Mesenchymal MAPKAPK2/HSP27 drivesintestinal carcinogenesisAna Henriquesa, Vasiliki Koliarakia,1, and George Kolliasa,b,1

aDepartment of Immunology, Biomedical Sciences Research Centre “Alexander Fleming,” 16672 Vari, Greece; and bDepartment of Physiology, MedicalSchool, University of Athens, 11527 Athens, Greece

Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved May 2, 2018 (received for review April 4, 2018)

Mesenchymal cells in the microenvironment of cancer exertimportant functions in tumorigenesis; however, little is known ofintrinsic pathways that mediate these effects. MAPK signals, suchas from MAPKAPK2 (MK2) are known to modulate tumorigenesis,yet their cell-specific role has not been determined. Here, westudied the cell-specific role of MK2 in intestinal carcinogenesisusing complete and conditional ablation of MK2. We show thatboth genetic and chemical inhibition of MK2 led to decreasedepithelial cell proliferation, associated with reduced tumor growthand invasive potential in the Apcmin/+ mouse model. Notably, thisfunction of MK2 was not mediated by its well-described immuno-modulatory role in immune cells. Deletion of MK2 in intestinalmesenchymal cells (IMCs) led to both reduced tumor multiplicityand growth. Mechanistically, MK2 in IMCs was required forHsp27 phosphorylation and the production of downstream tumor-igenic effector molecules, dominantly affecting epithelial prolifer-ation, apoptosis, and angiogenesis. Genetic ablation of MK2 inintestinal epithelial or endothelial cells was less effective in com-parison with its complete deletion, leading to reduction of tumorsize via modulation of epithelial apoptosis and angiogenesis-associated proliferation, respectively. Similar results were obtainedin a model of colitis-associated carcinogenesis, indicating amesenchymal-specific role for MK2 also in this model. Our findingsdemonstrate the central pathogenic role of mesenchymal-specificMK2/Hsp27 axis in tumorigenesis and highlight the value ofmesenchymal MK2 inhibition in the treatment of cancer.

colorectal cancer | MAP kinases | stromal cells | tumor microenvironment

Colorectal cancer (CRC) is the fourth most common cancerworldwide (1) and results from cumulative effects of multipleand sequential mutations (2). One of the most frequently mu-tated genes in CRC is the adenomatous polyposis coli (APC)gene, which is a tumor suppressor involved in multiple physio-logical functions as it controls the levels of β-catenin. Mutationsin the APC gene result in familial adenomatous polyposis (FAP),while somatic APC mutations also appear to be an early event inthe development of sporadic CRCs (3). Mice carrying a mutationin the APC gene (Apcmin/+ mice) develop multiple intestinaltumors (Min), mainly in the small intestine and serve as a mousemodel for spontaneous colorectal tumorigenesis (4). Differencesin tumor location between mice and human patients can beexplained by the difference in the number of stem cell divisionsin the colon and small intestine between the two species (5).Colitis-associated cancer (CAC) is a subtype of CRC, associatedwith the previous presence of chronic intestinal inflammation.Despite the differences in pathogenic sequence between the twoforms of cancer, there is also a significant overlap in the geneticand molecular pathways leading to their pathogenesis (6).The p38 mitogen-activated protein kinase (MAPK) pathway

regulates multiple cellular responses to stress and plays an impor-tant role in tissue homeostasis, immune signaling, and cancer (7). Inthe intestine, recent work in mice has shown that p38α plays a tu-mor suppressive role in the early stages of CAC, by maintaining thehomeostasis of the intestinal epithelium. In contrast, once the tu-mor has formed, p38α contributes to the survival and proliferation

of tumor cells, enhancing tumor growth (8, 9). Due to the in-volvement of the p38 MAPK pathway in important cellular func-tions across several organs and its implication in differentpathologies, efforts were spent for the development of efficientp38 MAPK inhibitors. However, the use of p38 inhibitors has ledto systemic side effects mostly associated with increased toxicity.For this reason, MK2 (MAPK-activated protein kinase-2), a ki-nase downstream of p38, has been proposed as a potential al-ternative target to p38 inhibition (10, 11). MK2 deletion wouldleave intact important feedback loops, such as p38α–TAB1, andantiinflammatory effects of downstream targets such as MSK1and MSK2. Therefore, MK2 inhibitors could reproduce thebeneficial effects of p38 inhibitors potentially sparing the ac-companying side effects (11, 12).MK2 is activated in response to a variety of stimuli, including

stress, inflammatory signals, and DNA damage. Depending onthe stimuli, MK2 regulates the phosphorylation, mRNA stability,and expression of various proteins involved in actin remodeling(13), cell migration (14, 15), and immune responses (16), while ithas also been reported to be involved in the regulation of cellcycle and apoptosis (17). The role of MK2 in inflammation isthe most extensively studied. It is mediated through the posttran-scriptional control of proinflammatory cytokines, such as TNFand IL-6, and has been shown to play an important role invarious inflammatory diseases in vivo, such as LPS-inducedsepsis, arthritis, pancreatitis, atherosclerosis, skin and airwayinflammation, and neuroinflammation (18, 19). MK2 is also

Significance

Although MK2 inhibition has been proposed as a therapy incancer, its exact role, as well as the cellular and molecularmechanisms underlying it, in the intestine is not known. Here,we show that complete MK2 deletion leads to decreased epi-thelial cell proliferation, associated with reduced tumor growthand invasive potential in the Apcmin/+ and colitis-associatedcancer model. Notably, this function of MK2 is not mediatedby its well-described immunomodulatory roles in inflammatorycells. Instead, MK2 modulates tumor progression mainly viamodulating mesenchymal-specific Hsp27-mediated activationof protumorigenic mediators. Our results advance our un-derstanding of mesenchymal MAPK signaling in intestinalcancer progression and demonstrate the value of MK2 in-hibition in the treatment of cancer.

Author contributions: A.H., V.K., and G.K. designed research; A.H. and V.K. performedresearch; A.H., V.K., and G.K. analyzed data; A.H. and V.K. wrote the paper; and V.K. andG.K. cosupervised the study.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: koliaraki@fleming.gr or kollias@fleming.gr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1805683115/-/DCSupplemental.

Published online May 29, 2018.

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the main kinase that phosphorylates Hsp27, which mediatescytokine expression and has been further implicated in theregulation of cell proliferation, apoptosis, and migration.Hsp27 is often found up-regulated and phosphorylated incancer, and it has been associated with poor prognosis (20).The role of MK2 in cancer is not as well studied as in in-flammation; in vivo data using MK2 knockout mice have shownthat it plays an important role in the initiation and the earlystages of chemically induced skin tumorigenesis in mice,mainly through the regulation of proinflammatory cytokineexpression and the stabilization of the tumor suppressor pro-tein p53 (21). Recently, it was also shown that mice deficient inMK2 were resistant to the development of azoxymethane(AOM)/dextran sulfate sodium (DSS)-induced CAC, althoughthe exact cellular and molecular mechanism underlying thisphenotype remained unclear (22). In the present study, we haveaddressed the cell-specific pathophysiological role of MK2 in in-testinal carcinogenesis. Notably, we reveal an important protu-morigenic role for MK2 in the intestine, mediated throughHsp27 activation and downstream expression of tumorigenic ef-fector molecules in intestinal mesenchymal cells.

ResultsGeneration and Characterization of Complete and Conditional MK2-Deficient Mice. To investigate the role of MK2 in intestinal tu-morigenesis and the cellular and molecular mechanisms underlyingits functions, we generated a conditional MK2 knockout mousestrain, in which loxP sites flanked exons 2–5 of the mouseMapkapk2gene (MK2f/f mice). Crossing mice carrying this targeted allele withDeleter-Cre mice (23) also led to the generation of completeknockout mice, referred to as MK2D/D (D, deleted) (SI Appendix,Fig. S1A). Screening by Southern blot (SI Appendix, Fig. S1B) andPCR (SI Appendix, Fig. S1C) was used to select the respectivefounder for each mouse line. Both MK2 complete and conditionalknockout mice developed normally and did not exhibit obviousphenotypic defects. Deletion of MK2 in the intestine of MK2D/D

mice was confirmed both by qRT-PCR (SI Appendix, Fig. S1D) andWestern blot analysis (SI Appendix, Fig. S1E). Moreover, consistentwith published data (13, 16), MK2D/D mice produced less TNF bythioglycollate-elicited peritoneal macrophages (TEPMs) after LPS

stimulation in vitro (SI Appendix, Fig. S1F) and displayed reducedserum levels of TNF in response to systemic LPS/D-Gal adminis-tration (SI Appendix, Fig. S1G).

MK2 Promotes Tumor Progression in the Intestine. To examine therole of MK2 in spontaneous intestinal carcinogenesis, we crossedMK2D/D mice with the Apcmin/+ mouse model (4) and analyzedthe effects of MK2 deletion on tumor development. Initialexperiments were performed using littermate controls, andthen age-matched controls were used for further analysis.Apcmin/+MK2D/D mice displayed increased survival (Fig. 1A)and a significant decrease both in tumor incidence (Fig. 1B)and the number of macroscopically visible polyps in the in-testine compared with Apcmin/+MK2+/+ controls at 22 wk of age(Fig. 1C and SI Appendix, Fig. S2A). In addition to their re-duced number, tumors in Apcmin/+MK2D/D mice were also sig-nificantly smaller in size (Fig. 1 D and E). Moreover, increasedspleen size, a common characteristic of the Apcmin/+ mouse relatedto tumor-associated anemia and cancer progression (24), wassignificantly ameliorated in Apcmin/+MK2D/D mice (Fig. 1F), in-dicating decreased disease severity, which further correlated withthe increased lifespan of these mice.Detailed histopathological analysis during the course of the

disease revealed that both Apcmin/+MK2D/D and Apcmin/+MK2+/+

mice developed a comparable number of microadenomas atweek 8 (Fig. 1G). However, in Apcmin/+MK2D/D mice, these wereconsiderably smaller in size (Fig. 1H and SI Appendix, Fig. S2B).Similarly, in later time points (12, 16, and 22 wk), micro-adenomas and adenomas were indeed present in the small in-testine of Apcmin/+MK2D/D, but they were fewer and smaller incomparison with their age-matched controls and, thus, rarelymacroscopically visible, consistent with our initial observation(Fig. 1 G and H and SI Appendix, Fig. S2 B and C). In contrast,Apcmin/+MK2+/+ mice displayed adenocarcinomas/carcinomas at22 wk of age, which penetrated the muscularis mucosae and in-vaded into the muscularis propria, characteristics that were ab-sent in Apcmin/+MK2D/D mice (Fig. 1 I and J). Overall, these datasuggest that MK2 plays an important tumor-promoting role inthe intestine through the regulation of cancer progression,growth, and invasive potential.

Fig. 1. Genetic and chemical inhibition of MK2 reducestumor load in Apcmin/+ mice. (A) Kaplan–Meier survivalcurve of Apcmin/+MK2+/+ (n = 25) and Apcmin/+MK2D/D

(n = 20). Tumor incidence (B), tumor number (C), aver-age tumor size (D), tumor size distribution (E), andspleen weight (F) in 22-wk-old Apcmin/+MK2+/+ (n = 20)and Apcmin/+MK2D/D (n = 27) mice and normal controls(n = 3). Data represents mean ± SEM. Average micro-adenoma/adenoma number per mouse (G) and averagesize of microadenomas/adenomas (H) at 8-, 12-, 16-, and22-wk-old Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice.Data represents mean ± SEM (n = 8 per genotype andtime point). Quantification of adenocarcinomas permousein 22-wk-old of Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice(I) and representative image of an adenocarcinoma in 22-wk-old of Apcmin/+MK2+/+ mice (J). (Scale bar: 25 μm.) Tu-mor number per mouse (K), average tumor size (L), andaverage spleen weight (M) in Apcmin/+ + PHA 767491 (n =14) mice and respective Apcmin/+ control with vehicle(dH2O) mice (n = 13), and Apc

min/+ + PF-3644022 (n = 14)mice and its respective Apcmin/+ with vehicle (0.5% meth-ylcellulose and 0.025%Tween 80) (n= 16) at 16wkof age.Data represents mean ± SEM. n.s., not significant; *P ≤0.05; **P < 0.01; ***P < 0.001.

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Chemical Inhibition of MK2 Attenuates Intestinal Tumorigenesis in theApcmin/+ Mouse Model. MK2 has been proposed as an alternativetherapeutic target to p38 inhibition, which could reproduce thebeneficial effects of p38 inhibitors without the accompanyingside effects (12). To further investigate whether pharmacolog-ical inhibition of MK2 would have comparable effects to itsgenetic ablation, we employed two commercially available MK2inhibitors, PHA-767491 and PF-3644022 (25, 26). Initially, weverified the efficiency of PHA-767491 in the inhibition of TNFproduction after LPS stimulation in thioglycollate-elicitedperitoneal macrophages (TEPMs) (SI Appendix, Fig. S3A).We also confirmed its effect on TNF levels in the serum ofmice, treated with LPS/D-Gal. i.p. administration of the in-hibitor at 30 mg/kg, 1 h before LPS/D-Gal challenge, efficientlyinhibited production of TNF (SI Appendix, Fig. S3B). Similartreatment with the inhibitor led to increased survival 24 h afterLPS/D-Gal administration, comparable to MK2D/D mice (SIAppendix, Fig. S3C), and in agreement to what has previouslybeen reported (16). The in vivo efficacy of PF-3644022 has beenpreviously described (26).

We next examined the effect of chemical MK2 inhibition ontumorigenesis of the Apcmin+ mouse model. Apcmin/+ mice re-ceived PHA-767491 or PF-3644022 inhibitors and respectivevehicles, daily for 7 wk, starting at 8 wk of age. Mice receivingeither of the inhibitors showed a significant reduction in both thenumber of macroscopically visible tumors and their size, incomparison to their respective control groups (Fig. 1 K and L).Spleen weight was significantly reduced only in the case of thePF-3644022 inhibitor, indicating a more efficient amelioration ofdisease severity (Fig. 1M). These results are in agreement withthe genetic requirement of MK2 in intestinal tumorigenesis andprovide preclinical proof of concept for the potential of MK2inhibition in the treatment of CRCs.

MK2 Regulates Proliferation and Apoptosis in the Intestine of Apcmin/+

Mice. As MK2 regulates many different cellular processes thatcould affect cancer progression, we next examined key parametersrelated to the tumorigenic process in the intestine, such as epi-thelial cell proliferation and apoptosis. Proliferation, whichwas assessed by BrdU incorporation, was significantly lower in

Fig. 2. MK2 deficiency in Apcmin/+ leads to deregulation of proliferation, apoptosis, and angiogenesis without affecting inflammation in the intestine. (A)Representative immunohistochemical staining for BrdU in crypts and BrdU, pHH3, and CC3 in size-matched tumors of 8- and 22-wk-old Apcmin/+MK2+/+ andApcmin/+MK2D/D mice, respectively. (B) Quantification of BrdU-positive cells per crypt in 8-wk-old MK2+/+, MK2D/D, Apcmin/+MK2+/+, and Apcmin/+MK2D/D mice.Quantification of BrdU-positive cells, pHH3-positive cells, and CC3-positive cells in size-matched tumors of 22-wk-old Apcmin/+MK2+/+ and Apcmin/+MK2D/D

mice. Data represents mean ± SEM (n = 5–8 mice per genotype). (C) Western blot analysis of tumor lysates from 16-wk-old Apcmin/+MK2+/+ and Apcmin/+MK2D/D

mice. β-actin was used as a loading control. Data represent mean ± SEM (n = 6 mice per genotype). (D) Quantification of inflammatory cell infiltration of CD45+

leukocytes, CD45+CD11b+F4/80+ macrophages, CD45+CD11b+Gr1+ neutrophils, and CD45+CD4+ and CD8+ T cells was performed by FACS, in the small intestine of12-wk-old MK2+/+, MK2D/D, Apcmin/+MK2+/+, and Apcmin/+MK2D/D mice, and tumors from 22-wk-old Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice. Data representsmean ± SEM (n = 4–5 mice per genotype). Representative immunohistochemical staining of CD31+ microvessels (E) and quantification of CD31+ cells in size-matched tumors of 22-wk-old Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice (F). Data represents mean ± SEM (n = 5 mice per genotype). n.s., not significant; ***P <0.001. (Scale bars: A, columns 1, 3, and 4, and E, 50 μm; A, column 3, 100 μm.)

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Apcmin/+MK2D/D mice both in normal crypts and size-matchedmicroadenomas/adenomas at all time points examined (Fig. 2A and B and SI Appendix, Fig. S4 B and C). Similarly, phospho-Histone H3 (pHH3), a mitotic marker, was also reduced insize-matched tumors of the Apcmin/+ MK2D/D mice (Fig. 2 Aand B). Interestingly, proliferation was similar betweenMK2+/+

and MK2D/D mice, not carrying the Apcmin mutation, and be-tween MK2D/D and Apcmin/+MK2D/D (Fig. 2B and SI Appendix,Fig. S4A), indicating a possible role also in tumor initiation.Cell death, assessed by TUNEL staining, was similar in size-

matched tumors of Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice(SI Appendix, Fig. S4 D and E). Interestingly, however, immu-nohistochemistry for cleaved caspase 3 (CC3) showed a signifi-cant reduction of CC3-positive cells in Apcmin/+MK2D/D incomparison with size-matched tumors of Apcmin/+MK2+/+ mice(Fig. 2 A and B). Western blot analysis of Apcmin/+MK2+/+ andApcmin/+MK2D/D tumors confirmed these results and showed asignificant reduction in CC3 in MK2-deficient mice (Fig. 2 A–C).These results show a deregulation of cellular processes impli-cated in tumor growth, including proliferation and apoptosis. Ofthese, the significant decrease in proliferation is directly associ-ated with reduced tumorigenesis. Reduced CC3-mediated apo-ptosis is probably not causally linked to tumorigenesis but ratherreflects the suppressed growth and differential tissue context ofthe MK2-deficient tumors.

MK2 Deletion Modulates Angiogenesis, but Not Inflammation inApcmin/+ Tumors. Since MK2 plays a crucial role in the regula-tion of proinflammatory gene expression and the modulation ofimmune responses (16), we also measured immune cell in-filtration in the small intestine of 12-wk-old healthy MK2+/+ andMK2D/D and Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice and tu-mors of 22-wk-old Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice. Cellpreparations from the small intestine and tumors were stainedwith combinations of antibodies to identify activated macrophages(CD45+CD11b+F4/80+), neutrophils (CD45+CD11b+Gr1+),CD4+ (CD45+CD4+), and CD8+ T cells (CD45+CD8+), andquantifications were performed by flow cytometry analysis (SIAppendix, Fig. S5 A and B). There was no significant differencebetween Apcmin/+MK2D/D and Apcmin/+MK2+/+ mice in thenumbers of any of the above populations (Fig. 2D), indicatingthat MK2 does not regulate tumorigenesis through modulationof immune responses in the intestine.We also assessed whether regulation of adenoma formation by

MK2 was associated with a deregulation in angiogenesis byperforming immunohistochemistry with anti-CD31 and anti-CD34 antibodies. Indeed, CD31+ and CD34+ blood vessels weresignificantly less in tumors of the Apcmin/+MK2D/D mice incomparison with size-matched controls (Fig. 2 E and F and SIAppendix, Fig. S4 F and G). These results suggest an importantrole of MK2 in angiogenesis and neovascularization during in-testinal tumor development.

MK2 in Nonhematopoietic Cells Is Responsible for Tumor Promotion.To identify the cell type responsible for MK2’s tumor-promotingfunction in the intestine, we generated bone marrow chimeras byreconstituting Apcmin/+MK2+/+ and Apcmin/+MK2D/D recipientswith bone marrow from either MK2+/+ or MK2D/D donors.Apcmin/+MK2+/+ recipients, reconstituted with either MK2+/+ orMK2D/D bone marrow, did not show any significant difference ineither tumor multiplicity (Fig. 3A) or size (Fig. 3B). However, boththe number and size of tumors of Apcmin/+MK2D/D recipients thatreceived either MK2+/+ or MK2D/D bone marrow was signifi-cantly lower in comparison with the respectively transplantedApcmin/+MK2+/+mice. No difference could be observed between thetwo Apcmin/+MK2D/D recipient groups (Fig. 3 A and B). Accordingly,spleen size in the different groups followed their pathogenic pat-terns (Fig. 3C). Further histopathological analysis of tumors showed

an increased proportion of invasive adenocarcinomas/carcinomas inApcmin/+MK2+/+ mice receiving either MK2+/+ or MK2D/D bonemarrow, in contrast to Apcmin/+MK2D/D recipients, where signifi-cantly fewer adenocarcinomas/carcinomas could be observed (Fig.3D). These results demonstrate that MK2 regulates tumor multi-plicity, growth, and invasive potential in the Apcmin/+ mouse modelthrough its function in the nonhematopoietic cell compartment.

MK2 in Intestinal Epithelial and Endothelial Cells Contributes toTumor Progression Through Regulation of Apoptosis and Angiogenesis,Respectively. We next examined the cell-specific role of MK2 in theApcmin/+ mouse model by assessing its function in two major stromalintestinal populations, epithelial and endothelial cells. To this end,we crossed mice carrying the floxed MK2 allele (MK2f/f) with thetissue-specific Cre strains, Villin-Cre (27) and Tie1-Cre (28), on theApcmin/+ background to achieve cell-specific ablation of MK2 inintestinal epithelial (Apcmin/+MK2IECko) and endothelial cells(Apcmin/+MK2ECko), respectively. Deletion efficiency were verifiedin both mouse lines (Apcmin/+MK2IECko and Apcmin/+MK2ECko) byWestern blot/PCR analysis (SI Appendix, Fig. S6 A–D). Next, weanalyzed tumor development in these mice but found no differencesin either the incidence or the number of macroscopically visibletumors in comparison with their respective littermate controls(Apcmin/+MK2f/f) (Fig. 4A). However, both Apcmin/+MK2IECko andApcmin/+MK2ECko mice displayed a significant decrease in tumorsize (Fig. 4B). Additionally, histological grading of tumors showedthat Apcmin/+MK2IECko mice developed significantly less adenocar-cinomas/carcinomas compared with Apcmin/+MK2f/f mice, whiletheir development was similar between Apcmin/+MK2ECko andApcmin/+MK2f/f mice (Fig. 4C).Subsequently, we examined the effect of IEC- and EC-specific

MK2 deletion on cell proliferation, apoptosis, and angiogenesisin intestinal tumors. Immunohistochemical analysis showed asignificant decrease in the number of BrdU+ proliferating cells inApcmin/+MK2ECko, but not Apcmin/+MK2IECko size-matched micro-adenomas/adenomas (Fig. 4 D and E). On the contrary, apoptosis

Fig. 3. MK2 in the stroma is responsible for its tumor-promoting function.Tumor multiplicity (A), average tumor size (B), spleen weight (C), and ade-nocarcinoma incidence (D) in the intestine of the following bone marrowchimera groups: MK2+/+ > Apcmin/+MK2+/+ (n = 19), MK2D/D > Apcmin/+MK2+/+

(n = 19),MK2D/D > Apcmin/+MK2D/D (n = 20), andMK2+/+ > Apcmin/+MK2D/D (n =18) at 20 wk of age. Data refer to the cumulative results of two independentexperiments. Data represents mean ± SEM. n.s., not significant; **P < 0.01;***P < 0.001.

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assessed by CC3 staining was significantly increased only in size-matched tumors of Apcmin/+MK2IECko mice (Fig. 4 F and G). Thiswas further verified by Western blot analysis of isolated IECs fromApcmin/+MK2f/f and Apcmin/+MK2IECko mice, which showed increasedlevels of CC3, but no difference in other apoptosis and proliferationmarkers, such as Bak and PCNA, respectively (Fig. 4H). CD31+

microvessels were also significantly less in size-matched tumors ofApcmin/+MK2ECko mice (Fig. 4 I and J), which is consistent with re-duced angiogenesis in Apcmin/+MK2D/D mice. These results show thatneither epithelial nor endothelial MK2 alone is responsible for therole of MK2 in intestinal carcinogenesis, but they rather contribute totumor progression through regulating intestinal epithelial cell apo-ptosis and enhancing angiogenesis and proliferation, respectively.

MK2 Regulates Activation of Hsp27 in Mesenchymal Cells.One of themost important downstream mediators of MK2’s function is heatshock protein 27 (Hsp27), which is phosphorylated by MK2 inresponse to a variety of stimuli and strongly associates withcancer progression and metastasis (20, 29). Western blot analysisof Apcmin/+MK2+/+ and Apcmin/+MK2D/D tumors showed a sig-

nificant reduction in Hsp27 phosphorylation in the MK2-deficient background (Fig. 5A), which was further verified byimmunohistochemistry (Fig. 5B). Interestingly, p-Hsp27 stainingwas predominantly located in the stroma including the smoothmuscle layer, both in the normal mucosa and in tumors (Fig. 5B).For a more quantitative insight into the cell types that show

Hsp27 activation in the intestine, we performed FACS analysisof both normal and cancerous tissue from Apcmin/+MK2+/+ andApcmin/+MK2D/D mice (SI Appendix, Fig. S7). FACS analysis ofwild-type and Apcmin/+ mice showed that p-Hsp27 staining wasfound mainly in cells of the stromal compartment, which isnegative for the lineage-specific markers EpCAM, CD45, andTer119 (Lin−). CD31+ cells, and specifically podoplanin (PDPN)negative cells, showed increased phosphorylation of Hsp27, in-dicating specific activation in blood endothelial cells (BECs)(Fig. 5 C and D and SI Appendix, Fig. S7). However, deletion ofMK2 did not affect phosphorylation of Hsp27 in these cells,in contrast to what has been previously described for endothelial-specific regulation of Hsp27 activation by MK2 (Fig. 5 C andD) (30, 31).

Fig. 4. MK2 in intestinal epithelial and endothelial cells contribute to intestinal tumor growth and progression. Number of tumors (A), average tumor size(B), and quantification (C) of adenocarcinoma per mouse in 20-wk-old littermate controls Apcmin/+MK2f/f (n = 19) and Apcmin/+MK2IECko (n = 27) mice (Left),and littermate controls Apcmin/+MK2f/f (n = 15) and Apcmin/+MK2ECko (n = 14) mice (Right). Representative immunohistochemical staining of BrdU (D) andquantification for BrdU-positive cells (E) in size-matched tumors of 20-wk-old Apcmin/+MK2IECko and respective littermate Apcmin/+MK2f/f, Apcmin/+MK2ECko andrespective controls Apcmin/+MK2f/f mice. Data represents mean ± SEM (n = 5 mice per genotype). Representative immunohistochemical staining for CC3 (F)and quantification (G) for CC3-positive cells in size-matched tumors of 20-wk-old littermate Apcmin/+MK2IECko and respective littermates Apcmin/+MK2f/f,Apcmin/+MK2ECko and respective littermates Apcmin/+MK2f/f mice. Data represents mean ± SEM (n = 5 mice per genotype). Representative immunohistochemicalstaining (H) and quantification (I) for CD31-positive cells in size-matched tumors of 20-wk-old Apcmin/+MK2IECko and respective littermate Apcmin/+MK2f/f mice,Apcmin/+MK2ECko and respective controls Apcmin/+MK2f/f mice. Data represents mean ± SEM (n = 5 mice per genotype). (J) Western blot analysis of intestinalepithelial cells lysates from the small intestine of 12-wk-old littermate Apcmin/+MK2f/f and Apcmin/+MK2IECko mice (n = 3 mice per genotype). β-actin was used as aloading control. n.s., not significant; *P ≤ 0.05; **P < 0.01; ***P < 0.001. (Scale bars: D, 100 μm; F and H, 50 μm.)

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Interestingly, Hsp27 phosphorylation was particularly high inthe CD31−α-SMA+ mesenchymal compartment (Fig. 5E). De-letion of MK2 led to a significant decrease of p-Hsp27 inα-SMA–positive cells both in the normal and the cancerous tis-sue (Fig. 5E), revealing a hitherto unknown cell specificity forHsp27 activation by MK2 in the intestine. Immunohistochemicalcostaining with α-SMA further confirmed that Hsp27 was foundactivated in α-SMA+ cells in the intestine (Fig. 5F). In agreementwith these data, staining with antibodies against phospho-Hsp27has revealed a stromal pattern of Hsp27 activation in humantumors (32). These results indicate that MK2 regulates activationof Hsp27 in intestinal mesenchymal cells, both in homeostasisand cancer in the intestine.

MK2 in Mesenchymal Cells Promotes Intestinal Tumorigenesis. SinceHsp27 is preferentially expressed in α-SMA+ mesenchymal cellsand regulated by MK2, we next examined the pathophysiologicalrole of IMC-specific MK2 in the Apcmin/+ model by crossingMK2 conditional knockout mice with Twist2-Cre mice (33).Deletion efficiency was verified by Western blot analysis (SIAppendix, Fig. S6E). Apcmin/+MK2IMCko mice displayed a signif-icant reduction in both the number and size of tumors, as well as inspleen weight, in comparison with their littermate Apcmin/+MK2f/f

controls at 4 mo of age (Fig. 6 A and B). Tumors from theApcmin/+MK2IMCko showed a decrease in proliferation measured

by BrdU staining (Fig. 6 D and E), whereas there was an increasein apoptosis analyzed by CC3 staining (Fig. 6 D and F). Angio-genesis (Fig. 6 D and G) and Hsp27 phosphorylation were alsoreduced (Fig. 6D). These results show that deletion of MK2 inIMCs mimics in several features the phenotype of the completeknockout Apcmin/+MK2D/D mice.

MK2 Deletion Leads to Deregulation of Downstream Pathwaysin Intestinal Mesenchymal Cells, Which Affects Mesenchymal toEpithelial Communication. MK2 has been previously implicatedin the regulation of mesenchymal cell function. More specifi-cally, MK2 in fibroblasts promotes pulmonary fibrosis by medi-ating myofibroblast differentiation (34–36). Therefore, we initiallyexamined whether deletion of MK2 resulted in reduced fibroblastdifferentiation. We performed immunohistochemical staining forα-SMA in sections from Apcmin/+MK2+/+ and Apcmin/+MK2D/Dmice, but we did not detect any difference in mean signal intensityof α-SMA between size-matched tumors of the two genotypes (SIAppendix, Fig. S8A). Quantification with FACS analysis alsoshowed no difference in the number of α-SMA+ cells either in thenormal mucosa or in the cancerous tissue (SI Appendix, Fig. S8B),suggesting that MK2 does not regulate fibroblast differentiation inthe intestine.Next, we examined whether deletion of MK2 affected

Hsp27 activation and secretion of soluble mediators that could

Fig. 5. MK2 regulates phosphorylation of HSP27 in mesenchymal cells in vivo. (A) Western blot analysis of p-Hsp27 in tumors lysates from 16-wk-oldApcmin/+MK2+/+ and Apcmin/+MK2D/D mice. (Left) β-actin was used as a loading control. (Right) Densitometric analysis of p-Hsp27 protein relative to β-actin.Data represent mean ± SEM (n = 6 mice per genotype) (p-Hsp27 antibody from Cell Signaling, no. 2406). (B) Representative immunohistochemistry forp-Hsp27 staining in size-matched tumors of 22-wk-old Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice (p-Hsp27 antibody from Cell Signaling, no. 2406). RepresentativeFACS analysis (C), quantification by cell percentage and mean fluorescent intensity (MFI) (D) of p-Hsp27 expression in BECs in the normal mucosa of MK2+/+ andMK2D/D mice, and in tumors of Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice. Data represents mean ± SEM (n = 4 mice per genotype) (p-Hsp27 antibody from CellSignaling, no. 2406). (E) Representative FACS analysis and quantification of p-Hsp27 expression (MFI and by cell percentage) in α-SMA+ cells, in the normal mucosaof MK2+/+ and MK2D/D mice, and in tumors of Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice. Data represents mean ± SEM (n = 4 mice per genotype)(p-Hsp27 antibody from Cell Signaling, no. 2406). (F) Colocalization of p-Hsp27 and α-SMA in FFPE of small intestine and its respective higher magnification fromMK2+/+ mice (Left) and tumor sections and its respective higher magnification from Apcmin/+MK2+/+ mice (Right) (p-Hsp27 antibody from Cell Signaling, no. 9709).n.s., not significant; *P ≤ 0.05; **P < 0.01. (Scale bars: B, 25 μm; F, 50 μm.)

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influence epithelial/cancer cell differentiation, function, and me-tastasis upon various stimuli. To accomplish this, we isolated andcultured IMCs from 4-wk-old Apcmin/+MK2+/+ and Apcmin/+MK2D/D

mice, and treated them with known MK2 and Hsp27 inducers, suchas IL-1β, TNF, and TGF-β (36, 37), which are increased in tumorsand act in a paracrine way to induce fibroblast recruitment andactivation (38). All stimuli led to increased phosphorylation ofHsp27 in IMCs, which was abrogated in MK2-deficient cells (Fig.7A and SI Appendix, Fig. S9A). Accordingly, MK2-deficient cellsproduced less cytokines (IL-6), chemokines (MIP2), and MMPs(MMP9) in response to the above inducers (Fig. 7 B and C). Toidentify if effector molecule expression was indeed regulated byHsp27 activation downstream of MK2, we also used the specificHsp27 inhibitor KRIBB3 in vitro (39). Preincubation of Apcmin/+

IMCs with the Hsp27 inhibitor, followed by induction with IL-1β,TNF, and TGF-β, led to a decrease in Hsp27 phosphorylation (SIAppendix, Fig. S9B) and a significant reduction in the production ofcytokines (IL-6), chemokines (MIP2), and MMPs (MMP9) (Fig. 7D and E). These results show that MK2 is an important kinasedownstream of various stress-related stimuli in intestinal mesen-chymal cells, where it regulates the phosphorylation of Hsp27 andthe induction of tumor-promoting molecules.To further evaluate the importance of MK2-mediated in-

duction of effector molecules in IMCs and their function asparacrine mediators of epithelial cell activation, we performedcocultures of IMCs and intestinal organoids, both originatingfrom Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice. To accomplishthis, we plated IMCs and allowed them to create a monolayerbefore adding an equal number of intestinal crypts in Matrigeland culturing them in the absence of R-spondin 1. We also in-duced IMCs with IL-1β, TNF, and TGF-β for 8 h before additionof intestinal crypts. We then measured organoid number andsize, 5 d after plating. The number of organoids in MK2-deficientconditions was significantly lower in comparison with wild-type

conditions (Fig. 7F and SI Appendix, Fig. S10A). Induction withIL-1β and TNF led to increased organoid size, which was abro-gated in Apcmin/+MK2D/D cells (Fig. 7G and SI Appendix, Fig.S10A). Similarly, when Apcmin/+MK2+/+ and Apcmin/+MK2D/D

IMCs, induced with the above stimuli, were cocultured withApcmin/+ single tumor cells, there was a statistically significantdecrease in the number and size of tumor organoids (SI Ap-pendix, Fig. S10 B–D). These results suggest that MK2 anddownstream Hsp27 in intestinal mesenchymal can modulateepithelial cell functions in a paracrine way.

Mesenchymal MK2 Promotes Tumorigenesis in CAC, Without AffectingColitis Susceptibility. We next analyzed whether the stromal-specific functions of MK2 were also playing a role in anothermodel of intestinal carcinogenesis, the AOM/DSS model of CAC.Application of this protocol to MK2-deficient mice (MK2D/D) andtheir littermate controls (MK2+/+) showed that MK2D/D miceexhibited a significant decrease in the number of macroscopicallyvisible tumors in agreement with Ray et al. (22) (Fig. 8A). How-ever, in contrast to Ray et al. (22) MK2-deficient mice did developtumors, which were also smaller in size in comparison with wild-type mice (Fig. 8B). This is possibly due to our use of littermateand cohoused mice in these experiments, which allows for multipleand more accurate analysis. Histological analysis showed no dif-ference in inflammatory or tissue damage indices between the twogroups (Fig. 8C). FACS analysis of inflammatory cell infiltration intumors also did not show significant differences in CD45+, F4/80+,Gr-1+, and CD3+ cell infiltrates between MK2D/D mice and theirlittermates controls (Fig. 8D).To further assess whether the difference in tumorigenesis was

due to a reduced inflammatory response to DSS treatment, wealso subjected MK2D/D and their littermate controls to the acutemodel of DSS colitis (40). BothMK2D/D andMK2+/+mice displayedsimilar weight loss during the experiment, and no difference in

Fig. 6. Mesenchymal-specific genetic deletion of MK2 protects against tumorigenesis in the Apcmin/+ model. Tumor number (A), average tumor size andtumor size distribution (B), and spleen weight (C) in 16-wk-old Apcmin/+MK2f/f (n = 9) and Apcmin/+MK2IMCko (n = 7) mice. Data represents mean ± SEM. (D)Representative immunohistochemical staining for BrdU, CC3, CD31, and p-Hsp27 in size-matched tumors of 16-wk-old Apcmin/+MK2f/f and Apcmin/+MK2IMCko

mice. (Scale bars: column 1, 100 μm; 2 and 3, 50 μm; 4, 25 μm.) Quantification of BrdU+ (E), CC3+ (F), and CD31+ (G) cells in size-matched tumors of these mice(n = 5 mice per genotype). Data represents mean ± SEM. n.s., not significant; *P ≤ 0.05; **P < 0.01; ***P < 0.001.

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colitis index at the end of the protocol (Fig. 8 E and F). Takentogether, these results suggest that the tumor-promoting role ofMK2 in CAC, as in the Apcmin/+ model, is independent of itsproinflammatory function.Next, to further examine the cellular basis of the tumor-

promoting role of MK2, we crossed mice carrying the floxedMK2 allele (MK2f/f) with Lysozyme-Cre (LysM-Cre) (41), Villin-Cre (27), Tie1-Cre (28), and Twist2-Cre (33) mice to achieve cell-specific ablation of MK2 in myeloid cells (MK2Myeko) (SI Ap-pendix, Fig. S6 G–I), intestinal epithelial cells (MK2IECko),endothelial cells (MK2ECko), and intestinal mesenchymal cells(MK2IMCko), respectively, and subjected them to the AOM/DSSprotocol of CAC. From the different MK2 conditional knockoutmice only MK2IMCko mice showed a significant reduction in thenumber of tumors at the end of the protocol, while MK2IECko,MK2ECko, and MK2IMCko showed a reduction in tumor size incomparison with their respective littermate controls (Fig. 8 Gand H). Colitis score (Fig. 8I) did not show any difference in anyof the conditional knockout lines, in agreement with the phe-notype of MK2-deficient mice. These data suggest an importantprotumorigenic role for MK2 in stromal cells, and especiallyIMCs, during CAC, similar to its role in the Apcmin/+ model.

DiscussionIn this study, we show that the kinase MK2 has an importanttumor-promoting role in the intestine, which is associated withtumor growth and progression. The p38 kinase is the main up-stream regulator of MK2 and intestinal epithelial-specific p38

deletion has been shown to have both protumorigenic andantitumorigenic properties during CAC. Chemical inhibition ofp38 in established colon tumors leads to less tumor load, char-acterized by reduced Hsp27 phosphorylation (9, 42). In ourstudy, the use of MK2 inhibitors after tumor initiation led tosimilar results, such as reduced tumor number and size, whilephosphorylation of Hsp27 was also significantly decreased intumors of MK2-deficient mice. Our results, therefore, suggestthat MK2 mediates the protumorigenic properties of p38 andthat the potential use of MK2 inhibitors could prove a promisingalternative to p38 inhibition in intestinal cancer, and potentiallyother types of cancer (9, 18, 43).MK2 has been extensively studied for its role in inflammation

through the regulation of activation and production of proin-flammatory and antiinflammatory cytokines in immune cells(19). Accordingly, deletion of MK2 in vivo leads to decreasedimmune responses in several models of inflammatory diseases(18). In DSS colitis and AOM/DSS-induced CAC, MK2 knock-out mice have been shown earlier to express lower levels ofproinflammatory and antiinflammatory cytokines, which wasassociated with resistance to both colitis and carcinogenesis.The regulation of macrophage function by MK2 was proposedas a possible mechanism leading to this phenotype; however,experiments were not performed in littermate controls, Mk2−/−

did not develop any tumors, and restoration of MK2 in mac-rophages did not affect resistance of knockout mice to thismodel (22, 44). In the present study, we show that inflammatory in-filtration in MK2D/D tumors is unaffected in both the Apcmin/+ model

Fig. 7. MK2 and p-HSP27 regulate protumorigenic mediator secretion and subsequent epithelial activation. (A) Representative images of immunoblotdetection of p-MK2, MK2, and p-Hsp27 in whole protein extracts from Apcmin/+MK2+/+ and Apcmin/+MK2D/D primary IMC cultures after induction with IL-1β(10 ng/mL) (Left), TNF (10 ng/mL) (Center), and TGF-β1 (10 ng/mL) (Right) at the indicated time points. β-actin was used as a loading control. Data representsone of three independent experiments performed. (B) IL-6 and MIP2 were measured by ELISA in supernatants from Apcmin/+MK2+/+ and Apcmin/+MK2D/D

primary IMC cultures after induction IL-1β, TNF, and TGF-β1 for 24 h. Data represents mean ± SEM of one from four experiments performed in triplicates. (C)MMP9 and MMP2 zymography detection in supernatants from Apcmin/+MK2+/+ and Apcmin/+MK2D/D primary IMC cultures after induction with IL-1β, TNF, andTGF-β1 for 24 h. (D) IL-6 and MIP2 were measured by ELISA in supernatants from Apcmin/+ primary IMC cultures with and without prior incubation ofKRIBB3 upon induction of IL-1β, TNF, and TGF-β1 for 18 h. Data represents mean ± SEM from four experiments performed in triplicates. (E) MMP9 andMMP2 zymography detection in supernatants from Apcmin/+ primary IMC cultures with and without prior incubation of Hsp27 inhibitor (KRIBB3) after in-duction with IL-1β, TNF, and TGF-β1 for 18 h. Data represents one of four independent experiments performed. Organoid count (F) and organoid size (G) ofintestinal organoids cocultured with IMCs both from Apcmin/+MK2+/+ and Apcmin/+MK2D/D mice. IMCs were preincubated with IL-1β, TNF, and TGF-β1 for 8 h.Data represents mean ± SEM from one of three experiments performed in triplicates. n.s., not significant; *P ≤ 0.05; **P < 0.01; ***P < 0.001.

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and the AOM/DSS models of intestinal cancer. Moreover, thetumor-promoting role of MK2 is independent of its function in cellsof the hematopoietic compartment, including immune cells, as shownby bone-marrow transfer experiments and the use of myeloid-specific knockouts, respectively. In addition, MK2 deletion doesnot affect acute colitis in carefully controlled experiments. This isin contrast to its known role as an important inflammatory mod-ulator and suggests a more significant stromal-specific role at leastin epithelial cancers.The cell-specific role of MK2 has been studied mainly

through in vitro experiments, and focus has been placed on itsrole in cancer cells themselves, besides immune cells (14, 15,45). The generation of MK2 conditional knockout mice that wereport here can be thus useful for the evaluation of the path-ophysiological significance of MK2’s function in different celltypes. Our results show that MK2 in epithelial cells plays a rolein intestinal cancer, as its deletion leads to reductions of bothtumor size and invasive potential in both the Apcmin/+ andAOM/DSS models. This phenotype is similar to the inducibleepithelial-specific deletion of p38 after CAC development,where reduced tumor load is associated with increased apo-ptosis, reduced proliferation, and production of proinflammatorymediators (9). Accordingly, our epithelial-specific MK2 knockoutmice also show increased apoptosis. Surprisingly, however, epi-thelial MK2 deletion does not mimic the effect of its completedeletion in Apcmin/+ MK2-deficient mice, where apoptosis is de-creased, indicating that MK2-regulated epithelial apoptosis is notcausally linked to tumor progression in this model. Therefore, theeffect of MK2 in other cell types could play more important rolesin driving tumorigenesis in the intestine.In vitro and ex vivo studies have also suggested that MK2 has

important functions in endothelial cells. Although it does notaffect normal vascularization, it plays a role in arterial devel-opment and could affect neovascularization in disease, such ascancer (46). Our results, indeed, show that tumors of Apcmin/+

MK2-deficient mice show reduced angiogenesis. Deletion ofMK2 in endothelial cells led to reduced tumor size, which wasassociated with reduced angiogenesis and proliferation in tu-mors. This suggests that intrinsic MK2-regulated pathways inendothelial cells affect tumor progression in this model. Similar

reduction in tumor size but not multiplicity was also observedin the AOM/DSS model of CAC. Possible functions thatMK2 could regulate include VEGF and IL-1β−mediated tubeformation and cell migration (30, 47, 48).MK2 has also been implicated in the function and differenti-

ation of myofibroblasts mainly during lung fibrosis (34–36). Ourresults showed that deletion of MK2 in intestinal mesenchymalcells had the most profound of all cell types effect on tumormultiplicity and size, both in the Apcmin/+ and CAC models, andwas associated with decreased epithelial proliferation, increasedapoptosis, and decreased angiogenesis. In our study, the numberof α-SMA-positive cells was similar between wild-type and MK2-deficient mice, suggesting that the role of MK2 in myofibroblastdifferentiation is possibly tissue dependent. Interestingly, wefound that α-SMA+ cells, including smooth muscle cells andmyofibroblasts, were the cells that showed the highest levels ofphosphorylated Hsp27 both in the normal mucosa and tumors,and that this property was dependent on MK2 expression. Thisis consistent to previous reports on the expression pattern ofHsp27 in CRC, as well as its coexpression with α-SMA in CRClung metastases (32). Interestingly, phosphorylation of Hsp27,which is increased in blood endothelial cells, is not influencedby MK2 deletion, which indicates that the function of the p38/MK2/Hsp27 pathway is cell and tissue dependent. Induction ofmesenchymal cells with various MK2 and Hsp27 inducers fur-ther supported a MK2-dependent functional property of thispathway in mesenchymal cells. These and possibly other stimuli,which are abundant in the tumor microenvironment, induce theactivation of MK2 and Hsp27 and subsequently the down-stream production of cytokines, chemokines, and MMPs, whichmodulate the tumor microenvironment and signal throughIECs or cancer cells to induce tumor cell differentiation, sur-vival, and growth. They also suggest that mesenchymal cellshow immune-like responses, which are regulated by pathwaystraditionally functioning in immune cells.In conclusion, we show that the kinase MK2 regulates tumor

growth and progression in the intestine and could thus serve asa potential therapeutic target and a promising alternative top38 inhibition. We have further delineated its cellular andmolecular mechanism of action, which is mediated predominantly

Fig. 8. Genetic deletion of MK2 reduces tumor load in CAC. Number of macroscopically visible tumors per mouse (A), average tumor size (B), and colitis score(C) was measured in MK2D/D (n = 7) and MK2+/+ (n = 5) littermate controls at day 60 of the CAC model. Data are presented as mean ± SEM from one of threeindividual experiments performed, except in the case of tumor size graph, which is cumulative from three individual experiments. (D) Inflammatory cellinfiltration in colonic tumors of MK2+/+ and MK2D/D mice on day 60 of the experimental protocol. Quantification of CD45+ leukocytes, CD45+CD11b+F4/80+

macrophages, CD45+CD11b+Gr1+ neutrophils, and CD45+CD3+ T cells was performed by FACS. Data represents mean ± SEM (n = 3–4 mice per genotype). (E) Bodyweight loss ofMK2+/+ andMK2D/D during acute DSS colitis. (F) Colitis score were measured at day 8 after DSS administration. Data represent mean± SEM from oneof two experiments performed (n = 5–10mice per genotype). Tumor number (G), average tumor size (H), and colitis score (I) was measured inMK2Myeko,MK2IECko,MK2ECko, and MK2IMCko mice in comparison with MK2f/f littermate controls on day 60 after AOM/DSS administration. Data represent mean ± SEM from one ofthree (forMK2Myeko,MK2IECko, andMK2IMCkomice) or two (forMK2ECkomice) experiments performed (n = 7–13mice per genotype). Data represents mean ± SEM.n.s., not significant; **P < 0.01; ***P < 0.001.

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through its regulation of mesenchymal-specific effector moleculeproduction and is further supported by functions in epithelial andendothelial cells. These functions reflect the complexity of tissuemicroenvironments and the interplay between different cell typesin driving disease pathogenesis, while they highlight the impor-tance of mesenchymal cells in cancer progression.

Materials and MethodsDeleter-Cre (23), LysM-Cre (41), Tie1-Cre (28), and Villin-Cre (27) mice have beenpreviously described. Apcmin/+ (4) and Twist2-Cre (33) mice were purchased fromthe Jackson Laboratory. All experiments were approved by the InstitutionalCommittee of Protocol Evaluation in conjunction with the Veterinary ServiceManagement of the Hellenic Republic Prefecture of Attika. Details of generationand screening of complete and conditional MK2 knockout mice, animal models,and animal procedures are provided in SI Appendix, Supporting Materials and

Methods. Procedures for the isolation of IECs, ECs, TEPMs, IMCs, organoids, andcocultures are further elaborated in SI Appendix, Supporting Materials andMethods. Immunohistochemistry/immunofluorescence, ELISA, FACS, qRT-PCR,Western blot analysis, and statistical analysis were performed using standardprocedures detailed in SI Appendix, Supporting Materials and Methods.

ACKNOWLEDGMENTS. We thank Anna Katevaini, Dimitra Papadopoulou,Lida Iliopoulou, and Spiros Lalos for technical assistance in histopathology;Dr. D. Gumucio, Dr. I. Forster, Dr. R. Fassler, and Dr. K. Rajewsky for providingVillin-Cre mice, LysM-Cre mice, Tie1-Cre mice, and Deleter-Cre mice, respec-tively; our colleague Maria Apostolaki (deceased December 10, 2010), whodesigned and generated the MK2f/f and MK2D/D mouse strains; and theInfrafrontierGR infrastructure (cofunded by the European Regional Devel-opment Fund and Greek NSRF 2007–2013) for providing mouse hosting andphenotyping facilities. This work was supported by FP7 Advanced ERC grantMCs-inTEST Grant Agreement 340217 (to G.K.).

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