Pro-Oncogenic Role of Alternative P38 Mitogen-Activated Protein Kinases P38g and P38d, Linking Inﬂammation and Cancer in Colitis-Associated Colon Cancer

Published OnlineFirst September 12, 2014; DOI: 10.1158/0008-5472.CAN-14-0870

Cancer Molecular and Cellular Pathobiology Research

Pro-Oncogenic Role of Alternative p38 Mitogen-Activated Protein Kinases p38g and p38d, Linking Inﬂammation and Cancer in Colitis-Associated Colon Cancer

Paloma del Reino1, Dayanira Alsina-Beauchamp1, Alejandra Escos 1,Ma Isabel Cerezo-Guisado1, Ana Risco1, Noelia Aparicio1, Rafal Zur1, Marian Fernandez-Estevez 1, Elena Collantes2, Jose Montans3, and Ana Cuenda1

Abstract p38 MAPK signaling has been implicated in the regulation of processes leading to cancer development and progression. Chronic inflammation is a known risk factor for tumorigenesis, yet the precise mechanism of this association remains largely unknown. The related p38aMAPK (MAPK14) proteins p38g (MAPK12) and p38d (MAPK13) were recently shown to modulate the immune response, although their role in tumorigenesis remains controversial and their function in inflammation-associated cancer has not been studied. We analyzed the role of p38g and p38d in colon cancer associated to colitis using the azoxymethane/dextran sodium sulphate (AOM/DSS) colitis-associated colon cancer model in wild-type (WT), p38g-, p38d-, and p38g/ d-deficient (p38g/d / ) mice. We found that p38g/d deficiency significantly decreased tumor formation, in parallel with a decrease in proinflammatory cytokine and chemokine production. Analysis of leukocyte populations in p38g/d / mouse colon showed less macrophage and neutrophil recruitment than in WT mice. Furthermore, WT chimeric mice with transplanted p38g/d / bone marrow had less tumors than WT mice transplanted with WT bone marrow, whereas tumor number was significantly increased in p38g/d / chimeric mice with WT bone marrow compared with p38g/d / mice transplanted with p38g/d / bone marrow. Together, our results establish that p38g and p38d are central to colitis-associated colon cancer formation through regulation of hematopoietic cell response to injury, and validate p38g and p38d as potential targets for cancer therapy. Cancer Res; 74(21); 1–11. 2014 AACR.

Introduction that chronic inflammation creates a favorable environment for Cancer and chronic inflammation are intimately associated CAC initiation and for tumor growth promotion and progres- (1), for example, in inflammatory bowel diseases (IBD) such sion (3). Noxious compounds released during chronic colon fl as ulcerative colitis and Crohn disease, which confer in ammation are, thus, proposed to damage DNA and/or alter an increased risk for colorectal cancer, one of the most cell proliferation or survival, thereby promoting oncogenesis fi common fatal malignancies worldwide (2). Patients with (3, 4). Immune cells, which often in ltrate tumors and pre- long-standing ulcerative colitis are particularly prone to coli- neoplastic lesions, produce a variety of cytokines and chemo- fl tis-associated colon cancer (CAC), the major cause of death in kines that propagate a localized in ammatory response; they these patients (2). The mechanisms that link these chronic also enhance premalignant cell growth and survival by acti- inflammatory states to colorectal cancer development are vating signaling pathways such as NF-kB or MAPKs (3, 5). fl nonetheless largely unknown. Experimental evidence suggests Within this group, the p38MAPKs are central to in ammatory processes and to the production of proinflammatory molecules that contribute to CAC pathogenesis (6). The p38MAPK group has four members, p38a (MAPK14), 1Department of Immunology and Oncology, Centro Nacional de Biotec- nología/CSIC, Madrid, Spain. 2Servicio de Anatomía Patologica, Hospital p38b (MAPK11), p38g (MAPK12), and p38d (MAPK13), which Universitario La Paz, Madrid, Spain. 3Centro Anatomopatologico, Camino share very similar protein sequences; they are activated by de Vinateros, Madrid, Spain. dual phosphorylation mediated primarily by the MAPK kinases Note: Supplementary data for this article are available at Cancer Research (MKK)3 and MKK6 in response to a range of cell stresses and to Online (http://cancerres.aacrjournals.org/). inflammatory cytokines (6, 7). The most abundant, best-char- P. del Reino and D. Alsina-Beauchamp contributed equally to this article. acterized isoform is p38a, also the p38MAPK most frequently fi Corresponding Author: Ana Cuenda, Centro Nacional de Biotecnología/ referred to in the literature. Studies using tissue-speci c con- CSIC, Madrid 28049, Spain. Phone: 34-91-585-5451; Fax: 34-91-372- ditional knockouts of p38a mice showed the importance of 0493; E-mail: [email protected] this isoform in the inflammatory response, both in cultured doi: 10.1158/0008-5472.CAN-14-0870 macrophages in vitro (8) and for development of skin and 2014 American Association for Cancer Research. gut inflammation in vivo (9, 10). p38a also mediates

www.aacrjournals.org OF1

Reino et al.

inflammation in IBD; it is highly phosphorylated and active al and EU guidelines, with the approval of the Centro Nacional in the inflamed intestinal mucosa of patients with IBD de Biotecnología Animal Ethics Committee. (11, 12). The role of p38a in colitis was confirmed in exper- Colorectal cancer associated with colitis was induced as imental mouse models, and p38a/bMAPK inhibitors have been described previously (27). Briefly, 10- to 14-week-old mice were tested in clinical trials (10, 13–16). The precise contribution injected i.p. with AOM (10 mg/kg; Sigma-Aldrich). On day 5 of other p38MAPK isoforms in colitis and in CAC has none- after injection, mice were treated with 2% DSS (MW 36,000- theless not been yet characterized. 50,000 Da; MP Biomedicals) in drinking water for 5 consecutive Studies in mice deficient in p38g, p38d, or both showed that days, followed by 16 days administration of normal drinking these kinases are essential for the innate immune response water. This DSS treatment was repeated for two additional (17–20). Combined deletion of p38g and p38d (p38g/d) impairs cycles. During the course of the experiment, mice were mon- proinflammatory cytokine production in macrophages and itored for body weight, diarrhea, and macroscopic bleeding. dendritic cells (DC) in response to the bacterial lipopolysac- On days 63 or 105 of the regime, mice were killed, the colon charide (LPS) by controlling steady-state levels of TPL2 (tumor removed, washed with PBS, and opened longitudinally for progression locus 2), the MKK kinase that mediates ERK1/2 analysis. pathway activation in these cells; ERK1/2 are MAPK with a In colitis experiments, mice were treated for 5 days with central role in cytokine production (18, 21). p38g/d / mice are 3% DSS (w/v) in drinking water and then allowed to recover less sensitive than controls to LPS-induced septic shock and, in for additional days as indicated (see text and figures). Mice a mouse collagen–induced arthritis model, p38g/d deficiency were monitored for signs of distress and rectal bleeding. greatly reduced symptom severity and joint damage compared with wild-type (WT) mice (18, 20). In both experimental Histologic analysis and scoring models, p38g/d / mice produced lower levels of cytokines Histologic analysis was performed on hematoxylin and such as IL1b and TNFa (18, 20). eosin (H&E)–stained colon sections. Inflammation was In addition to their critical role in inflammation, p38g and scored by the following criteria (28): 0, no evidence of p38d have tumorigenic functions in certain contexts (22). inflammation; 1, low level of inflammation with scattered p38d / mice show reduced susceptibility to skin carcinogen- infiltrating mononuclear cells (1–2 foci only); 2, moderate esis, suggesting a protumorigenic role for p38d (23). p38g inflammation with multiple foci; 3, high level of inflammation regulates oncogenic proteins involved in colorectal cancer withincreasedvasculardensityandmarkedwallthickening; development, such as K-Ras, which is mutated in 50% of colon and 4, maximal severity of inflammation with transmural cancers (24). Nonetheless, studies in immortalized mouse leukocyte infiltration and loss of goblet cells. The ulceration embryonic fibroblasts and in K-Ras–transformed cells lacking score was graded as 0, absence of ulcers; 1, few scattered p38g or p38d indicated that these kinases can also inhibit lesions; 2, 1 or 2 ulcers; 3, 3 or 4 ulcers; and 4, more than 4 tumor development by regulating processes associated with ulcers—extensive ulceration. malignant cell transformation such as proliferation, contact For IHC, formalin-fixed, paraffin-embedded colon sec- inhibition and/or migration (25). tions were stained with antibodies to PCNA (proliferating These findings suggest that p38g and p38d are implicated in cell nuclear antigen), F4/80, and Gr-1. Anti-rabbit 633 (Sig- linking tumor promotion and/or progression and inflamma- ma), anti-rat Alexa Fluor 647 (Molecular Probes), and anti- tion. Here, we used the azoxymethane/dextran sodium sul- mouse Cy5 (The Jackson Laboratory) were used as secondary phate (AOM/DSS) mouse model to compare CAC development antibodies. Slides were mounted for fluorescence with in WT mice and in mice lacking p38g, p38d, or both. We found Hoechst-containing mounting medium (Sigma) and ana- that blockade of p38g/d expression, either in the whole mouse lyzed with a TCS SP5 Microscope (Leica). Proliferation and or in hematopoietic cells, largely suppressed colon tumor apoptosis were determined by IHC in deparaffinized sec- formation in AOM/DSS–induced CAC. In addition, p38g and tions using anti-PCNA and TUNEL (terminal deoxynucleo- p38d promoted an inflammatory response in the colon by tidyl transferase–mediated dUTP nick end labeling) staining, regulating cytokine and chemokine production and, thus, respectively. The percentage of PCNA-positive cells was leukocyte recruitment. Our study shows the important pro- quantified from at least 20 crypts per mouse and divided oncogenic role of p38g and p38d in a yet unidentified signaling by the total cell number in each crypt. TUNEL-positive cells pathway for development of colitis-associated colon tumors, were counted on slides from 30 random fields per mouse and confirms p38g and p38d as potential targets for colon from three different mouse colons. cancer treatment. Immunoblot analysis Cells or distal colon sections were lysed in buffer A Materials and Methods (50 mmol/L Tris-HCl pH 7.5, 1 mmol/L EGTA, 1 mmol/L Mice and experimental models EDTA, 0.15 mol/L NaCl, 1 mmol/L sodium orthovanadate, Mice lacking p38g (MAPK12), p38d (MAPK13), and p38g/d 10 mmol/L sodium fluoride, 50 mmol/L sodium b-glycer- have been described previously (26). All mice were backcrossed ophosphate, 5 mmol/L pyrophosphate, 0.27 mol/L sucrose, onto the C57BL/6 strain for at least nine generations. Mice 0.1 mmol/L phenylmethylsulphonyl fluoride, and 1% (v/v) were housed in specific pathogen-free conditions, and all Triton X-100) plus 0.1% (v/v) 2-mercaptoethanol and com- animal procedures were performed in accordance with nation- plete proteinase inhibitor cocktail (Roche). Lysates were

OF2 Cancer Res; 74(21) November 1, 2014 Cancer Research

p38g and p38d in Colitis-Associated Colon Cancer

centrifuged (13,000 g,15minutes,4C), supernatants re- mouse) into the tail vein of lethally irradiated (10 Gy) WT or moved, quick frozen in liquid nitrogen, and stored at 80C. p38g/d / female recipients. After 60 days, mice were For immunoblot analysis, 50 mg protein samples were AOM/DSS–treated as above to induce acute colitis or CAC. resolved in SDSPAGE and transferred to nitrocellulose mem- The extent of bone marrow repopulation was determined by branes, blocked (30 minutes) in TBST buffer [50 mmol/L PCR analysis for the Y chromosome–linked Sry gene in geno- Tris/HCl pH 7.5, 0.15 mol/L NaCl, 0.05% (v/v) Tween] with mic DNA extracted from blood samples as well as immunoblot 10% (w/v) dry milk, then incubated in TBST buffer with 10% analysis to detect p38g and p38d in splenocytes (20). (w/v) dry milk, and 0.5 to 1 mg/mL antibody [2 hours, room temperature (RT) or overnight, 4C]. Protein was detected using Statistical analysis horseradish peroxidase–conjugated secondary antibodies and Differences in tumor numbers and volume, in inflammation the enhanced chemiluminescence reagent (Amersham Pharma- score, and in ulcers size were analyzed by the Mann–Whitney cia Biotech), and using fluorescently labeled secondary anti- U test and the c2 test. Other data were processed using the bodies (Invitrogen) and the Odyssey infrared imaging system. Student t or the one-tail Mann–Whitney tests. Data were expressed as mean SD. A P value of <0.05 was considered Gene-expression analysis statistically significant. All data in this study were analyzed cDNA for real-time quantitative PCR (qPCR) was generated statistically; for clarity of the figure, some nonsignificant from 1 mg total RNA using the High Capacity cDNA Reverse differences are not indicated. Transcription Kit (Applied Biosystems) in an 8 mL final reac- tion volume. Real-time qPCR reactions were performed in triplicate using 3 mL/well of two serial dilutions (1/50 and Results 1/500) of each cDNA, 0.3 mmol/L of each primer, and 1x p38g and p38d deletion decreases colitis-associated Fluocycle SYBR Green Mix for real-time qPCR (Genycell-Euro- tumor incidence Clone) in a volume of 5 mL in MicroAmp Optical 384-well plates To study the role of p38g and p38d in CAC cancer, we used a (Applied Biosystems). PCR reactions were carried out in an ABI protocol that combines the carcinogen AOM with DSS-induced PRISM 7900HT (Applied Biosystems) and results were ana- colitis (Supplementary Fig. S1A). AOM/DSS induces tumors in lyzed by the comparative Ct method (DDCt) using SDS v2.2 the distal colon of rodents and is commonly used to elicit software. X-fold induction in mRNA expression was quantified colorectal cancer in experimental animals (27). When this relative to unstimulated WT samples, and 18S, b-actin or protocol was applied to adult WT, p38g-, p38d-, and p38g/ GAPDH mRNAs were used as housekeeping genes. Primer d-deficient mice, all lost weight and AOM/DSS–induced mor- sequences are listed in the Supplementary Table S1. tality was similar in all genotypes (Supplementary Fig. S1B). Fifteen weeks after AOM injection, mice were euthanized, Isolation of intestinal epithelial cells and flow-cytometry colons resected, and colon length and tumor number mea- analysis sured. Length decreased similarly in all mouse groups For crypt isolation, the colon was removed, flushed with ice- (Supplementary Fig. S1B) and all AOM/DSS–treated mice cold Hank balanced salt solution (HBSS; Gibco, Invitrogen), developed tumors regardless of genotype; tumor incidence and incubated in HBSS with antibiotics (15 minutes, RT). The was 100% in WT mice and between 80% and 87% in other colon was cut into 0.5-cm pieces, which were incubated in mouse groups. Tumors were located mainly in the distal to HBSS with 8 mmol/L EDTA (15 minutes, 37C) and then in middle colon (Fig. 1A), which is the predominant site of human HBSS; crypt epithelial cells were dissociated by repeated colorectal tumors (27). Tumors from all knockout mice were vigorous shaking. Tissue debris was removed with a cell histopathologically indistinguishable from those of WT mice. strainer (100 mm) and crypts collected by centrifugation All tumors were composed of crypts and glands, consistent (200 g, 15 minutes, 4C). with a well-differentiated adenocarcinoma, and did not pen- Cell pellets were resuspended in 10 mL HBSS with etrate the muscularis mucosa in any case (Fig. 1B). In contrast, 0.4 mg/mL dispase (Life Technologies) and 10,000 U/mL p38g / , p38d / , and p38g/d / mice showed a significant penicillin/streptomycin, and incubated (30 minutes, 37C). decrease in tumor numbers and in total tumor volume per FBS was added at 5% (v/v) final concentration, and tissue mouse compared with WT mice; the decrease in p38g/d / debris was removed by sequential filtering through 100, 70, mice was more pronounced than in the other genotypes and 40 mm cell strainers. Cells were collected by centrifu- (Fig. 1C and D). gation (150 g,10minutes,4C). Colon cells were stained Individual tumor size distribution and average tumor size with combinations of fluorescence-labeled antibodies to the were similar in all genotypes, although p38g / and p38g/d / cell surface markers CD45, CD4, CD8, Ly6G, and F4/80, and mice had slightly fewer large tumors than p38d / and WT analyzed in a FACScalibur cytometer (BD Biosciences). The mice (Fig. 1E). Reduced tumor incidence with no apparent profiles obtained were analyzed with FlowJo software (BD change in size suggests a role for p38g and p38d early in colon þ Biosciences); leukocytes were gated as CD45 cells. tumor initiation or development. AOM-induced DNA damage was similar in WT and p38g/d / mouse colon (Supplemen- Bone marrow transplant tary Fig. S1C), suggesting that differences in tumorigenesis Bone marrow cells were obtained from femurs of WT or between these mice were not due to a distinct initial response p38g/d / male mice (20) and were injected (4 106 cells/ to AOM. We analyzed mouse colons immediately after the final

www.aacrjournals.org Cancer Res; 74(21) November 1, 2014 OF3

Reino et al.

Figure 1. Reduced incidence of CAC cancer in p38g/d / mice. WT, p38g / , p38d / , and p38g/d / mice were treated with AOM/DSS, and colon tumors were analyzed at week 15. A, representative colons showing tumors in the distal region (arrow). B, representative H&E- stained colon sections showing colon tumors; bars, 200 mm. C, tumors were counted at week 15. Dot, a single mouse. D, total tumor volume calculated by summing all tumor volumes for a given mouse. Dot, a single mouse; ns, not signiﬁcant; , P 0.05; , P 0.01. E, histogram showing tumor size distribution in C and D. F, tumor average size was the mean of tumor volumes for a given mouse. Results are mean SD (n ¼ 15–20 mice/group); ns, not signiﬁcant.

DSS treatment round, 9 weeks after tumor initiation. No higher p38d protein levels than WT mice, whereas p38g protein macroscopic tumors were observed in any genotype; however, expression was similar in WT and p38d / mice. After AOM/ histologic analysis of colon sections from all genotypes showed DSS treatment, p38d mRNA and protein expressions were signs of neoplastic lesions, including adenomas and adeno- higher in p38g / than in WT mice, but p38d / mice showed carcinomas, and diffused inflammation of colon mucosa only mRNA upregulation for p38g (Supplementary Fig. S2), (Supplementary Fig. S1D). Average tumor size was similar in which indicates that p38g modulated p38d expression and vice p38g / , p38d / , p38g/d / , and WT mice, and histologic versa at the transcriptional level. These results might explain analysis of neoplastic lesions showed that tumors from knock- the partial effect in tumor development in single- compared out mice were indistinguishable from those of WT mice with double-knockout mice. We therefore use p38g/d / mice (Supplementary Fig. S1D and S1E). for further analysis. The intermediate phenotype in p38g / and p38d / compared with WT and p38g/d / mice (Fig. 1C and D) indicates Effect of p38g/d in chronic and acute colon partial isoform redundancy, with neither predominating; this inflammation overlap has been reported (18, 20) and might account for the Repeated DSS administration to mice leads to chronic limited effects we observed in single knockout mice. We tested inflammation, a main cause of colon tumorigenesis after the mouse groups for altered p38g or p38d expression in colon, AOM/DSS treatment. Analyses of colon sections from using qPCR and immunoblot analyses. p38g/d / mice did not p38g/d / and WT mice at 9 weeks after CAC induction and express p38g or p38d, and WT mice expressed both kinases after the last DSS cycle showed tissue damage in both groups, (Supplementary Fig. S2). Untreated p38g / mice showed with ulcerate regions in which crypts were lost (Fig. 2A). There

OF4 Cancer Res; 74(21) November 1, 2014 Cancer Research

p38g and p38d in Colitis-Associated Colon Cancer

Figure 2. p38g and p38d deletion have no major impact on chronic inflammation and decreased acute inflammation. A, representative H&E staining of WT and p38g/d / mouse colon showing ulcers (arrow) 9 weeks after initiation of AOM/DSS treatment; bars, 100 mm. Mean ulcer size and inflammation score were quantified (see Materials and Methods). Results show mean SD (n ¼ 6 mice/group); ns, not significant. B, colon extracts (50 mg) from control or AOM/DSS–treated WT and p38g/d / mice were immunoblotted with antibodies to phospho and total STAT3. Representative blots from three independent experiments. C, at 9 weeks after initiation of AOM/DSS treatment, relative mRNA expression was determined by qPCR for indicated genes in WT (black) and p38g/d / (white) mouse colon and normalized to 18S and GAPDH mRNA. Data, mean SD (n ¼ 5–8); ns, not significant; , P 0.05, relative to WT mice. D, WT and p38g/d / mice were treated with 3% DSS for 5 days. Colon samples were examined at times indicated. Representative H&E-stained colon sections analyzed at indicated times. Ulcers and inflammation areas are marked by black and red lines, respectively. E, mean ulcer size and inflammation score were quantified. Data, mean SD (n ¼ 5–6); ns, not significant; , P 0.05; , P < 0.01.

were no significant differences in ulcer size or inflammation a potential mechanism contributing to decreased tumorigen- score between genotypes (Fig. 2A). esis. We treated p38g/d / and WT mice with a single 5-day Key carcinogenesis-associated inflammatory molecules are DSS cycle, followed by 5, 10, or 15 days of normal drinking water also upregulated in DSS-induced chronic inflammation. We (30). There were no significant differences in weight loss or analyzed COX-2, STAT-3, IL10, IL6, IL1b, and TNFa expres- colon length between p38g/d / and WT mice (Supplemen- sions, as well as levels of hepatocyte growth factor (HGF), tary Fig. S3). Histologic analysis on different days after initi- whose expression by intestinal fibroblasts regulates colon ation of DSS treatment–associated p38g/d deletion with a tumor formation (29). After treatment, IL10, IL1b, IL6, HGF significant reduction in areas of ulceration and a moderate mRNA expression and STAT-3 phosphorylation and protein decrease in inflammation score compared with WT mice expression were similar in both genotypes (Fig. 2B and C). (Fig. 2D and E). COX-2 and TNFa mRNA levels were nonetheless significantly lower in treated p38g/d / than in WT mice, in accordance p38g/d deletion affects intestinal epithelial cell with the reduced tumor number in p38g/d-deficient mice proliferation and apoptosis compared with controls. To analyze the response of intestinal epithelial cells (IEC) to As we found no major impact of p38g/d deletion on chronic acute inflammation, we measured IEC proliferation and apo- inflammation, we analyzed whether this deletion affected ptosis, which are basic parameters of the tumorigenic process acute colon inflammation in p38g/d / mice and was, thus, in the intestine (3). We used immunofluorescent staining of

www.aacrjournals.org Cancer Res; 74(21) November 1, 2014 OF5

Reino et al.

These ﬁndings show that p38g/d enhances IEC proliferation and increases their resistance to apoptosis.

Reduced innate immune response in p38g/d-deficient mouse colon Consistent with the decreased ulcer size and inflammation score observed in p38g/d / mice compared with WT mice in the acute inflammation model (Fig. 2E), DSS treatment induced lower expression of the inflammatory mediators TNFa, IL1b, IL6, and COX-2, in p38g/d / than in WT mice. IL12p35, IFNb, IL23p19, and IL17A expressions were higher in p38g/d / mice, whereas IL10 and IL12p40 expressions were similar to WT (Fig. 4A). Transcription of inflammatory cytokines is largely mediated by NF-kB and MAPK signaling pathways (18, 21, 30), and p38g/d regulates cytokine production by modulating ERK1/2 activation in macrophages and DCs (18, 20). We therefore assessed the activation of several signaling pathways in mouse colon. DSS treatment weakly activated all three major MAPK pathways, c-Jun N-terminal kinase (JNK), p38a, and ERK1/2, as well as the canonical NF-kB signaling pathway (Fig. 4B). p38g/d deficiency did not affect DSS-induced transient activation of p38a or JNK1/2, as determined by immunoblot with phospho-specific antibodies (Fig. 4B). DSS-induced proteolysis of the NF-kB inhibitor IkBa was also unaffected by p38g/d deficiency (Fig. 4B, Supplementary Fig. S5A). In contrast, ERK1/2 phosphorylation was substan- / Figure 3. p38g/d / mice show decreased IEC proliferation and increased tially reduced in p38g/d colon extract and in IEC compared apoptosis. WT and p38g/d / mice were treated with 3% DSS. Colon with WT (Fig. 4B and C, Supplementary Fig. S5B). This was samples were examined at times indicated. A, immunofluorescence of accompanied by reduced expression of the ERK1/2 upstream WT and p38g/d / mouse colon sections on day 5 of DSS treatment to þ fi examine proliferating cells (PCNA). PCNA cells (red) were counted; activator TPL-2 (Fig. 4B), which supports our nding that TPL- >20 crypts per mouse were scored. Results show mean SD (n >3 2 protein expression is regulated by p38g/d (18). The results mice/group); , P 0.001 relative to WT mice. Nuclei are Hoechst suggest that, in the absence of p38g/d, a decreased inflamma- stained (blue). B, relative Ki67 mRNA expression in distal colon of 5-day tory response is associated with reduced ERK1/2 pathway / DSS-treated WT and p38g/d mice was determined by qPCR and activation. normalized to GAPDH mRNA. Data, mean SD (n ¼ 6); , P 0.05 relative to WT mice. C, apoptosis in mouse colons was evaluated by TUNEL In addition to impaired activation of signaling pathways staining (red) on days 2 and 5 after DSS administration. Apoptotic cells involved in cytokine production, reduced leukocyte infiltration were counted; 30 fields per mouse were usually scored. Nuclei are might cause the reduced inflammation and cytokine produc- Hoechst stained (blue). Results, mean SD (n >3 mice/group); , P 0.05 tion in DSS-treated p38g/d / mouse colon. To test this and , P 0.01 relative to WT mice. possibility, we analyzed hematopoietic cell recruitment after DSS treatment. Quantification of mRNA expression in colon PCNA to analyze IEC proliferation in p38g/d / and WT mice extracts showed that the production of chemokines implicated at several times after DSS treatment. At day 5 of DSS treatment, in macrophage (MCP1) and neutrophil migration (KC, MIP-2; when inflammation peaks (30), the majority of crypts appeared ref. 31) was higher in WT compared with p38g/d / mice after normal in WT and p38g/d / mice (Fig. 3A). Quantification of DSS treatment (Fig. 5A), whereas chemokine receptor levels immunofluorescence data showed significant decreases in were similar in both (Supplementary Fig. S6A). colon epithelial crypt cell proliferation in p38g/d / compared Phenotypic characterization of colon-infiltrating inflamma- with WT mice (Fig. 3A). Gene-expression analysis of Ki67, tory cells from control and DSS-treated colons showed a associated with cell proliferation, showed significantly lower significant increase in macrophages and neutrophils in WT mRNA levels in p38g/d / compared with WT mice (Fig. 3B). compared with p38g/d / mice (Fig. 5B), consistent with the To test whether IEC apoptosis differs between p38g/d / reduced inflammation. These findings were confirmed by and WT mice, we used in situ TUNEL staining of colon sections immunohistochemical analysis of colons from DSS-treated þ in early stages of DSS treatment. Colon sections from mice (Supplementary Fig. S6B). The percentages of CD4 and þ p38g/d / and WT mice were prepared 2 and 5 days after CD8 T lymphocytes nonetheless showed no variation DSS treatment; quantification showed significantly higher between genotypes (Fig. 5B). We also examined the percentage þ frequencies of TUNEL-positive cells in p38g/d / mice, located of CD127 Treg (regulatory T cell) cells, because they are mainly in epithelium and mucosa (Fig. 3C). Immunoblot involved in intestinal inflammation (32). After DSS treatment, analysis of apoptotic proteins confirmed higher apoptosis p38g/d / colon had a slightly lower percentage of Treg cells levels in p38g/d / than in WT IEC (Supplementary Fig. S4). than WT mice, whereas Treg cells in the thymus (where these

OF6 Cancer Res; 74(21) November 1, 2014 Cancer Research

p38g and p38d in Colitis-Associated Colon Cancer

Figure 4. p38g/d deletion reduces DSS-induced cytokine production and ERK1/2 activation. A, relative mRNA expression was determined by qPCR for indicated genes in DSS-treated WT and p38g/d / mouse distal colon and normalized to 18S and GAPDH mRNA. Data, mean SD (n ¼ 5–6); , P 0.05, relative to WT mice in the same conditions. B, colon extracts (50 mg) from WT and p38g/d / mice, DSS-treated for indicated times, were immunoblotted with the indicated antibodies. C, WT and p38g/d / IECs (50 mg) were immunoblotted with antibodies to phosphorylated or total ERK1/2. B and C, representative blots are shown from three independent experiments (n ¼ 6 mice/ condition).

cells are generated) are similar in p38g/d / and WT mice were treated by the AOM/DSS CAC cancer protocol, (Supplementary Fig. S6C and S6D). These findings suggest that WT!p38g/d / and WT!WT had more tumors than p38g and p38d modulate the procarcinogenic local environ- p38g/d / !WT and p38g/d / !p38g/d / mice (Fig. ment and inflammation in CAC. 6C).Tumorsfromallchimericmicewerehistologically similar (Fig. 6D). These data show that p38g/d expression p38g/d in hematopoietic cells modulate colon tumor in hematopoietic cells is critical for production of inflam- formation matory mediators and cell recruitment that lead to colon As hematopoietic and non-hematopoietic cells in colon tumor development, and indicate that absence of p38g and express p38g and p38d (Fig. 4 and Supplementary Fig. S2; p38d in the hematopoietic compartment is responsible for refs. 18, 20), decreased tumor formation in p38g/d / mice decreased tumorigenesis in p38g/d / mice. could be due to indirect effects caused by surrounding inflammatory cells, rather than to changes in intrinsic properties of IEC. To identify the cell compartment respon- Discussion sible for decreased tumorigenesis in p38g/d / mice, we Cancer and chronic colon inflammation are intimately generated reciprocal bone marrow chimeric mice by trans- associated; however, the links between these processes remain planting WT bone marrow into lethally irradiated p38g/d / to be completely characterized. In this study, we addressed (WT!p38g/d / )orWT(WT!WT) hosts, and p38g/d / the role of p38g and p38d kinases in colorectal carcinogenesis bone marrow into p38g/d / (p38g/d / !p38g/d / )or and intestinal inflammation. We evaluated whether p38g WT (p38g/d / !WT) mice. Mice reconstituted with p38g/ and p38d deletion interfered with CAC development, and d / bone marrow had lower levels of DSS-induced cyto- found that the combined deletion of these p38MAPK isoforms kines and chemokines and of cell recruitment than WT bone led to a marked reduction in tumor incidence in the AOM/ marrow–reconstituted mice (Fig. 6A and B). When mice DSS–induced colorectal carcinogenesis model. Lack of p38g

www.aacrjournals.org Cancer Res; 74(21) November 1, 2014 OF7

Reino et al.

Figure 5. Reduced macrophage and neutrophil recruitment in p38g/d / mice. A, relative mRNA expression for indicated genes in DSS-treated WT and p38g/d / mouse colon determined by qPCR and normalized to GAPDH mRNA. Data, mean SD (n ¼ 5–6). B, colon crypt cells from 0- to 5-day DSS-treated WT and p38g/d / mice were stained with anti-CD45, anti-CD4, anti-CD8, anti-Ly6G, and anti-F4/80 antibodies and the percentage of positive cells analyzed by flow cytometry. Representative profiles are shown. þ C, CD45 cells were gated and þ þ percentages of CD4 , CD8 , þ þ Ly6G , and F4/80 cells analyzed by flow cytometry. Data, mean SD (n >20 mice/condition); , P 0.05; , P 0.01; , P 0.001.

or p38d alone had a partial effect on tumor incidence, as WT mouse colon contrast with findings for LPS-stimulated each of these p38 isoforms might modulate expression of the TPL2 / macrophages (36, 37), indicating a certain TPL2 other, and also to redundancy of p38g and p38d functions independence in p38g/d regulation of cytokine production. (18, 20). The number of myeloid cells, neutrophils, and macrophages Study of the mechanisms of p38g/d action in the CAC model recruited to the colon after inflammation induction was also showed that p38g/d deletion had no significant effect on late lower in p38g/d / than in WT mice. In contrast, TPL2 tumor progression. Deletion did not alter tumor size, indicat- deficiency does not affect macrophage and neutrophil infil- þ þ ing that p38g/d act on early tumor promotion in this model. tration (33). Recruitment of CD4 or CD8 T cells, which Comparison of p38g/d / and WT mice showed alterations also infiltrate colon in patients with IBD (38), was similar in in the innate immune response, important for initiation of p38g/d / and WT genotypes; however, p38g/d / mice had inflammation. Production of proinflammatory mediators such slightly fewer Treg cells in the colon than WT mice, as well as as the cytokines TNFa, IL1b, and IL6 and the chemokines increased production of IL17A, a Th17 cell marker. This is þ MCP1, KC, and MIP-2 was reduced in p38g/d / mice. consistent with data for the Apcmin/ /TPL2 / model (32), in The effect of p38g/d deficiency on DSS-induced colitis in which TPL2 deficiency led to decreased Treg and increased some ways resembled that described in TPL2 / mice (33). Th17 cell recruitment to the colon, which enhanced inflam- p38g/d / colon shows a severe reduction in TPL2 protein. mation and tumorigenesis (32). Paradoxically, p38g/d / mice Similar to p38g/d, TPL2 is necessary for optimal TNFa, IL6, and had less colon inflammation– and colitis-associated colon IL1b production in DSS-treated mice. The changes in IL12p35, cancer than WT mice, possibly explained by the reduction IFNb, MCP1, and MIP-2 mRNAs in p38g/d / colon coincide in infiltrating macrophages and neutrophils in p38g/d / with observations in p38g/d / and TPL2 / macrophages mice. DSS-induced colitis is mediated by macrophages, the (18, 34, 35). The changes in IL23p19 and chemokine receptor main cells responsible for inflammation in this model, and mRNA expression in DSS-treated p38g/d / compared with is T-cell independent (39). Macrophages and neutrophils

OF8 Cancer Res; 74(21) November 1, 2014 Cancer Research

p38g and p38d in Colitis-Associated Colon Cancer

Figure 6. WT chimeric mice with p38g/d / bone marrow show reduced CAC incidence. A, relative mRNA expression was determined by qPCR for indicated genes in 5-day DSS-treated chimeric mouse colon and normalized to GAPDH mRNA. Data, mean SD (n ¼ 6–9); ns, not significant; , P 0.05. B, colon crypt cells from 5-day DSS-treated chimeric mice were stained with anti-CD45, anti-Ly6G, and anti-F4/80 antibodies and the percentage of positive cells analyzed by flow cytometry. Data, mean SD (n >10 mice/condition); , P 0.05. C, chimeric mice were AOM/ DSS–treated to induce CAC and colon tumors analyzed at week 15. Tumors were counted and total volume determined by summing all volumes for a given mouse. Dot, a single mouse; ns, not significant; , P 0.05; , P 0.01. D, representative H&E-stained colon sections showing colon tumors; bars, 200 mm. are essential for tumor progression and microenvironment (3). and thus, recruitment of activated macrophages and neutro- Although their precise role in tumor development has not been phils. These cells are the main source of COX-2, reactive oxygen clearly established, macrophage infiltration into colon has species, and reactive nitrogen intermediates that can cause been correlated with colon carcinogenesis (40), and neutrophil DNA damage and mutations in IEC (3). We cannot rule out the depletion using neutralizing anti-Ly6G antibodies markedly possibility that p38g and p38d have additional roles in tumor reduces CAC (41). These findings are supported by our obser- progression, as they regulate IEC proliferation and apoptosis, vations that reduced macrophage and neutrophil recruitment and modulate activation of signaling pathways and expression paralleled a CAC decrease in p38g/d / mouse colon. of genes involved in tumor progression, such as p53. The Bone marrow transfer experiments demonstrated that mechanisms by which p38g/d regulates IEC apoptosis and p38g/d expression in hematopoietic cells is the main contrib- proliferation remain unclear, although lamina propria myeloid utor to colon tumor formation. These studies indicated that cells in CAC-induced mice produce cytokines that stimulate decreased tumor formation in p38g/d / mice is due mainly to proliferation of adjacent premalignant IEC (3). p38g/d deletion a direct effect of the degree of surrounding inflammation, impairs production of the proinflammatory cytokines TNFa rather than to effects on intrinsic IEC homeostasis. We thus and IL1b in colon as well as in macrophages and DCs in hypothesize that p38g and p38d expression in hematopoietic response to TLR4 ligand (18), which could affect IEC prolifer- cells is critical for colon tumor initiation; these isoforms ation. p38g/d might protect against IEC apoptosis by regulat- control expression of inflammatory cytokines and chemokines ing activation of the ERK1/2 pathway, also an important

www.aacrjournals.org Cancer Res; 74(21) November 1, 2014 OF9

Reino et al.

regulator of cell survival (42). As in p38g/d / macrophages Authors' Contributions Conception and design: P. del Reino, D. Alsina-Beauchamp, A. Cuenda and DCs (18), ERK1/2 activation in response to DSS treatment / Development of methodology: P. del Reino, D. Alsina-Beauchamp, A. Escos, is reduced in p38g/d compared with WT colon, although we Ma.I. Cerezo-Guisado cannot exclude the possibility that p38g/d also protects the Acquisition of data (provided animals, acquired and managed pati- – ents, provided facilities, etc.): P. del Reino, D. Alsina-Beauchamp, intestinal epithelium via unknown non ERK1/2-dependent Ma.I.Cerezo-Guisado,A.Risco,N.Aparicio,M.Fernandez-Estevez, mechanisms. Our data also suggest that p38g/d promote IEC E. Collantes, A. Cuenda survival and protect the colon lining from chemically induced Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P. del Reino, D. Alsina-Beauchamp, A. Escos, Ma.I. damage . Because IEC apoptosis is one of the mechanisms by Cerezo-Guisado, A. Risco, N. Aparicio, R. Zur, M. Fernandez-Estevez, E. Collantes, / which DSS can induce colitis, our findings in p38g/d IEC J. Montans, A. Cuenda apoptosis and colon ulcer size nonetheless appear contradic- Writing, review, and/or revision of the manuscript: P. del Reino, D. Alsina- Beauchamp, A. Escos, Ma.I. Cerezo-Guisado, A. Risco, N. Aparicio, R. Zur, tory. A possible explanation is the lower cytokine production A. Cuenda by p38g/d / mice, because increased production of cytokines Administrative, technical, or material support (i.e., reporting or orga- a fl nizing data, constructing databases): D. Alsina-Beauchamp, A. Risco such as TNF in the colon impedes resolution of in ammation Study supervision: A. Cuenda and leads to tissue destruction (43). Also, the higher production Other (assessed histologic samples): E. Collantes of IFNb, which promotes epithelial regeneration (43), by p38g/d / mice could lead to a decreased ulcer size. fi Acknowledgments In the AOM/DSS model, TPL2-de cient mice develop more The authors thank Drs. A. Gonzalez-García and J.J. Sanz-Ezquerro for helpful tumors than WT mice due to increased HGF expression by discussions, L. Almonacid for help with qPCR, A. Alvarez-Barrientos (STAB, intestinal fibroblasts (29). Our results show that p38g/d / Universidad de Extremadura) for support with immunofluorescence analysis, fi / g the antibody puri cation teams (DSTT, University of Dundee) coordinated by H. mice do not resemble TPL2 mice, indicating that the p38 / McLauchlan and J. Hastie for antibody generation and purification, and C. Mark d signaling promotes CAC independently of TPL2. Our findings for editorial assistance. describe a major role for p38g and p38d in CAC pathogenesis, and indicate that they modulate inflammation and the procarcinogenic environment, which influences epithelial cell Grant Support This work was supported by The Spanish Ministry of Economy and Compe- proliferation and apoptosis in CAC. Because clinical trials tiveness (MINECO; BFU2010-19734) and La Marato TV3 Foundation (82031). P. using p38a inhibitors have yielded contradictory results in del Reino and D. Alsina-Beauchamp received MINECO FPI fellowships. Ma.I. Cerezo-Guisado received an FIS Sara Borrell award from the Instituto de Salud patients with Crohn disease and have proven minimally effec- Carlos III. R. Zur receives a La Caixa Foundation fellowship. tive in human disease (13, 15), the implication of p38g/d in The costs of publication of this article were defrayed in part by the colitis and colon tumor development suggests these p38 iso- 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 forms as an alternative therapeutic target in IBD and in CAC. fact.

Disclosure of Potential Conﬂicts of Interest Received March 22, 2014; revised July 18, 2014; accepted August 12, 2014; No potential conﬂicts of interest were disclosed. published OnlineFirst September 12, 2014.

References 1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. 10. Otsuka M, Kang YJ, Ren J, Jiang H, Wang Y, Omata M, et al. Distinct Cell 2011;144:646–74. effects of p38alpha deletion in myeloid lineage and gut epithelia in 2. Feagins LA, Souza RF, Spechler SJ. Carcinogenesis in IBD: potential mouse models of inflammatory bowel disease. Gastroenterology targets for the prevention of colorectal cancer. Nat Rev Gastroenterol 2010;138:1255–65, 65 e1–9. Hepatol 2009;6:297–305. 11. Docena G, Rovedatti L, Kruidenier L, Fanning A, Leakey NA, 3. Grivennikov SI. Inflammation and colorectal cancer: colitis-associated Knowles CH, et al. Down-regulation of p38 mitogen-activated neoplasia. Semin Immunopathol 2013;35:229–44. protein kinase activation and proinflammatory cytokine pro- 4. Meira LB, Bugni JM, Green SL, Lee CW, Pang B, Borenshtein D, et al. duction by mitogen-activated protein kinase inhibitors in DNA damage induced by chronic inflammation contributes to colon inflammatory bowel disease. Clin Exp Immunol 2010;162: carcinogenesis in mice. J Clin Invest 2008;118:2516–25. 108–15. 5. Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, et al. 12. Zhao X, Kang B, Lu C, Liu S, Wang H, Yang X, et al. Evaluation of p38 NF-kappaB functions as a tumour promoter in inflammation-associ- MAPK pathway as a molecular signature in ulcerative colitis. J Prote- ated cancer. Nature 2004;431:461–6. ome Res 2011;10:2216–25. 6. Cuenda A, Rousseau S. p38 MAP-kinases pathway regulation, func- 13. Hommes D, van den Blink B, Plasse T, Bartelsman J, Xu C, Macpher- tion and role in human diseases. Biochim Biophys Acta 2007;1773: son B, et al. Inhibition of stress-activated MAP kinases induces clinical 1358–75. improvement in moderate to severe Crohn's disease. Gastroenterol- 7. Remy G, Risco AM, Inesta-Vaquera FA, Gonzalez-Ter an B, Sabio G, ogy 2002;122:7–14. Davis RJ, et al. Differential activation of p38MAPK isoforms by MKK6 14. Hollenbach E, Neumann M, Vieth M, Roessner A, Malfertheiner P, and MKK3. Cell Signal 2010;22:660–7. Naumann M. Inhibition of p38 MAP kinase- and RICK/NF-kappaB- 8. Kang YJ, Chen J, Otsuka M, Mols J, Ren S, Wang Y, et al. Macrophage signaling suppresses inflammatory bowel disease. FASEB J 2004; deletion of p38alpha partially impairs lipopolysaccharide-induced 18:1550–2. cellular activation. J Immunol 2008;180:5075–82. 15. Schreiber S, Feagan B, D'Haens G, Colombel JF, Geboes K, 9. Kim C, Sano Y, Todorova K, Carlson BA, Arpa L, Celada A, et al. The Yurcov M, et al. Oral p38 mitogen-activated protein kinase inhi- kinase p38 alpha serves cell type-specificinflammatory functions in bition with BIRB 796 for active Crohn's disease: a randomized, skin injury and coordinates pro- and anti-inflammatory gene expres- double-blind, placebo-controlled trial. Clin Gastroenterol Hepatol sion. Nat Immunol 2008;9:1019–27. 2006;4:325–34.

OF10 Cancer Res; 74(21) November 1, 2014 Cancer Research

p38g and p38d in Colitis-Associated Colon Cancer

16. ten Hove T, van den Blink B, Pronk I, Drillenburg P, Peppelenbosch MP, 30. Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, et al. van Deventer SJ. Dichotomal role of inhibition of p38 MAPK with SB IKKbeta links inflammation and tumorigenesis in a mouse model of 203580 in experimental colitis. Gut 2002;50:507–12. colitis-associated cancer. Cell 2004;118:285–96. 17. Ittner A, Block H, Reichel CA, Varjosalo M, Gehart H, Sumara G, et al. 31. Wang D, Dubois RN, Richmond A. The role of chemokines in intestinal Regulation of PTEN activity by p38delta-PKD1 signaling in neutrophils inflammation and cancer. Curr Opin Pharmacol 2009;9:688–96. confers inflammatory responses in the lung. J Exp Med 2012;209: 32. SerebrennikovaOB,TsatsanisC,MaoC,GounarisE,RenW, 2229–46. Siracusa LD, et al. Tpl2 ablation promotes intestinal inflammation 18. Risco A, del Fresno C, Mambol A, Alsina-Beauchamp D, MacKenzie and tumorigenesis in Apcmin mice by inhibiting IL-10 secretion and KF, Yang HT, et al. p38gamma and p38delta kinases regulate the Toll- regulatory T-cell generation. Proc Natl Acad Sci U S A 2012;109: like receptor 4 (TLR4)-induced cytokine production by controlling E1082–91. ERK1/2 protein kinase pathway activation. Proc Natl Acad Sci 33. Lawrenz M, Visekruna A, Kuhl A, Schmidt N, Kaufmann SH, Steinhoff U S A 2012;109:11200–5. U. Genetic and pharmacological targeting of TPL-2 kinase ameliorates 19. Gonzalez-Teran B, Cortes JR, Manieri E, Matesanz N, Verdugo A, experimental colitis: a potential target for the treatment of Crohn's Rodríguez ME, et al. Eukaryotic elongation factor 2 controls TNF-alpha disease? Mucosal Immunol 2012;5:129–39. translation in LPS-induced hepatitis. J Clin Invest 2013;123:164–78. 34. Gantke T, Sriskantharajah S, Ley SC. Regulation and function of TPL- 20. Criado G, Risco A, Alsina-Beauchamp D, Perez-Lorenzo MJ, Escos A, 2, an IkappaB kinase-regulated MAP kinase kinase kinase. Cell Res Cuenda A. Alternative p38 mitogen-activated protein kinases are 2011;21:131–45. essential for collagen-induced arthritis. Arthritis Rheum 2014;66: 35. Bandow K, Kusuyama J, Shamoto M, Kakimoto K, Ohnishi T, Matsu- 1208–17. guchi T. LPS-induced chemokine expression in both MyD88-depen- 21. Arthur JS, Ley SC. Mitogen-activated protein kinases in innate immu- dent and -independent manners is regulated by Cot/Tpl2-ERK axis in nity. Nat Rev Immunol 2013;13:679–92. macrophages. FEBS Lett 2012;586:1540–6. 22. Risco A, Cuenda A. New Insights into the p38gamma and p38delta 36. Kakimoto K, Musikacharoen T, Chiba N, Bandow K, Ohnishi T, Mat- MAPK Pathways. J Signal Transduct 2012;2012:520289. suguchi T. Cot/Tpl2 regulates IL-23 p19 expression in LPS-stimulated 23. Schindler EM, Hindes A, Gribben EL, Burns CJ, Yin Y, Lin MH, et al. macrophages through ERK activation. J Physiol Biochem 2010;66: p38delta Mitogen-activated protein kinase is essential for skin tumor 47–53. development in mice. Cancer Res 2009;69:4648–55. 37. Rowley SM, Kuriakose T, Dockery LM, Tran-Ngyuen T, Gingerich AD, 24. Qi X, Pohl NM, Loesch M, Hou S, Li R, Qin JZ, et al. p38alpha Wei L, et al. Tumor progression locus 2 (Tpl2) kinase promotes antagonizes p38gamma activity through c-Jun-dependent ubiquitin- chemokine receptor expression and macrophage migration during proteasome pathways in regulating Ras transformation and stress acute inflammation. J Biol Chem 2014;289:15788–97. response. J Biol Chem 2007;282:31398–408. 38. Marsal J, Agace WW. Targeting T-cell migration in inflammatory bowel 25. Cerezo-Guisado MI, del Reino P, Remy G, Kuma Y, Arthur JS, Gallego- disease. J Intern Med 2012;272:411–29. Ortega D, et al. Evidence of p38gamma and p38delta involvement in 39. Axelsson LG, Landstrom E, Goldschmidt TJ, Gronberg A, Bylund- cell transformation processes. Carcinogenesis 2011;32:1093–9. Fellenius AC. Dextran sulfate sodium (DSS) induced experimental 26. Sabio G, Arthur JS, Kuma Y, Peggie M, Carr J, Murray-Tait V, et al. colitis in immunodeficient mice: effects in CD4(þ) -cell depleted, p38gamma regulates the localisation of SAP97 in the cytoskeleton by athymic, and NK-cell depleted SCID mice. Inflamm Res 1996;45: modulating its interaction with GKAP. Embo J 2005;24:1134–45. 181–91. 27. Neufert C, Becker C, Neurath MF. An inducible mouse model of colon 40. Popivanova BK, Kostadinova FI, Furuichi K, Shamekh MM, Kondo T, carcinogenesis for the analysis of sporadic and inflammation-driven Wada T, et al. Blockade of a chemokine, CCL2, reduces chronic colitis- tumor progression. Nat Protoc 2007;2:1998–2004. associated carcinogenesis in mice. Cancer Res 2009;69:7884–92. 28. Neurath MF, Vehling D, Schunk K, Holtmann M, Brockmann H, Helisch 41. Shang K, Bai YP, Wang C, Wang Z, Gu HY, Du X, et al. Crucial A, et al. Noninvasive assessment of Crohn's disease activity: a com- involvement of tumor-associated neutrophils in the regulation of parison of 18F-fluorodeoxyglucose positron emission tomography, chronic colitis-associated carcinogenesis in mice. PLoS ONE 2012; hydromagnetic resonance imaging, and granulocyte scintigraphy with 7:e51848. labeled antibodies. Am J Gastroenterol 2002;97:1978–85. 42. Balmanno K, Cook SJ. Tumour cell survival signalling by the ERK1/2 29. Koliaraki V, Roulis M, Kollias G. Tpl2 regulates intestinal myofibroblast pathway. Cell Death Differ 2009;16:368–77. HGF release to suppress colitis-associated tumorigenesis. J Clin 43. Neurath MF. Cytokines in inflammatory bowel disease. Nat Rev Invest 2012;122:4231–42. Immunol 2014;14:329–42.

www.aacrjournals.org Cancer Res; 74(21) November 1, 2014 OF11

Pro-Oncogenic Role of Alternative p38 Mitogen-Activated Protein Kinases p38 γ and p38δ, Linking Inflammation and Cancer in Colitis-Associated Colon Cancer

Paloma del Reino, Dayanira Alsina-Beauchamp, Alejandra Escós, et al.

Cancer Res Published OnlineFirst September 12, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-14-0870

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2014/09/12/0008-5472.CAN-14-0870.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2014/10/15/0008-5472.CAN-14-0870. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.