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Oncogene. Author manuscript; available in PMC Sep 28, 2012.
Published in final edited form as:
PMCID: PMC3460638
NIHMSID: NIHMS405024
Loss of tumor progression locus 2 (tpl2) enhances tumorigenesis and inflammation in two-stage skin carcinogenesis
KL DeCicco-Skinner,1,3 EL Trovato,1,2,3 JK Simmons,2 PK Lepage,2 and JS Wiest2
1Department of Biology, American University, Washington, DC, USA
2Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
Correspondence: Dr JS Wiest, Laboratory of Cancer Biology and Genetics, National Institutes of Health, National Cancer Institute, 37 Convent Drive, Bethesda, MD 20892, USA. wiestj/at/mail.nih.gov
3These authors contributed equally to this work.
Tumor progression locus 2 (Tpl2) is a serine/threonine kinase in the mitogen-activated protein kinase signal transduction cascade known to regulate inflammatory pathways. Previously identified as an oncogene, its mutation or overexpression is reported in a variety of human cancers. To address its role in skin carcinogenesis, Tpl2−/− or wild-type (WT) C57BL/6 mice were subjected to a two-stage dimethylbenzanthracene/12-O-tetradecanoylphorbol- 13-acetate (TPA) mouse skin carcinogenesis model. Tpl2−/− mice developed a significantly higher incidence of tumors (80%) than WT mice (17%), as well as a reduced tumor latency and a significantly higher number of total tumors (113 vs 6). Moreover, Tpl2−/− mice treated with TPA experienced significantly higher nuclear factor kappaB (NF-κB) activation, edema, infiltrating neutrophils and production of proinflammatory cytokines than did WT mice. We investigated the role of the p38, JNK, MEK and NF-κB signaling pathways both in vitro and in vivo in WT and Tpl2−/− mice by using inhibitors for each of these pathways. We confirmed that the proinflammatory effect in Tpl2−/− mice was due to heightened activity of the NF-κB pathway. These studies indicate that Tpl2 may serve more as a tumor suppressor than as an oncogene in chemically induced skin carcinogenesis, with its absence contributing to both tumorigenesis and inflammation.
Keywords: Tpl2, skin cancer, inflammation, MAP3K8, NF-κB
Tumor progression locus 2 (Tpl2), also known as Cot/MAP3K8, is a serine/threonine kinase in the mitogen-activated protein kinase (MAPK) signal transduction cascade (Aoki et al., 1991). As a member of the MAP kinase kinase kinase (MAPKKK) family, it resides upstream of the MAPKK MEK1/MEK2 and MAPK ERK1/ERK2. In non-stimulated cells, Tpl2/Cot associates with nuclear factor-kappaB (NF-κB) precursor protein, p105, rendering Tpl2 inactive and preventing the proteolysis of p105 (Beinke et al., 2003). Tpl2 is activated in response to various proinflammatory stimuli such as lipopolysaccharide (LPS), tumor necrosis factor (TNF) and CD40 ligand. Following activation, IκB kinase phosphorylates p105, releasing Tpl2. p105 is subsequently degraded into p50 by the proteasome, allowing p50 to dimerize with other NF-κB family members and translocate to the nucleus, wherein NF-κB can regulate over 400 genes (Beinke et al., 2004). In addition, the Tpl2 liberated from p105 can phosphorylate substrates such as MEK1, leading to ERK activation and activation of diverse genes involved in growth, differentiation and inflammation. Moreover, protein kinase B (AKT) physically interacts with and phosphorylates Tpl2 (Kane et al., 2002). This phosphorylation is required for activation of the IκB kinase complex and nuclear translocation of the p65 subunit of NF-κB (Kane et al., 2002). Although Tpl2 is involved in numerous other signaling pathways, the molecular mechanism of Tpl2 regulation has not been fully elucidated.
The role of Tpl2 in tumorigenesis was first identified by a provirus integration site in MoMuLV-induced rodent T-cell lymphomas and mouse mammary tumor virus-induced mammary carcinomas (Patriotis et al., 1993). Overexpression of Tpl2 activates ERK, JNK and p38 MAPK pathways, as well as transcription factors such as nuclear factor of activated T cells and NF-κB (Patriotis et al., 1994; Salmeron et al., 1996; Tsatsanis et al., 1998; Belich et al., 1999; Chiariello et al., 2000). Activation of Tpl2 contributes to cellular transformation and tumorigenesis through overexpression and mutation, as Tpl2 activity was reported in a number of cancers including breast, endometrial, thymomas, lymphomas, lung, Hodgkin’s disease and nasopharyngeal carcinoma (Salmeron et al., 1996; Ceci et al., 1997; Sourvinos et al., 1999; Tsatsanis and Spandidos, 2000; Clark et al., 2004). Moreover, truncation of Tpl2 after the penultimate exon transforms cells in culture and is highly oncogenic in animals (Ceci et al., 1997; Chiariello et al., 2000). However, recent evidence suggests that in some settings Tpl2 may serve a tumor suppressor role. Tpl2−/− mice, when crossed with the T-cell receptor transgene, develop a high incidence of T-cell lymphomas, whereas wild-type (WT) mice remain cancer free (Tsatsanis et al., 2008). The participation of Tpl2 in a de novo chemically induced cancer model system has not been tested.
Squamous cell carcinoma (SCC) is among the most commonly diagnosed cancers in the United States. (Jemal et al., 2009). The two-stage skin cancer model is the prototype system for investigating SCC development, as carcinomas appearing in this protocol evolve in a manner similar to that of SCC in humans (Glick et al., 2007). In the two-stage model, mice are initiated with 7,12-dimethylbenz(a)anthracene (DMBA) and repeatedly administered the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Typically, benign papillomas arise within 10–15 weeks, and a small percentage progresses to locally invasive SCC. The preponderance of tumors contain mutations in the H-ras oncogene.
Despite the significant functions that Tpl2 has in eliciting cellular responses, Tpl2 knockout animals appear phenotypically normal (Dumitru et al., 2000). All their immune organs and ratio of immune system cellular subsets are normal, as is their antibody response to T-cell-dependent and -independent antigens (Dumitru et al., 2000). However, Tpl2−/− mice have impaired host defense against Toxoplasma gondii, reduced parasite clearance and decreased interferon-γ production, suggesting that Tpl2 may be a regulator of helper T cells (Watford et al., 2008). Moreover, genetic evidence using Tpl2−/− mice suggests that the Tpl2 signaling pathway may have both pro- and anti-inflammatory functions. As a proinflammatory mediator, Tpl2 positively regulates the expression and transport of TNF-α in response to LPS. Consequently, Tpl2−/− animals reportedly display low levels of TNF-α in response to LPS and are resistant to endotoxin shock (Dumitru et al., 2000). Similarly, ablation of Tpl2 has been shown to be beneficial in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and pancreatitis (Hu et al., 2006). However, in some circumstances, Tpl2 can function in an anti-inflammatory manner through its inhibition of proinflammatory cytokines (Sugimoto et al., 2004; Tomczak et al., 2006). Thus, depending on the cell type and stimuli used, Tpl2 can have different effects as an immunomodulatory agent.
In this report, we demonstrate that Tpl2−/− mice have a significantly higher incidence of tumor development and malignant progression in the mouse skin model of multistage carcinogenesis. Keratinocytes from Tpl2−/− mice have an accelerated cell cycle demonstrated in vivo through heightened Ki67 and grafting experiments. Moreover, Tpl2−/− mice treated with TPA have increased levels of edema, neutrophil infiltration, proinflammatory cytokines and NF-κB activation compared with control mice. In summary, we suggest that Tpl2−/− mice have a heightened inflammatory state in response to TPA, possibly contributing to their increased susceptibility to develop chemically induced skin carcinogensis.
Tpl2−/− mice form more skin tumors
C57BL/6 mice are relatively resistant to skin tumor induction (DiGiovanni et al., 1984). In DMBA/TPA skin tumor induction experiments on a C57BL/6 background, Tpl2−/− mice developed significantly higher numbers of tumors than did WT controls, and, within 22 weeks, nearly 80% of Tpl2−/− mice formed tumors, compared with only 17% of WT mice (Figure 1a). Some Tpl2−/− mice were killed before 52 weeks because of tumor size, explaining the slight dip in tumor incidence in Tpl2−/− mice between weeks 27 and 34. All tumors required DMBA/TPA treatment, as treatment with DMBA or TPA alone did not produce any tumors in either genotype (data not shown). In addition, the average number of tumors per mouse was higher in the Tpl2−/− genotype (Figure 1b). Tpl2−/− mice averaged 4–5 tumors per animal, compared with one tumor per mouse in WT animals. Between weeks 15 and 20, several papillomas coalesced, to be reported the following week as the animal having larger papillomas, explaining the slight decrease in the number of tumors/mouse. No obvious differences in tumor size were observed between the genotypes.
Figure 1
Figure 1
Two-stage skin carcinogenesis in Tpl2−/− mice and their wild-type (WT) controls. Mice were treated once with 100 μg DMBA/200 μl acetone and promoted by treating with 10 μg/200 μl TPA twice a week from weeks (more ...)
Tpl2 influences tumor growth in both the tumor and stromal compartments
The contribution of the Tpl2 gene to enhanced skin tumor formation was tested by grafting nude mice with v-rasHa-transduced keratinocytes from Tpl2−/− or WT mice mixed with fibroblasts from either genotype. Mice grafted with keratinocytes and fibroblasts from WT animals developed small tumors (less than 100mm3), whereas grafts containing mixed phenotypes (WT keratinocytes plus Tpl2−/− fibroblasts or Tpl2−/− keratinocytes plus WT fibroblasts) developed tumors approximately twice as large as the nude mice that received only WT cells. Mice receiving both Tpl2−/− fibroblasts and Tpl2−/− keratinocytes developed tumors approximately nine times larger than the mice containing WT keratinocytes and fibroblasts (Figure 1c). These results indicated that Tpl2 influences tumor development in both tumor and stromal compartments.
Skin papillomas progress in the absence of Tpl2
All tumors underwent a histological examination to determine phenotype and progression. Out of the 30 C57BL/6 WT animals, there were a total of five papillomas, only one of which converted into an SCC. Conversely, Tpl2−/− mice had a total of 113 skin tumors, of which 95 were benign papillomas, 8 were sebaceous adenomas, 8 were SCC and 2 were of a mixed sebaceous/squamous phenotype (Table 1).
Table 1
Table 1
Summary of histological examination from the two-stage carcinogenesis experiment
Tpl2−/− keratinocytes have increased proliferation
The proliferative capability of TPA-treated WT and Tpl2−/− skin was determined by measuring Ki67, a nuclear antigen present in all phases of the cell cycle except G0 (Rijzewijk et al., 1989). Tpl2−/− mice had two times higher Ki67 staining, even in the absence of TPA treatment, and both genotypes had increased Ki67 staining following TPA treatment (Figures 2a and b).
Figure 2
Figure 2
Ki67 proliferation marker is increased in mice lacking Tpl2. The number of Ki67-positive keratinocytes per millimeter epidermis was counted in five randomly selected regions from each slide. Representative photomicrographs at ×40 magnification (more ...)
The inflammatory response is enhanced in TPA-treated Tpl2−/− skin
A number of studies suggest that the inflammatory response to tumor promoters contributes to skin tumor susceptibility (Coussens and Werb, 2002). Therefore, we assessed the inflammatory response of Tpl2−/− mice and WT animals to acute exposure to TPA. Although Tpl2−/− mice showed no differences in increased macrophage infiltration (data not shown), there was a rapid and enhanced infiltration by neutrophils as assessed through myeloperoxidase (MPO) staining (Figure 3a). There were 35 times as many neutrophils in TPA-treated Tpl2−/− mouse skin than in WT mice within 12 h as assessed through an MPO assay (Figure 5d). In addition, Tpl2−/− mice had higher numbers of CD3+T cells and mast cells (Figures 3b and c). Sections from Tpl2−/− and WT mice were also stained for basophils and eosinophils, but no major differences were found between genotypes (data not shown).
Figure 3
Figure 3
Immunohistochemical analysis of inflammatory cells in wild-type (WT) and Tpl2 −/− mice. TPA-treated skin (4, 8 or 12 h) from WT and Tpl2 −/− mice were stained for the neutrophil marker myeloperoxidase (a), the mast cell (more ...)
Figure 5
Figure 5
Tpl2−/− mice display heightened inflammatory responses due to exaggerated NF-κB signaling. (a) Primary keratinocytes were transiently transfected with a NF-κB reporter plasmid construct and treated with TPA or vehicle for (more ...)
TPA-treated Tpl2 −/− mice have an elevated expression of inflammatory cytokines
TNF-α, interleukin (IL)-12, IL-1β and IL-5 levels were measured in TPA-treated Tpl2−/− and WT mouse skin to compare the effect of Tpl2 ablation (Figures 4a–d). All cytokines were below detection limits in untreated WT and Tpl2−/− mice. TPA treatment induced TNF-α levels rapidly in the skin of both genotypes, peaking at 4 h in Tpl2−/− mice and at 8 h in WT mice. At 8 and 12 h after TPA treatment, Tpl2−/− mice had slightly lower levels of TNF-α than Tpl2−/− mice. In contrast, IL-12, IL-1β (at 4 h) and IL-5 were significantly higher in TPA-treated Tpl2−/− mice compared with WT animals.
Figure 4
Figure 4
Proinflammatory cytokine concentrations in wild-type (WT) and Tpl2 −/− mice. TPA-treated skin samples (4, 8 or 12 h) from WT and Tpl2 −/− mice were collected and cytokine concentrations measured. Concentrations less than (more ...)
p38, JNK and MEK pathways are not primarily responsible for the heightened inflammation found in Tpl2−/− mice
Tpl2 has been associated with various inflammatory pathways, including p38, JNK, MEK and NF-κB. To assess whether dysregulation in one or more of these pathways may contribute to the elevated inflammatory cell subsets and proinflammatory cytokines present in Tpl2−/− mice, we used inhibitors specific for each of these pathways in vitro and in vivo to detect differences between genotypes. In vitro, there were no significant differences in the basal levels of p38, JNK and MEK pathways between the two genotypes (Supplementary Figure 1). Furthermore, there were no differences between the two genotypes on TPA stimulation. Inhibitors specific for p38, JNK or MEK pathways were equally effective in blocking this induction in both WT and Tpl2−/− mice as evidenced by analyzing downstream genes for each of these pathways (Supplementary Figure 2). In addition, TPA treatment alone induced edema equally in WT and Tpl2−/− mice in vivo and this induction was abrogated to a similar extent when either genotype was subjected to inhibitors (Supplementary Figure 3).
The loss of Tpl2 activates NF-κB signaling in keratinocytes
A dual luciferase NF-κB reporter assay was used to measure differences in NF-κB basal activity in keratinocytes from WT and Tpl2−/− mice, and after treatment with TPA (Figure 5a). We found Tpl2−/− mice had two times higher basal activity of NF-κB than WT mice. Treatment with TPA magnified these differences, resulting in three times higher NF-κB activation in Tpl2−/− keratinocytes than WT cells. Treatment with the NF-κB inhibitor, SN50, reduced the NF-κB response by 34% in WT keratinocytes but only by 14% in Tpl2−/− keratinocytes. In addition, TPA-treated Tpl2−/− keratinocytes were found to have lower levels of NF-κB inhibitory proteins (IκB-α and IκB-β) and increased p65 nuclear translocation (Supplementary Figure 4).
Blocking NF-κB decreases edema in wild-type mice but not in Tpl2−/− mice
To confirm the differences in NF-κB signaling between WT and Tpl2−/− mice in an in vivo setting, mouse ears were treated with TPA with or without SN50 treatment, and edema measurements were recorded over a 3 h period. TPA treatment induced edema in both genotypes, although to a greater extent in Tpl2−/− mice (41% induction at the 3 h time point compared with 32%). Treatment with SN50 blocked this induction in WT mice, bringing the edema levels to control values. In contrast, the same concentration of SN50 in Tpl2−/− mice was not able to block the edema, as evidenced by ear thickness measurements and hematoxylin and eosin staining (Figures 5b and c).
Neutrophil infiltration is blocked in WT mice receiving an NF-κB inhibitor but is not diminished in Tpl2−/− mice
TPA treatment induces neutrophil infiltration to a much greater extent in Tpl2−/− mice compared with WT animals (Figure 3a). An MPO activity assay was performed to quantify differences between neutrophil numbers in TPA-treated WT or Tpl2−/− mice, alone or in the presence of SN50. Although untreated WT and Tpl2−/− mice had comparable MPO activity, Tpl2−/− mice treated with TPA had 35 times greater MPO activity than WT mice. Treatment of Tpl2−/− mice with TPA plus SN50 reduced MPO activity to basal levels in WT animals but had no significant effect in Tpl2−/− mice (Figure 5d).
Chemically induced skin carcinogenesis using DMBA as an initiator and TPA as a tumor promoter is the prototype system for SCC induction (Yuspa, 1998). Typically, mice will develop benign papillomas within 10–15 weeks, with conversion of squamous papillomas to carcinomas occurring spontaneously at a low frequency (Quintanilla et al., 1986). The Tpl2 gene, previously identified as an oncogene, is involved in a variety of cellular functions, including inflammatory processes and immune function. However, the function of this protein in a de novo inducible cancer model has not been tested. Using a two-stage skin carcinogenesis model, Tpl2−/− mice developed a significantly higher incidence of tumors, developed them with reduced latency and had a significantly higher number of tumors per mouse.
To assess whether Tpl2−/− mice have a higher proliferative capacity, possibly contributing to their increased rate of papilloma formation, we measured Ki67 staining on TPA-treated skin from Tpl2−/− and WT mice. Previous studies suggest that Tpl2 can accelerate the cell cycle in T lymphocytes by promoting the G1 to S transition (Velasco-Sampayo and Alemany, 2001). However, we found that the absence of Tpl2 heightened the proliferative capacity of keratinocytes, both innately and after TPA stimulation. A skin-grafting protocol was used to further determine the in vivo phenotype of keratinocytes and fibroblasts from Tpl2−/− vs WT mice. In agreement with our other data, we found that nude mice grafted with keratinocytes and fibroblasts from Tpl2−/− mice developed significantly larger tumors than did mice grafted with keratinocytes and fibroblasts from WT animals or by grafting mixed genotypes. Mice receiving keratinocytes and fibroblasts from both genotypes developed tumors that were intermediate in size compared with the animals receiving only knockout or only WT cells. This suggests that both Tpl2−/− keratinocytes and Tpl2−/− fibroblasts contribute to the highly increased tumorigenic response of these animals.
Several reports suggest that inflammation in the microenvironment may contribute to the development or progression of skin cancer (Mueller, 2006; de Visser et al., 2006). Therefore, we analyzed the recruitment of various inflammatory cell types and proinflammatory cytokines in TPA-treated mouse skin. We found that Tpl2−/− mice had significantly higher numbers of neutrophils, achieving levels 35 times greater than control animals within 12 h. In addition, the skin of Tpl2−/− mice had significant increases in the number of mast cells and CD3+ cells. The infiltration of mast cells and neutrophils may contribute to skin tumor initiation and progression, as others have reported an increase in these cell types in skin cancer (Mueller, 2006). In addition, mast cells are often found below the regions of hyperplastic epithelium in chemically induced skin carcinogenesis and may contribute to the activation of stroma that supports tumor growth. Moreover, heightened and continual recruitment of neutrophils is associated with progression to invasive SCCs (Mueller, 2006).
Neutrophil and mast cells may also secrete cytokines contributing to a tumor-promoting environment. We found an induction in several of these cytokines in TPA-treated skin from Tpl2−/− mice, including IL-1β, IL-5 and IL-12. As previously reported, induction of IL-1β by neutrophils can serve to recruit macrophages and activate fibroblasts in the skin (Mueller, 2006). In addition, IL-1β causes the infiltration and activation of leukocytes, resulting in enhanced tumor growth and angiogenesis. IL-1β-overexpressing cells significantly increase cancer cell growth in a xenograft model and activation of IL-1β induces numerous angiogenic factors, including VEGF-A, MMP-9, IL-8 and MCP-1 (Kimura et al., 2007). Furthermore, induction of these factors was blocked by an NF-κB inhibitor and also by the knockdown of p65, suggesting NF-κB as a major contributor to IL-1β-dependent tumorigenesis and angiogenesis.
We also found inductions in IL-12 protein levels in Tpl2−/− mice. This agrees with previous literature showing that challenge with CpG-DNA or Helicobacter hepaticus in Tpl2−/− macrophages showed significantly increased IL-12 production compared with their WT counterparts (Sugimoto et al., 2004; Tomczak et al., 2006). The role of IL-12 in tumorigenesis is complex. Many reports have shown decreased IL-12 production to be associated with tumors, including those of colon, kidney and lung (Brunda et al., 1993; Tahara et al., 1994; Colombo et al., 1996). However, recent reports using two-stage skin carcinogenesis protocols found a reduction in papilloma formation and papilloma to carcinoma conversion in IL-12 knockout mice, suggesting IL-12 as a promoter of chemically induced skin tumorigenesis (Sharma et al., 2009).
Contrary to previous reports showing a defect in TNF-α production and transport when LPS is used as a stimulus, in our system the two genotypes produced comparable levels of TNF-α, although the induced TNF-α level was slightly lower in Tpl2−/− skin. The ability to still produce TNF-α in Tpl2−/− mice may be important because TNF-α is critical for the early stages of tumor promotion, and TNF-α−/− mice resist developing benign and malignant skin tumors in response to chemically induced skin carcinogenesis (Moore et al., 1999).
The heightened inflammation found in Tpl2−/− mice may be initiated by aberrant inflammatory signaling pathways. Numerous inflammatory pathways are associated with Tpl2, including ERK, JNK, p38 and NF-κB (Patriotis et al., 1994; Salmeron et al., 1996; Tsatsanis et al., 1998; Belich et al., 1999; Chiariello et al., 2000). Although we cannot rule out the possibility that ERK, JNK and p38 may contribute to the inflammatory and tumorigenic state of Tpl2−/− mice, inhibitors specific to each of these pathways in vitro and in vivo demonstrated that the heightened inflammatory response in Tpl2−/− mice was primarily due to a dysregulation in NF-κB activation and signaling. Basal NF-κB activity levels of Tpl2−/− mice are two times greater than those of control mice. Stimulation with TPA magnifies this difference to a greater extent. Moreover, whereas treatment with an NF-κB inhibitor could block edema and neutrophil infiltration in WT mice, the inhibitor was unable to block the exaggerated NF-κB response in Tpl2−/− mice. The role of NF-κB in skin homeostasis is complex, as both overactivation or inhibition of NF-κB signaling in keratinocytes reportedly leads to inflammation (Sur et al., 2008). However, NF-κB ablation in keratinocytes can also promote formation of SCCs in response to DMBA/TPA treatment (Karin, 2006; Sur et al., 2008). The manner in which the loss of Tpl2 contributes to an exaggerated NF-κB activation in a skin cancer model is currently being investigated.
The ability of Tpl2 to serve an anti-inflammatory role in our model system contradicts other reports. Cell type and stimulus may determine how Tpl2 serves proinflammatory or anti-inflammatory functions. Previous reports found Tpl2 to be proinflammatory when LPS, CpG or anti-CD3/CD28 were the stimuli, functioning through toll-like receptors. TPA signals through protein kinase C (PKC), a multigene family of serine/threonine kinases (Griner and Kazanietz, 2007). Six isoforms of PKC are expressed in mouse keratinocytes, namely, α, δ, ε, μ, η and ζ (Reddig et al., 1999). Each isoform contributes differently to normal keratinocyte growth and differentiation, and, if inappropriately regulated, certain isoforms are associated with disease pathogenesis. As previously reported, mice overexpressing PKC-ε have enchanced susceptibility to carcinoma formation in a chemically induced skin cancer model (Jansen et al., 2001). TPA-treated mice overexpressing PKC-α exhibit extensive neutrophilic inflammation and heightened NF-κB nuclear translocation (Cataisson et al., 2005). It is currently unknown if one or more PKC isoforms are dysregulated in Tpl2−/− mice and this warrants further investigation.
Our studies reveal a previously unknown role of Tpl2 in chemically induced skin carcinogenesis. In this setting, Tpl2 appears to serve more as a tumor suppressor than as an oncogene. In addition, a heightened inflammatory response, directed primarily by NF-κB, in Tpl2−/− mice may contribute to the enhanced susceptibility to skin carcinogenesis.
Wild-type and transgenic mice
Male and female Tpl2−/− mice were engineered as previously described (Ceci et al., 1997). C57BL/6 WT mice were generated from the same colony as Tpl2−/− mice. All mice were bred and maintained at the NIH animal facility (Bethesda, MD, USA). Tpl2−/− status was regularly confirmed by PCR. All animal work was performed following NIH guidelines under an approved animal protocol.
Tumor initiation/promotion studies
A total of 31 Tpl2−/− mice and 30 C57BL/6 WT mice of 8–10 weeks of age were initiated with 7,12-dimethylbenz(a)anthracene (DMBA; 100 μg/200 μl acetone) on shaved dorsal skin. Promotion began 1 week later with twice weekly application of TPA painted on the skin (10 μg/200 μl acetone) and continued for 20 additional weeks. The number and size of tumors were recorded weekly. Three control groups (n=5) receiving only acetone, DMBA or TPA were maintained for both genotypes. All groups were matched for age and sex. Tumor-bearing animals were individually housed to avoid injury to the tumor sites. Animals were killed 52 weeks after the date of initiation, or at an earlier time point if the animal was deemed moribund by the veterinary staff.
Grafting cells on athymic nude mice
Tpl2−/− or WT primary keratinocytes isolated from newborn mice were infected with the v-rasHa retrovirus, trypsinized and used for grafting on day 8 as described previously (Lichti et al., 2008). Briefly, 20 athymic nude mice of 8 weeks of age were divided into four groups and a graft site was prepared on their backs. Mice received 7 million keratinocytes (either WT or Tpl2−/−) mixed with 7 million fibroblasts (either WT or Tpl2−/−). The four groups of mice received the following combinations: (1) WT keratinocytes plus WT fibroblasts, (2) Tpl2−/− keratinocytes plus WT fibroblasts, (3) WT keratinocytes plus Tpl2−/− fibroblasts or (4) Tpl2−/− keratinocytes plus Tpl2−/− fibroblasts. Determination of tumor measurements began 1 week after grafting and continued once a week for 28 days. Tumor volume was calculated using the height multiplied by the length along the spinal column and the width at right angles to the length. Data are expressed as means±s.e.m. of approximate tumor volume in mm3.
Immunohistochemistry
Mouse skins were fixed in 4% paraformaldehyde. Tissue sections from Tpl2−/− and C57BL/6 mice were stained with hematoxylin and eosin, deparaffinized, rehydrated, subjected to antigen retrieval and quenched for endogenous peroxidase activity. Anti-MPO primary rabbit polyclonal antibody (1:2000) or anti-CD3 goat antibody (1:400) was applied for 30 min. Negative controls were obtained by substitution of the primary antibody with buffer solution. Following washing, the labeled avidin-biotinylated enzyme complex technique was used for amplification of primary antibody binding and 3,3-diaminobenzidine was applied for visualization. Sections were melanin-bleached and counterstained with Gill’s hematoxylin. The sections were dehydrated through graded alcohols, immersed in xylene and mounted with coverslips. Ki67 staining on mouse sections was performed by the Pathology and Histotechnology Laboratory at the NCI (Frederick, MD, USA). Representative areas were photographed using an Olympus DP70 (Olympus, Center Valley, PA, USA) digital camera.
Transfection and luciferase reporter assay
The NF-κB-luciferase plasmid DNA was a gift from Dr SH Yuspa (National Cancer Institute, National Institutes of Health). Primary keratinocytes were transfected using Lipofectamine 2000 according to the manufacturer’s protocol (Invitrogen Life Technologies, Carlsbad, CA, USA). Transient cotransfection with firefly and Renilla luciferases was conducted for 48 h before treatment with TPA (Cataisson et al., 2005). Keratinocytes receiving the NF-kB inhibitor SN50 (Calbiochem, La Jolla, CA, USA) were pretreated for 30 min before TPA treatment and again in conjunction with TPA. At 6 h after treatment with TPA, cells were rinsed twice in phosphate-buffered saline and harvested in reporter lysis buffer (Promega, Madison, WI, USA). Luciferase activity was measured by using the dual luciferase reporter assay kit (Promega) according to the manufacturer’s protocol. The ratio of firefly to Renilla luciferases was calculated after each assay, and this value was used as the normalized luciferase activity. The results are shown as mean ratios of quadruplicates±s.d. of two independent experiments.
MPO assay
Pinna from WT and Tpl2−/− mice treated with acetone, TPA (2 μg/10 μl acetone), SN50 (10 mg/ml) or SN50+TPA were used for the MPO assay. Animals receiving SN50 (Calbiochem) were pretreated with inhibitor for 30 min before TPA treatment. The assay was performed as previously reported (Cataisson et al., 2005). Briefly, samples were homogenized in potassium phosphate buffer (pH, 6.0) containing 0.5% hexadecyltrimethylammonium bromide, sonicated and freeze-thawed three times. The suspension was centrifuged at 35 000 g for 30 min at 4 °C, at which point 20 μl of supernatant was added to 300 μl of potassium phosphate buffer containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. Changes in optical density were monitored at 460nm at 25 °C.
Immunoblot analysis
Primary keratinocytes and fibroblasts were prepared from Tpl2−/− and C57BL/6 mouse pups at 1–2 days of age as previously described and plated in six-well dishes (Lichti et al., 2008). Cells receiving inhibitor during treatment were pretreated topically for 30 min. At the time of treatment, cells received vehicle control (DMSO), TPA only (10ng/ml) or TPA+5 10 or 20μM of the JNK inhibitor SP600125 or the MEK inhibitor U0126 (Calbiochem). Cells were treated with the p38 inhibitor SB203580 (Calbiochem) at concentrations of 5 or 10μM. All lysates were collected 5, 15 or 30 min after treatment. Total protein lysates were prepared from primary keratinocyte cultures using M-PER reagent (Thermo Fisher Scientific, Rockford, IL, USA) containing complete protease inhibitor (Roche, Indianapolis, IN, USA) and Halt phosphatase inhibitor (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. Proteins were separated using 12% Criterion XT Bis-Tris gels (Bio-Rad, Hercules, CA, USA), followed by immunoblotting. Blots were incubated in 5% BSA-TBST for 24 h at 4 °C with 1:1000 dilutions of primary rabbit antibodies purchased from Cell Signaling Technology (Danvers, MA, USA): phospho-ATF-2, phospho-c-Jun, phospho-ERK1/2, p38, ATF-2, c-Jun, ERK or β-actin. Anti-rabbit HRP (1:2000) was used as the secondary antibody (Cell Signaling Technology).
Detection of inflammatory cytokines
In total, 48 Tpl2−/− or WT mice were shaved on their dorsal epidermis at 13 weeks of age. Mice were treated with either 10μg of TPA/0.2 ml acetone (Sigma-Aldrich, St Louis, MO, USA) for 4, 8, 12, 24 or 48 h, or acetone alone. Cytokine concentrations in mouse skin were measured by Linco Research (St Charles, MO, USA).
In vivo experimentation using pharmacological inhibitors
Ears from Tpl2−/− and WT mice were pretreated with topical administration of the inhibitors SB203580 (3.7 mg/ml), SP600125 (4.0 mg/ml), U0126 (3.8 mg/ml) and SN50 (10.0 mg/ml) 30 min before TPA treatment. At time point 0, mouse ears were treated topically with TPA alone (2 μg TPA/10 μl acetone), inhibitor alone, inhibitor+TPA or with acetone control (n=4 per group). Edema was measured with digital calipers before pretreatment and every hour after time point 0 for 3 h. At the third hour, mice were killed by CO2 asphyxiation, and treated pinna were collected, sectioned and used for hematoxylin and eosin and MPO staining.
Statistical analysis
Cell proliferation, cytokine levels, MPO assay, grafting and skin carcinogenesis experiments were analyzed through two-way ANOVA using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). P-values <0.05 were considered significant. In vivo edema measurements within the same genotype were analyzed by repeated-measures ANOVA. In vivo edema measurements between genotypes were analyzed by two-way ANOVA with LSD post hoc analyses using SPSS 13.0 software. P-values <0.01 were considered significant.
Supplementary Material
Supplementary Figure 1
Supplementary Figure 2
Supplementary Figure 3
Supplementary Figure 4
Acknowledgments
We thank Dr Jyotsna Pandey for her assistance with confocal microscopy. This work was supported by the NCI/NIH Intramural Research Program.
Footnotes
Conflict of interest
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
  • Aoki M, Akiyama T, Miyoshi J, Toyoshima K. Identification and characterization of protein products of the cot oncogene with serine kinase activity. Oncogene. 1991;6:1515–1519. [PubMed]
  • Beinke S, Deka J, Lang V, Belich MP, Walker PA, Howell S, et al. NF-kappaB1 p105 negatively regulates TPL-2 MEK kinase activity. Mol Cell Biol. 2003;23:4739–4752. [PMC free article] [PubMed]
  • Beinke S, Robinson MJ, Hugunin M, Ley SC. Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IkappaB kinase-induced proteolysis of NF-kappaB1 p105. Mol Cell Biol. 2004;24:9658–9667. [PMC free article] [PubMed]
  • Belich MP, Salmerón A, Johnston LH, Ley SC. TPL-2 kinase regulates the proteolysis of the NF-kappaB-inhibitory protein NF-kappaB1 p105. Nature. 1999;397:363–368. [PubMed]
  • Brunda MJ, Luistro L, Warrier RR, Wright RB, Hubbard BR, Murphy M, et al. Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J Exp Med. 1993;178:1223–1230. [PMC free article] [PubMed]
  • Cataisson C, Pearson AJ, Torgerson S, Nedospasov SA, Yuspa SH. Protein kinase C alpha-mediated chemotaxis of neutrophils requires NF-kappa B activity but is independent of TNF alpha signaling in mouse skin in vivo. J Immunol. 2005;174:1686–1692. [PubMed]
  • Ceci JD, Patriotis CP, Tsatsanis C, Makris AM, Kovatch R, Swing DA, et al. Tpl-2 is an oncogenic kinase that is activated by carboxy-terminal truncation. Genes Dev. 1997;11:688–700. [PubMed]
  • Clark AM, Reynolds SH, Anderson M, Wiest JS. Mutational activation of the MAP3K8 protooncogene in lung cancer. Genes Chromosomes Cancer. 2004;41:99–108. [PMC free article] [PubMed]
  • Chiariello M, Marinissen MJ, Gutkind JS. Multiple mitogen-activated protein kinase signaling pathways connect the Cot oncoprotein to the c-jun promoter and to cellular transformation. Mol Cell Biol. 2000;20:1747–1758. [PMC free article] [PubMed]
  • Colombo MP, Vagliani M, Spreafico F, Parenza M, Chiodoni C, Melani C, et al. Amount of interleukin 12 available at the tumor site is critical for tumor regression. Cancer Res. 1996;56:2531–2534. [PubMed]
  • Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002;420:860–867. [PMC free article] [PubMed]
  • de Visser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nat Rev Cancer. 2006;6:24–37. [PubMed]
  • DiGiovanni J, Prichett WP, Decina PC, Diamond L. DBA/2 mice are as sensitive as SENCAR mice to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate. Carcinogenesis. 1984;5:1493–1498. [PubMed]
  • Dumitru CD, Ceci JD, Tsatsanis C, Kontoyiannis D, Stamatakis K, Lin JH, et al. TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway. Cell. 2000;103:1071–1083. [PubMed]
  • Glick A, Ryscavage A, Perez-Lorenzo R, Hennings H, Yuspa S, Darwiche N. The high-risk benign tumor: evidence from the two-stage skin cancer model and relevance for human cancer. Mol Carcinog. 2007;46:605–610. [PubMed]
  • Griner EM, Kazanietz MG. Protein kinase C and other diacylglycerol effectors in cancer. Nat Rev Cancer. 2007;7:281–294. [PubMed]
  • Hu Y, Green N, Gavrin LK, Janz K, Kaila N, Li HQ, et al. Inhibition of Tpl2 kinase and TNFalpha production with quinoline-3-carbonitriles for the treatment of rheumatoid arthritis. Bioorg Med Chem Lett. 2006;16:6067–6072. [PubMed]
  • Jansen AP, Verwiebe EG, Dreckschmidt NE, Wheeler DL, Oberley TD, Verma AK. Protein kinase C-epsilon transgenic mice: a unique model for metastatic squamous cell carcinoma. Cancer Res. 2001;61:808–812. [PubMed]
  • Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer Statistics 2008. CA Cancer J Clin. 2009;59:225–249. [PubMed]
  • Kane LP, Mollenauer MN, Xu Z, Turck CW, Weiss A. Akt-dependent phosphorylation specifically regulates Cot induction of NF-kappa B-dependent transcription. Mol Cell Biol. 2002;22:5962–5974. [PMC free article] [PubMed]
  • Karin M. Nuclear factor-kappaB in cancer development and progression. Nature. 2006;441:431–436. [PubMed]
  • Kimura YN, Watari K, Fotovati A, Hosoi F, Yasumoto K, Izumi H, et al. Inflammatory stimuli from macrophages and cancer cells synergistically promote tumor growth and angiogenesis. Canc Sci. 2007;98:2009–2018. [PubMed]
  • Lichti U, Anders J, Yuspa SH. Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat Protoc. 2008;3:799–810. [PubMed]
  • Moore RJ, Owens DM, Stamp G, Arnott C, Burke F, East N, et al. Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis. Nat Med. 1999;5:828–831. [PubMed]
  • Mueller MM. Inflammation in epithelial skin tumours: old stories and new ideas. Eur J Cancer. 2006;42:735–744. [PubMed]
  • Patriotis C, Makris A, Bear SE, Tsichlis PN. Tumor progression locus 2 (Tpl-2) encodes a protein kinase involved in the progression of rodent T-cell lymphomas and in T-cell activation. Proc Natl Acad Sci USA. 1993;90:2251–2255. [PubMed]
  • Patriotis C, Makris A, Chernoff J, Tsichlis PN. Tpl-2 acts in concert with Ras and Raf-1 to activate mitogen-activated protein kinase. Proc Natl Acad Sci USA. 1994;91:9755–9759. [PubMed]
  • Quintanilla M, Brown K, Ramsden M, Balmain A. Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature. 1986;322:78–80. [PubMed]
  • Reddig PJ, Dreckschmidt NE, Ahrens H, Simsiman R, Tseng CP, Zou J, et al. Transgenic mice overexpressing protein kinase C delta in the epidermis are resistant to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 1999;59:5710–5718. [PubMed]
  • Rijzewijk JJ, van Erp PE, Bauer FW. Two binding sites for Ki67 related to quiescent and cycling cells in human epidermis. Acta Derm Venereol. 1989;69:512–515. [PubMed]
  • Salmeron A, Ahmad TB, Carlile GW, Pappin D, Narsimhan RP, Ley SC. Activation of MEK-1 and SEK-1 by Tpl-2 protooncoprotein, a novel MAP kinase kinase kinase. EMBO J. 1996;15:817–826. [PubMed]
  • Sharma SD, Meeran SM, Katiyar N, Tisdale GB, Yusuf N, Xu H, et al. IL-12 deficiency suppresses 12-O-tetradecanoylphorbol-13- acetate-induced skin tumor development in 7,12-dimethylbenz(a)anthracene-initiated mouse skin through inhibition of inflammation. Carcinogenesis. 2009;30:1970–1977. [PMC free article] [PubMed]
  • Sourvinos G, Tsatsanis C, Spandidos DA. Overexpression of the Tpl-2/Cot oncogene in human breast cancer. Oncogene. 1999;18:4968–4973. [PubMed]
  • Sugimoto K, Ohata M, Miyoshi J, Ishizaki H, Tsuboi N, Masuda A, et al. A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA-induced IL-12 production and Th cell differentiation. J Clin Invest. 2004;114:857–866. [PMC free article] [PubMed]
  • Sur I, Ulvmar M, Toftgaård R. The two-faced NF-kappaB in the skin. Int Rev Immunol. 2008;27:205–223. [PubMed]
  • Tahara H, Zeh HJ, III, Storkus WJ, Pappo I, Watkins SC, Gubler U, et al. Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo. Cancer Res. 1994;54:182–189. [PubMed]
  • Tomczak MF, Gadjeva M, Wang YY, Brown K, Maroulakou I, Tsichlis PN, et al. Defective activation of ERK in macrophages lacking the p50/p105 subunit of NF-kappaB is responsible for elevated expression of IL-12 p40 observed after challenge with Helicobacter hepaticus. J Immunol. 2006;176:1244–1251. [PubMed]
  • Tsatsanis C, Patriotis C, Tsichlis PN. Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-kappaB. Oncogene. 1998;17:2609–2618. [PubMed]
  • Tsatsanis C, Spandidos DA. The role of oncogenic kinases in human cancer. Int J Mol Med. 2000;5:583–590. [PubMed]
  • Tsatsanis C, Vaporidi K, Zacharioudaki V, Androulidaki A, Sykulev Y, Margioris AN, et al. Tpl2 and ERK transduce antiproliferative T cell receptor signals and inhibit transformation of chronically stimulated T cells. Proc Natl Acad Sci USA. 2008;105:2987–2992. [PubMed]
  • Van Acker GJ, Perides G, Weiss ER, Das S, Tsichlis PN, Steer ML. Tumor progression locus-2 is a critical regulator of pancreatic and lung inflammation during acute pancreatitis. J Biol Chem. 2007;282:22140–22149. [PubMed]
  • Velasco-Sampayo A, Alemany S. p27kip protein levels and E2F activity are targets of Cot kinase during G1 phase progression in T cells. J Immunol. 2001;166:6084–6090. [PubMed]
  • Watford WT, Hissong BD, Durant LR, Yamane H, Muul LM, Kanno Y, et al. Tpl2 kinase regulates T cell interferon-gamma production and host resistance to Toxoplasma gondii. J Exp Med. 2008;205:2803–2812. [PMC free article] [PubMed]
  • Yuspa SH. The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis. J Dermatol Sci. 1998;17:1–7. [PubMed]