signaling pathways are essential for TNF-α-promoted pulmonary metastasis of murine colon cancer cells
Article first published online: 29 DEC 2005
Copyright © 2005 Wiley-Liss, Inc.
International Journal of Cancer
Volume 118, Issue 11, pages 2758–2764, 1 June 2006
How to Cite
Choo, M.-K., Sakurai, H., Koizumi, K. and Saiki, I. (2006), TAK1-mediated stress signaling pathways are essential for TNF-α-promoted pulmonary metastasis of murine colon cancer cells. Int. J. Cancer, 118: 2758–2764. doi: 10.1002/ijc.21734
- Issue published online: 14 MAR 2006
- Article first published online: 29 DEC 2005
- Manuscript Accepted: 2 NOV 2005
- Manuscript Received: 21 JUN 2005
- Ministry of Education, Culture, Sports, Science and Technology, Japan. Grant Numbers: 15790040, 16022224, 16023225
- The Uehara Memorial Foundation, Tokyo Biochemical Research Foundation
- First Bank of Toyama Foundation
We have recently established a TNF-α-promoted metastasis model, in which the ability to metastasize to the lung was enhanced by stimulation of cultured colon 26 cells with TNF-α before intravenous inoculation. To investigate intracellular events in metastatic cascades of TNF-α-treated cancer cells, we have focused on the stress signaling pathways to c-Jun N-terminal kinase (JNK) and p38. Treatment with a specific inhibitor, SP600125 or SB203580, in vitro suppressed TNF-α-induced migration and pulmonary metastasis. Activation of endogenous TAK1, a mitogen-activated protein kinase (MAP3K) regulating the JNK and p38 MAPK pathways, was induced rapidly by TNF-α, and co-transfection of TAK1 with its activator protein TAB1 stimulated activation of JNK and p38 MAPKs, which led to activation of the transcription factor AP-1. The activation of stress signaling pathways by TAK1 resulted in enhanced migration to fibronectin in vitro and metastasis to the lung in vivo without affecting cell proliferation in vitro and tumor growth in vivo. Moreover, knockdown of endogenous TAK1 using small interfering RNA (siRNA) suppressed the TNF-α-induced JNK/p38 activation, migration and pulmonary metastasis. These results indicate that TAK1-mediated stress signaling pathways in cancer cells are essential for TNF-α-promoted metastasis to the lung. © 2005 Wiley-Liss, Inc.
The theory of a functional relationship between inflammation and cancer is not new. Two millennia ago, the Greek doctor Galen noticed a link between inflammation and cancer, and Virchow hypothesized in 1863 that the origin of cancer was a site of chronic inflammation.1, 2, 3 Over the past 10 years, the relationship between inflammation and cancer has become more widely accepted. Inflammatory cells and cytokines found in tumors are more likely to contribute to tumor growth, progression and immunosuppression than they are to mount an effective host anti-tumor response.1, 2, 3, 4, 5 Therefore, inflammation is responsible for the development of cancers, in organs such as the liver, esophagus, stomach, large intestine and urinary bladder.2
The proinflammatory cytokine tumor necrosis factor-α (TNF-α) is a key mediator in inflammation. Despite the name, TNF-α is important in early events in tumorigenesis, regulating a cascade of cytokines, chemokines, adhesion molecules, extracellular proteases and proangiogenic molecules.1, 6 In fact, it has been demonstrated that TNF-α expression is increased in the serum of cancer patients.7, 8 TNF-α also plays a critical role in tumor progression. Injection of TNF-α in mice results in an enhancement of metastasis accompanied by tumor cell extravasation with increased expression of adhesion molecules, in which the action of TNF-α is not distinguishable between host cells or cancer cells.9, 10 Kitakata et al. demonstrated that the action of TNF-α toward host cells is essential for metastasis using TNF-RI-deficient mice.11 We have recently established a TNF-α-promoted metastasis model, in which the ability to metastasize to the lung was enhanced by stimulation of cancer cells with TNF-α in vitro before intravenous inoculation.12
TNF-α triggers several signaling pathways such as mitogen-activated protein kinases (MAPKs) and NF-κB pathways.13, 14, 15 In this study, we focused on the stress-activated protein kinase (SAPK)/MAPK cascades, including c-Jun N-terminal kinase (JNK) and p38 pathways. JNK and p38 MAPKs have been reported to be related to metastatic properties, including migration, invasion and expression of MMPs and uPA in some carcinomas such as gastric, colon and breast cancer cell lines.16, 17, 18 However, functional upstream pathways to these kinases in cytokine-induced metastasis remain to be elucidated. Among members of the mitogen-activated protein kinase (MAP3K) family, transforming growth factor-β-activated kinase 1 (TAK1) has been proposed to be a critical regulator of the rapid activation of JNK/p38 MAPKs and IκB kinase (IKK) signaling pathways in response to the various cellular stimuli, including TNF-α, interleukin-1 (IL-1), lipopolysaccharide and T-cell receptor ligation.19, 20, 21, 22, 23, 24, 25, 26, 27 In the JNK and p38 MAPK cascades, TAK1 phosphorylates MAPK kinase MKK4 and MKK3/6, respectively.28, 29 TAK1 is a unique MAP3K whose kinase activity is controlled by specific TAK1-binding proteins, TAB1, TAB2 and TAB3.22, 23, 25, 26, 27, 30, 31, 32, 33 TAB1 induces TAK1 kinase activity potently compared to TAB2/TAB3.23, 30 We have recently reported that phosphorylation at Thr-187 is involved in the TNF-α-induced activation of TAK1.23
The significance of TNF-α in metastatic cascades is not negligible. Reports have proved that TNF-α enhances metastatic properties in many cancer cell lines.34, 35, 36, 37 The in vitro TNF-α stimulation model we have recently developed provides a new way to characterize the importance of signaling molecules in cancer cells for cytokine-induced metastasis. Several signaling pathways related to enhanced metastasis have been studied; however, the contribution of TAK1 to tumor metastasis has never been investigated. In this study, we found that TAK1 is a major MAP3K, regulating the TNF-α-induced activation of JNK/p38, and that the TAK1-mediated stress signaling pathways play a critical role in TNF-α-enhanced pulmonary metastasis.
Material and methods
Antibodies and reagents
Anti-phospho-TAK1 (Thr-187) antibody was reported previously.23 Briefly, it was generated by immunizing rabbits with a synthetic phospho-peptide corresponding to amino acids 180–194 of human TAK1. The sequence of the peptide antigen is as follows: NH2-CDIQTHM[pT]NNKGSAA-COOH. Anti-phospho-p38 (Thr-180/Tyr-182), anti-phospho-p44/42 ERK (Thr-202/Tyr-204), anti-phospho-ATF2 (Thr-69/Thr-71) and ATF2 (20-F1) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against TAK1 (M-579), TAB1 (C-20), TAB2 (K-20), p38 (C-20), JNK (FL), ERK1 (C-16) and PCNA (PC-10) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Recombinant human TNF-α was purchased from GenzymeTECHNE (Minneapolis, MN) SP600125 and SB203580 were purchased from Calbiochem (San Diego, CA).
Cell culture and transfection
Colon 26 cells were maintained in RMPI-1640 medium (GIBCO BRL, Life Technologies, NY) supplemented with 10% fetal bovine serum, 2 mM of L-glutamine, 100 units/ml of penicillin and 100 μg/ml of streptomycin. Cultures were kept at 37°C in a humidified atmosphere of 5% CO2/95% air. Colon 26 cells were transfected using LipofectAMINE and PLUS reagents (Invitrogen Life Technologies, CA). Expression vectors for Flag-tagged TAK1 and HA-tagged TAB1 were reported previously.21, 30 The total amount of DNA was adjusted to 1 μg with their empty vectors. After 24-hr post transfection, cells were subjected to immunoblotting, migration and metastasis assays.
Six-week-old specific pathogen-free female BALB/c mice were purchased from Japan SLC (Hamamastu, Japan). The mice were maintained in the Laboratory for Animal Experiments, Institute for Natural Medicine, Toyama Medical and Pharmaceutical University, under laminar air flow conditions with a 12 hr light–dark cycle at a temperature of 22–25°C. The mice were used according to institutional guidelines.
Total RNA was isolated from lung tissues with Isogen reagent (Nippon gene, Japan) according to the manufacturer's instructions. First strand cDNA was synthesized using an oligo(dT)18 primer and Superscript II reverse transcriptase (Invitrogen Life Technologies, CA). The reaction profile was 42°C for 50 min, followed by 70°C for 15 min. The cDNA was mixed with Taqman universal PCR master mix and Taqman probe and primers (Applied Biosystems, Foster City, CA). Real-time quantitative RT-PCR was performed using an ABI Prism 7700 sequence detection system (Applied Biosystems). Conditions for the reaction were 2 min at 50°C, 10 min at 95°C and then 40 cycles of 15 s at 95°C and 1 min at 60°C. To determine the relative RNA levels within samples, standard curves for PCR were prepared by using the cDNA from 1 sample and making a 5-fold serial dilution. The results for TNF-α were normalized to those for GAPDH from the same sample.
The migration assay of colon 26 cells was performed in Transwell cell culture chambers (Costar, Cambridge, MA) as reported previously with some modifications.38 Polyvinylpyrrolidone-free polycarbonate (PVFP) filters (8.0 μm pore size, Nuclepore, CA) were precoated with 1 μg of fibronectin (Asahi Technoglass, Tokyo, Japan) on the lower compartment. Cells were harvested with trypsin-EDTA, washed with serum-free RPMI 1640 medium and resuspended in RPMI 1640 medium with 0.1% BSA. The cell suspension (1 × 104/100 μl) was added to the upper compartment and incubated for 4–6 hr at 37°C in a 5% CO2 atmosphere. The filters were stained with hematoxylin and eosin, and the cells that migrated onto the lower surface were counted under the microscope in 5 predetermined fields at a magnification of 400×. Each assay was performed in triplicate.
Colon 26 cells were harvested with trypsin-EDTA, washed with serum-free RPMI 1640 medium and resuspended in cold PBS. The cell suspension (3 × 104/200 μl) was implanted by intravenous injection. The mice were sacrificed on day 14 after the tumor inoculation and the lungs were removed. The metastatic tumor colonies in the lungs were enumerated microscopically.
Small interfering RNAs
TAK1-siRNA was designed at iGENE Therapeutics (Tsukuba, Japan) and synthesized at Hokkaido System Science (Sappora, Japan). The target sequence was as follows: TAK1: UCCUGAACUUCGAAGAGAUCGACUA. Control siRNA (Qiagen, Chatsworth, CA) was as follows: UUCUCCGAACGUGUCACGU. Colon 26 cells were transfected with siRNAs in a final concentration of 20 nM, using LipofectAMINE and PLUS reagents (Invitrogen Life Technologies, CA). At 72-hr post-transfection, cells were stimulated with TNF-α.
After stimulation or transfection, whole cell lysates were prepared with lysis buffer (25 mM HEPES pH 7.7, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mMβ-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 10 μg/ml aprotinin and 10 μg/ml leupeptin). Cell lysates or immunoprecipitates described later were resolved by SDS-PAGE and transferred to an Immobilon-P nylon membrane (Millipore, Bedford, MA). The membrane was treated with BlockAce (Dainippon pharmaceutical, Suita, Japan) overnight at 4°C and probed with antibodies against phospho-TAK1, TAK1, TAB1, phospho-p38, phospho-ERK, phospho-ATF2, p38, ERK, ATF2, JNK and PCNA. The primary antibodies were detected using horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (DakoCytomation, Glostrup, Denmark) and visualized with the ECL system (Amersham Biosciences, Piscataway, NJ).
Cell lysates were diluted with an equal volume of dilution buffer (20 mM HEPES, pH 7.7, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 10 μg/ml aprotinin and 10 μg/ml leupeptin). After centrifugation, lysates were incubated with anti-TAK1 antibody on ice for 1.5 hr and then rotated with Protein G-Sepharose (Amersham Bioscience, Piscataway, NJ) at 4°C overnight. The Sepharose beads were washed 3 times with wash buffer (1:1 mixture of whole cell lysate buffer and dilution buffer)
In vitro kinase assay
The in vitro kinase assay with GST-c-Jun was carried out in 30 μl of reaction buffer containing 20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 μM ATP, 2 mM DTT, 20 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 1 μg of GST-c-Jun and [γ-32P]ATP (0.05 MBq) at 30°C for 30 min. The reaction mixtures were resolved by SDS-PAGE followed by autoradiography.
Dual luciferase assay
Dual reporter assays were performed as previously described.21 Briefly, pAP-1 Luc (Stratagene, CA) and pRL-EF (Elongation Factor) 1α vector (a gift from Dr. M. Tsuda) reporter plasmids were cotransfected using LipofectAMINE and plus reagents (Invitrogen Life Technologies, CA). AP-1 luciferase activity was determined by using a Dual-Luciferase Reporter Assay System (Promega, MD). The AP-1 transcriptional activity was normalized on the basis of pRL-EF-1α promoter-driven Renilla luciferase activity.
The significance of differences between groups was determined by applying Student's two-tailed t test.
TNF-α mRNA expression in lung after intravenous injection of colon 26 cells
To investigate the pathogenic changes in metastasized tissues, we injected colon 26 cells intravenously and measured the TNF-α mRNA expression in lungs, using real-time RT-PCR (Fig. 1). Three hours after the injection, TNF-α mRNA expression was significantly increased. The expression rapidly declined to near the basal level at 12 hr. But, it increased slightly again at 24 hr after injection, and this level was maintained until 72 hr. This result suggests that cancer cells injected intravenously via the tail are exposed to TNF-α especially in the early phase of metastasis.
Role of JNK and p38 in TNF-α-promoted metastasis
TNF-α triggers several intracellular signaling pathways, including MAPKs and NF-κB pathways.13, 14, 15 Here, we focused on the stress-responsive JNK and p38 MAPKs, which have been shown to be associated with enhanced expression of metastasis-related genes, invasion and metastasis in some cancer cell lines.16, 17, 18 Therefore, we first tried to confirm the metastatic activities of these kinases using specific inhibitors. In vitro kinase assays and immunoblotting showed that TNF-α-induced activation of JNK, p38, ATF-2 in colon 26 cells and the activation was inhibited selectively by their inhibitors (Fig. 2a). SP600125, a JNK inhibitor, specifically suppressed TNF-α-induced activation of JNK and ATF-2, but not phosphorylation of p38α, in a dose-dependent manner. Conversely, SB203580, a p38 inhibitor, showed a selective inhibition of TNF-α-induced autophosphorylation of p38α without affecting activation of the JNK/ATF-2 pathway. In addition, these inhibitors did not affect cell proliferation even in the presence of TNF-α (data not shown). We next studied the contribution of JNK and p38 to the metastatic ability of colon 26 cells. We recently showed that TNF-α is capable of inducing migration to fibronectin in vitro and metastasis to the lung in vivo.12 Cells were pretreated with these inhibitors for 15 min, and then stimulated with TNF-α for 6 hr in vitro. The treated cells were subjected to migration and lung metastasis assays. The increase in migration was inhibited by SP600125 or SB203580 in a dose-dependent manner (Fig. 2b). Moreover, TNF-α-promoted lung metastasis was also abrogated by treatment with these inhibitors in vitro (Fig. 2c), indicating that TNF-α-induced pulmonary metastasis is mediated through the JNK/p38 stress signaling pathways.
Metastatic ability is enhanced by overexpression of TAK1 and TAB1
Several MAP3Ks regulating JNK/p38 signaling pathways have been identified, such as TAK1, apoptosis signal-regulated kinase 1 (ASK1), MAPK/extracellular signal-regulated kinase 1 (MEKK1), and mixed-lineage kinase 3 (MLK3).23, 24, 39, 40, 41 It is still controversial that which MAP3K is the main contributor in TNF-α-induced JNK/p38 pathways; however, we have shown that knockdown of endogenous TAK1 by RNA interference significantly blocked the TNF-α-induced, but not osmotic stress-induced, rapid activation of JNK/p38 in HeLa cells.23 Therefore, we focused on TAK1 to characterize the functional significance of JNK/p38 activation and cytokine-induced metastasis of murine colon cancer cells.
To investigate the role of TAK1 in tumor metastasis, we first evaluated the effect of transient expression of the active form of TAK1. The transfection efficiency at 24-hr post-transfection was at least 50%, which was estimated by the FACS analysis of enhanced green fluorescence protein (EGFP) (data not shown). Colon 26 cells were transfected with expression vectors for TAK1 and its activator protein TAB1. At 24 hr after the transfection, phosphorylation of TAK1 at Thr-187, a critical residue for activation identified recently, was detected on immunoblotting with a phospho-specific anti-TAK1 antibody. Activation of TAK1 stimulated signaling pathways leading to JNK and p38 (Fig. 3a). In contrast, classical MAPK, extracellular signal-regulated kinase (ERK), was not significantly influenced by TAK1 and TAB1. In addition, AP-1-binding site-driven luciferase expression was significantly increased by TAK1 and TAB1 (Fig. 3b).
We next evaluated the effects of TAK1 and TAB1 on the metastatic ability of colon 26 cells. At 24 hr after the cotransfection of TAK1 and TAB1, cells were subjected to migration and lung metastasis assays. The overexpression did not affect cell proliferation in vitro and tumor growth in vivo as monitored by measuring the incorporation of BrdU and the subcutaneous injection (data not shown). Cells expressing the activated form of TAK1 were seeded in the fibronectin-coated chamber and incubated for 6 hr, and those cells that migrated onto the lower surface of the filter were counted (Fig. 3c). TAK1 significantly increased migration compared to mock-transfected control. This correlated with the increase in the formation of colonies in the lung. Fourteen days after intravenous injection of the cells, the mice were sacrificed and the tumor colonies in the lung were counted. About a 50% increase in tumor colonies was observed, showing the metastasis-inducing activity of TAK1 (Fig. 3d).
Down-regulation of metastatic propertiesby suppression of TAK1
The increased metastasis caused by overexpression of TAK1 raises the possibility that TAK1-mediated stress signaling pathways play a crucial role in TNF-α-promoted lung metastasis. To address this issue, we at first determined whether endogenous TAK1 is activated in response to TNF-α by immunoblotting with the phospho-TAK1 antibody. Figure 4a shows that TNF-α induced a rapid and transient phosphorylation of TAK1 at 2 min, which peaked at 5 min after the stimulation. The activation of TAK1 preceded the subsequent activation of JNK and p38 downstream. Phosphorylation of TAB1 and TAB2 was also detected as a change in mobility on SDS-PAGE, which was detected from 5 min after the stimulation and assumed to be mediated through the p38-mediated feedback control of TAK1.42
To elucidate the metastatic potential of TAK1, we tried to down-regulate the endogenous TAK1 expression using RNA interference. At 72-hr post-transfection of TAK1-siRNA, endogenous TAK1 had disappeared without affecting the expression of JNK, p38 and ERK. The knockdown did not affect cell proliferation for 3 days culture in vitro (data not shown). The down-regulation of TAK1 was well correlated with the impaired activation of JNK and p38 (Fig. 4b). In contrast, constitutive phosphorylation of ERK was not affected by siRNA. These results support our previous finding that TAK1 is a major MAP3K regulating the TNF-α-induced JNK and p38 signaling pathways in HeLa cells.23
Finally, we evaluated the effects of TAK1-siRNA on cytokine-induced metastasis. Colon 26 cells were treated with TNF-α for the last 6 hr of the 72-hr transfection, and then subjected to assays of migration and lung metastasis. The TNF-α-induced enhancement of migration was significantly suppressed by down-regulation of TAK1 (Fig. 5a). Moreover, knockdown of TAK1 resulted in suppression of TNF-α-induced metastasis as well as basal metastasis (Fig. 5b). In addition, similar results were obtained in the pulmonary metastasis of colon 26 cells expressing a dominant negative mutant of TAK1, TAK1-K63W (data not shown).
During the metastatic cascade, there are many pathogenic changes, including inflammation, which are associated with the over-secretion of cytokines such as TNF-α.43 We confirmed that the expression of TNF-α was induced in metastasized lung after intravenous injection of colon 26 cells, especially in the early phase (Fig. 1). The proinflammatory cytokine TNF-α has a wide range of biological activities, being involved in apoptosis, inflammation, cell proliferation and cell differentiation.14, 34 Numerous reports have also documented that TNF-α is responsible for tumor progression, including metastasis. Here, we investigated intracellular events in TNF-α-induced metastatic cascades, using an in vitro cytokine stimulation model, and found a significant role for the TAK1-mediated stress signaling pathways to JNK and p38 in cancer cells.
TAK1 was originally identified as a MAP3K, which can be activated by TGF-β, bone morphological protein (BMP).19 Rather, TAK1 has recently been classified as a stress response MAP3K regulating JNK, p38 and IKK-NF-κB pathways from the receptors of TNF-α, IL-1 and microbial pathogens.19, 20, 21, 22, 23, 24, 25, 26, 27 Both the gain of function and loss of function experiments demonstrated that TAK1 plays a critical role in TNF-α-promoted migration to fibronectin and lung metastasis of colon 26 cells. In the transfection experiment, the expression and phosphorylation of TAK1 were transient and maintained for only 2 days in culture, and the downstream pathways were also activated during these days (data not shown). Even though the expression of TAK1 in lung tumor was not confirmed, from the findings of in vitro study, TAK1 might play a crucial role in the early stage of metastasis in vivo. It should be noted that knockdown of endogenous TAK1 suppressed not only TNF-α-induced metastasis but also the basal metastasis of untreated cells. The cells injected intravenously might be exposed to TNF-α intrapulmonary in the early phase of metastasis (Fig. 1). Therefore, it is possible that cancer cells defective in TAK1 signaling are unable to induce responses to TNF-α in the lung. Tomita et al. recently reported that metastasized tumors of renal cell carcinoma in the lung spontaneously regressed in TNF-RI-deficient mice with less neovascularization, indicating that responses to TNF-α in host cells such as endothelial cells are essential for supporting tumor metastasis.44 We demonstrated in this study that TNF-α-elicited responses in cancer cells, especially the TAK1 stress signaling, are essential for the development of tumor metastasis.
We confirmed that the JNK and p38 pathways are essential for TNF-α-induced metastasis, using their specific inhibitors (Fig. 2). We demonstrated the selectivity of SP600125 in the JNK and ATF-2 assays. SB203580 partially phosphorylation of p38α, but not activation of JNK and phosphorylation of ATF-2. It has been shown that the activation of p38α is triggered by the MKK-dependent and independent pathways.45 Autophosphorylation of p38α is involved in the MKK-independent pathway, indicating that it is one of the parameters for a p38 kinase activity. To elucidate the inhibitory effect of SB203580 directly on an intracellular p38 activity, we tried to detect phosphorylation of MAPKAPK-2, a substrate for p38α; however, it was not detected in colon 26 cells (data not shown). Therefore, identification of transcription factors regulated by JNK/p38α signaling pathways will help our understanding of the role of TAK1-mediated stress signaling pathways in metastasis. The results from the overexpression and knockdown experiments of TAK1 support significance of the TAK1-mediated JNK and p38α pathways in the TNF-α-promoted metastasis.
Recently, Greten et al. demonstrated that the IKK-NF-κB pathway is linked to colitis-associated cancer.46 TNF-α is involved in the pathogenesis of Crohn's disease (CD), and anti-TNF-α agents are effective clinical therapeutics.47, 48, 49 Mutation of Nod2, a susceptibility gene in CD, potentiates NF-κB activity.50, 51 Interestingly, Nod2 is able to associate with TAK1 through its leucine-rich repeat region and modulates TAK1-induced NF-κB activation.52 In addition, TAK1 plays a role in host defense against bacterial infection by receiving signals from Toll-like receptors.26 We originally reported the NF-κB activating potential of TAK1, and that TAK1 activates IKK directly.20 In fact, we observed that TNF-α induced the activation of IKK, and overexpression of TAK1 induced a slight activation of NF-κB in colon 26 cells (data not shown). Therefore, these findings raise the possibility that the TAK1-IKKβ pathway plays a role in inflammation-induced progression of colon cancers.
Since TAK1 has been widely accepted as an MAP3K regulating IL-1-induced signaling pathways, it could also play a role in IL-1-induced metastatic properties in vivo.24, 25, 32 Moreover, inflammatory cells that have infiltrated the tumor regulate its progression. Tumor-associated macrophages (TAMs) and lymphocytes have been reported to be positive regulators of tumor progression.53, 54 TAK1 is known as a signaling intermediate in macrophages and T lymphocytes, suggesting a possible role for TAK1 in host inflammatory responses in the tumor mass.26, 27 We demonstrated in Figure 1 that TNF-α mRNA was induced in lung after injection of colon 26 cells. Identification of the cells expressing TNF-α at the mRNA and protein levels will provide the role of activation of TAK1 in TNF-α production.
In summary, we found that the activation of TAK1-mediated stress signaling pathways in colon 26 cells is involved in TNF-α-promoted experimental tumor metastasis. To further confirm the metastatic potential of TAK1, it is necessary to employ a spontaneous metastasis model by inoculating cancer cells orthotopically. Characterization of the activation of TAK1 in primary tumors and identification of its target genes controlling metastatic processes will also reveal the novel functional significance of TAK1 in inflammation-induced tumor progression.
We are grateful to Drs. Masaaki Tsuda and Takahisa Sugita for providing plasmid DNAs.
- 23Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-β-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2. J Biol Chem 2005; 280: 7359–68., , , , .
- 48New concepts in anti-tumor necrosis factor therapy for inflammatory bowel disease. Rev Gastroenterol Disord 2005; 5: 10–18..