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Molecular mechanisms specific to colitis-associated cancers have been poorly characterized. Using comparative whole-genome expression profiling, we observed differential expression of epiregulin (EREG) in mouse models of colitis-associated, but not sporadic, colorectal cancer. Similarly, EREG expression was significantly upregulated in cohorts of patients with colitis-associated cancer. Furthermore, tumor-associated fibroblasts were identified as a major source of EREG in colitis-associated neoplasms. Functional studies showed that Ereg-deficient mice, although more prone to colitis, were strongly protected from colitis-associated tumors. Serial endoscopic studies revealed that EREG promoted tumor growth rather than initiation. Additionally, we demonstrated that fibroblast-derived EREG requires ERK activation to induce proliferation of intestinal epithelial cells (IEC) and tumor development in vivo. To demonstrate the functional relevance of EREG-producing tumor-associated fibroblasts, we developed a novel system for adoptive transfer of these cells via mini-endoscopic local injection. It was found that transfer of EREG-producing, but not Ereg-deficient, fibroblasts from tumors significantly augmented growth of colitis-associated neoplasms in vivo. In conclusion, our data indicate that EREG and tumor-associated fibroblasts play a crucial role in controlling tumor growth in colitis-associated neoplasms.
Inflammatory bowel disease (IBD) is a present health problem believed to have a complex genetic and environmental etiology that can severely compromise the life quality of affected individuals of all ages. IBD involves an overactive immune response in genetically predisposed individuals driven by the bacterial flora (1–3). Aside from the symptomatic disease burden caused by relapsing intestinal inflammation, IBD patients are threatened by long-term complications including increased susceptibility to colorectal cancer (CRC) development. The elevated risk for colitis-associated cancer (CAC) is well documented for ulcerative colitis (UC) (4), and more recent studies provided evidence of an increased intestinal cancer development in individuals with Crohn’s disease (CD) (5). Notably, the risk for CAC in IBD patients appears to be influenced by the inflammatory activity as well as the extent of colitis and duration of the disease. In fact, the severity of inflammation is a key risk factor for progression to colorectal neoplasia in UC (6). Even though the molecular mechanisms that explain how chronic inflammatory states contribute to the development of CAC are not fully understood, experimental models of colitis-associated tumorigenesis have suggested that chronic inflammation can facilitate tumor initiation and progression. In particular, it has been suggested that increased levels of reactive oxygen or nitrogen species may elevate the risk for mutations in intestinal epithelial cells (IEC) of the colon (7).
In humans, CACs share several characteristics with sporadic CRCs, which represent the most frequent group within the heterogeneous variety of CRCs. CAC and sporadic CRC also differ strikingly in numerous features (6). In sporadic CRC, healthy epithelium can typically turn into CRC in the adenoma-carcinoma sequence spanning several years. In contrast, CAC precursor lesions are often flat and multifocal, and such lesions may grow more rapidly.
Experimental data on molecular mechanisms that can modulate growth of CAC and sporadic CRC are increasing (8, 9). Numerous variables seem to be involved in influencing the development of intestinal neoplasms including the bacterial flora (10, 11). In addition, there are striking differences along the GI tract itself with respect to the cellular composition and the structure of the intestinal wall. Different regional susceptibilities for intestinal tumors are well known, but not sufficiently understood.
To improve translational relevance, our study focused on a direct comparison between experimental CAC and experimental sporadic CRC originating from the same rectosigmoid region of the lower GI tract that is the most frequent location for CAC and sporadic CRC in humans.
The molecular basis for characteristics specific to CAC as compared with sporadic CRC has not previously been clarified, although mechanisms such as different order of mutations in the multistep process of colon carcinogenesis have been hypothesized in humans (6). For the differential molecular analysis of CAC and sporadic CRC, we performed comparative whole-genome expression profiling between CAC and sporadic CRC in experimental mouse models and evaluated the potential role of differentially expressed candidate genes. Strikingly, functional studies including experiments with gene-deficient mice identified epiregulin (EREG) as a predominant regulatory factor for growth of CAC.
To uncover molecules specifically involved in the pathogenesis of CAC, we studied tumorigenesis in experimental models of CAC and sporadic CRC that mimic key characteristics of human CRC (12–14). Whereas tumors in the Apcmin/+ model (where Apc indicates adenomatous polyposis coli) of sporadic CRC typically exhibit exophytic, pedunculated polyps, flat and multifocal tumors are characteristically observed in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of CAC by endoscopy (Figure (Figure1).1). To identify molecular mechanisms giving rise to such different growth patterns, we profiled gene expression in both models using high-density microarray analysis (HDMA). Tumor growth was monitored by repetitive mini-colonoscopy, and tissue was harvested when tumors reached a size covering between one-fourth and up to one-half of the colonic circumference, i.e., representing an endoscopic size score of 4 (15).
The cellular and molecular microenvironment of the intestinal wall has marked regional differences along the gastrointestinal (GI) tract; all tumors, including those from the Apcmin/+ model, were obtained from the distal part of the colon. To minimize stage-related differences in transcriptional profiles, all tumors were of the same size. Finally, to reduce the influence of different cellular compositions within the colonic wall, we used purified IEC from the distal colon as a control.
Whole-genome expression screening was performed with Affymetrix-GeneChips after purification of total RNA from tumors and respective control IEC. Hierarchical clustering as visualized by heat map analysis revealed groups of genes with similar expression patterns (Figure (Figure2A).2A). Expression profiles were then subjected to groupwise comparison, and advanced significance analysis between the tumors from both models revealed 924 probe sets (603 down, 321 up) with significance criteria of P < 0.05 after the Benjamini-Hochberg correction and at least a 2-fold change. The distribution of genes was visualized with a volcano plot (Figure (Figure2B).2B). The 25 genes with the greatest up or down differential expression showed a fold change (FC) ranging from 6 to 40 (Figure (Figure2C).2C). Among the genes with higher expression in the CAC model were several immune response–related genes such as IgI-V1, IgkC, or Arg1, which might be induced by immune cell infiltration. In addition, genes that have been related to tumor progression such as Malat1 and Klf5 were elevated in CAC. Among probe sets showing lower expression in the CAC model were several genes related to cell structure organization such as Pfn2, Actn1, and Mark1. Next, we performed Gene Ontology (GO) Enrichment Analysis using the list of differentially expressed upregulated genes, which identified GO terms such as cell proliferation, response to wounding, inflammatory response, and EGF binding in the CAC model compared with the sporadic CRC model (Figure (Figure2D).2D). In contrast, GO categories such as WNT receptor signaling pathway or TGF-β receptor signaling were not significantly enriched suggesting that no major differences exist in these pathways between the 2 models.
To further investigate differences, we performed significance analysis between the microarrays from the CAC model and control epithelia as well as the sporadic CRC model and control epithelia. This analysis revealed a high number of probe sets with differential expression (P < 0.05; FC > 2) (Figure (Figure2E2E and Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI63748DS1). By comparing these gene lists, we found overlapping gene expression patterns, as shown by Venn diagrams, and identified 387 probe sets that were exclusively upregulated in CAC but not in sporadic CRC (Figure (Figure2F).2F). We further refined our analysis by comparing the lists of probe sets “differentially upregulated in CAC model only” with the gene list “upregulated in CAC model versus sporadic CRC model” (Figure (Figure2,2, B, E, and F). We obtained another list of genes, in which EREG, a member of the EGF family, ranked at position 3 of the most upregulated genes specific to CAC (Table (Table1).1). Of note, EREG was also identified on the list of top induced probe sets when a direct comparison between both tumor entities was performed (Figure (Figure2C).2C). Thus, EREG was differentially expressed between CAC and controls, CAC and sporadic CRC, but not sporadic CRC and controls (Figure (Figure2,2, B and E). Surprisingly, other EGF-like ligands were not differentially expressed in the comparative analysis between the 2 models (Supplemental Figure 1), suggesting selective differential regulation of the EREG gene. Subsequent validation experiments using a larger panel of mouse tumors and control tissue by quantitative PCR (qPCR) confirmed the observations from HDMA.
To investigate the potential relevance of EREG for human CRC, we next studied tissue from different stages of tumor development including sporadic and colitis-associated high grade dysplasias (HGDS) and sporadic CRC as well as CAC (Figure (Figure3,3, A and B). Strikingly, the expression of EREG, as assessed by immunohistochemistry, was highly significantly elevated in colitis-associated dysplasias and carcinoma as compared with colonic tissue from control patients (P < 0.01). In contrast, the expression of EREG in specimens from sporadic tumor development was lower, although still elevated at the average level compared with normal colonic tissue. Of note, EREG expression appeared to be more heterogenous in sporadic as compared with colitis-associated neoplasms. Consistently, quantitative analysis of EREG expressing cells revealed a significant increase in colitis-associated neoplasms (UC-associated HGDs and cancer) as compared with sporadic neoplasms (sporadic HGDs and cancer) (Figure (Figure33C).
Additional differences between sporadic and UC-associated neoplasms were noted with regard to the cellular source of EREG production. In particular, we detected that EREG expression in colitis-associated tumors was dominated by tumor stromal cells, whereas EREG expression in sporadic tumors was often found in patches of epithelial cells that showed basolateral EREG enrichment. Taken together, these findings highlighted the potential functional relevance of EREG in UC-associated tumor lesions in humans.
Since data using gene expression profiling and immunohistochemistry were consistent with a potential regulatory role of EREG in colitis-associated tumors, we next addressed the functional role of this growth factor in experimental tumorigenesis. In these studies, we took advantage of Ereg–/– mice and performed experiments using the AOM/DSS model of colitis-associated tumor growth. Strikingly and in marked contrast to previous studies on sporadic intestinal tumorigenesis in the Apcmin/+ small intestinal model (16), Ereg–/– mice were strongly protected from tumor development in the mouse AOM/DSS colonic model. This observation was verified by in vivo imaging of integrin αVβ3, macroscopic inspection, high-resolution endoscopy, and histopathological analysis of H&E-stained cross sections (Figure (Figure4,4, A–E). In fact, endoscopic scoring demonstrated a significant difference in the tumor load between Ereg–/– mice and WT controls (P < 0.01). Of note, although we detected a difference in tumor numbers (Figure (Figure4D),4D), the striking difference in tumor load (Figure (Figure4E)4E) was particularly caused by significant differences in tumor sizes between WT and Ereg–/– mice (Figure (Figure5).5). To analyze the kinetics of tumor development in both groups, we performed serial endoscopies in longitudinal studies directly addressing tumor growth over time. Whereas early neoplastic lesions were detected around days 15 to 20 in both groups, tumor load analysis showed an increasing difference between WT and Ereg–/– mice at later time points (Figure (Figure5,5, A and B). In fact, endoscopic analyses suggested that tumor growth is inhibited in Ereg–/– mice at later time points, resulting in slower growth rates and a persistent majority of small tumors compared with the composition of tumor load in WT controls. In WT mice, almost one-fourth of all tumors were large (sizes 4 or 5) at day 80. In contrast, large tumors were not observed in Ereg–/– animals by day 80 and very small tumors (size 1) accounted for more than 50% of all tumors in Ereg–/– mice even at day 80 (Figure (Figure5C).5C). Thus, the strong reduction in tumor load over time in Ereg–/– mice is due to a reduction of large tumors, suggesting a key role of EREG in tumor growth promotion rather than initiation.
As previous studies demonstrated a direct link between inflammatory activity and tumor growth in experimental colitis-associated tumorigenesis (17, 18), we next addressed EREG expression during DSS-induced colonic inflammation. Consistent with previous findings showing a strong temporary increase in intestinal EREG levels in acute colitis (19), we noted repeated more than 10-fold upregulation of EREG expression during DSS cycles, mimicking flares of increased activity in IBD (Figure (Figure6A).6A). Interestingly, we observed a slight increase of average EREG expression over time, suggesting a marked and repeated EREG induction by flares in chronic intestinal inflammation. Although Ereg–/– mice were more susceptible to acute DSS colitis than WT controls (16), no significant differences in colonic inflammation between these mouse strains were found during chronic DSS colitis (Supplemental Figure 2). Thus, although EREG expression is induced by inflammation, this protein does not seem to importantly regulate activity of chronic intestinal inflammation.
To explore possible alternative functions of EREG, we performed studies on the role of this protein in epithelial wound healing. In initial studies, we used an in vitro assay for cell migration in which a mouse colonic epithelial cell line (CMT93) was grown to confluence before gaps of defined size were scratched into the cell layer. Subsequently, cells were cultured in the presence or absence of EREG followed by assessment of gap sizes. Strikingly, wound gap closure in the presence of EREG was significantly accelerated (about 50% of remaining gap size after 18 hours compared with untreated cells), suggesting a prominent role of EREG during reconstitution after epithelial injury (Figure (Figure6,6, B and C). To study whether EREG also contributes to intestinal wound gap closure in vivo, we generated epithelial defects in the distal colon of Ereg–/– mice and control animals by use of the biopsy forceps in mini-endoscopy. Wound healing was monitored by serial endoscopy (20). Consistent with our data from in vitro experiments, intestinal wound healing was significantly delayed in Ereg–/– mice compared with controls, indicating a major regulatory function of EREG in epithelial wound healing in vivo (Figure (Figure6,6, D and E). Consistently, studies with heterozygous lacZ-Ereg-reporter mice demonstrated the accumulation of EREG-producing cells around the wound bed in response to in vivo wounding in the distal colon (Figure (Figure66F).
To directly demonstrate effects of EREG on epithelial cell proliferation, full-thickness rectosigmoidal pieces were incubated with mock or recombinant EREG and stained for Ki67 (Figure (Figure7,7, A and B). EREG treatment led to a significant increase of epithelial cell proliferation (P < 0.05). To test whether EREG could directly increase the number of IECs and promote growth of colon tumors, we applied recombinant EREG to CMT93 cells and early colon tumors in vivo. Interestingly, we observed a significant increase in epithelial cell numbers after incubation in the presence of EREG, providing further evidence for an increase in proliferation mediated by EREG (P < 0.05). Of note, the number of cells showed a dose-dependent increase in the presence of EREG at 48 hours compared with control cells (Figure (Figure7C).7C). We next injected EREG or other EGF-like ligands into WT mice during AOM/DSS treatment and performed endoscopic tumor screening at day 30 (Figure (Figure7D).7D). We chose animals with similar tumor load and performed local injections of EREG, EGF, amphiregulin (AREG), or equal volumes of PBS. The injections were applied during endoscopy using a flexible syringe device near the base of similar sized small tumors every 3–4 days. Tumor growth was monitored by endoscopy, and mice with EREG injections displayed a significantly accelerated increase in tumor load compared with mice that had obtained PBS injections (P < 0.05). In contrast, other EGFR ligands such as EGF and AREG were less potent and did not result in a significant increase of tumor load as compared with the PBS control group (Figure (Figure7,7, E and F).
In summary, these observations suggest that colitis-associated EREG expression promote not only intestinal wound healing, but also tumor growth.
The cellular source of EREG has been assigned to epithelial and submucosal cells during acute inflammation (16). To identify the cellular source of EREG in the AOM/DSS model of CAC, we took advantage of the LacZ reporter gene knockin of Ereg-deficient mice and performed X-gal stainings in heterozygous animals. It was found that the majority of cells that stained positive for EREG in AOM/DSS tumors were stromal cells (Figure (Figure8A).8A). In addition, we detected some EREG-expressing epithelial cells. As fibroblasts display a frequent cell type in the tumor microenvironment that may produce regulatory molecules (21), we next addressed the possibility that cancer-associated fibroblasts are the major cellular source of EREG production in AOM/DSS tumors.
To test this hypothesis, we performed consecutive stainings of cross sections from AOM/DSS-tumors of heterozygous lacZ-Ereg-reporter mice by using both X-gal staining and confocal immunohistochemistry. Here. we could show that over two-thirds of all EREG-expressing cells and virtually all EREG-expressing stromal cells colocalized with fibroblast markers including vimentin (VIM) and fibroblast-specific protein-1 (FSP1, also known as S100A4) (Figure (Figure8,8, A and B). A similar expression pattern could be demonstrated for PDGFR-β (Figure (Figure8A).8A). In addition, approximately 60% of EREG-expressing stromal cells could be colocalized with α-SMA (Figure (Figure8A),8A), indicating an activated myofibroblastic phenotype in a substantial number of EREG-expressing cells in colitis-associated tumors. To verify whether tumor-associated fibroblasts represent also a major cellular source of EREG in CAC in humans, in an additional series of experiments, we stained samples from patients with UC-associated colorectal neoplasms with antibodies against fibroblast markers and EREG. In congruence with our observations from the mouse model of CAC, EREG-expressing stromal cells in human CAC demonstrated coexpression with the fibroblast marker VIM (Figure (Figure8C),8C), suggesting that EREG-producing tumor-associated fibroblasts are present in both murine and human colitis-associated neoplasms.
To further characterize EREG-producing cells in colitis-associated neoplasms, we next isolated fibroblasts from AOM/DSS tumors of heterozygous lacZ-Ereg-reporter mice. The purity of these fibroblast cultures was assessed by immunocytochemical analysis, which revealed a high number of cells expressing proteins related to tumor-associated fibroblasts including VIM (>90%), FSP1 (>90%), PDGFR-β (90%), versican (VCAN, also known as chondroitin sulfate proteoglycan 2 or NG2) (85%), and α-SMA (80%). Strikingly, EREG expression was seen in more than 75% of the cells in these fibroblast cultures, as demonstrated by X-gal stainings (Figure (Figure8,8, D and E).
Whereas lacZ staining was useful to localize and count the number of EREG-expressing cells in murine cell culture, we took advantage of real-time qPCR to quantify the expression of EREG in tumor fibroblasts. Remarkably, these cells produced high amounts of EREG and a significant, more than 10-fold difference in EREG production was noted between tumor-associated fibroblasts and IEC (Figure (Figure8F).8F). Of note, the addition of recombinant EREG to tumor fibroblasts significantly increased EREG production, suggesting the presence of additional self-perpetuating autocrine and paracrine loops in these cells (Figure (Figure88F).
In subsequent experiments, we aimed at the identification of signaling pathways inducing EREG expression in tumor-associated fibroblasts. It was found that tumor-associated fibroblasts produce high baseline levels of EREG, which could be further enhanced by in vitro stimulation with LPS or TNF-α (Figure (Figure8G).8G). As TNF-α and bacterial proteins such as LPS are thought to play a major role in IBDs (1, 22), these findings suggested that microbial products and proinflammatory molecules released during intestinal inflammation are important inducers of local EREG production by tumor-associated fibroblasts in colitis-associated neoplasms.
In subsequent studies, we analyzed whether tumor fibroblast–derived EREG may affect growth of IEC. Accordingly, mouse IEC (cell line CMT93) were grown in the presence of conditioned medium from AOM/DSS tumor fibroblasts purified from WT and Ereg–/– mice, respectively. Strikingly, the conditioned medium obtained from the WT tumor fibroblasts resulted in a significant increase in the epithelial cell number after 48 hours, whereas the conditioned medium from Ereg–/– cells did not induce significant changes in cell numbers (Figure (Figure9A).9A). These findings indicated that fibroblast-derived EREG favors proliferation of IEC.
In subsequent studies, we addressed the possibility that EREG derived from tumor fibroblasts augments growth of colitis-associated neoplasms in vivo. To further evaluate the in vivo relevance of fibroblast-derived EREG production for the growth of CAC in the AOM/DSS model, we intended to set up studies with adoptive transfer of tumor-derived fibroblasts. Accordingly, we developed an adoptive transfer system of fibroblasts via endoscopically guided local injections into small tumors (endoscopic tumor score 1). In a first set of experiments, we validated this injection technique and injected portions of 50 μl of a fibroblast suspension from heterozygous lacZ-Ereg-reporter mice (105 cells) into tumors of WT mice. At day 2 after injection, tumors were harvested and stained for X-gal to assess EREG expression. These studies demonstrated not only the presence of transferred fibroblasts surrounding the tumor tissue, but also showed EREG expression in transferred cells within the tumor stroma (Figure (Figure9,9, B and C).
We next treated Ereg–/– mice with AOM and 1 cycle of DSS and performed endoscopic tumor screening at day 30 to ensure equal average tumor scores in all groups. We then injected Ereg–/– or WT fibroblasts from AOM/DSS-treated mice into small tumors of these Ereg–/– mice during endoscopy every 5 days. An additional group of WT mice did not receive tumor-associated fibroblasts and served as a control group. In consecutive studies, changes in tumor load were monitored endoscopically in all 3 groups of animals (Figure (Figure9,9, D–F). AOM/DSS-treated Ereg–/– mice repetitively injected with Ereg–/– fibroblasts showed significantly slower tumor growth as compared with AOM/DSS treated Ereg+/+ (WT) mice without fibroblast injections (P < 0.05). However, Ereg-deficient mice given WT fibroblasts showed significantly augmented tumor growth as compared with Ereg-deficient mice given Ereg-deficient fibroblasts (Figure (Figure9,9, E and F), suggesting that EREG from tumor-associated fibroblasts induces tumor growth in vivo. Furthermore, Ereg–/– animals repetitively treated with WT tumor fibroblasts showed no significant differences compared with AOM/DSS-treated WT control mice, suggesting that adoptive transfer of EREG-producing but not EREG-deficient tumor-associated fibroblasts is sufficient to abrogate the differences in tumor growth between WT and Ereg-deficient mice. In summary, these observations indicate that colitis-associated EREG expression by tumor fibroblasts can promote tumor growth in the AOM/DSS model in vivo.
EREG belongs to the EGF-like family of growth factors that can signal through of ERBB receptors, which can activate several pathways potentially involved in tumor growth such as ERK (also known as ERK1/2, MAPK3/1, or p44/p42-MAPK), p38-MAPK, and STAT3 pathways (23). Thus, we tested a number of downstream targets potentially involved in EREG signaling in our experimental model of CAC. Our data showed that EREG causes a pronounced activation of ERK in IEC in vitro (Figure (Figure10A).10A). In contrast, other major pathways related to tumor development including p38-MAPK and STAT3 showed little or no changes upon stimulation with EREG (data not shown).
To functionally address the role of ERK as a potential downstream effector molecule for the EREG-induced promotion of tumor growth, we tested whether the ERK inhibitors U0126 and PD98059 could counteract EREG-mediated effects on IEC and tumors. Indeed, while EREG significantly enhanced the proliferation of colonic epithelial cells (P < 0.01), this effect was completely abrogated by ERK inhibitors (Figure (Figure10B),10B), suggesting that ERK signaling controls EREG-mediated effects on epithelial cell proliferation. Furthermore, we detected that high levels of ERK phosphorylation in the AOM/DSS model could be blocked by incubation with these specific inhibitors despite the presence of EREG. These findings are consistent with ERK acting as the downstream effector of EREG-mediated tumor growth promotion in vitro and in vivo (Figure (Figure10C).10C). To address the in vivo relevance of this observation, we performed repetitive local injections into the base of small tumors during endoscopy and monitored tumor growth serially (Figure (Figure10D).10D). Interestingly, EREG injections resulted in accelerated tumor growth, while specific ERK inhibition with U0126 significantly suppressed tumor development (Figure (Figure10,10, E and F). Of note, the same ERK inhibitor was markedly less potent in the Apcmin/+ model in sporadic colorectal tumorigenesis, as this inhibitor caused no significant reduction of tumor load in the Apcmin/+ model (Figure (Figure10,10, E and F).
Thus, our data shows that interference with ERK signaling can block EREG-mediated growth promotion of experimental CAC.
In the present study, we aimed to identify genes specifically involved in the development of CAC as opposed to sporadic CRC. By comparative gene expression analysis in murine models of CAC and CRC, we demonstrated that the protein EREG is specifically enriched in CAC and provided functional evidence that EREG functions as a key tumor growth promoter. Our findings are particularly remarkable considering that EREG-deficient mice develop more severe acute experimental colitis (16), excluding the possibility that EREG controls tumor growth by directly regulating colitis activity. Furthermore, we noted augmented expression of EREG in the colon of patients with IBD and colitis-associated dysplasia or CAC, highlighting the potential relevance of EREG for CAC growth in humans. In contrast, EREG expression in samples from sporadic tumor development was significantly lower and highly variable (24). To our knowledge, EREG is therefore the first epithelial growth factor to be recognized that preferentially controls growth of colitis-associated tumors.
In studies looking at the expression of EREG in the AOM/DSS model of colitis, we noted that EREG is repeatedly induced by flares in chronic colitis. In addition, tumor fibroblasts were identified as the major cellular source of EREG production in experimental colitis and patients with CAC. Functionally, EREG was shown to promote wound healing in IEC and experimental colitis in vivo by facilitating cell migration and activating proliferation of epithelial cells. Thus, our observations provide further evidence for a close association among intestinal injury, EREG induction, and EREG-mediated mucosal healing, which is consistent with recent work demonstrating high EREG production during intestinal injury (19, 25). In this context, it will be interesting to study a potential relationship between EREG and WNT5A in future studies, as WNT5A-dependent formation and subdivision of wound channels into crypts upon wounding has been very recently shown to be important during colonic crypt regeneration (26).
Several recent publications suggested that the inflammatory degree dictates the risk for colitis-associated tumor development (27, 28) Specifically, tumor development in the AOM/DSS model has been linked to STAT3 signaling in IEC induced by proinflammatory mediators such as IL-6 and IL-11 (29, 30). VEGF (31) and TNF receptor signaling events (32) also influence CAC growth in this model. Our findings highlight yet another key regulatory pathway mediated by EREG that drives tumor growth in experimental colitis. In contrast to the above mediators, however, EREG appears to specifically control inflammation-associated tumor growth in the AOM/DSS model rather than sporadic tumor growth in the Apcmin/+ model.
Tumor-associated fibroblasts were identified as key producers of EREG in experimental colitis–associated neoplasms and patients with CAC. The proinflammatory cytokine TNF-α was shown to be a potent inducer of EREG production, suggesting that TNF-α produced during experimental colitis (33) and human UC (34) drives EREG expression. Furthermore, the TLR4 ligand LPS markedly augmented EREG production. Of note, Tlr4–/– mice are more susceptible to experimental colitis, but are also protected from colitis-associated tumor development (35, 36). Another mechanism for EREG induction in colitis-associated neoplasms might involve hypomethylation, although we could not detect hypomethylation of the Ereg promoter (data not shown). Furthermore, EREG was a potent inducer of its own expression, suggesting the existence of autoregulatory loops augmenting EREG levels.
The role of cancer-associated fibroblasts in tumorigenesis has not been fully clarified yet. However, previous studies reported that such fibroblasts include subpopulations that are highly heterogeneous (37, 38). Furthermore, it was found that these cells have pleiotropic functions and may favor tumor promotion via secreted growth and angiogenic factors such as IL-8 (39), VEGF (31, 40), FGF-2 (41), and SDF-1 (42). Additional publications have highlighted a crucial role of cancer-associated fibroblasts in inflammation-associated carcinogenesis of the skin and stomach, respectively (43, 44). In contrast, the role of these cells in colitis-associated tumorigenesis has not been previously addressed. Here, we demonstrated that EREG is expressed by tumor-associated fibroblasts in the AOM/DSS model and patients with colitis-associated neoplasms. As the acquisition of a myofibroblastic phenotype is a common feature for tumor-associated fibroblasts (45), we analyzed the relationship between α-SMA–expressing fibroblasts and EREG expression in the AOM/DSS model. It was found that EREG expression is not limited to α-SMA–expressing tumor-associated fibroblasts, which is in line with studies showing that the tumor-promoting properties of tumor-associated fibroblasts are not mandatorily linked to α-SMA expression (37, 43). Subsequently, we took advantage of a newly developed adoptive transfer system to demonstrate that EREG producing, but not EREG-deficient, tumor-associated fibroblasts augment tumor cell growth in experimental colitis–associated neoplasms. These findings underline what we believe is a novel role of tumor-associated fibroblasts in colitis-associated neoplasms by directly stimulating cell proliferation via EREG production.
EREG has previously been shown to induce EGFR phosphorylation and cell proliferation in IEC (19, 23), and such EGFR signaling is known to induce ERK activation (27, 46, 47). Studies on the signaling events induced by EREG activation showed induction of ERK upon stimulation of IEC with EREG. Functional studies using ERK blockade revealed that ERK is essential for EREG-mediated induction of epithelial cell proliferation, suggesting that ERK and EGFR induce tumor cell growth. However, we cannot rule out that other ERBB family receptors may be involved in EREG signaling, since EREG can also signal via other ERBB molecules (48). Furthermore, selective targeting of EGFR signaling has been recently suggested to augment tumor growth in the AOM/DSS model (27), consistent with the idea that other ERBB receptors might be involved in EREG signaling. Nevertheless, ERK inhibitors suppressed the proliferative effects of EREG on epithelial cells in the AOM/DSS model in vivo, highlighting a crucial role of ERK for EREG function. Interestingly, ERK activation has recently been reported to drive sporadic intestinal tumorigenesis in Apcmin/+ mice (49), and EGFR blockade resulting in ERK inhibition has been successfully applied in Apcmin/+ mice in the intestine (50). However, our study indicates that ERK inhibition is substantially more potent in the AOM/DSS model of CAC, suggesting that ERK blockade might be particularly effective as a novel therapeutic strategy for patients with CAC.
The EREG/ERK pathway appears to be particularly relevant for inflammation-associated colorectal tumors, as mucosal inflammation results in marked production of potent EREG inducers such as LPS and TNF-α. Subsequently, EREG may further augment its own production by an autoregulatory feedback loop in tumor-associated fibroblasts, thereby explaining the particular relevance of EREG for inflammation-associated neoplasms. Thus, our data suggest that ERK activation links EREG to the growth of CAC. The functional relevance of these findings was underlined by the observation that growth of experimental CAC could be induced by transfer of EREG-producing tumor-associated fibroblasts or recombinant EREG via ERK activation, while EREG deficiency substantially suppressed tumor growth. Thus, inhibition of EREG function and blockade of EREG production by tumor-associated fibroblasts emerges as a promising innovative approach for the blockade of colitis-associated tumorigenesis. As patients with CAC show markedly higher levels of EREG than controls, these results open new avenues for therapy of this disease.
Ereg–/– mice (B6.129S6-Eregtm1Dwt) are genetically altered by a β-geo cassette knocked into the first exon of the Ereg gene, as previously described (16). Our studies included animals from P.-L. Tharaux′ colony of Ereg–/– mice (Laboratory for Optics and Biosciences & Cardiovascular Research Center, Inserm Ecole Polytechnique, Paris, France). Ereg–/– mice had been bred congenic onto C57BL6/J for more than 10 generations. C57BL/6J-Apcmin/+/J and control mice on the same genetic background were obtained from the animal facilities of the Johannes Gutenberg-Universität Mainz and the Medical Clinic 1, Friedrich-Alexander-Universität Erlangen-Nürnberg. All mice were kept under specific pathogen–free conditions.
Human specimens were obtained from the Institute of Pathology in Bayreuth and the Medical Clinic 1, Friedrich-Alexander-Universität Erlangen-Nürnberg, where they had been taken from routine colonoscopic samples. The analysis included 47 samples (non-IBD control patients, n = 7; sporadic HGD, n = 12, sporadic CRC, n = 11; colitis-associated HGD, n = 9; CAC, n = 8).
For investigations into experimental colitis–associated tumorigenesis, a single i.p. injection of AOM (10 mg/kg) (Sigma-Aldrich) was followed by 3 cycles of DSS administration, with each cycle comprising 1 week of 2.5% (w/v) DSS (MP Biomedicals) in drinking water followed by 2 weeks of regular water (14). For studies of sporadic tumorigenesis, the Apcmin/+ model was used (12). Tumor development was monitored by the Colorview endoscopic system (Karl Storz), as previously described (15). Chronically relapsing colitis was induced by 3 cycles of 2.5% DSS, as described above.
Tumors were harvested at size 4 in endoscopic scoring (15). All tumors and control epithelia that were included in HDMA originated from the lower sixth of the colon. Epithelial purification was performed as previously described (51) with slight modifications. In brief, distal colonic specimens were incubated in prewarmed HBSS supplemented with EDTA (2 mM), EGTA (1 mM), DTT, and FCS (1%) with gentle agitation at 37°C for 20 minutes. The epithelial purity was routinely at 80%–85%, as assessed by microscopic quantitative analysis.
For RNA purification, tumors and purified distal colon epithelium were placed in RNeasy lysis buffer (QIAGEN), disrupted, and homogenized using a master mill before being processed by using RNeasy Mini Kits (QIAGEN).
Whole-genome transcriptome analysis was performed with 5 μg high quality total RNA per sample at the HDMA core facility of the Johannes Gutenberg-Universität in Mainz. Data analysis was performed with ArrayAssist and PathwayArchitect software (Agilent Technologies). Details about microarray analysis have been deposited under accession number GSE43338 at NCBI GEO.
Multispectral fluorescence imaging was done with the Maestro imaging system (Intas). Integrin αVβ3 expression could be visualized after i.v. injection of 100 μl fluorescence dye integriSense (Visen Medical).
Histopathological analysis was performed on paraffin-embedded tissue after H&E staining. Immunofluorescence of cryosections or paraffin-embedded tissue was done using the TSA Cy3 system (PerkinElmer) (17). Primary antibodies were goat anti-human EREG (polyclonal; R&D), rabbit anti-human FSP1/S100A4 (polyclonal; Lifespan Bioscience), rabbit anti-human PDGFR-β (clone 28E1; Cell Signaling), rabbit anti-mouse α-SMA (polyclonal, Abcam), and rat anti-mouse Ki67 (Dako). Rabbit anti-human VIM was used as an AF488-conjugated antibody (Cell Signaling). Nuclei were counterstained with DAPI (Invitrogen), and fluorescence analysis was performed with an IX70 (Olympus) or SP5 (Leica). LacZ staining on tumors or purified cells was performed as previously described (16). For quantitative analyses, 4–5 representative high power fields (×40) per sample were scored in a blinded fashion.
For fibroblast isolation, colonic tumors were cut into small pieces and incubated with complete medium including antibiotics and antimycotics for 15 minutes at 37°C. After 2 washes, tumor pieces were incubated with an enzyme cocktail made of trypsin (2.5 mg/ml; Difco), collagenase IV (400 U/ml), and DNase1 (350 U/ml) for 30 minutes at 37°C. The supernatant containing the cell suspension was removed and filled with complete medium. The remaining tissue was incubated with enzyme cocktail for another 30 minutes at 37°C. If necessary, the tissue was further disintegrated by passing through a 23-gauge needle. Next, all cell suspensions were combined and filtered through a 100-μm cell strainer. After additional washes, cells were cultured in tissue culture flasks and medium was renewed after 1 day and subsequently when necessary. Adhering fibroblasts grew to confluence within some days.
Proteins were purified from intestinal specimens with the M-PER mammalian protein extraction reagent according to the manufacturer’s recommendations (Pierce Biotechnology). Western blotting was performed using the ECL Analysis System (GE Healthcare), as previously described (17). Antibodies were from Cell Signaling Technology (anti-p44/42-MAPK, anti phospho-p44/42-MAPK) or Santa Cruz Biotechnology Inc. (anti-ACTB).
For intestinal wound-healing studies in vivo, mucosal lesions of 800 μm in diameter were generated with a biopsy forceps during mini-endoscopy (20). The wound-healing process was followed by endoscopic evaluations every 1–2 days until the lesion had been resolved.
For the analysis of cell migration in vitro, murine IEC (CMT93) were grown to confluence in serum-free medium. Then, standardized gaps of approximately 650 μm in diameter were scratched into the cell layer using a pipette tip, and the cell-culture system was supplemented as specified. The process of wound gap closure was monitored and quantified by microscopy (IX70; Olympus).
For in vitro proliferation studies, murine IEC (CMT93) were seeded at almost confluence using equal numbers and grown in the presence of various concentrations of recombinant EREG (R&D) or ERK-inhibitors PD98059 (Merck) or U0126 (Merck) and cell numbers were counted at different time points. For in vitro stimulation experiments, TNF-α and IL-6 were purchased from Immunotools; LPS and lipoteichoic acid (LTA) were from Invivogen.
For ex vivo tumor studies, freshly harvested tumors were cut into pieces and equally distributed over tissue culture wells, where they were cultured, as specified (Figure (Figure10),10), i.e., in the presence of EREG and /or different ERK inhibitors.
For fibroblast transfer studies, tumors were induced in Ereg–/– or WT mice by AOM (10 mg/kg) and 1 cycle of DSS (2.5%). At day 30, all animals were endoscopically screened, and then mice with early tumors were equally assigned to the experimental groups. Starting at day 30, local injections were performed as previously described (20, 32). In detail, fibroblasts were injected into growing tumors via a mini-injection needle introduced through the working channel of the endoscope. The fibroblast transfer was performed with 105 cells per injection at volumes of 50 μl, and 4 applications were done within 15 days. The change of tumor load was monitored by endoscopy.
For other in vivo studies, EREG (200 ng, R&D), EGF (200 ng, Immunotools), AREG (200 ng, R&D), or U0126 (20 ng, Merck) was locally injected 4 times in 10 days at volumes of 50 μl.
Significance analysis of normally distributed data was performed using 1-tailed Student’s t test. Error bars represent SD. To reduce the false discovery rate in microarray data, P value correction by Benjamini-Hochberg was applied. P values of less than 0.05 and 0.01 were considered significant and highly significant, respectively.
Animal studies were performed in accordance with German law and with approval from Landesuntersuchungsamt Rheinland-Pfalz, (Koblenz, Germany) and Regierung Mittelfranken (Ansbach, Germany). The collection of human intestinal biopsies was approved by Ethik-Kommission der Medizinischen Fakultät der Friedrich-Alexander-Universität, Erlangen-Nürnberg. All subjects provided informed consent prior to their participation in the study.
We thank P.-L. Tharaux (Paris, France) for sending Ereg–/– animals from his colony. The authors appreciate helpful discussions with U. Luxemburger and M. Kozlowski (Mainz, Germany) regarding microarray analysis. We thank A. Nikolaev (Mainz, Germany), I.-Zöller-Utz, D. Fröba, and S. Wallmüller (Erlangen, Germany) for excellent technical assistance. This study was supported by grants from United European Gastroenterology (to C. Neufert, top abstract prize 2011; M.F. Neurath, research prize 2008), by ELAN-Fonds and IZKF-Fonds of Friedrich-Alexander-Universität Erlangen-Nürnberg (to C. Neufert), and by the DFG (GK1043/2, KFO257 to C. Becker and M.F. Neurath).
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2013;123(4):1428–1443. doi:10.1172/JCI63748.