ERK5 Is a Critical Mediator of Inflammation-Driven Cancer

The idea that the immune system influences tumorigenesis was largely overlooked until epidemiologic studies identified chronic infection and inflammation as major risk factors for developing various types of cancers (1). Furthermore, in cancers where there is no evidence for an underlying inflammatory reaction before tumor formation, malignant cells, unlike their normal counterparts, can attract leukocytes (1). As a result, immune cells create a microenvironment that supports not only the de novo development of tumors by promoting the survival and sustained proliferation of cancer cells, but also many aspects of cancer progression, including malignant transformation and angiogenesis (2). In addition, the resistance of tumor cells to chemotherapeutic agents appears to be enhanced by macrophages (3). Consequently, cancer-related inflammation (CRI) is now recognized as one hallmark of cancer (4).

Central to the inflammatory condition associated with tumorigenesis is a complex network of chemokines and cytokines produced by both malignant and myeloid cells in the microenvironment (2). Chemokines and cytokines mediate their effect via the activation of oncogenic transcription factors that have been found constitutively activated in many types of cancers (5). For example, through the activation of nuclear factor κB (NFκB), interleukin (IL) 1α signaling via the IL1 receptor–MyD88 complex establishes a positive feedback loop to reinforce the expression of protumorigenic factors in Ras-expressing keratinocytes (6). Collectively, these observations have advanced our molecular understanding of the protumorigenic function of chemokines and cytokines in vivo. In contrast, very little is known about the pathogenic signaling mechanisms that initiate the inflammatory reaction.

Mitogen-activated protein kinases (MAPK) are components of core signaling pathways essential for controlling a plethora of cellular processes. In particular, the extracellular-regulated protein kinase 5 (ERK5) is a nonredundant member of this family, which has been implicated in vascular integrity during development and in angiogenesis associated with the formation of tumor xenografts (7). Furthermore, a novel potent and specific ATP-competitive inhibitor of ERK5, XMD8-92, was recently shown to suppress tumor growth through reduced cell proliferation in mice bearing xenografts (8). These findings are highly relevant to human cancer considering that elevated expression of MAPK/ERK kinase 5 (MEK5) and ERK5 in human epithelial tumors correlates with unfavorable prognosis, that includes shorter disease-free intervals, increased risk of metastasis, and resistance to chemotherapy (9). However, the interpretation of results obtained from xenograft models can be limited due to immune deficiency of the host. Consequently, a causal relationship between ERK5 and tumorigenesis remained to be rigorously established.

Here, we have developed compound mutant mice with a defect in erk5 gene expression in epidermal keratinocytes. Using the two-stage chemical carcinogenesis protocol to recapitulate in situ the stepwise formation of tumors in the presence of an intact immune system, we have discovered that ERK5 is an essential component of the inflammatory response of the skin associated with tumor formation and maintenance. Accordingly, we demonstrate that anti-ERK5 therapy combined with anti-mitotics more effectively regressed tumors than each treatment alone.

Mice carrying the erk5^fl allele and the K14-CreER^T2 transgene were identified by PCR on genomic DNA isolated from tail and tissues, as previously described (10, 11). The mouse strains were maintained in a pathogen-free facility at the University of Manchester. All animal procedures were performed under license in accordance with the UK Home Office Animals (Scientific Procedures) Act (1986) and institutional guidelines. In particular, careful clinical examination of the mice was carried out to allow detection of deterioration of their physical condition associated with the progression of tumors. Animals showing signs of distress were sacrificed before any further deterioration in condition occurred.

Mice were backcrossed into an FVB background. Six-week old littermates were subjected to intraperitoneal injection of a nontoxic amount of tamoxifen (200 μg) every day for 5 days. Two weeks later, the shaved backs of the mice received a single application of 7,12-dimethyl-benzanthracene (DMBA; 25 μg). One week after DMBA, the back skin was treated biweekly with 12-O-tetradecanoylphorbol 13-acetate (TPA; 6 μg) for 20 weeks. In Fig. 1E, mice were exposed weekly to a single dose of DMBA (30 μg) for 29 weeks. Tumor burden was recorded (blind counts and photographs) every week for the duration of the experiment. For the tumor regression studies, mice were subjected to the DMBA/TPA protocol for 10 to 16 weeks before intraperitoneal injection of tamoxifen (200 μg; every day for 5 days) followed a week later by three topical applications of 4-hydroxy-tamoxifen (4-OHT; 100 μg) on alternating days. Alternatively, XMD8-92 (CRUK Manchester Institute, Manchester, United Kingdom; 25 μmol/L) was administered topically every other day (three times, weekly) until the end of the experiments. Age-matched mock-treated (acetone) animals were used as controls. Doxorubicin (1.25 mg/kg per mouse) was administered intravenously weekly for 4 consecutive weeks, 1 week after the end of 4-OHT dosing or 1 week after the beginning of XMD8-92 therapy. Biweekly TPA treatment was continued for the duration of all regression experiments. When this was combined with XMD8-92, XMD8-92 was applied 30 minutes before TPA. The number of tumors larger than 3 mm in size was recorded (blind counts) every week for the duration of the experiment.

Skin biopsies were removed and floated dermis-down in 0.25% trypsin-EDTA overnight at 4°C. Epidermises were then separated from the dermis and analyzed as required.

HaCat cells were cultured in DMEM containing 10% FBS, 1% penicillin/streptomycin, and 1% glutamine. Where indicated, the cells were pretreated for 30 minutes with 25 μmol/L XMD8-92 and/or 10 μmol/L caspase-1 inhibitor (Ac-YVAD-CHO; Enzo Life Sciences).

Proteins were extracted from cells in RIPA buffer containing inhibitors of proteases and protein phosphatases. Extracts (20 μg) were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to an Immobilon-P membrane (Millipore, Inc.). The membranes were incubated with 3% nonfat dry milk or BSA for 60 minutes and probed overnight at 4°C with antibodies (1:1,000) to ERK5 (Upstate), human IL1β (R&D Systems), mouse IL1β (R&D Systems) or cleaved caspase 1 (Cell Signaling Technology). Tubulin (Sigma) or actin (Sigma) were used to monitor protein loading. Immunecomplexes were detected by enhanced chemiluminescence with immunoglobulin G coupled to horseradish peroxidase as the secondary antibody (GE Healthcare).

Endogenous ERK5 was immunoprecipitated from cellular or epidermal extracts in the triton lysis buffer (TLB; 20 mmol/L Tris pH 7.4, 137 mmol/L NaCl, 2 mmol/L EDTA, 1% Triton X-100, 25 mmol/L β glycerophosphate, 10% glycerol, 1 mmol/L orthovanadate, 1 mmol/L phenylsulphonyl fluoride, 10 μg/mL leupeptin, and 10 μg/mL aprotinin) using an antibody to ERK5 (Upstate). Immunecomplexes were washed twice with TLB and twice with kinase buffer (25 mmol/L HEPES pH 7.4, 25 mmol/L β-glycerophosphate, 25 mmol/L MgCl₂, 2 mmol/L DTT, and 0.1 mmol/L orthovanadate) before being incubated at 30°C for 20 minutes in kinase buffer containing 50 μmol/L [γ-³²P] ATP (10 Ci/mmol) and 1 μg of GST-MEF2C. The reactions were terminated by addition of Laemmli sample buffer. Radioactivity incorporated into the recombinant protein was quantified after SDS-PAGE by PhosphoImager analysis.

Release of mature IL1β into culture medium was measured using a specific sandwich-type enzyme-linked immunosorbent assay (ELISA; Ready Set Go; Human Interleukin-1 β kit; eBioscience) according to the manufacturer's instructions.

Cellular or epidermal extracts were prepared in caspase buffer (10 mmol/L HEPES pH 7.5, 150 mmol/L NaCl, 2 mmol/L EDTA containing 0.5% NP40). Extracts were incubated with 100 μmol/L YVAD-AMC caspase-1–specific fluorogenic substrate (Enzo Life Sciences) for 1 hour. Cleavage of the substrate was measured by spectrofluorometer.

Freshly isolated skin biopsies and tumors were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Five-micrometer thick sections were cut. For histologic analysis, sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, sections were deparaffinized, rehydrated, and treated in boiling sodium citrate buffer (10 mmol/L pH 6.0) for 10 minutes to unmask antigens. Endogenous peroxidase activity was quenched by treating the slides with 0.3% hydrogen peroxide for 10 minutes. Sections were blocked in PBS containing 3% to 10% goat serum and 0.1% Triton X-100 for 1 hour at room temperature before being incubated overnight at 4°C with primary antibodies (1:100) to ERK5 (Eurogentec), IL1β (R&D Systems) or MPO (Thermo Scientific). The following day, the slides were rinsed in PBS and incubated at room temperature for 1 hour with secondary biotinylated antibodies. The slides were processed using the ABC Detection Kit (Vector Laboratories). The presence of the antigens was revealed using the Vector SG (gray) or diaminobenzidine (DAB; brown) peroxidase substrate kit (Vector Laboratories) and counterstained with nuclear red or with hematoxylin (blue). For bromodeoxyuridine (BrdUrd) staining, the mice were injected with 500 mg/kg BrdUrd and sacrificed 1 hour or 2 hours later. Immunohistochemistry was performed using a mouse monoclonal antibody to BrdUrd (Dako). For mast cell staining, sections were incubated for 5 minutes in toluidine blue stain [0.1% toluidine blue O (Sigma), 1% NaCl, pH 2.5]. All slides were viewed using the Axioplan2 microscope.

Total RNA was isolated from cells or epidermises using the TRIzol reagent and cDNA synthesis carried out, as previously described (12). Quantitative real-time PCRs were performed using the SYBR Green I Core Kit (Eurogentec). Sequences of the forward and reverse primers are indicated in Supplementary Table S1. PCR products were detected in the ABI-PRISM 7700 sequence detection systems (Applied Biosystems). Results were analyzed using the 2^−ΔΔG method. The level of expression of mRNA was normalized to actin mRNA in mouse tissues or GAPDH and Pgk1 mRNA in HaCat cells.

Statistical significance was determined using the two-tailed Student t test or ANOVA (one- or two-way) depending on the type of data. *, P < 0.05 was taken to be statistically significant. ns indicates no statistical difference. For all mouse tissue experiments, images are representative of cohorts of at least three mice.

Mice lacking erk5 die before birth (13). To circumvent this early lethality and permit the study of the pathologic function of ERK5, we developed a mouse model in which the erk5 gene was flanked with LoxP sites [referred to as the flox (fl) allele; 10]. Homozygous erk5^fl mice expressing Cre fused to a mutated form of the ligand-binding domain of the estrogen receptor (CreER^T2) under the control of the keratin 14 (K14) promoter (14), were generated. Immunoblot analysis demonstrates that this system allows the specific ablation of ERK5 in the epidermis following injection of the mice with tamoxifen (Fig. 1A and B). Immunostaining of skin sections with an antibody to ERK5 confirmed the absence of ERK5 in the epidermis of K14-CreER^T2;erk5^fl/fl mice treated with tamoxifen (Fig. 1C). We verified that a control IgG antibody produced no staining under the same experimental conditions (data not shown). The loss of ERK5 in adult mice did not cause any obvious morphologic defect, suggesting that ERK5 was not required for skin homeostasis (Fig. 1C). In subsequent experiments, erk5^fl/fl and K14-CreER^T2;erk5^fl/fl animals injected with tamoxifen will be referred to as erk5^wt and erk5^Δepidermis animals, respectively.

To examine the role of ERK5 in tumorigenesis, mice were subjected to the classic two-stage chemical carcinogenesis protocol, which gives rise to skin papillomas (15). This simple and highly reproducible system is particularly suited to explore the mechanism of epithelial cancer-associated inflammation because the repeated treatment of the mouse skin with TPA recapitulates a persistent and nonresolving inflammation that promotes the clonal expansion of keratinocytes harboring oncogenic Ha-ras mutation (caused by a single, cutaneous application of the genotoxic carcinogen DMBA; ref. 16). Importantly, resolution of inflammation via cessation of TPA treatment causes significant inhibition of tumor growth, as well as frequent spontaneous tumor regression (16). We discovered that tumor burden (number of tumors per mouse) was markedly decreased in the absence of ERK5 (Fig. 1D). In fact, papillomas that developed on the back skin of erk5^Δepidermis animals were genotypically wild-type. This was predicted because deletion of floxed genes following tamoxifen-induced Cre expression does not occur with 100% efficiency.

In addition, we observed that the malignant conversion of papillomas, which occurred at a frequency ≤5% to 10%, was restricted to erk5^wt mice. However, it is possible that malignant tumors in the erk5^Δepidermis group may have remained undetected, simply because of the low overall total tumor number. Therefore, to assess the role of ERK5 in malignancy, we compared the effect of the repeated treatment of erk5^wt and erk5^Δepidermis animals with DMBA alone (Fig. 1E). This complete carcinogenesis protocol leads directly to the formation of predominantly invasive squamous cell carcinomas (SCC; ref. 17). As with the DMBA/TPA model (Fig. 1D), the absence of ERK5 caused a marked decrease in tumor burden upon exposure to DMBA (Fig. 1E). SCC formation was significantly delayed in erk5-deleted skin, with the majority of tumors detected in erk5^Δepidermis mice significantly smaller than in wild-type littermates (Fig. 1E). Together, these results clearly demonstrate that ERK5 is required for the development of malignant skin cancer.

To test whether the resistance of ERK5-deficient mice to tumorigenesis could be attributed to a cell proliferation defect, we performed a short time–course analysis of TPA exposure. Epidermal cell proliferation was maximally induced 24 hours after a single application of TPA on the dorsal skin of the wild-type mice, as demonstrated by a 10-fold increase in the number of BrdUrd-positive cells (Fig. 2A and B). This was significantly impaired in the absence of ERK5 (Fig. 2A and B). However, 6 hours after TPA exposure, we did not observe any marked difference in the number of BrdUrd-positive cells between wild-type and ERK5-deficient epidermises (Fig. 2B). Instead, by this time, the mutant skin displayed a noticeably lower number of infiltrating cells in the dermis compared with wild-type controls (Fig. 2C). This indicated that the proliferative defect displayed by erk5-deleted keratinocytes was preceded by a failure of the mutant epidermis to respond to inflammatory stimulation.

To confirm the requirement of ERK5 in mediating the inflammatory response of the skin to TPA, we examined the effect of epidermal erk5 gene deletion on neutrophil infiltration into the dermis. Neutrophils rapidly migrate to the skin exposed to TPA and impaired neutrophil recruitment is sufficient to prevent TPA-induced cutaneous inflammation, hyperproliferation, and papilloma formation (18, 19). Accordingly, the number of neutrophils (MPO-positive cells) in skin sections of erk5^wt mice increased 10-fold 6 hours after TPA treatment and 16-fold after 24 hours (Fig. 2D). This effect was significantly reduced in the absence of ERK5, with only a 4- to 5-fold increase in neutrophil number detected in erk5^Δepidermis animals exposed to TPA for 6 or 24 hours (Fig. 2D). A similar defect was observed in skin sections of mice treated with DMBA/TPA for 20 weeks (25-fold increase in neutrophil number in wild-type skin, 5- to 6-fold increase in mutant skin).

Two major neutrophil chemoattractants produced by keratinocytes, namely chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL2, can trigger the recruitment of neutrophils in the mouse skin (20). We confirmed that TPA induced a transient upregulation of CXCL1 and CXCL2 transcripts in the epidermis, with a maximum after 6 hours (Fig. 2E). This effect was abolished in the absence of ERK5, consistent with decreased number of neutrophils trafficking through the dermis of the mutant skin (Fig. 2D and E). Furthermore, erk5^Δepidermis mice were resistant to cutaneous chronic inflammation caused by repeated long-term TPA exposure (Fig. 2F). The establishment of chronic inflammation in the wild-type skin subjected to the DMBA/TPA protocol for 10 and 20 weeks is demonstrated by the accumulation of resident mast cells in the dermis before papilloma formation, and in the tumor stroma (Fig. 2F). Together, our results are consistent with evidence that neutrophil recruitment is a prerequisite to the establishment of a chronic inflammatory microenvironment and that mice deficient in mast cells are resistant to skin carcinogenesis (21).

To further characterize the requirement of ERK5 in CRI, we analyzed the expression pattern of a panel of inflammatory mediators implicated in tumorigenesis. We found that ERK5 was required for TPA-induced IL1α and IL1β mRNA expression and for the upregulation of cyclooxygenase-2 (COX-2), a downstream effector of IL1 (22) (Fig. 3A). Prostaglandin E2 (PGE2) is the predominant COX-2–derived product in the skin. It acts by binding to its cognate G-protein–coupled receptors, namely EP1-4. Although all EP receptors, except EP3, participate in skin tumor development, genetic analyses have suggested an essential role of EP2 in tumorigenesis (23). Here, we confirmed that TPA increased the expression of EP1 and EP2, but not EP3, mRNA in the epidermis (Fig. 3A and Supplementary Fig. S1). Consistent with its very low level of expression in the skin (24), the EP4 transcript was undetectable (data not shown). More importantly, TPA-induced EP2 expression was abolished in the absence of ERK5 (Fig. 3A), whereas no marked difference was detected in the level of EP1 between wild-type and erk5-deleted epidermises, under basal or stimulated conditions (Supplementary Fig. S1). Likewise, TPA-mediated increased TNFα and IL6 mRNA expression was independent of ERK5 (Fig. 3A), thereby demonstrating the selective requirement of ERK5 signaling in controlling a specific subset of inflammatory mediators.

These results establish ERK5 as a novel regulator of cutaneous inflammation via the upregulation of neutrophil chemoattractants and of the IL1–COX-2 pathway. CXCL-mediated dermal recruitment of neutrophils and COX-2–generated PGE2 signaling through the EP2 receptor constitute potentially important mechanisms by which ERK5 contributes to skin tumorigenesis.

IL1α and IL1β are synthesized as pro-forms. While IL1α is active as a membrane precursor, caspase-1 mediated cleavage of pro-IL1β is required for IL1β to be active as a secreted form (25). Once cleaved, IL1β is quickly released into the microenvironment (26). We found that the intracellular level of cleaved IL1β was significantly lower in the mutant epidermis compared with wild-type samples (Fig. 3B). Consistent with the requirement of ERK5 for mediating the proteolytic cleavage of pro-IL1β, erk5-deleted epidermises were resistant to TPA-induced caspase-1 activation (Fig. 3C). Elevated caspase-1 activity in the wild-type skin exposed to TPA is indicative of rapid turn-over of the pro-form and subsequently, secretion of the cleaved fragment. This dynamic process explains why no difference was detected in the level of IL1β protein expressed in the wild-type epidermis between basal conditions and following exposure to TPA (Fig. 3B), despite our previous evidence that TPA increased the expression of the transcript (Fig. 3A).

The secreted form of IL1, mostly IL1β, contributes to the recruitment of leukocytes (mainly neutrophils and macrophages) but also, acts on stromal and infiltrating cells to induce the production of inflammatory cytokines, including IL1β itself, to sustain and propagate the inflammatory reaction (27). Accordingly, a transient increase in the number of IL1β-positive cells was detected in the dermis of the skin exposed to TPA (Fig. 3D). Chronic cutaneous inflammation caused by long-term DMBA/TPA treatment correlated with the presence of a population of IL1β-positive cells permanently residing in the dermis (Fig. 3D). In contrast, and consistent with impaired IL1β secretion, no infiltrating IL1β-positive cells were detected in the dermis of the mutant skin exposed to short-term TPA treatment or subjected to the two-stage chemical carcinogenesis protocol for 20 weeks (Fig. 3D). On the basis of these results, we concluded that ERK5-dependent release of IL1β in the epidermis was required for promoting a secondary inflammatory reaction in the dermis.

To confirm that increased IL1β expression in the epidermis was a primary response to ERK5 activation in keratinocytes exposed to TPA, we undertook an analysis of ERK5 function in HaCat cells, a human immortalized keratinocyte cell line, to reproduce in vitro the immune response defect associated with the epidermal loss of ERK5 in vivo. We demonstrated that TPA stimulation induced phosphorylation of ERK5, as indicated by an electrophoretic mobility shift of the protein analyzed by immunoblot, and increased ERK5 activity (Fig. 4A). This was prevented by preincubating the cells with the pharmacologic inhibitor of ERK5, XMD8-92 (Fig. 4A). Using this model system, we confirmed the requirement of ERK5 in mediating TPA-induced upregulation of IL8 (the functional homolog of murine CXCL1/2), and IL1β, but not TNFα, mRNA (Fig. 4B). Next, HaCat cells were incubated with a caspase-1 inhibitor (YVAD-CHO) to prevent pro-IL1β from being cleaved and secreted (Fig. 4C). Under these conditions, we could detect TPA-mediated increased expression of pro-IL1β protein (Fig. 4C). This was prevented by preincubating the cells with XMD8-92 (Fig. 4C). Likewise, inhibition of ERK5 significantly reduced the amount of IL1β released in the media from cells treated with TPA (Fig. 4D). Accordingly, ERK5 activation was required for both TPA-induced increased caspase-1 activity (Fig. 4E) and the proteolytic cleavage of caspase-1 to its active form (p20; Fig. 4F). Together, these results firmly established that ERK5 was essential for IL1β production by keratinocytes and revealed a novel role for ERK5 in the regulation of IL1β signaling and IL8 expression in human cells.

To investigate whether our findings could be translated into a useful approach for cancer treatment, we examined the phenotypic consequence of the epidermal loss of ERK5 expression in skin tumors. We found that, under low tumor burden, the loss of ERK5 in neoplastic keratinocytes halted tumor growth induced by biweekly exposure to TPA (Fig. 5A). This was associated with a reduction in the number of proliferating cells labeled with BrdUrd, together with a collapse of tumor architecture (Fig. 5B). To determine whether an anti-ERK5 therapy could also block the growth of more advanced tumors, a similar experiment was performed in cohorts of animals exhibiting a high tumor burden before disrupting ERK5 function by either erk5 gene deletion or XMD8-92 treatment (Fig. 5C and D). Firstly, we demonstrated that XMD8-92 effectively blocked TPA-induced ERK5 activation in the skin (Supplementary Fig. S2A). Moreover, like genetic deletion, pharmacologic inhibition of ERK5 activity caused by exposing the skin to XMD8-92 prevented increased epidermal cell proliferation (Supplementary Fig. S2B), cells from infiltrating the dermis (Supplementary Fig. S2C), and increased caspase-1–mediated IL1β cleavage (Supplementary Fig. S2D and S2E) in response to TPA stimulation.

As expected, the number of papillomas larger than 3 mm in size increased over the time course of TPA treatment. Although this was not prevented by the genetic loss of erk5, representative pictures show that tumors were noticeably smaller in erk5^Δepidermis than in erk5^wt mice (Fig. 5C). Similarly, exposure of the mouse skin to XMD8-92 affected tumor development with quantitative evidence of halted tumor growth (Fig. 5D). In contrast with the genetic strategy that permits the specific loss of ERK5 in keratinocytes, XMD8-92 will inhibit ERK5 activity in all cell types present in the epidermal and dermal compartments of the skin, including immune cells. This could explain the stronger effect of the inhibitor on tumor growth. However, we cannot exclude the possibility that the compound may exert some of its effect by partially inhibiting additional protein kinases involved in the inflammatory reaction.

Many effective anticancer regimens use more than one therapeutic agent. Therefore, we sought to explore the possibility that targeting ERK5 signaling could enhance the effectiveness of existing chemotherapeutic drugs. We used a subtherapeutic dosing regimen of chemotherapy (doxorubicin; 1.25 mg/kg weekly intravenous injection for 4 consecutive weeks) that exerted a limited effect on tumor growth (Fig. 6A and B), comparable with that caused by erk5 gene deletion in mice harboring a high tumor burden (Fig. 5C). Importantly, mice subjected to this low-dose doxorubicin did not display transient gastrointestinal side effects as exhibited by littermates receiving high-dose doxorubicin (2.5 mg/kg weekly intravenous injection for 4 consecutive weeks). We found that epidermal erk5 gene deletion or XMD8-92 therapy combined with doxorubicin had an additive effect in preventing tumor growth, with evidence of tumor regression indicated by a decreased number of large tumors (Fig. 6A and B). This is substantiated by the demonstration that the functional disruption of ERK5 signaling in mice concurrently treated with doxorubicin severely affected tumor architecture and markedly reduced the number of proliferating cells in the tumors (Fig. 6C).

On the basis of our previous results, we examined whether the effectiveness of anti-ERK5 therapy in regressing existing tumors was attributable to the requirement of ERK5 for sustained inflammation in the skin. This hypothesis was confirmed by evidence that the loss of ERK5 expression (Fig. 7A) or activity (Fig. 7B) was associated with a significant reduction in the number of neutrophils (MPO-positive cells) infiltrating the neoplastic epidermis (E) and the tumor stroma (S), independently of doxorubicin treatment. This correlated with a decreased number of mast cells (toluidine blue–stained cells) present in the tumor stroma (Fig. 7A and B). These results are consistent with evidence that persistent neutrophil and mast cells recruitment is required for tumor maintenance (16). Furthermore, tumors displayed strong and diffuse IL1β staining in the papilloma epithelium (E), indicative of IL1β secretion in the tumor (Fig. 7A and B). This was accompanied by an influx of IL1β-positive cells in the tumor stroma (S; Fig. 7A and B). A reduction in both, IL1β secretion and the number of IL1β-positive cells was observed following the functional disruption of ERK5 signaling, thereby demonstrating the requirement of ERK5 for continued IL1β production in established tumors (Fig. 7A and B). Importantly, none of these inflammatory markers were affected by doxorubicin treatment alone.

Together, these results indicate that neutralizing inflammation by blocking ERK5 in existing tumors represents a novel strategy by which to treat CRI. In addition, we demonstrate that combining anti-ERK5 therapy with chemotherapeutic agents augments the antitumorigenic effect of anti-ERK5 treatment while concurrently lowering the therapeutic threshold of these agents, consequently reducing the associated side effects.

We have discovered that epidermal expression of ERK5 is required for mediating inflammation in the skin. Furthermore, we have demonstrated that inactivation of ERK5 in epidermal keratinocytes prevented inflammation-driven tumorigenesis, with evidence that ERK5 is critical for the development of skin SCC. SCC is one of the most common types of human nonmelanoma skin cancer (28). The lesions arise from keratinocytes in sun-exposed areas of the epidermis and are characterized by an increased risk of metastasis compared with basal cell carcinomas. Therefore, these findings are highly relevant because elevated MEK5/ERK5 expression in human epithelial tumors correlates with unfavorable prognosis (9). In addition, inflammation is a hallmark of most, if not all, cancer (4). In particular, besides ultraviolet light, inflammatory skin diseases also predispose patients to developing cutaneous SCC (28). Accordingly, targeting CRI has become one of the best options by which to advance cancer treatment. This therapeutic strategy is already supported by evidence that tumor burden can be reduced in mice in which key mediators of the immune response are blocked by genetic or pharmacologic means (2). Importantly, several cytokine antagonists are being tested in phase II clinical trials with some encouraging results (2).

The idea that ERK5 provides a mechanistic link between inflammation and cancer originated from our observation that the migration of neutrophils and mast cells to the skin, after short and long-term exposure to TPA, was impaired in the absence of epidermal ERK5. Likewise, the functional inhibition of ERK5 in the neoplastic epidermis correlated with a significant reduction in the number of infiltrating neutrophils and mast cells in all tumor compartments. The recruitment of neutrophils in inflamed skin is regulated by chemokines, notably CXCL1 and CXCL2, which signal through the same receptor, CXCR2. Like erk5 gene deletion, CXCR2 deficiency was shown to suppress inflammation-driven tumorigenesis (18, 19) and it was postulated that CXCR2-driven neutrophil recruitment stimulates epithelial cell proliferation in the skin (19). Furthermore, depletion of Ly6G⁺ cells, which include neutrophils, was associated with increased tumor cell death (19). Therefore, it is reasonable to propose that the upregulation of CXCL1 and CXCL2 constitutes an important mechanism by which ERK5 triggers the neutrophilic inflammation required for tumor cell proliferation and viability.

The causal relationship between ERK5 and inflammation is further supported by the demonstration that epidermal ERK5 controls the expression of a specific subset of proinflammatory mediators currently under preclinical therapeutic investigation for the treatment of human cancer (2). In particular, the functional loss of ERK5 in keratinocytes prevented TPA-induced upregulation of IL1α, IL1β, and COX-2 transcripts. However, in contrast with malignant mesothelioma cell lines (29), we found no evidence that epidermal ERK5 was required for IL6 gene expression. This may reflect a cell-specific function of ERK5 at various stages of malignant transformation. Likewise, no difference was observed in the level of TNFα mRNA between wild-type and ERK5-deficient epidermis under basal or stimulated conditions. Mice lacking TNFα have been reported to be resistant to the development of skin tumors induced by DMBA/TPA (30, 31). However, TNF receptor deficiency does not prevent neutrophil infiltration in the epidermis exhibiting increased PKCα activity (32). Therefore, our findings are consistent with the idea that TNFα does not contribute to ERK5-mediated recruitment of neutrophils in the skin exposed to inflammatory stimuli.

Recently, autocrine activation of NFκB through an IL1 receptor–MyD88 axis has emerged as an essential positive feedback loop that reinforces the expression of protumorigenic factors in Ras-transformed keratinocytes (6). Importantly, targeted deletion of the MyD88 gene in keratinocytes conferred a significant resistance of the skin to DMBA/TPA-induced tumorigenesis, thereby confirming the important contribution of epidermal IL1 signaling in cancer (6). However, although transformation by oncogenic Ras induced both IL1α and IL1β mRNA, IL1β was undetectable in the culture supernatants (6). Therefore, we can speculate that increased caspase-1 activity, caused by elevated ERK5 signaling in keratinocytes, independently of oncogenic mutation, may be sufficient to trigger a nonresolving inflammatory condition that increases cancer risk. This raises the interesting possibility of potential prophylactic applications for anti-ERK5 therapy in cancer development. Furthermore, consistent with the requirement of ERK5 for caspase-1–mediated cleavage of pro-IL1β, tumor regression associated with the epidermal loss of ERK5 was accompanied with the depletion of secreted IL1β in the papilloma epithelium. It is well established that IL1β can act on stromal cells (fibroblasts and inflammatory cells) to induce the production of a cascade of inflammatory cytokines but also, adhesion molecules and angiogenic factors to increase tumor invasiveness and metastasis (27, 33). Therefore, impairing tumoral IL1β signaling via anti-ERK5 therapy may also yield therapeutic results in the prevention of metastatic progression.

Collectively, genetic evidence that the functional loss of ERK5 in keratinocytes causes, simultaneously, the downregulation of several inflammatory mediators overexpressed in various human cancers, suggest that an anti-ERK5 therapy may have the potential to be clinically more efficacious than therapies directed against individual inflammatory mediators. Furthermore, the pathway organized in a typical three-tiered kinase (MAPKKK, MAPKK, and MAPK) cascade offers multiple potential targets, highly suitable for drug design. In particular, an alternative therapeutic strategy targeting MEK5, instead of ERK5, may allow the specific inhibition of pathologic ERK5 activation with reduced undesirable side effects. Indeed, while the systemic administration of XMD8-92, a selective and potent ATP-competitive inhibitor of ERK5, had no apparent adverse effects in mice, the specific loss of ERK5 signaling in endothelial cells during embryogenesis or in adult mice resulted in lethality as a consequence of vascular instability (8, 34). A similar approach has been used for the ERK1/2 MAPK signaling pathway, where most current efforts target their upstream activators, namely MEK1/2, rather than inhibiting ERK1/2 directly. A clinical study involving patients with cancer has confirmed decreased ERK1/2 phosphorylation in tumors and several partial remissions when MEK1/2 were inhibited (35). Hence, the rationale for MAPK kinase blockade in humans is quite clear.

To conclude, we propose that ERK5 is part of a core signal transduction pathway that drives the expression of a specific subset of inflammatory mediators in epithelial cells that can act in an autocrine and paracrine manner to initiate an immune response. As such, anti-ERK5 therapy provides a new opportunity to neutralize inflammation associated with cancer.

No potential conflicts of interest were disclosed.

Conception and design: K.G. Finegan, C. Tournier

Development of methodology: K.G. Finegan, A.M. Jordan

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.G. Finegan, C.C. Davies

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.G. Finegan, D. Perez-Madrigal, C. Tournier

Writing, review, and/or revision of the manuscript: K.G. Finegan, A.M. Jordan, C. Tournier

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K.G. Finegan, D. Perez-Madrigal, C. Tournier

Study supervision: K.G. Finegan, C. Tournier

Other (synthesis of compounds used in the study): J.R. Hitchin

Other (provision of chemical matter used to obtain the key results): A.M. Jordan

The authors thank Peter March (Bioimaging Facility, University of Manchester) for very helpful advice with the microscopy and the staff at the University of Manchester Biological Services Facility for looking after the mice.

This work was supported by grants mainly from Worldwide Cancer Research (10-0134) and partly from Cancer Research UK (C18267/A11727) and the Wellcome Trust (097820/Z/11/B).

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.